VIROLOGY

Dr. O'Callaghan MID LECTURES #48 to #52 I. INTRODUCTION, OVERVIEW, AND BRIEF HISTORY (Read Only; Do Not Memorize) Viruses are the major pathogens on the planet Earth and infect virtually every life form from man to animals to insects to plants to microorganisms such as bacteria. There are hundreds, perhaps thousands, of viruses that can infect humans and animals and the outcome of these infections can vary from subclinical (inapparent) infection, to mild disease (common cold; febrile disease), to moderately severe disease (mononucleosis; influenza) to life-threatening disease (hemorrhagic fever; AIDS; rabies) to chronic disease (hepatitis type B) to cancer (lymphoma; hepatocellular carcinoma). The mechanisms of transmission, pathogenesis and outcome of infection vary tremendously for viruses of man and animals. Often one virus can cause a variety of diseases depending on the route of entry, the efficiency of host defense systems and the immunological state of the host; also a given disease or clinical syndrome, such as the common cold, viral pneumonia, viral encephalitis, etc. may be caused by any one of several different viruses. Thus, it is NOT helpful to name a virus for the disease it may cause (such as "common cold virus"). Viruses have victimized mankind since the beginning of recorded history, and the ancient writings of the Egyptians and the Chinese describe diseases that resemble smallpox and poliomyelitis. Indeed, examination of the skeletal remains and mummified remains of Egyptian kings from 5000 BC reveals lesions identical to those of smallpox. Throughout history humans and their animals have suffered from diseases, centuries later shown to be smallpox, yellow fever, rabies, etc. Viral diseases have affected the course of history and the attitude and behavior of man. Yellow fever was a scourge in many parts of the world for centuries and caused great hardships to African trading ships of the l7th and l8th century. In this century, yellow fever retarded the construction of the Panama Canal, which was not constructed until the mosquito was controlled. Because of rabies in animals, man feared animals such as the fox, the bat, and canines. The great influenza pandemics of 1918 affected the population of the world and accounted for millions of deaths--more than the bullets of World War I. If you doubt these relationships between disease and human attitude and behavior, consider how herpes virus type 2 has affected sexual behavior in the l980's and how society today is less tolerant of bathhouses for male homosexuals as Human Immunodeficiency Virus spreads and enters other groups such as prostitutes and heterosexual persons. Although there is some evidence that vaccination was practiced before Jenner's time, Edward Jenner (1749-1823) was the first scientist to record experiments with viral material in 1796 to 1798. His observation that dairymaids, who had acquired cowpox by milking diseased cows, were immune to smallpox lead to the development of the first "vaccine" (vaccinia virus used to induce immunity to smallpox). In 1840, Jacob Henle hypothesized small agents invisible to the light microscope may exist. Louis Pasteur (1822-1895) worked with rabies late in his distinguished career. He developed a vaccine for rabies virus by passaging the agent in the brain of rabbits from one animal to the next. He was not aware that the agent was a "virus". In 1879, Adolph Mayer (1843-1942) showed that a disease he named Tobacco Mosaic Disease (dark and light spots on leaves) could be transmitted mechanically via the sap of diseased plants. In l892 the Russian scientist Dimitri Ivanofsky (1864-1920) demonstrated that the causative agent of tobacco mosaic disease could pass through filters that retarded bacteria . (Feb. 12, 1892.The sap of leaves infected with tobacco mosaic disease retains its infectious properties even after filtration through Chamberland filter candles) His work was not appreciated at the time and he did NOT realize that the "agent" could replicate. In l898, the Dutch scientist Martinus Beijerinck (1851-1931) repeated these experiments and ruled out a bacterial etiology. He called the agent the "contagium vivum fluidum" and showed that it multiplied within the plant even after dilution (it could replicate!!). Thus a "filterable agent" existed that could multiply in the tissues and cause disease, but did not multiple on artificial medium as bacteria can. In l898, Friedrich Loeffler and Paul Frosch , German scientists trained by Robert Koch, demonstrated that foot-and-mouth disease (member of Picornaviridae family) was caused by a filterable agent that could be passaged from animal to animal but not grown on artificial media. First demonstration that a virus caused disease in animals. In l901, Walter Reed (U. S. Army) and his team demonstrated that yellow fever was spread by the mosquito and eventually that the agent was a virus. First human virus to be described. His assistants Dr. Carroll and Dr. Lazear contracted severe yellow fever; Dr. Carroll survived, but Dr. Lazear died. V. Ellerman and O. Bang (1908) showed the cell free transmission of leukemia in chickens, and Peyton Rous (1911) showed a transmissible, filterable agent caused sarcomas in chickens. This virus is called Rous Sarcoma Virus and is the RNA tumor virus (Retroviridae) in which a viral oncogene (vSRC) was shown to have a counterpart in normal cells (cSRC) in 1976 by Harold Varmus and Michael Bishop (Nobel Prize work for discovery of SRC oncogene).

In l9l5 Frederick Twort (British) discovered bacteriophage by observing that bacterial monolayers exhibited "glassy" spots which could be transferred. In l922 Felix d'Herelle (Canadian working at Pasteur inst. In Paris) "rediscovered" bacteriophage, developed a plaque assay for infectious virus, coined the term "bacteriophage", and explained concepts of host range and attachment. In l928-31: Ernest W. Goodpasteur introduced the use of avian embryos (eggs) for propagating viruses. Virology developed as a science as experimental pathologists began to demonstrate that specimens from patients with various diseases would cause specific cytopathology when inoculated into chick embryonated eggs or cell cultures. As the science of growing cells in culture developed, basic knowledge of viruses as pathogenic agents expanded. Isolated viruses were characterized because of the development of the electron microscope and biochemical techniques. Much of the early knowledge of viral biochemistry came from the study of the bacteriophage by Delbruck, Hershey, Chase, and others. A few of the milestones in the development of virology include: 1929 1933 1935 1936 1939 1940 1941 1941 1942 1946 1940s 1949 C. Vinson & A. W. Petre (USA) show that TMV is mainly composed of protein W. Smith, C. Andrews, and P Laidlaw discover and isolate human Influenzavirus. Wendell Stanley; Crystallizes Tobacco Mosaic Virus (Nobel prize in 1946) F. C. Bawden & N. W. Pirie show chemical composition of TMV as 95% protein and 5% RNA G. Kausche, P. F. Ankuch, and E. Ruska first to see a virus (TMV) in the electron microscope. Max Delbruck describes replication of bacteriophage. J. Bernal & I. Fankuchen : X-ray studies on TMV show rod-like structure of protein. George Hirst showed agglutination of RBC by viruses; this allowed rapid detection of many viruses. J. Bittner described mouse mammary tumor viruses and its transmission by milk. K. Sanford & W. Earle (NIH) grow cell culture from single cell; help develop cell culture. Cell culture techniques developed allow cultivation many viruses John Enders, Tom Weller, & P. Robbins demonstrate poliovirus multiplication in non-neural tissues; allowed development of poliovirus vaccines (Nobel Prize work). 1950s Jim Watson (USA) and Francis Crick (Brit) solve structure of DNA (Nobel prize work) 1951 Max Theiler receives Nobel Prize for development of Yellow Fever virus vaccine 1952 George Gey develops cell line known as HeLa cells from human cancer tissue 1952 A. Hershey & M. Chase only the DNA of bacteriophage required for replication. DNA is infectious. 1952 N. Zinder and J. Lederberg discover transduction (bacteriophage transmits bacterial gene). 1954 John Enders, Tom Weller, and Frederick Robbins receive Nobel Prize for cultivation of poliovirus. 1956 H. Fraenkel-Conrat; A Gierer and G. Schramm show that pure TMV RNA is infectious. 1957 A. Issacs and J. Lindenmann discovered interferon. 1957 John Colter showed RNA is the genome of an animal virus and the RNA per se may be infectious. 1958 Joshua Lederberg receives Nobel Prize for work on molecular genetics. 1962 D. Caspar and A Klug show that isometric viruses have icosahedral type of symmetry 1967 T. Diener and W. Raymer elucidate nature of plant Viroids as small non-encoding RNA molecules. 1968 Werner & Gertrude Henle show EBV causes infectious mononucleosis 1969 Max Delbruck, Alfred Hershey, and Salvador Luria receive Nobel Prize for work biochemistry of virus infection and transformation by DNA tumor viruses. 1970 H. Temin and D. Baltimore discovered reverse transcriptase and confirmed Temin's postulation That viral RNA of a Retrovirus is copied to a DNA template by the viral enzyme. Nobel prize 1975 1970 P. Duesberg and P. Vogt show an oncogene is in Rous Sarcoma Virus. 197l D. Nathans et al. use restriction enzymes to map viral DNAsNobel Prize in 1978. 1970's Recombinant DNA technology explodes. Techniques for cloning and sequencing genes developed. 1975 Howard Temin & D. Baltimore (reverse transcription) & R. Dulbecco (tumor virology): Nobel Prize 1976 Harold Varmus & Mike Bishop showed viral (vSRC) oncogene has cellular counterpart (cSRC). 1976 W. Fiers sequences RNA genome of bacteriophage MS2first genome to be sequenced 1976 Baruch Blumberg receives Nobel Prize for work on Hepatitis B Virus. he discovered HBsAg. 1976 D. Carleton Gajdusek receives Nobel Prize for work on Kuru (later shown to be a "prion") 1977 F. Sanger et al sequence 1X DNA genome of bacteriophage phiX174. 1977 Philip Sharp and Rich Roberts et al discover RNA splicing while investigating Adenovirus replication. Their work changes our concept of gene organization. 1977 Last case of Smallpox (in Somali in a person named Ali Maolin) 1978 W. Fiers et al and B. Reddy et al sequence 2X DNA genome of SV40 tumor virus. 1978 R. Erikson SRC oncogene shown to encode a protein kinase. 1978 Daniel Nathans, Hamilton Smith, and Werner Arber receive Nobel Prize for work on restriction enzymes. Arber had proposed their existence, Smith has isolated restriction enzyme and Nathans used a restriction enzyme to map the genome of a DNA tumor virus called SV40.

1980's 1982 1980 1980

R. Gallo et al. isolate human Retroviruses (HTLV-I & II). I. I. Miyoshi and Y. Hinuma show that Gallos HTLV-1 is human cancer virus. Smallpox claimed to be eradicated by W.H. O. Paul Berg & Walter Gilbert (USA) and F. Sanger : Nobel Prize for developing methods to clone and sequence DNA. 1982 Aaron Klug receives Nobel Prize for work on structure of viruses. 1982 D. Panicali & E. Paoletti use vaccinia virus as a vector to express foreign genes to proteins. 1982 J. Summers & W. Mason show Reverse Transcriptase involved in Hepatitis B virus replication. 1982-85: L. Montagnier (French) and R. Gallo (NIH) groups credited for the isolation of "AIDS virus". 1982 Stan Prusiner proposes role for the Prion (small proteinaceous infectious unit) in slow disease. 1990's Oncoproteins of some DNA tumor viruses interact with cellular "tumor suppressor" proteins. 1986 K. Wang; A. Kos; Chin et al show Hepatitis D has Viroid-like structure. 1986 P. Powell-Abel et al Use transgenic plants to obtain resistance to the plant virus TMV. 1989 Choo et al discover Hepatitis Type C virus (Flavivirus-like virus) by molecular techniques. 1990s New herpesvirus discovered and associated with Kaposi sarcoma 1990's Molecular techniques to identify receptors for various animal viruses. Viral genes introduced as transgenes in transgenic animals. Molecular data being applied to understand viral diseases. 1990s Molecular techniques used to introduce human genes into viruses as vectors as a means to repair genetic diseases. Plant virus genes introduced into plants to make plants resistant to viruses. 1990s Protease inhibitors developed to treat AIDS; Coreceptors for HIV-1 discovered. New approaches for drugs to treat AIDS based on X-ray crystallography and molecular studies.

SOME NOBEL PRIZE LAUREATES

1997 1996 Stanley Prusinerprions: theory and years of work Peter Doherty & Rolf Zinkernagel-viral immunology (MHC restriction for CTL to kill virus infected cell). 1993 Kary Mullis invention of PCR methodology 1993 Phillip Sharp and Richard RobertsmRNA splicing and genes organized as exons 1989 Michael Bishop and Harold Varmus: cellular origin of retroviral oncogenes 1988 James Black, Gertrude Elion, & George Hitchings: drug treatment; one drug later became AZT. 1982 Aaron Klug: virus structure and structure of macromolecules. 1980 Paul Berg: Recombinant DNA 1980 Walter Gilbert & Frederick Sanger: DNA sequencing methods 1978 Werner Arber, Daniel Nathans, & Hamilton Smith: discovery and use of restriction enzymes 1976 Baruch Blumberg: hepatitis B; so called Australian antigen as a marker for hepatitis B 1976 D. Carleton Gajdusek: work on Kuru as a slow unconventional viral disease. 1975 David Baltimore, Renato Dulbecco, & Howard Temin: Tumor and molecular virology 1969 Max Delbruck, Alfred Hershey, & Salvador Luria: genetics & replication of viruses. 1966 Peyton Rous: for tumor virology research conducted in 1906-1911!!!! 1962 Francis Crick, James Watson, & Maurice Wilkins: structure of DNA 1954 John Enders, Thomas Weller, & Frederick Robbins: growth of poliovirus in non-neural tissue cells. 1951 Max Theiler: work on yellow fever virus vaccine. Much of our present day understanding of the molecular biology of gene structure and gene regulation, membrane biochemistry, molecular genetics, tumor biology, etc. has come from the study of viruses. For example, research on viruses has led to the discoveries that messenger RNA molecules are spliced and that two key mechanisms for "cancer" are either the mutation of proto-oncogenes to generate oncogenes or the interaction of a viral oncoprotein with a tumor suppressor protein to inactivate the function of this protein. DEFINITION AND TYPES OF VIRUSES Viruses are small, obligate, intracellular parasites that are composed of a nucleic acid ( genome) enclosed within a protein coat ( capsid) and replicate by use of the host cell synthetic machinery. Animal viruses are those that infect and replicate in the cells of animals, including man. Some animal viruses may also replicate in insects such as ticks, mosquitoes, etc. Viruses are a heterogenous class of infections agents ranging in size from approximately l5 to 300 nm (a nanometer is 0.00l micron or l x l0- 9 meter). Viruses are particulate (particles) in nature and have a highly organized structure, which is maintained by noncovalent bonding of viral structural proteins to each other and to the genome. Viruses vary considerably in morphology, chemical composition, and their effect on host cells, yet they all have certain distinctive features, which are unique to viruses. Viruses contain a single type of nucleic acid , either DNA or RNA, as their genetic material ( genome), which is surrounded by a protein coat or shell called the capsid. They multiply only in living cells and are dependent on the host cell's biosynthetic and energy-

generating machinery for reproduction. Some viruses (NOT all) contain enzymes, but these are concerned chiefly with genome translation and replication, i.e. RNA and DNA polymerase, DNAses, etc. Viruses do not contain functional ribosomes, however one family of RNA viruses, the Arenaviridae, contain ribosomes, which are accidentally incorporated during the maturation of the virus. Thus the key features of a virus particle (virion) are that it is a small particle with a highly organized architecture, that it has a genome comprised of RNA or DNA (not both types of nucleic acid), and that the genome is enclosed in a protein shell called the capsid. Some viruses have an envelope (membrane-like covering derived from a cellular membrane). A virus does not have intrinsic motility, cannot respond to stimuli, and cannot "grow" or replicate in the usual sense. Viruses are said to be "filterable agents" because their small size allows them to pass through filters that retard bacteria.

KEY PROPERTIES OF A VIRUS

SMALL: Vary In diameter From 20 To 200 nm (Nanometer = 1x10- 9 M) Obligate Intracellular Parasite, Need A Live Cell For Replication PARTICLE: Particulate In Nature; Not A Liquid Or Jelly-Like Mass Highly Organized Architecture With A Symmetrical Structure: Composed of 1. Nucleic Acid = Genome is either DNA or RNA 2. Capsid = protein shell: Symmetry is either Helical, Icosahedral, or Complex 3. Envelope: SOME viruses, Not All, Have An Envelope; Lipid bilayer from cell membrane REPLICATE by use of cellular enzyme systems and cellular derived precursors. Some (NOT all) viruses have endogenous viral-encoded enzyme(s) within the particle. Replicate by use of cellular enzymes and organelles; cell provides energy & precursors Viral genome is copied to viral mRNA and viral genome is replicated MOST (few exceptions) viruses encode one or more proteins with enzyme function(s)
VIRUS WORLD: Viruses infect animals, insects, plants, and microbes (bacteriophage). Virus classification changes frequently (to hassle students and allow virologists to write new textbooks). Presently viruses are classified into >184 Genera, of which ~161 are classified into 54 different Families. Thus 23 genera are not yet placed into Families. Changes daily. NATURE OF VIRUSES Positive Sense 1X RNA Viruses Negative Sense 1X RNA Viruses Reverse Transcribing (RNA to DNA) Double-stranded RNA Viruses 2X DNA Viruses 1X DNA Viruses FAMILIES 17 families 7 families 3 Families 6 Families 17 Families 5 Families NUMBER OF GENERA 43 Genera (+19 genera not yet in family) 22 Genera 14 Genera 21 genera 52 Genera (2 not yet in family) 16 genera

"UNCONVENTIONAL VIRUSES" OF PLANTS, MAN, AND ANIMALS Evidence indicates that "agents" even smaller and less complex than conventional viruses do exist. These agents are Viroids and Prions. "UNCONVENTIONAL AGENTS" OF PLANTS: VIROIDS A VIROID is a small piece of infectious RNA that is a circular 1X RNA molecule that has a highly organized secondary structure of loops and hairpins with areas of single-strandedness and double-strandedness. The size of this RNA molecule is only 246 to 375 nucleotides.. The VIROID RNA molecule does NOT encode any protein, but uses plant cell enzymes (DNA-dependent RNA polymerase II) to replicate the VIROID RNA molecule. Several dozen viroids have been identified and they have been shown to cause disease in plants. Examples are l) Chrysanthemum stunt viroid, 2) Citrus exocortis viroid, 3) Potato spindle tuber viroid, 4) Coconut cadang-cadang viroid, 5) Tomato planta macho viroid, etc. These infectious RNA molecules actually cause major diseases of great economic importance. Viroids do NOT act as messenger RNAs (do NOT encode protein) and are NOT RNA copies of plant cell DNA sequences (genes).

VIROIDS replicate by a complex mechanism in the plant cell by being copied to a complementary piece of RNA (G copied to C; A copied to U; etc.). The VIROID is said to be "PLUS RNA" while its complementary RNA copy is said to be NEGATIVE RNA (anti-viroid RNA). Plant cells have enzymes (DNA-dependent RNA polymerase II) capable of being used by the VIROID RNA (PLUS) to generate a complementary ("NEGATIVE" or "MINUS" RNA) multimeric RNA intermediate which is then copied to generate multimeric VIROID (PLUS) RNA which is then cleaved to yield individual molecules of viroid (plus) RNA. Thus Viroids are pathogenic, very small RNA molecules that cause diseases in plants . They behave genetically since a substitution of nucleotides within the molecule at key locations can modify virulence (ability to produce disease in the plant). Viroids MAY (??) interfere with proper splicing of cellular messenger RNAs. Thus, the Viroid is a very tiny RNA molecule (only 246 to 375 nt) that initiates disease without the requirement of being translated to protein. Some researchers speculate that Viroid-like agents may exist in man and animals and cause diseases such as slowly developing dementias and CNS disorders. Viroids have a "genetic system" only in the sense that mutations in specific ribonucleotides can affect the virulence of the VIROID. Some mutations result in greater virulence, while other mutations may reduce virulence. Viroids are transmitted from plant to plant by man and his tools. Some VIROIDS can replicate in a large number of plant species, while other Viroids have a limited host range. Viroids take a large toll on many crops and are economically important as some damage tomatoes, potatoes, flowering plants. A few VIROIDS have been shown to have "self-cleaving" enzymatic activity--that is the VIROID RNA molecule can cleave its own RNA multimeric units to monomeric units "UNCONVENTIONAL VIRUSES" OF MAN AND ANIMALS: "SLOW VIRAL DISEASES" "Slow viral diseases" refers to a group of "viral diseases" that have a prolonged incubation period (time required for the disease to appear after the initial infection). Often these diseases affect the central nervous system and are fatal. Slow viral diseases may be caused by "conventional viruses" such as the measles virus (subacute sclerosing panencephalitis), the rubella virus (progressive rubella panencephalitis), etc. Also, some "slow viral diseases" of man and animals are due to "Unconventional Agents" which do not have the properties of conventional viruses. These "Unconventional Agents" are NOT viruses and have remarkable properties such as: Remarkably stable and resistant to many chemical and physical agents that destroy conventional viruses. No recognizable morphology similar to conventional viruses; are NOT "particles" per se. No antigenicity (do not induce interferon or antibody or lymphocyte response). Extremely small in size. Molecular composition and "replication" are not fully understood yet. Appear to be protein encode by HOST CELL GENE that becomes "altered" in nature. The best known of these "Unconventional Agents" are those that cause Kuru (man; kuru = trembling)), Creutzfeldt-Jacob disease (man), Scrapie (sheep), and Mink Encephalopathy (mink). These agents are filterable (small in size; pass through a filter that retards bacteria), can replicate (able to increase in titer in the host), and cause disease with an extremely long incubation period ("slow disease"), which are a group of chronic, progressive, always fatal infections of the CNS of man and animals, known as the subacute spongiform encephalopathies. Some have been adapted to replicate and cause disease in animals such as scrapie in the mouse or hamster and Creuzfeldt-Jacob ("CJ") in lower primates. "Subacute Spongiform Encephalopathy" refers to several lethal neurodegenerative diseases that have similar clinical and pathological features and causative agents: These diseases are: Scrapie of sheep and goats: Scrapie is the prototype of Subacute Spongiform Encephalopathies Bovine Spongiform Encephalopathy (BSE = "mad cow disease") Mink and Feline encephalopathy Wasting disease of deer and elk Kuru of humans (Fore tribe in the highlands of New Guinea; transmitted by ritualistic cannibalism) Creutzfeldt-Jakob Disease (CJD) of humans (10% are autosomal-dominant transmission; 90% are sporadic). Gerstsmann-Straussler-Scheinker syndrome (GSS) of humans (form of CJD) Fatal Familial Insomnia (FFI) syndrome of humans Basic lesion in all of these diseases is a progressive vacuolation in neurons, and to lesser extent in astrocytes and oligodendrocytes, an extensive astroglial hypertrophy and proliferation, and finally a spongiform change in the GRAY MATTER of the CNS. Patient shows ataxia; brain has characteristic plaque lesions and extracellular collections of proteinaceous material. CDJ has been transmitted iatrogenically (cornea transplants; injection of growth hormone from human pituitaries).

1987: Mad Cow Disease occurred in U. K. due to cattle being fed bonemeal from sheep with scrapie. Transmission of BSE to macaque primates suggests that BSE may have been transmitted to humans!! Asypical CJD cases in human in U.K. indicate transmission from BOVINE to HUMAN. CJD occurs in human (1 per million); Can be transmitted via neurosurgery, corneal transplants, etc. GSS syndrome and FFI syndrome are TWO FAMILIAL forms of CJD due to an autosomal dominant genes. Mutation in cellular gene encoding a brain protein can lead to production of the PRION PROTEIN. 1982: Stan Prusiner proposed the PRION Hypothesis: Unconventional Agent is an altered cellular protein (NO genome) called the PRION, an ISOFORM of a normal brain protein of 27,000 to 35,000 encoded by cellular gene. Spongiform disease is due to the accumulation of an altered, malfolded form of this cellular protein in the brain. The protein called PrP c can change its conformation to an ISOFORM ( PrP Sc) that is highly resistant to proteases, etc. Prusiner awarded Nobel Prize in 1997 for pioneering work & theory. Thus these PRION diseases are caused by the CONVERSION of a cellular brain protein to an altered form of the same protein called the PRION that is malfolded, highly stable, not readily degraded, and in the environment is resistant to conditions of heat, chemicals, and radiation that kills conventional viruses. In general, disease may result by mutation in endogenous protein that converts PrP c to PrPSc (a genetic autosomal dominant disease; some cases of CJD; cases of FFI and GSS)) or by infection (exposure) with exogenous PRION such as by cornea transplant (CJD), eating contaminated meat (Mad Cow) or even human flesh (kuru). Brains of "scrapie infected: animals show SCRAPIE-ASSOCIATED FIBRILS (SAF's) composed of PRIONS. A PRION is an "infectious protein" of 33,000 to 35,000 encode by a HOST GENE on HUMAN CHROMOSOME #20 (Chromosome #2 in the mouse). The "normal" protein is present in membranes of neurons, but the PRION is an "altered conformational" form ( "ISOFORM") of the normal cell protein that somehow mediates disease. PRION protein can somehow cause its NORMAL COUNTERPART PROTEIN to become altered and lead to disease. Thus, the PRION is said to be "infectious" HOW CAN THE PRION BE MADE AND REPLICATE ?: THEORY: PRION protein can interact with the NORMAL BRAIN PROTEIN (its counterpart) and cause it to SWITCH CONFORMATION to become the PRION ISOFORM which is highly resistant to proteolysis. Perhaps, a heterodimer structure is formed. This Switching Of Conformation mediated by the PRION then occurs exponentially in the brain leading to a build up of PRION PROTEIN that interferes with brain function and normal neuron morphology. PRION does NOT contain a GENOME (no nucleic acid). Experiments using PRION TRANSGENES in mice and hamsters (transgenic rodents) support the PRION THEORY. VIRINO HYPOTHESIS: "Virino" term refers to hypothetical agent composed of a nucleic acid (genome) enclosed in a host-encoded protein. This hypothesis states that there must be a nucleic acid, which has yet to be discovered, and it induces a protein that somehow disrupts regulation of cellular function(s). VIROID: An infectious RNA that does NOT encode a protein yet can cause disease in plants. Could there be a VIROID-LIKE AGENTS of humans? Hepatitis Delta Virus is actually similar to VIROID in structure (circular, small RNA molecule) but it encodes TWO PROTEINS from one OPEN READING FRAME, but the Delta Agent requires Hepatitis B Virus as a HELPER for its maturation. PRION THEORY: An altered form (ISOFORM) of a cellular protein that can interact with its "normal" cellular counterpart (normal protein) and somehow convert the normal protein to the altered form which is highly resistant to proteolysis, builds up in the brain, & causes the lesions and abnormal CNS function. II. STRUCTURE OF ANIMAL VIRUSES (Real Viruses): N ucleic acid genome contains the information necessary for virus replication, surrounded by a protective protein shell called the capsid. Genome plus the capsid is referred to as the nucleocapsid, and for some animal viruses the nucleocapsid is the complete virus particle or virion. Some viruses have an envelope composed of lipid and viral-specified glycoproteins. Envelopes have small spike-like glycoprotein structures termed peplomers, on their surfaces; these are transmembrane proteins. ENVELOPE is a lipid-bilayer structure derived from a CELL MEMBRANE and is modified to have VIRAL-ENCODED glycoproteins inserted as TRANSMEMBRANE structures called PEPLOMERS. Some virus contains enzymes within the VIRION. PROPERTIES OF ANIMAL VIRUSES SMALL: 20 to 200 nm (nanometer = 1xl0 -9 Meters) OLIGATE INTRACELLULAR PARASITE-- needs a live cell for its replication. PARTICLE: Particulate in nature; HIGHLY ORGANIZED ARCHITECTURE: Symmetrical structure 1. Nucleic Acid = Genome 2. Capsid = protein shell with a special symmetry 3. Envelope: Not All Viruses Have An Envelope Replicate Using Cellular Biosynthetic Systems; Key Enzymes For Genome Replication = Viral Encoded Some Viral Particles (Virion) Contain Enzymes

A. GENOME: Genome is either RNA or DNA; either single-stranded (lX) or double-stranded (2X). Most DNA viruses have a genome of a one linear, double-stranded DNA molecule. Genome of Papovaviridae is a 2X circular configuration; Parvoviridae consists of a linear, single, lX strand of DNA. Genome of Type B hepatitis virus, Hepadnaviridae, is a circular DNA molecule that is partially 2X and partially lX. RNA viruses have great variability in the nature of the RNA genome: some RNA viruses (e.g. rabies, mumps, measles, and polio) have one molecule of lX RNA as the genome; some viruses have several molecules of lX RNA as the genome such as the influenzaviruses (Orthomyxoviridae; 8 different molecules of lX RNA); some viruses such as those in the Reoviridae family have l0 to l2 pieces of 2X (double-stranded) RNA as the genome. The size (molecular weight) of animal virus genomes varies greatly. Parvoviridae possess a lX DNA molecule with a MW of l.5 x l0 6 daltons enough to code for 3 to 4 small proteins. The 2X DNA genome of the Poxviridae is l60 to 200 million daltons (megadaltons = million daltons = md), and the viral genome may encode for 200 or more proteins. DNA genomes vary greatly in size, strandedness (lX or 2X), and conformation (linear or circular). Genomes of RNA viruses vary in size also: Picornaviridae members have a lX RNA genome of 2.5 md which encodes for l2 to l5 viral proteins (structural + nonstructural), while Colorado tick fever virus (Reoviridae) has a genome of l2 pieces of 2X RNA with a total M.W. of approximately l5 md. FAMILY ENVELOPE CAPSID GENOMIC DNA POXVIRIDAE YES complex 2X, linear, 160 md; some are crosslinked PAPOVIRIDAE NO COS 2X, circular, 5 md; supercoiled HERPESVIRIDAE YES ICOS 2X, linear 100 md may form isomers ADENOVIRIDAE NO ICOS 2X, linear 23 md; viral protein at each 5end PARVOVIRIDAE NO COS 1X, linear; 1.8 md; hairpin ends HEPADNAVIRIDAE HBsAg complex Partially 2X; 1.5 md; protein linked to minus strand Replicates via RNA using Reverse Transcriptase PROPERTIES OF RNA GENOMES (DO NOT MEMORIZE) RNA FAMILY RNA Molecule(s) SIZE (nt)) FEATURES OF GENOME Replicate PICO 1X One 7,431 VPg Protein at 5' end; Poly A tail PLUS CALICI 1X One 8,400 Protein at 5' end; Poly A tail PLUS FLAVI 1X One 10,862 Cap at 5' end PLUS RHABDO 1X One 11,162 Negative TOGA 1X One 11,700 Cap at 5' end; Poly A tail PLUS FILO 1X One 12,700 Negative PARAMYXO 1X One 16,600 Negative CORONA 1X One 20,000 Cap at 5 end; Poly A tail PLUS RETRO 1X Diploid 8.500-10,000 Cap at 5' end; t RNA; Poly A tail Retro ARENA 1X Two 7,500; 3,500 2 RNA molecules Negative BUNYA 1X Three 8,480; 4,460; 900 3 Circular RNAs Negative ORTHOMYXO 1X Eight (2,341 to 892) 8 molecules of RNA Negative REO 2X 10 to 12 (4,500 to 1,198) 5' cap on + strand Negative ASTRO 1X One 7,500 PLUS DELTAVIRUS 1X Circular 1,700 NEEDS HELPER HBV need HBV B. CAPSIDS The capsid is the protein shell that contains and protects the viral genome. The genome together with the capsid is called the nucleocapsid . For viruses that do NOT have an envelope, the nucleocapsid is the complete virus particle (VIRION). The capsid is comprised of viral structural protein(s) that are present in repeated copies; these viral protein molecules are the chemical units (protomers) that bind to each other by non-covalent bonds (hydrogen bonds, hydrophobic bonds, ionic bonds, van der Waals forces) and form the capsid. The capsid protein molecules (protomers) are encoded by the viral genome, and the size and overall architectural symmetry of the capsid is determined by the primary amino acid sequence of the viral protomers. Capsids have a highly ordered architecture and exhibit symmetry. Three types of symmetry are: l) Helical, 2) Icosahedral (Cubic Symmetry), and 3) Complex (Unknown) Symmetry. CAPSID SYMMETRY OF ANIMAL VIRUSES 1. HELICAL SYMMETRY: ALL viruses whose CAPSID has HELICAL SYMMETRY are 1X RNA viruses and ALL have an envelope.

2. ICOSAHEDRAL SYMMETRY: Many viruses were shown to be ISOMETRIC--spherical in appearance and the three axes in each direction are equal. The CAPSID of these isometric viruses was shown to have ICOSAHEDRAL symmetry. Some DNA viruses and some RNA viruses have ICOSAHEDRAL SYMMETRY. Some viruses with an ICOSAHEDRAL capsid are enveloped, some lack an envelope. 3. COMPLEX SYMMETRY (really means we do NOT understand the symmetry). If the CAPSID is said to have COMPLEX SYMMETRY, it simply means that we do not yet understand the nature of the symmetry or the exact organization as to how the viral polypeptides are arranged to form the capsid. The nature of the capsid structure of the POXVIRIDAE, RETROVIRIDAE, and HEPADNAVIRIDAE is not understood.

l. HELICAL SYMMETRY : The capsid is comprised of many copies of a single (one) species of structural viral protein (protomer), which binds to each other (protein-protein noncovalent bonding) and to the lX RNA genome (RNA-protein noncovalent bonding) to form a helical structure that appears cylindrical in shape in the electron microscope. PROTEIN-PROTEIN bonding and the PROTEIN-RNA bonding are by NON-COVALENT bonds. HELICAL CAPSID has a single axis of rotational symmetry; the diameter is determined by the nature of the protein-protein interactions of the viral protomers (single species of structural protein), and the length is dependent on the molecular size (length) of the lX RNA genome. ALL viruses with helical capsids have 1X RNA as the genome and are ALL are enveloped viruses. In some cases, the virus has a "FRAGMENTED GENOME, comprised of several viral RNA molecules. For example, the GENOME of the ARENAVIRIDAE is comprised of TWO 1X RNA molecules, each being in a SEPARATE HELICAL CAPSID. The BUNYAVIRIDAE GENOME is comprised of THREE MOLECULES of 1X RNA, each in a separate HELICAL CAPSID that is comprised of the SAME CAPSID PROTEIN. The ORTHOMYXOVIRIDAE (Influenzavirus Type A) has a GENOME of EIGHT MOLECULES of 1X RNA, each RNA molecule being in a separate HELICAL CAPSID. a) length of the nucleocapsid (capsid + RNA genome), determined by the length of the viral RNA molecule. b) diameter of the nucleocapsid, determine by the size of the capsid protein c) pitch = which is the distance from one turn of the helical structure to the next turn of the helical capsid. Helical capsids are comprised of only one species of viral polypeptide (protein) which is repeated many times and which forms NONCOVALENT bonds of RNA-capsid protein and capsid Protein-Capsid Protein FAMILIIES OF RNA VIRUSES WITH HELICAL CAPSIDS (capsid has helical symmetry) Orthomyxoviridae 8 capsids 30 to l00 nm in length 60,000 MW protein Paramyxoviridae l capsid l,000 nm in length 56,000 MW protein Rhabdoviridae l capsid 3500 nm in length 50,000 MW protein Bunyaviridae 3 capsids 200 nm, 5l0 nm, 700 nm 23,000 MW protein Arenaviridae 2 capsids 640 nm and 1300 nm 65,000 MW protein Coronaviridae l capsid 120 nm length 60,000 MW protein Filoviridae l capsid 800 nm length (to l4,000 nm) 40,000 MW protein ICOSAHEDRAL SYMMETRY : Type of CUBIC SYMMETRY used by ISOMETRIC capsids. This capsid appears quasi-spherical (ISOMETRIC; all axes from the center appear as equal like a SPHERE), but have regular features that indicate they are polyhedra rather than spheres. These viruses are sometimes termed isometric since they have identical linear dimensions along their axes. The type of symmetry for a "spherical virus" (ISOMETRIC) is limited to cubic symmetry because the three coordinate directions in space are identical. Of the FIVE possible types of Platonic POLYHEDRA that exhibit CUBIC SYMMETRY, the ICOSAHEDRON is the TYPE USED BY VIRUSES. Of the Five polyhedra with cubic symmetry, the icosahedral shell is the most efficient and stable form that can be constructed from identical basic units, and this is the only type of polyhedral structure that has been identified in viruses. An icosahedron is a solid figure that has 20 faces, each being an equilateral triangle, and 12 vertices or corners. An ICOSAHEDRON has 20 faces, 30 EDGES, 12 VERTICES . An ICOSAHEDRON exhibits AXES of 2-fold, 3-fold and 5-fold symmetry. 15 AXES of 2-fold symmetry: Located at each edge. View at each edge, you can turn the Icosahedron 180 degrees and it appears identical. 10 AXES of 3-fold symmetry: Located in center of each of the 20 faces. View from center of each face, you can turn 120 degrees and it appears identical. 6 AXES of 5-fold symmetry: located at each VERTEX. View from each VERTEX, you can turn an icosahedron 72 degrees and it appears identical. Some viruses with an ICOSAHEDRAL CAPSID are DNA viruses (Herpesviridae, Adenoviridae, Papovaviridae, Parvoviridae); some are RNA Viruses (e.g. Picornaviridae, Reoviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae). Some have an ENVELOPE; some do NOT have an ENVELOPE. Often the viral GENOME (RNA or DNA) is condensed and forms the central portion or core located within the capsid. The genome may be associated with internally located viral protein(s) of one or more species of viral protein to form a core. Thus the genome is folded and condensed within the capsid structure. The icosahedral shell may be composed of repeated copies of one species of viral protein for some viruses such as Rubella (Togaviridae) etc., or of repeated copies of different viral proteins for other viruses such as poliovirus (Picornaviridae), Herpesviridae, etc. See PowerPoint slides. The structural units that form the shell may be a single protomer or several different protomers tightly bonded (noncovalently).

ICOSADELTRHEDRAL SYMMETRY : An ICOSADELTAHEDRON is an icosahedron whose 20 faces are subdivided into subtriangles. Icosahedral capsid architecture becomes very complicated as many viruses as each face of an icosahedron may be further subdivided into a given number of subtriangles. This is called the TRIANGULATION NUMBER (T number) . For example, the capsid of the Togaviridae (rubella virus) has a T Number of 4, thus each of the 20 faces is subdivided into FOUR TRIANGLES. The HERPESVIRIDAE capsid has a T Number of 16, thus each face is divided into 16 triangles. CAPSOMERS: Morphological Subunits Of Icosahedral (Icosadeltahedral) Capsids: The clustering of these proteins (structural units) results in the formation of capsomers which are morphological subunits that can be observed in the electron microscope. The number of capsomers depends on the arrangement of viral proteins. The number of capsomers is helpful in virus identification. For example, the Adenoviridae capsid has 252 capsomers, the Herpesviridae capsid has l62 capsomers, the Papovaviridae capsid has 72 capsomers, etc., yet all are icosahedral capsids. Icosahedral viruses may /may not have an envelope, icosahedral viruses may contain RNA or DNA. 3. COMPLEX SYMMETRY Some viruses (e.g. Poxviridae) have a very complex symmetry, which has not been resolved at this time. C. ENVELOPE: An envelope is a MEMBRANE structure that surrounds the capsid (nucleocapsid) of some viruses. The envelope is derived from a "modified" portion of a cellular membrane such as the plasma membrane (many viruses), the endoplasmic reticulum membrane (e. g. Arenaviridae) the Golgi membrane (e. g. Bunyaviridae), or nuclear membrane (Herpesviridae). Inserted into the "modified area" of the cell membrane (destined to become the viral envelope) are viral glycoproteins that are TRANSMEMBRANE GLYCOPROTEINS that will be organized as spike-like structures called PEPLOMERS. Under the ENVELOPE of some enveloped (not all) viruses is another structure called the MATRIX PROTEIN. The lipid bilayer nature of the viral envelope makes the viral envelope susceptible to being disrupted by detergents, soaps, and disinfectants. ALL viruses with a HELICAL CAPSID have an envelope. Some, but not all, viruses with an icosahedral capsid have an envelope. Several families of animal viruses with an icosahedral capsid do not have an envelope and exist as naked nucleocapsids. This envelope is derived from the host cell and is acquired by the virus as it matures and emerges from the cell. The lipid composition of the viral envelope reflects the lipid composition of the host cell membranes. During infection viral-specific proteins and glycoproteins are inserted into the membranes and these areas of the modified cell membrane are destined to form viral envelopes. All enveloped viruses are sensitive to lipid solvents and can be inactivated by ether, chloroform, and other lipid solvents. Thus, the envelope is a unit membrane type of structure. All animal viruses obtain their envelope from a cell membrane such as the nuclear membrane (Herpesviridae), Golgi (Bunyaviridae, Coronaviridae), E.R. (Arenaviridae) or plasma membrane (Orthomyxoviridae, Paramyxoviridae, Retroviridae, Rhabdoviridae, etc). The acquisition of the envelope structure of some viruses such as the Poxviridae is quite complex and involves several steps. D. PEPLOMERS: Viral envelopes have specialized structures associated with them called Peplomers. Peplomers are spike-like structures that protrude from the envelope and are clusters of viralencoded glycoprotein(s) that are transmembrane in nature. These glycoproteins are transmembrane proteins that have a small cytophilic portion (internal) that binds to the viral capsid protein (or Matrix = M protein; see below), a very hydrophobic portion that extends through the lipid bilayer (transmembrane), and a large external portion that has the oligosaccahride chains. The external portion contains the sites to which oligosaccharide chains are covalently attached. Usually, oligosaccharides chains are "N-linked", which involves covalent bonding of the oligosaccharides to certain Asparagine residues in the amino acid sequence (Asparagine - X - Serine or Threonine). In some viruses, some of the oligosaccharide side chains are also "O-linked" to serine or to threonine. The number of oligosaccharide chains varies for different viral glycoproteins; most range from 2 to 7 oligosaccharides per protein. Some, such as the peplomer of AIDS virus (HIV-1) has numerous oligosaccharide side chains. The glycosylation of the peplomer protein is mediated by cellular enzymes and cellular organelles (Endoplasmic reticulum and Golgi compartments). The protein per se is viral-encoded. The process of Glycosylation was actually defined by study of viral glycoproteins. The viral protein enters the ER due to a SIGNAL SEQUENCE and has "simple" oligosaccharides added by cellular enzymes and then is transported through the Golgi compartments for processing and addition of other oligosaccharides by cellular enzymes. The peplomers of animal viruses vary in size and morphology. They range in overall size from 4 nm to 20 nm; most appear to be a spike-like structure, some appear club-shaped, and the peplomers of Coronaviridae are large 20 nm petal-shaped structures. Some viruses (e. g. Orthomyxoviridae, Herpesviridae) have more than one type of peplomer. Peplomers of the envelope are vitally important to the virus and mediate several key steps in the virus replication cycle. Examples are:

a) Attachment: One peplomer type is the attachment apparatus of the virion and it interacts with specific receptor molecules on the surface of cells. Also, many enveloped viruses agglutinate red blood cells because the peplomer binds to RBC's and a lattice of virus-RBCs is formed; this allows a Hemagglutination test to be used to detect and quantitate the virus and an HI (Hemagglutination Inhibition) test to be used to detect and measure antibody to antigenic sites on the peplomer. Thus, the tropism (host range and type of cells and organs susceptible to the virus) of an ENVELOPED virus is determined by the nature of the attachment peplomer; the virion cannot attach to cells that lack virus-specific receptor sites. b) Penetration : A peplomer of the ENVELOPED mediates the penetration of the virus into the cell. In some cases, the viral glycoprotein that comprises the peplomer must be cleaved by a cellular proteinase to "activate" the peplomer. This is a factor in the tropism of the virus. For some viruses, there is only species of peplomer and it mediates BOTH Attachment and Penetration. Some viruses have TWO or more species of pepolmers and one peplomer mediates Attachment, while a second peplomer species mediates Penetration. For example, mumps virus (Paramyxoviridae) has two types of peplomers called the HN peplomer and the F peplomer. The HN peplomer mediates ATTACHMENT, while the F peplomer mediates Penetration by a fusion mechanism. The F peplomer must be CLEAVED into two components by a host cell surface protease in order for the mumps virus to enter the host cell. Thus, for a given cell to be infected by mumps virus, the cell must have the correct RECEPTOR for the HN peplomer to ATTACH and the cell must have the "correct" surface protease to cleave the F peplomer for Penetration to occur. c) Neuraminidase: Some viruses (influenzavirus, mumps, etc.) have a viral-encoded neuraminidase on one peplomer. This viral enzymatic activity digests sialic acid and thus is a virulence factor since this activity allows the virus to digest mucus present in the upper respiratory tract, allowing the virus to reach the surface epithelial cells... Some Coronaviruses have a peplomer with enzymatic activity related to neuraminidase. d) Cytopathology : Some peplomers help determine the cytopathic effects (CPE) of infection. The peplomers becomes embedded in the plasma membranes of the infected cells and cause the cells to "fuse" and form syncytium. Thus, "giant cells" observed in tissues and cells infected with some viruses formed because virus peplomers were inserted in-to the plasma of infected cells. Also, the inserted peplomer may be a target for antibodies and/or T lymphocytes, which can then attack and lyse the infected cell. e) Hemagglutination : As mentioned above, some enveloped viruses can Hemagglutinate RBCs due to peplomer. This allows the development of both a Hemagglutination Assay to detect and quantitate the virus particles and a Hemagglutination Inhibition (HI) Assay to detect antibody (that blocks hemagglutination) in the sera of patients. The H. I. assay is an important assay in diagnostic virology. e) Hemolysis: Some peplomers interact with RBCs and lyse them. f) Maturation: Peplomers as transmembrane glycoproteins play a role in virus maturation. Their presence in the membrane helps "vector" the nucleocapsid to the specific area of the cell membrane so that interaction of the peplomer protein and nucleocapsid (or M protein) can trigger the process of envelopment (the cell membrane with the embedded peplomer proteins will become the viral envelope). E. SPECIALIZED COMPONENTS OF SOME VIRUSES (See PowerPoint Slides) 1. MATRIX PROTEIN (M Protein): Some (NOT all) enveloped viruses have a thick layer of a viral Matrix Protein (M protein) that lies beneath the envelope. The M protein lies between the nucleocapsid and the envelope structure; it gives rigidity to the virion and serves a role in the maturation process. In the maturation process, the M protein "recognizes" areas of the cellular membrane that contain transmembrane glycoproteins (peplomers) and the M protein binds to the small portion of the glycoproteins protruding into the cytoplasm; later, the viral nucleocapsid(s) will bind to the M protein and this triggers the envelopment process. Some viruses that have an M protein layer are Orthomyxoviridae, Rhabdoviridae, Paramyxoviridae, etc. 2. ENZYMES: Many, but not all, viruses contain one or a few viral "structural proteins" (in the virion per se) that have enzymatic activities important in virus replication. These enzymes do NOT make up the capsid itself but are usually packaged into the virion. Neuraminidase is actually located on one PEPLOMER species such as the NA Peplomer of Influenzavirus Type A. Some examples are: l) ALL Negative-Stranded RNA viruses contain an RNA-dependent-RNA polymerase activity that copies the genomic RNA to synthesize viral messenger RNAs. 2) Neuraminidase is an enzyme located on one peplomer type of some RNA viruses. 3) ALL Retroviridae virions contain several enzymatic activities (RNA dependent DNA polymerase), RNase H, viral protease, and integrase) 4) Poxviridae contain several enzymatic activities important in viral DNA replication and transcription of DNA to mRNAs. 5) Reoviridae contain an RNAdependent RNA polymerase and viral encoded enzymes that add the 5' cap to viral mRNA and methylate mRNA. 6) Influenzaviruses contain an endoribonuclease) that cleaves the 5' cap end of cell mRNA to yield a l4 nucleotide sequence used as a primer for viral mRNA synthesis as well as an RNA-dependent RNA

polymerase 7). Hepadnaviridae (Hepatitis B virus) contain an RNA-dependent DNA polymerase (reverse transcriptase) as these DNA viruses replicate via an RNA intermediate by a unique mechanism. The above is a discussion of viral proteins in the VIRION that are enzymes. Virtually every virus has genes that encode for nonstructural proteins (present only in infected cell during replication), which have one or few enzymatic activities. 3. FIBERS OF ADENOVIRIDAE: At each of the l2 VERTICES of the Adenoviridae capsid there is a long, thin fiber (NOT called a peplomer) protruding out composed of viral protein; this filamentous projection is responsible for hemagglutination of RBC and attachment of virus to the cell, but it does NOT contain the major neutralization antigenic site. The Adenovirus FIBER IS NOT a peplomer. 4. CELLULAR RIBOSOMES IN ARENAVIRIDAE: During Arenaviridae envelopment, cellular ribosomes become enveloped within the virion. These 20 nm ribosome structures give the virus the appearance of having "grains of sands" when thin cross-sections of virus are examined in the electron microscope; hence the viruses are called "arena" viruses (arena = sand). These entrapped ribosomes and their constituent ribosomal RNAs do NOT play a role in virus replication 5. M2 Channel Protein Of Influenzavirus Type A: Influenzavirus Type A virions encode an M2 channel protein that is inserted into the ENVELOPE of the virion. Copies of this M2 protein form a CHANNEL in the virion envelope and also are inserted into cell membranes of the infected cell. This channel allows H+ ions to enter the virion as part of the UNCOATING process within lysosomes or endosomes. The anti-viral drug to treat Influenzavirus Type A infections is AMANTADINE HCl; this drug interacts with the M2 channel protein and PREVENTS UNCOATING by blocking the H+ ions from entering and lowing the pH. III. CLASSIFICATION OF ANIMAL VIRUSES : An International Committee on Taxonomy of Viruses is responsible for classifying and naming viruses. Goal is to classify viruses into Families, Subfamilies, and Genera on the basis of physical, biochemical, morphological and antigenic properties such as: l. Nucleic Acid: Type (RNA or DNA), size, structure (linear, circular, 1X or 2X), base composition, etc. 2. Morphology: Size, type of capsid symmetry, number of capsomers for the icosahedral viruses or diameter of the helix for helical viruses, presence or absence of envelope, unusual features. 3. Antigenic properties: Many viruses possess a common group-specific antigen as well as specific antigens for the various subgroups and serotypes. 4. Molecular Biology of Replication: Mechanisms of viral transcription, presence of viral enzymes and mechanisms of viral genome replication are all important parameters. 5. Homology of Viral Genomes: Exact relatedness of viruses will be determined by computer analyses of sequence data, etc. 6. Cytopathic effect, including inclusion body formation: Intranuclear inclusions are characteristic of infection with Herpesviruses, while Poxviruses produce intracytoplasmic inclusions, Some inclusion bodies contain virus particles, but others are "scars" produced during viral replication. Multinucleated giant cell formation is characteristic of certain Paramyxoviruses and Herpesviruses. Cell necrosis is the most prominent feature of adenovirus and poliovirus infections. Chromosome damage may occur with many viruses. A Family is a collection of genera with common characters and the ending of the name of a viral Family is viridae. The name of Subfamilies end in virinae. A virus Genus is a collection of species (serotypes) sharing common characters and the ending is virus. Efforts are being made to develop a Latinized, universal nomenclature. Individual viruses within a genera may vary considerably in antigenic properties and need not cross-react immunologically. As our knowledge of the structure, molecular biology, and biochemistry of viruses increases, virus classification is revised and expanded.

VIRUS CLASSIFICATION: MAJOR FAMILIES DNA VIRUSES RNA VIRUSES

1. 2. 3. 4. 5. 6. POXVIRIDAE HERPESVIRIDAE PAPOVAVIRIDAE ADENOVIRIDAE PARVOVIRIDAE HEPADNAVIRIDAE 1. PICORNAVIRIDAE 2. REOVIRIDAE 3. TOGAVIRIDAE 4. BUNYAVIRIDAE 5. ORTHOMYXOVIRIDAE 6. PARAMYXOVIRIDAE 7. RHABDOVIRIDAE 8. ARENAVIRIDAE 9. CORONAVIRIDAE 10. RETROVIRIDAE 11. CALICIVIRIDAE 12. FLAVIVIRIDAE 13. FILOVIRIDAE 14. ASTROVIRIDAE 15. DELTAVIRUS genus only)

Memory Help
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FAMILIES OF VIRUSES THAT INFECT HUMANS FAMILY SUBFAMILY GENUS EXAMPLE ENVELOPE DIAMETER GENOME CAPSID

Single-stranded RNA Viruses, Plus-stranded

Picornaviridae polioviruses, No 1X RNA ICOS Echoviruses Coxsackieviruses Aphthovirus foot-and-mouth disease Cardiovirus Mengo virus 28-30nm 7.2 - 8.4kb Hepatovirus hepatitis A virus Rhinovirus human rhinovirus 1A Parechovirus Some former Echoviruses ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Caliciviridae Calicivirus Norwalk virus No 1X RNA ICOS 30-38nm 7.5kb ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Astroviridae Astrovirus human astrovirus 1 No 1X RNA ICOS 28-30nm 7.9kb ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Coronaviridae Coronavirus human 229E virus Yes 1X RNA Helical 120-140nm 20-30kb ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Flaviviridae Flavivirus yellow fever virus Yes 1X RNA ICOS Pestivirus bovine virus diarrhea virus 45-60nm 9-12 kb Hepacivirus hepatitis C virus ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Togaviridae Alphavirus EEE, WEE Yes 1X RNA ICOS Rubivirus rubella virus 170nm 11kb ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Enterovirus

FAMILIES OF VIRUSES THAT INFECT HUMANS FAMILY SUBFAMILY GENUS EXAMPLE ENVELOPE DIAMETER GENOME CAPSID

Single-stranded RNA Viruses, Negative-stranded Paramyxovirinae Respirovirus parainfluenza virus 1 &3 Yes 1X RNA Helical Morbillivirus measles virus Rubulavirus mumps virus 150-300nm 16-20kb Unclassified Nipah and Hendra viruses Pneumovirinae Pneumovirus respiratory syncytial virus -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Rhabdoviridae Lyssavirus rabies virus Yes 1X RNA Helical Vesiculovirus vesicular stomatitis virus 75 x 150nm 13-16kb -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Filoviridae Filovirus Marburg virus Yes 1X RNA Helical 800 x 80nm 19kb -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Orthomyxoviridae Influenzavirus Influenza A + B viruses Yes 1X RNA Helical A,B 80-120nm 8 or 7 Influenzavirus C Influenza C virus pieces -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bunyaviridae Bunyavirus Bunyamwera virus Yes 1X RNA Helical Nairovirus Nairobi sheep disease 80-120nm 3 pieces Phlebovirus sandfly fever Sicilian virus Hantavirus Hantaan virus -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Arenaviridae Arenavirus LCM; Lassa fever Yes 1X RNA Helical 110-130nm 2 pieces Non-defined Deltavirus delta hepatitis agent Yes 1X RNA ICOS from HBV 1.7kb ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Unclassified Bornaviridae Borna disease virus yes 1X RNA ? 70 - 130 Paramyxoviridae

FAMILIES OF VIRUSES THAT INFECT HUMANS FAMILY SUBFAMILY GENUS EXAMPLE ENVELOPE DIAMETER GENOME CAPSID

Double-stranded DNA Viruses Chordopoxvirinae Orthopoxvirus vaccinia virus Yes 2X DNA Complex Parapoxvirus orf virus Avipoxvirus fowlpox virus Capripoxvirus sheeppox virus 220 x 190nm 130-375kbp Leporipoxvirus myxoma virus Suipoxvirus swinepox virus Molluscipoxvirus molluscum contagiosum virus Yatapoxvirus Yaba monkeypox virus -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Herpesviridae Alphaherpesvirinae Simplexvirus herpes simplex viruses Yes 2X DNA ICOS Varicellovirus varicella-zoster virus Betaherpesvirinae Cytomegalovirus human cytomegalovirus 150nm 124-235kbp Muromegalovirus mouse cytomegalovirus 1 Roseolovirus human herpesvirus 6B Gammaherpesvirinae Lymphocryptovirus Epstein-Barr virus Rhadinovirus HHV8 = Kaposi Sarcoma -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Adenoviridae Mastadenovirus human adenovirus 2 No 2X DNA ICOS Aviadenovirus fowl adenovirus 1 80-110nm 38kbp -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Papovaviridae Papillomavirus human wart viruses No 2X DNA ICOS Polyomavirus JC, BK, SV40 45-55nm 5-8kbp -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Single-stranded DNA Viruses Parvoviridae Chordoparvovirinae Parvovirus RA arthritis virus No 1X DNA ICOS Dependovirus adeno-associated virus 2 Erythrovirus human parvovirus B 19 20-26nm 5kbp Poxviridae

FAMILIES OF VIRUSES THAT INFECT HUMANS FAMILY SUBFAMILY GENUS EXAMPLE ENVELOPE DIAMETER Yes GENOME CAPSID

Hepadnaviridae

Partial 2X DNA 48nm 3.2kb ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------RNA Reverse Transcribing Viruses Retroviridae Alpharetrovirus Avian sarcoma YES (C Type) Two Complex Betaretrovirus Mouse mammary tumor YES B, D) 1X RNA Gammaretrovirus Murine leukemia C type Deltaretrovirus Human HTLV-1, 2 C Type Epsilonretrovirus Fish viruses Lentivirus HIV-1, HIV2, SIV L Type Spumavirus Human foamy -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Double-stranded RNA Viruses, Negative Stranded Reoviridae Orthoreovirus Orbivirus Coltivirus Rotavirus Aquareovirus reovirus 3 bluetongue virus 1 Colorado tick fever human Rotaviruses golden shiner virus No 80nm 2X RNA 10 to 12 pieces ICOS double capsids

DNA Reverse Transcribing Viruses Orthohepadnavirus Avihepadnavirus

hepatitis B virus duck hepatitis virus

ICOS

copies

HUMAN VIRAL PATHOGENS FAMILY Picornaviridae GENUS Enterovirus Parechovirus Rhinovirus Cardiovirus Aphthovirus Hepatovirus Calicivirus Astrovirus Alphavirus Rubivirus Flaviviridae Flavivirus EXAMPLES Poliovirus 1,2,3 Echoviruses 1 to 7, 9, 11 to 27, 29 to 33 Serotypes 1 to 100; 1A; 1B; Hanks Mostly animal pathogens; Mengo, EMC Foot and mouth disease viruses O, A, C, SAT 1 to 3, ASIA 1 Hepatitis type A virus Norwalk; Southampton; Snow Mountain; Hawaii; Taunton; Hepatitis type E virus Human Astroviruses 1 to 5 EEE; WEE; VEE; Sindbis; Chikungunya virus; ONyong-nyong virus; Ross River; Mayora virus; Igbo Ora virus; Barmah forest virus Rubella virus Yellow fever virus; Dengue viruses 1 to 4; St. Louis encephalitis virus; Japanese encephalitis virus; tick-borne encephalitis viruses such as Russian Spring Summer virus; European tick-borne virus; Kyasanur forest virus; OMSK virus; Powassan virus; louping ill virus Hepatitis C virus Animal pathogens such as hog cholera virus; bovine diarrhea virus Coronavirus 229-E; Coronavirus OC43; many pathogens of animals Likely human viruses that cause enteric and respiratory infections

Caliciviridae Astroviridae Togaviridae

Genus Unnamed Pestivirus Coronaviridae Coronavirus Torovirus

HUMAN VIRAL PATHOGENS FAMILY Paramyxoviridae Paramyxovirinae GENUS Respirovirus Rubulavirus Morbillivirus Unclassified Pneumovirus Vesiculovirus Lyssavirus Filovirus Influenzavirus A and B Influenzavirus C Bunyavirus Phlebovirus Nairovirus Hantavirus EXAMPLES Parainfluenzavirus types 1 and 3 Mumps virus; Parainfluenzaviruses types 2, 4a, 4b Measles virus; animal pathogens Nipah and Hendra viruses Respiratory syncytial virus Vesicular stomatitis virus Rabies virus; European bat viruses 1 and 2; Mokola virus; Duvenhage virus Marburg virus; Ebola virus subtype Zaire; Ebola virus subtype Sudan; Ebola virus subtype Reston (animals) Human influenzaviruses type A and B Human influenzavirus type C Bunyawera virus; Bwamba virus; Oriboca virus; Oropouche virus; Guama virus; La Crosse virus; Jamestown Canyon virus; California encephalitis virus; snowshoe hare virus; Tahyna virus Sandfly fever - Naples virus; sandfly fever - Sicilian virus; Rift Valley fever virus. Crimean - Congo hemorrhagic fever virus Hantaan virus; Seoul virus (hemorrhagic fever with renal syndrome); Sin Nombre virus (acute respiratory distress syndrome); Puumala virus (nephropathia epidemica) Lymphocytic choriomeningitis virus; Lassa fever virus; Machupo virus (Bolivian hemorrhagic fever); Junin virus (Argentine hemorrhagic fever); Guanarito virus (Venezuelan hemorrhagic fever)

Pneumovirinae Rhabdoviridae Filoviridae Orthomyxoviridae Bunyaviridae

Arenaviridae

Arenavirus

FAMILY Reoviridae

Hepadnaviridae Papovaviridae Adenoviridae Herpesviridae Alphaherpesvirinae

GENUS Orthoreovirus Orbivirus Rotavirus Coltivirus Orthohepadnavirus Papillomavirus Polyomavirus Mastadenovirus

HUMAN VIRAL PATHOGENS EXAMPLES Reoviruses 1 to 3 Orungo virus; Kemerovo virus; also many animal pathogens Group A and Group B human viruses Colorado tick fever virus; Eyach virus Human hepatitis B virus; hepatitis viruses of mammals Human wart viruses (60 serotypes) BK virus; JC virus; SV40 virus (all three can cause PML) Human adenoviruses Herpes simplex virus types 1 and 2; Simian herpes B virus Varicella -- zoster virus Human CMV Human herpesvirus 6A, 6B, and 7 Epstein Barr virus Human herpesvirus 8 (Kaposi Sarcoma herpesvirus) Human RA-1 virus (arthritis); animal viruses B19 virus Adeno-associated virus Smallpox virus; Vaccinia virus; monkeypox virus; cowpox virus ORF virus (contagious pustular dermatitis); pseudocowpoxvirus Birds Sheep Rabbits Pigs Molluscum contagiosum virus Yaba poxvirus; tanapox virus Avian sarcoma, etc C type Mouse mammary tumor virus: B and D Type Murine & feline leukemia, C Type HTLV-1 and 2 (human); C type Fish retroviruses HIV 1; HIV-2 (AIDS), SIV, L Type Human foamy virus Human hepatitis delta; defective agent (requires type B virus) Found in human associated with neuropsychiatric disorders

Simplexvirus Varicellovirus Betaherpesvirinae Cytomegalovirus Roseolovirus Gammaherpesvirinae Lymphocryptovirus Rhadinovirus Parvoviridae Parvovirus Erythrovirus Dependovirus Poxviridae:Chordopoxvirinae Orthopoxvirus Parapoxvirus Avipoxvirus Capripoxvirus Leporipoxvirus Suipoxvirus Molluscipoxvirus Yatapoxvirus Retroviridae Alpharetrovirus Betaretrovirus Gammaretrovirus Deltaretrovirus Epsilonretrovirus Lentivirus Spumavirus Not a Family Deltavirus Bornaviridae Bornavirus

VIRUS CLASSIFICATION: MAJOR FAMILIES (not all are included, only major families) DNA VIRUSES GENOME VIRION & CAPSID
1. 2. 3. 4. 5. 6. POXVIRIDAE HERPESVIRIDAE PAPOVAVIRIDAE ADENOVIRIDAE PARVOVIRIDAE HEPADNAVIRIDAE 2X linear DNA 175,000 to 375,000bp 2X linear DNA; 150,000-200,000 bp 2X Circular genome; 5,000 bp 2X linear DNA; 38,000 bp 1X hairpin DNA 5,000 bp Partially 1X; 3,200 bp multi-layered envelope; brick shaped; COMPLEX capsid Enveloped; Icosahedral capsid NO envelope; Icosahedral capsid NO envelope; Fibers at vertices of Icosahedral capsid NO envelope; Icosahedral capsid Envelope

VIRUS CLASSIFICATION: MAJOR FAMILIES RNA VIRUSES Memory Help GENOME

1. PICORNAVIRIDAE 2. REOVIRIDAE 3. TOGAVIRIDAE 4. BUNYAVIRIDAE 5. ORTHOMYXOVIRIDAE 6. PARAMYXOVIRIDAE 7. RHABDOVIRIDAE 8. ARENAVIRIDAE 9. CORONAVIRIDAE 10. RETROVIRIDAE 11. CALICIVIRIDAE 12. FLAVIVIRIDAE 13. FILOVIRIDAE 14. ASTROVIRIDAE 15. DELTAVIRUS genus only) PLEASE REMEMBER THAT BECAUSE O'CALLAGHAN PREFERS REFLECTIVE AUDIENCES CATNAPERS REPENT! COGITATE! FORGO FAILURE AND DISASTER 1X RNA, 1 molecule 2X RNA 10-12 pieces 1X RNA; 1 molecule 1X RNA; 3 molecules 1X RNA; 8 molecules 1X RNA; 1 molecule 1X RNA; 1 molecule 1X RNA; 2 molecules 1X RNA; 1 molecule 1X RNA; DIPLOID 1X RNA; ICOS 1X RNA; 1 molecule 1X RNA; 1 molecule 1X RNA; 1 molecule 1X RNA; circular

VIRION/CAPSID
NO envelope ICOS NO envelope; ICOS ENV; ICOS ENV; HELICAL ENV; HELICAL ENV; HELICAL ENV; HELICAL ENV; HELICAL ENV; HELICAL ENV; COMPLEX NO ENV; ICOS ENV; ICOS ENV; HELICAL NO ENV; ICOS defective

POLARITY OF REPLICATION
PLUS NEGATIVE PLUS NEGATIVE SOME ARE AMBISENSE NEGATIVE NEGATIVE NEGATIVE NEGATIVE (SOME ARE AMBISENSE) PLUS REVERSE: RNA TO DNA PLUS PLUS NEGATIVE PLUS DEFECTIVE

IV. ORIGIN OF VIRUSES : Several theories advanced to explain the origin of viruses. 1. "Non-Joiner Theory (Bioids): "Primal Bang" that initiated the formation of the Universe occurred approximately l6 billion years ago and much later the stars and planets were formed. Life on Earth developed by a process of molecular evolution that required 2.5 billion years. In this process, the "primordial thin soup" of simple molecules formed and these interacted to yield more complex molecules (crystal, etc.) and eventually yielded primitive self-replicating molecules in a sub-living state. These selfreplicating systems ("bioids") orchestrated their activities into a symbiotic system by joining together, and with time other primitive bioids joined ( "joiners") to eventually form a primitive cell. Even later, some "bioids" became joiners to give diversity and complexity to primitive cells. However, some systems were "non-joiners" and could be parasitic and eventually dependent on the living system for replication. Viruses were "non-joiners". 2. "Retrograde Evolution Theory": Viruses have evolved from free-living cells through a retrograde or regressive process - i.e., certain primitive life forms such as "prebacteria" became highly specialized parasites and lost most certain enzyme functions and primitive organelles because they were not needed, and eventually evolved into the molecular obligate intracellular parasites that we call viruses. 3. "Cellular Origin" : Viruses are derived from cellular genetic material which has ESCAPED from cellular control mechanisms and acquired the capacity to exist as entities that can be parasitic when introduced into the cell. Supporting facts: Retroviral oncogenes (vONCs) originate from cellular sequences (" proto-oncogenes" = cONCs). Retroviridae "provirus" (DNA copy of the viral RNA integrated into cellular chromosomes) can undergo genetic recombination with cellular genes and these "cellular DNA sequences" can be incorporated into the virus when the provirus is copied to RNA which becomes the genome of the Retroviridae

V. VIRAL CULTIVATION: Since viruses are obligate intracellular parasites and require living
cells for their replication, a limited number of culture systems are available. These include (l) animals, (2) embryonated eggs, and (3) cell culture. l. Animals were used in the earliest attempts to isolate and cultivate viruses. However, growing virus in the intact animal presents many problems - expense of animals and their maintenance, great individual variation in susceptibility from one animal to another, difficulty in preventing cross infections and mixed infections, limited ability to study certain aspects of infection in animals, interference from animal's immune system, etc. Animals offer advantages for certain studies such as tumor virology, viral immunology (study immune response to infection) study of disease pathogenesis, testing of viral vaccines, etc. 2. Avian Embryo ("Egg") : Fertilized avian egg (e.g. chicken, duck) is inoculated with virus specimen and the egg is incubated. Embryos (eggs) are monitored for development of cytopathology. Embryo may be used to isolate virus, culture virus, or titer virus (pock assay). Different tissues of the embryo may be inoculated: Allantoic cavity: 9-12 day embryos - A large yield of virus can be obtained from cells lining the allantoic cavity. Used for influenza, mumps and encephalitis viruses. Chorioallantoic Membrane (CAM): 9-12 day embryos - Poxviruses and some Herpesviruses form discrete lesions on the CAM, and can be quantitated and identified in this manner. Yolk Sac: 3-8 day embryos - Used for many neurotropic viruses. Amniotic Cavity: -9-12 day embryos. Used for primary isolation of influenza virus. A small hole or "window" is cut into the shell of the "egg" so that virus can be introduced; the egg is then incubated. 3. Cell Culture or "Tissue" Culture" : The science of growing fragments or organs of tissue in vitro or growing cells of one or more types in vitro. Earliest tissue cultures utilized slices of organs in a suitable nutrient medium, and cellular architecture and function could be maintained for a number of days in vitro. Later fragments of minced tissue were maintained in a suspension of nutrients, or explants of tissue were embedded in "plasma clots". Techniques used today are referred to as "cell culture". Cell culture offers many advantages in the cultivation of viruses: l) it is much cheaper, 2) culture is free of host factors that influence virus growth, 3) system is free of other microorganisms, 4) viral macromolecular synthesis can be followed with precision, 5) Individual cell type may be used as a lymphocyte or fibroblast, etc.; .6) radioisotopes may be used to label viral macromolecules and to study molecular aspects of virus infection. PRIMARY CULTURE: To establish a PRIMARY cell culture, the tissue or embryo is disassociated into individual cells by a mechanical mincing of the tissue followed by treatment with a proteolytic enzyme such as trypsin. The cells are suspended in suitable nutrient medium (e.g. a mixture of salts, vitamins, amino acids, buffer, and serum to provide growth factors) and placed in a test tube or flask. The cells will settle out, attach to the flat surface of the flask and begin to grown and divide until the cell sheet become confluent (a monolayer), forming a primary culture. Primary cell cultures contain a mixture of cell types, primarily epithelial or fibroblast-like; often fibroblastic cells will overgrow the epithelial types.

SECONDARY CULTURES: Primary cell cultures can be "subcultured" - that is harvested, fed with fresh medium, and divided into aliquots to establish daughter or secondary cultures. Usually these cells have a limited growth span and can only be subcultured a few times (3-8 subcultures) before cell death occurs. CELL STRAIN: Occasionally cells of a single type will survive repeated subcultures and will grow for 50-l00 subcultures. These are called " cell strains"; the cells of a cell strain maintain normal cellular architecture and chromosome complement, but are not immortal and will eventually die. An example is "WI 38" cells, human diploid embryonic fibroblasts, widely used in diagnostic virology and virus vaccine production. CELL LINE: A "cell line" is composed of cells that have undergone the "immortality event" and can be propagated in vitro indefinitely. Cell lines can be adapted to grow in "suspension culture" (a suspension of individual cells stirred by shaking on a rotary shaker or by a spinning magnet ["spinner culture"]). Cell lines grow very rapidly, are usually aneuploid (have an altered number of chromosomes) and exhibit other characteristics of malignant cells. Cell lines can originate from either normal tissue or from malignant tissue VI. DETECTION & QUANTITATION OF ANIMAL VIRUSES: Viruses detected by four general approaches: 1. Viruses As Physical Particles: "Live" (infectious) virus not required 2. Viruses As Macromolecules: "Live" Virus not required 3. Viruses As Biological Agents: Live virus required in plaque or pock assay and to assay disease in animal. 4. Viruses As Complex Antigens: Most immunological assays do not require infectious virus. l. Detection of Viruses as Physical Particles a): Electron Microscopy (EM) can be used to detect, and even quantitate, virus if it is present in concentrated amounts with limited amounts of debris. EM is not routinely used in Clinical Virology laboratories because it is time consuming and expensive, requires expertise, and specimen must contain a significant amount of virus. In specialized circumstances such as detection of Rotavirus (Reoviridae) in human fecal material and detection of Cytomegalovirus (Herpesviridae) in urine samples, some specialized laboratories may employ EM as a diagnostic tool. EM does not indicate whether virus observed was infectious or not. b) Hemagglutination: RBCs of some animals and/or human can be agglutinated by different viruses due to lattice formation when virus "join" cells by attaching to two cells. When a known amount of RBCs is mixed with a known amount of each dilution (l/l0, l/20, l/40, etc.) of virus sample, the virus can be detected and some general quantitation of the number of physical particles can be made. For example, l0 million Influenzavirus particles are required to give visible hemagglutination of 0.25 ml of l% RBC suspension; the observation that 0.25 ml of dilutions up to l/80 of a sample but not the l/l60 dilution give positive hemagglutination indicates that 0.25 ml of l/80 dilution contains at least l0 million viral particles; thus the undiluted sample contains 320 million particles per ml. Obviously, hemagglutination does not indicate whether the virus is infectious. The method is easy to do, rapid and inexpensive. Known antibody to the virus surface protein can be used in the Hemagglutination Inhibition test to confirm the identity of the virus. 2. Detection of Viruses as Infectious Agents (Biological Assays) a) Quantal type of assays (dilution to extinction). These methods vary for different viruses and vary from lab to lab. Quantal assays are those that detect and "quantitate" virus by measurement of animal death, animal infection (such as paralysis, cytopathic effects in cell culture; etc). The titer is usually expressed as the "50% infectious dose" (ID50% or LD50%), which is the greatest dilution of the virus sample that will cause the effect being monitored to occur in 50% of the animals or cultures. This quantal type of assay has many disadvantages since it does NOT give absolute amounts of virus. b) Enumerative types of assays:. Most precise since the effect being measured is the result of single, infectious viral particle. Plaque assay: a plaque is an area of cell cytopathology, usually lysis due to necrosis, on a monolayer of cells, which are infected, with a dilution of virus. Suitable dilutions of virus are used to infect a series of monolayer cell cultures, and after attachment of virus, nutrient medium in a semisolid form is added to each plate to prevent virus spreading so that virus later released from infected cells can spread only by cell to cell contact. The plates are incubated and 2 days to 6 days later (depending on the virus), the plates are examined and the plaques are counted. Virus titer is calculated and expressed in plaque-forming units per ml. The size, morphology, etc. of the viral plaques are useful markers in viral genetic experiments. A Plaque is the result of a SINGLE INFECTIOUS virus particle. The plaque assay is usually the most common assay to titer the amount of infectious virus. A plaque is the result of cell lysis and virus released from one cell on the monolayer will spread and infect and lyse neighboring cells, etc. The size of the plaque depends on the virus and host cell. Some viral mutants form plaques that differ in size and/or morphology POCK: Some viruses can also be titered on the chorioallantoic membrane (CAM) of the embryonated egg. The lesion induced is called a pock, which is an area of hyperplasia and hypertrophy containing mixed cell types. Morphology of the pock is useful in viral genetics. A POCK is due to a single infectious virus particle.

Some tumor viruses transform cells to an altered morphology and altered growth behavior. Can quantitate this biological activity by counting the areas of cells that grow rapidly (loss of contact inhibition); each area in which the cells pile up is called a focus and the titer of virus is expressed as focus-forming units per ml. 3. Detection of Viruses As Macromolecules (chemical assays)-New biotechnology. Examples would be detection by use of DNA hybridization with a radiolabeled cloned DNA to detect viral DNA in a specimen. This type of assay will become more common and will increase the sensitivity of detection. Can also use biochemical method to measure a viral specific enzyme such as reverse transcriptase (such as in HIV-1 infection). PCR is the method of recent years as it is so sensitive. Can be used to detect DNA or RNA ("reverse transcriptase PCR=RT-PCR). Polymerase Chain Reaction (PCR) can detect a tiny amount of nucleic acid in a specimen. For example, can detect the HIV-1 provirus (integrated DNA copy of the viral genome) by PCR. Method involves extraction of total DNA from specimen, add primers (primer is small nucleotide sequence which will bind to DNA in question), heat sample to "melt" DNA (2X DNA is denatured to 1X DNA strands). Reduce temperature and add bacterial DNA polymerase that functions at high temperature. The primers bind to each 1X strand of HIV-1 DNA and the DNA polymerase copies each strand from 1X to 2X. Repeat the cycle, denature the 2X DNA strands, lower temperature, add primer and DNA polymerase. In each cycle the number of HIV-1 DNA strands doubles. After several cycles in an automated machine, a few molecules are amplified a million fold and can be detected by hybridization method. 4. Detection of Viruses As Antigens: Immunological Approach Viruses are comprised of proteins in repeated copies and these are usually excellent antigens. Antigenic determinants exist on several different structural and nonstructural viral proteins. A variety of immunological methods to detect virus (viral or antibody to virus. An example of an immunological method to detect virus is use of fluorescent-labeled anti-rabies antibody to detect rabies virus (antigen) in the tissue of an animal. PAIRED SERA: used detect an INCREASE in ANTIBODY to the virus (viral antigen). Paired sera refers to TWO samples of the patients serum taken at two-week intervals. FIRST SERUM SAMPLE ("ACUTE" SERUM) is taken as soon as possible. SECOND SERUM SAMPLE ("CONVALESCENT" SERUM) is taken two weeks later. BOTH sera are assayed for ANTIBODY to the virus in question. A FOUR-FOLD RISE in ANTIBODY TITER is usually considered to be SIGNIFICANT. Some of the immunological assays are: a) Direct And Indirect Immunofluorescence : Used to detect VIRUS (Viral Antigen). Fluorescent-labeled anti-rabies antibody is used to detect rabies virus (viral antigen) in brain tissue of an animal. In DIRECT IF, the antibody to the viral antigen is tagged with a fluorescence dye. In the INDIRECT IF procedure, one adds antibody to the viral antigen firstly and then adds fluorescently tagged ANTIBODY TO IgG (antibody to antibody). Thus, in INDIRECT IF, one can use one Fluorescently-tagged antibody in numerous assays. b) Radio-Immuno-Assay: Radiolabeled viral antigen such as HBsAg is used in RIA to detect HBsAg in human serum by competition assay for anti-HBsAg. (Hepatitis Type B). In RIA to detect VIRUS or VIRAL ANTIGEN, (such as HBsAg), one sets up a competition assay to show that the specimen (viral antigen) will reduce the amount of radiolabeled antigen that reacts with antibody. Thus, the LEVEL OF REDUCTION of radiolabeled antigen that reacts with the antibody reflects the AMOUNT of viral antigen in the specimen. To detect antibody (such as anti-HBsAg), one can use a radiolabeled antigen and test whether the patient's sera has antibody that will react with the radioactive antigen. RIA is VERY SENSITIVE METHOD. c) Enzyme-Immuno Assay Antibody with enzyme linked to it is used to detect cytomegalovirus in urine of patient. The EIA may be used to detect either ANTIGEN or ANTIBODY. Detection of VIRUS ANTIGEN by EIA: The specimen is added to a micro-well plate that has ANTIBODY TO THE VIRUS IN QUESTION attached to the plastic well. After incubation, the well is washed. If the specimen contained the virus, it would react and bind to the Antibody. Then commercial ANTIBODY to the VIRUS tagged with a very stable ENZYME is added to each well. This ANTIBODY will react with bound virus. Then after washing, SUBSTRATE is added. If the patient's specimen (Virus Antigen) had bound, then the ENZYME-TAGGED Antibody would bind; thus, the ENZYME would be present to convert the SUBSTRATE to change color. Change in color indicates positive reaction. DETECTION OF ANTIBODY BY EIA: Add dilutions of patient's sera to well that have the VIRUS or VIRUS ANTIGEN bound (commercially available). If the sample has ANTIBODY, it will bind to the viral antigen. Wash wells. Add commercial anti-IgG antibody tagged with a stable enzyme. Incubate and then wash. Add SUBSTRATE. If the sera sample had antibody, the antibody to IgG with ENZYME would be bound and the substrate would change color. Determine the highest dilution of the ACUTE and the CONVALESCENT SERUM SAMPLE that react. If there is 4-FOLD rise in titer, it indicates SEROCONVERSION. d) Neutralization Assay: Usually used to detect ANTIBODY to a virus. Principle: Antibody will prevent or reduce plaque formation. Thus, testing for ANTIBODY in PAIRED SERA that will reduce plaques (plaque

reduction assay). Paired serum samples are diluted such as 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, etc; each dilution of each sample is reacted with a known amount of poliovirus type 1. After incubation, the amount of virus in each reaction is measured by plaque titration on human kidney cell monolayers. The results are: PLAQUE FORMING UNITS OF POLIOVIRUS ______________________________________________________________________________ Serum Dilution 1/4 1/8 1/16 1/32 1/64 1/128 ______________________________________________________________________________ Control (saline) 100 Acute Serum 80 100 100 100 100 100 Convalescent Serum 5 10 20 40 60 80 ______________________________________________________________________________ Interpretation: The patient seroconverted to poliovirus type 1 since the titer of antibody that neutralized the virus from 100 to 80 plaque-forming units was 1/4 in the Acute Serum but was 1/128 in the Convalescent. This is a 32-fold increase in antibody titer. e) Hemagglutination Inhibition (HI Assay): Principle is that ANTIBODY to VIRUS PROTEIN THAT MEDIATES Hemagglutination of RBCs will BLOCK formation of LATTICE FORMATION (hemagglutination). This is visible to the naked eye. EXAMPLE: Paired Serum samples are diluted and each sample is tested for its ability to block Influenzavirus Type A virus particles from causing RBCs to Hemagglutinate. This assay measures antibody to the virus by assaying for antibody that blocks virus hemagglutination. First add commercial Influenzavirus type A to each well that has a dilution of each serum sample. Incubate to allow any antibody present to the HA peplomer to react with the virus. Then add sample of washed RBCs and incubate in the cold room. If a serum dilution has anti-HA peplomer ANTIBODY, the antibody would PREVENT the virion from hem agglutinating the RBCs. Determine which dilution, if any, of ACUTE SERUM and CONVALESCENT SERUM prevented hemagglutination. Results of a sample H. I. test is shown here: ______________________________________________________________________________ Serum Dilution 1/4 1/8 1/16 1/32 1/64 1/128 1/256 ______________________________________________________________________________ Control (saline + Virus) Hem Acute Serum None Hem Hem Hem Hem Hem Hem Convalescent Serum None None None None None None Hem ______________________________________________________________________________ Interpretation: The 1/4 dilution of ACUTE SERUM prevented the Influenzavirus from causing Hem (Hemagglutination). However, in the CONVALESCENT SERUM, dilutions as high as 1/128 prevented HEM. TITER increased from 1/4 (Acute) to 1/128 (Convalescent), a 32 FOLD INCREASE IN ANTIBODY TITER. f) Complement Fixation Test : Usually used to detect Antibody. Principle is that reaction of an antigen (VIRUS or VIRAL Antigen) and ANTIBODY will allow complement to be "fixed" in the reaction. Thus one can add [RBCs coated with antibody to RBCs] and if the Ag-AB reaction had occurred, Complement would NOT be available to mediate LYSIS of the RBCs. If the Ag-AB react had NOT occurred, then free Complement would be available to LYSE the indicator [RBCs coated with anti-RBC AB]. Detection Of Antibody: The PAIRED SERUM Samples are first heated at 56C to inactivate complement in the sera. Dilutions of the Serum samples are made in wells. Add commercial VIRAL ANTIGEN and commercial COMPLEMENT. Incubate the plates. THEN, add the indicator of RBCs-coated with antibody to RBCs. IF the sample had antibody, then the commercial Complement would be fixed and would NOT be available to cause LYSIS (NO LYSIS) EXAMPLE: Test patient's sera for antibodies to mumps virus. Serum Dilution 1/4 1/8 1/16 1/32 1/64 1/128 1/256 ______________________________________________________________________________ Control: saline + Virus +C'+ [RBC-Ab] LYS Acute Serum None LYS LYS LYS LYS LYS LYS Convalescent Serum None None None None None None LYS ______________________________________________________________________________ Interpretation: In Acute Serum, only the 1/4 dilution had antibody to mumps that fixed Complement. But, in Convalescent Serum, dilutions as high as 1/128 had AB to mumps, thus the Complement was fixed and was NOT available to mediate lysis (LYS) of the RBCs. Four fold in titer of antibody to mumps virus. g) Latex Agglutination : Principle: virus-specific antibody can be chemically coupled to LATEX or POLYSTYRENE BEADS and patient's serum can be tested for VIRUS (viral antigen) that reacts with the AB-coated beads and caused a lattice formation (agglutination) that can be seen with the naked eye. For

example, LATEX AGGLUTINATION assays are used frequently to detect for Herpesvirus or Rotavirus in clinical specimen. REVERSE TECHNIQUE: It is also possible to couple a viral antigen to the BEADS and use the beads to detect ANTIBODY to a given virus, such as HIV-1. These methods are rapid and reasonably sensitive.

VII. DIAGNOSTIC VIROLOGY

Many of the above assays to detect VIRUS in a specimen from a patient or to show SEROCONVERSION in PAIRED SERA of a patient are used in the Diagnostic Virology Laboratory. CONFIRMED LABORATORY DIAGNOSIS: Ideally, the Virology Laboratory should make a CONFIRMED LABORATORY DIAGNOSIS by either isolating the virus from the patient's specimen and/or by demonstrating the presence of viral macromolecules in the specimen or by demonstrating a FOUR FOLD increase in virus specific antibody. Must always correlate the Lab Results with the clinical picture and history of the patient. Importance Of Confirmed Lab Diagnosis: Some Obvious Consideration 1. Allow anti-viral therapy to be initiated if an anti-viral drug is available. Such as for Influenzavirus type A, but NOT for Influenzavirus Type B; for several Herpesvirus infections, for HIV, etc. 2. Allow the physician to know how to "treat" the patient and watch for frequent complications, etc. If vaginal herpesvirus infection in a pregnant woman is confirmed, then cesarean delivery may be appropriate. 3. Avoid misuse of unnecessary antibiotic therapy. Many antibiotics are toxic and have side effects. 4. Public Health concern to community if virus is dangerous, highly transmissible, etc. Also, "new: viruses appear frequently and an alert laboratory can be important in the discovery of a "new: virus. FIVE KEY PREREQUISITES FOR A SATISFACTORY DIAGNOSTIC ASSAY 1. Speed 2. Simplicity 3. Sensitivity 4. Specificity 5. Cost Some Common Sense Comments About Specimen Collection: The correct specimen must be taken and taken at the "right time"--as soon as possible after the patient presents with symptoms. The nature of the specimen determined by the CLINICAL SIGNS and SYMPTOMS and knowledge of the likely of the pathogenesis of the likely viral agent. The epithelial surface of the likely portal of entry and the location of the site of maximal virus replication are key sources of specimens. important specimens in RESPIRATORY INFECTIONS and many generalized infections are nasal swab/throat swab. In enteric infections and many generalized infections feces is an important specimen. depending on the infection, swabs of genital tract, from the eye, or from vesicular lesions are important. Some viruses may be isolated from blood leukocytes so blood is an important specimen. biopsy or autopsy specimens may be taken by needle or knife; obviously, a specimen at autopsy should NOT be placed in fixative (e.g. formalin), which will inactivate the virus and prevent its cultivation. In cases of CNS involvement, CSF specimens are often very useful. Urine is not a frequent specimen but pronounced viruria is a feature of some infections such as CMV (cytomegalovirus, a Herpesviridae member) and mumps. The specimen must be kept cool and moist and MUST be transported to the LAB as soon as possible. aseptic technique to reduce bacterial contamination is important. in the case of many specimens such as feces, the specimen must be "processed" to remove debris, filtered to reduce bacterial contamination, treated with antibiotics, etc. LABORATORY APPROACHES IN VIRAL DIAGNOSIS 1. Direct Examination of Clinical Material 2. Isolation Of The Virus 3. Detection Of Viral Protein, Nucleic Acid, Macromolecules 4. Serological Tests 1. DIRECT EXAMINATION OF CLINICAL MATERIAL : Importantly, remember the patient's signs and symptoms and history when examining clinical material. A patient with a febrile rash and vesicles on the buccal mucosa is suspected to have measles. Thus, the detection of giant cells in the vesicle fluid (due to F peplomer on cell surface) would support the tentative diagnosis and would warrant use of a Fluorescent Antibody test to detect measles antigen in the specimen for a confirmed diagnosis. Histology: Examination of clinical material may reveal the types of CPE (cytopathic effects) such as formation of giant cells, the presence of intracytoplasmic inclusions or intranuclear inclusions or both, etc. Immunofluorescence: The Fluorescent Antibody (FA) test may be carried out on the clinical material to detect viral antigen. For example, to examine the brain of killed dog that bite a person to ascertain whether the impression smears of the dog's brain reacts with Fluorescent Antibody for rabies virus antigen. The

brain could also be processed for staining to detect Negri bodies (intracytoplasmic inclusions that are pathognomonic for rabies). FA tests may also be used to assay respiratory specimens for antigens of respiratory pathogens such as Influenzavirus, Respiratory Syncytial Virus (Pneumovirus), etc.

Electron Microscopic Detection: Several viruses first detected by EM examination of patient material. Viruses difficult to cultivate such as Astroviruses, some Coronaviruses, and Rotaviruses were first identified by EM. The FLAW is the lack of sensitivity as 10 MILLION VIRIONS (particles) per ml in the specimen required to allow a good chance of observing the virus by EM. Ultra-Thin Sectioning: Section specimen with a microtome and then stain (phosphotungstate or uranyl acetate) specimen and search for viral particles such as Herpes simplex in a fatal case of encephalitis. Not used frequently. Negative Staining: Mix heavy salt such as phosphotungstate specimen under correct pH and stain will penetrate into "spaces" of virus and will NOT react with the virus proteins per se (negative image). Thus, the electron-lucent virus will appear as "clear" while non viral areas "stain" darkly. Immuno-Electron Microscopy (IEM): Add antibody to specimen, then attempt to precipitate virus-AB complexes and then negatively stain. in the EM, the virus appears as "clumps". In the past, some UNKNOWN viruses were first identified by showing that sera of patients caused a specific viral particle to clump in the IEM technique. EIA: Enzyme Immuno Assay: Can use EIA (ELISA) method to search for viral antigen in clinical material. LATEX AGGLUTINATION: A rapid method, BUT the SENSITIVITY and SPECIFICITY are often low. False NEGATIVES may occur IF virus concentration is not high as lattice formation does not occur. RIA: Radio-Immuno Assay: Sensitive and useful method. Can be used to detect viral antigen or AB. used to detect HBsAg and anti-HBsAg. Disadvantage is that isotopes have limited life span of several months. 2. ISOLATION (Cultivation) AND SUBSEQUENT IDENTIFICATION OF THE VIRUS As noted, the type of specimen, its proper collection and processing, and the timing of taking the specimen are vital. Isolation of the virus may be made in the ANIMAL, AVIAN EMBRYO, or CELL CULTURE. ISOLATION IN THE ANIMAL: NOT usually done except in special cases and in major laboratories such as state health department laboratories, the CDC, etc. Animals used include mice, hamsters, guinea pigs, rats, rabbits, monkeys, etc. Age of the animal and route of inoculation are important. Example: Use of suckling mouse for Coxsackieviruses: Inoculate specimen S.C. and observe for a wide-spread myositis in skeletal muscle with flaccid paralysis (Coxsackievirus Type A; 29 serotypes) or focal myositis with spastic paralysis and necrotizing steatititis (inflammation of adipose tissue) for Coxsackievirus Type B (6 serotypes). AVIAN EMBRYO: As discussed above, various routes of inoculation into the "egg" may be done. Example: Throat specimen for suspected influenzavirus is inoculated in amniotic and allantoic sacs of 9-12 day embryo; several days later test for hemagglutination activity of embryo fluids and use specific antibody in H. I. test to identify specific subtype and strain of Influenzavirus. Embryo not used often today. CELL CULTURE: Most common approach. The Diagnostic Lab must maintain several cell types for virus isolation. Examples are: CELL LINES of heterploid HEp-2 and HeLa cells of human cancer origin; Vero, LLC-MK2, or BSC-1 cells are from normal monkey kidney and are also used. CELL STRAIN (limited life span): Diploid human embryonic fibroblast (HEF; human diploid fibroblasts =HDF) PRIMARY/SECONDARY CELL CULTURES: Primary monkey kidney cells (PMK). The specimen must be inoculated into cell culture and the cultures are monitored for CPE. Important aspects are time required for CPE to develop, nature of CPE such as lysis of cells, syncytium formation, inclusion formation in cytoplasm or nucleus or both. 3 DETECTION OF VIRAL MACROMOLECULES : Many molecular and biochemical methods are available to detect and identify viruses by detecting viral macromolecules. Nucleic Hybridization and PCR methodologies have become a major approach. For example, RT-PCR is VERY IMPORTANT in the detection of HIV-1. Used for many other viruses such as CMV, varicella zoster, enteric viruses as well as for viruses that are more difficult to cultivate such as Human Herpesvirus 6 (Roselovirus genus). Nucleic Acid Hybridization Assays: Southern Blot Hybridization; Dot Blot hybridization: In Situ Hybridization: PCR-In-Situ-Hybridization: Development of methods to use PCR in a tissue specimen to amplify a viral nucleic and then to detect the amplified viral nucleic acid by in situ techniques have been developed. 4. SEROLOGICAL METHODS Usually used to detect anti-viral ANTIBODY. Use of PAIRED SERA is vital in most all cases and it is important to demonstrate a FOUR RISE in AB titer. Recent development of tests to detect IgM antibody to a specific virus has allowed a more rapid interpretation of serological tests. Demonstration of IgM to a specific virus is indicative of very recent infection. IgM usually appears VERY EARLY, decreases by 1 to 2 months and is virtually not detectable by 3 months. Thus, detection of IgM to a specific virus indicates infection or recent infection. VIII. VIRAL REPLICATION: A typical replicative cycle can arbitrarily be divided into six steps common to all animal viruses: Attachment or adsorption, Penetration, Uncoating, Biochemical replication, Assembly or Maturation, and Release. It is important to understand the molecular aspects of viral replication in order to understand the mechanisms of viral disease, the action of viral chemotherapeutic agents, and the strategy

to develop new anti-viral drugs and vaccines. Attachment, Penetration and Uncoating are called the "early events" of virus infection.

l. ATTACHMENT Attachment of the VIRION to the receptor on a host cell is an electrostatic (ionic) attraction between certain macromolecules on the plasma membrane of the cell (cell receptor for the virus) and the viral attachment protein, which in the case of an ENVELOPED virus is a peplomer. In most viruses studied, there appears to be specific "receptor" macromolecules on the cell surface; the number of receptors varies, but in most cells there are l0,000 to 500,000 receptors per cell. The cell receptor concept is important as the presence of receptors determines tropism of the virus and the susceptibility of a given cell to a virus. For example, poliovirus will infect only primates (including humans) because sub-primates do not contain receptor. Attachment can occur at many temperatures, even in the cold at 0 C. Attachment does not require ATP per se and is an electrostatic interaction of the cell's receptor and the virion's attachment protein. CELL RECEPTORS for different viruses varies greatly. A given cell may NOT have a receptor for a given virus, thus this cell is NOT permissive for this virus at the level of attachment. A given cell type may have receptors for several different viruses. Some viruses use similar cellular macromolecules as the receptor. Co-Receptors: Some viruses (e/g HIV-1, Herpesviruses) require two receptors for entry. For example, HIV1 requires CD4 as the primary receptor and a Chemokine Receptor (either alpha or beta) as the Coreceptor. Some HIV-1 strains use the alpha chemokine receptor as co-receptor, some use beta chemokine receptor. 2. PENETRATION: Animal virus particle is taken into the infected cell. Several mechanisms of penetration exist and are utilized for different viruses. These mechanisms are: a) Viropexis b) Fusion--Only enveloped viruses can undergo fusion c) Direct Penetration-- a misnomer since we do not understand the process a. Endocytosis ("Viropexis") After the virus attaches to the cell receptor, the virion on the cell plasma membrane surface becomes enclosed in a cell membrane-bound vesicle , which transports the enclosed virion into the cell. This process is akin to phagocytosis. The membrane vacuole then interacts with cellular membrane structures called endosomes or migrates further into the cytoplasm and interacts with lysosomes. This interaction of the membrane structure containing the virion with either the endosomes or lysosome results in an internal fusion of the two cellular membranes provided the internal pH of the cellular organelles is approx. pH = 5. In the interaction or fusing of the cellular membrane structures, the virus envelope (if virus has an envelope) is removed and this starts the uncoating process. In the case of a nonenveloped virus, the virus capsid is somehow altered so that the internal viral genome can be released. Therefore a drug which alters the internal pH of the organelles (endosomes, lysosomes) may prevent virus uncoating but this drug may also act in blocking another step in virus replication. The antiviral drug Amantadine HCl is used to prevent and to treat Influenzavirus type A infections as Influenzavirus Type A has an M2 channel protein present in the virion envelope that forms a channel to allow H+ ions to enter at low pH and to mediate uncoating. Thus, if the M2 channel is blocked, the virus cannot undergo UNCOATING. b. Fusion : Some enveloped viruses interact with the cell surface membrane, and the viral envelope (a membrane structure) actually fuses with the plasma membrane of the cell. Thus, penetration occurs and uncoating is started in the process. Enveloped viruses that undergo fusion have specialized peplomers that mediate the process. In some cases (e. g. Orthomyxoviridae, Paramyxoviridae), the fusion peplomer must be cleaved at a specific site by a cellular protease for fusion to occur. This is a factor in tropism of the virus, and prevention of cleavage offers a possible target for antiviral drug development. The cleavage of the peplomer protein by a CELLULAR PROTEASE at the cell surface allows an internal hydrophobic domain of the peplomer protein to be exposed and this hydrophobic domain mediates the FUSION of the ENVELOPE and the CELL PLASMA MEMBRANE. Thus, this is a factor in tropism since only cells that can cleave the attached peplomer are capable of being infected (allowing the virus to enter by fusion). c. Direct Penetration: Some viruses appear to attach to the cell membrane and penetrate it directly, perhaps losing a portion of their capsid in the process. Adenoviruses attach to the cell membrane via their penton fibers, and appear to lose the fibers and the penton capsomeres as they pass through the cell membrane. The mechanism of the process of direct penetration is unknown 3. UNCOATING: Process in which the viral genome is converted to a state that it can be expressed. In most viruses, Uncoating involves removal of the envelope (if present) and for some viruses the viral capsid. In the case viruses with helical capsids (all have an envelope), some of the capsid protein or even most of the capsid protein monomers remain complexed to the viral lX RNA genome. As the viral genome is transcribed or replicated, a portion of the viral 1X RNA genome is exposed for the transcription or replication event.

For viruses that undergo Fusion, UNCOATING is part of the Penetration process. Some animal viruses have highly specialized processes of uncoating. For example, Reoviridae (have a double icosahedral capsid) are transported to the lysosome during penetration, but their uncoating involves removal of most of the outer capsid proteins, but the virus is never fully uncoated and the genomic RNA is transcribed within the inner capsid-core structure. Poxviridae are uncoated by a complex two-step process: l) preexisting cellular enzymes remove the outer layers of the virion releasing a "core" structure. 2) This core contains a DNA-dependent RNA polymerase that transcribes a portion of the viral genome, producing viral mRNA that codes for a number of new enzymes, including the one necessary for the final uncoating of the viral DNA. 4. Biochemical Replication The specific logistics and control mechanisms involved in the replication of viral components are quite diverse and vary from one virus to another. The MOLECULAR BIOLOGY of virus replication has given us much of our understanding of gene structure and gene regulation. Some viruses (ex. polio; Picornaviridae) are able to replicate in the cytoplasm of cells in which cellular RNA synthesis had been arrested, or in cells that have had their nucleic removed. Other viruses require not only intact cells, but also the active gene expression (RNA, DNA and protein synthesis) of the host cell. OVERVIEW: In general, after the processes of Attachment, Penetration, and Uncoating, the viral replicative cycle of a DNA Virus can be divided into 5 stages listed below. a). Immediate-early gene expression b). Early gene expression c). DNA Replication d) Late gene expression e) Capsid assembly and maturation; Envelopment if an enveloped virus a) IMMEDIATE-EARLY GENE EXPRESSION: Transcription of immediate early gene(s) and translation of immediate early viral mRNAs. In the case of DNA VIRUSES, usually one or a few genes are said to be IMMEDIATE EARLY (IE) genes as they are the very first to be transcribed to mRNAs. These mRNAs may undergo SPLICING in some cases and are synthesized by cellular DNA-dependent RNA Polymerase II (not fully true for Poxviridae) and the mRNAs are translated to Immediate-Early proteins that are REGULATORY FACTORS that bind to the promoters of other viral genes called the EARLY GENES and allow these early genes to be transcribed. Thus, the immediate-early proteins functions as TRANS-ACTING FACTORS that alone or together with CELLULAR TRANSCRIPTION FACTORS mediate gene regulation. Thus, a virus may not replicate in a certain cell type if the proper cellular TRANS-ACTING FACTORS are not present to interact with viral Trans-Acting Factors (Immediate Early Proteins) to mediate the "turn-on" of the early viral genes. These VIRAL and CELLULAR FACTORS serve in trans to bind to cis-acting elements within the promoter region of the viral genes. It should be noted that in some viruses (e.g. Herpesviridae), the Immediate early proteins also "down-regulate" the expression (transcription) of themselves (autoregulation). The IE proteins are capable of traveling from the cytoplasm (site of their synthesis by translation) to the nucleus as they have NLS (nuclear localization signal; specific amino acid sequence domain that allows entry into the nucleus). In some cases, the viral mRNAs or some of them may undergo SPLICING before being transported to the cytoplasm for translation b) EARLY GENE EXPRESSION : If the proper viral IE protein(s) and proper CELLULAR trans-acting factors are present, these factors can bind to the PROMOTERS of the EARLY genes and turn on their transcription by cellular DNA-dependent RNA polymerase II (transcriptase). The Early mRNAs may or may not undergo splicing (depends on mRNA and on virus). The Early mRNAs are translated to the early Proteins that serve several purposes. some Early proteins of some viruses are also regulatory factors that enter the nucleus and together with the IE proteins will function to turn on the Late genes. Some early proteins function in viral DNA replication as the viral DNA Polymerase and proteins that function in viral DNA replication. In some cases, some of the Early Proteins serve to inhibit cellular processes, serve as maturation factors for the virus, etc. c) REPLICATION OF THE VIRAL DNA GENOME: After the Early Proteins are made, viral DNA replication will begin. Viral DNA replication usually is mediated by a viral-encoded DNA polymerase and associated viral proteins. However, the Parvoviridae (genome is 1X DNA) and the Papovaviridae (2X DNA genome) use the cellular DNA polymerase, but viral proteins are associated with the DNA replication phase. Viral DNA replication starts at a specific locus (ORIGIN OF REPLICATION), and the mechanism of viral DNA replication is very complex and varies among the DNA viruses. d) LATE GENE EXPRESSION: Once viral DNA replication has started, the LATE GENES begin to be transcribed to mRNAs. In some viruses (e. g. Herpesviridae), the "turn-on" of Late Genes involves the IE regulatory proteins and some Early regulatory proteins and perhaps a variety of cellular factors. Most of the

Late Genes encode structural protein and in some viruses also encode factors that are needed for virus assembly and packaging of the viral DNA genome into a preformed immature capsid. The assembly of the capsid occurs by a self-assembly process involving non-covalent bonding.

The Replication of RNA Viruses varies greatly, but the RNA Viruses replicate by THREE Strategies: 1. Plus-Strand Strategy: Plus- stranded RNA viruses are RNA viruses whose genomic RNA serves as mRNA, at least in part. After UNCOATING, the viral genomic RNA is translated to yield viral protein. The virion does NOT contain an RNA-dependent RNA polymerase, but encodes for this RNA polymerase as a non-structural protein. The genome of Plus-Stranded RNA viruses per se is INFECTIOUS (naked genomic RNA transfected into cells will be replicated and virus particles will be produced). After translation of the genomic RNA, the Viral RNA polymerase copies the genomic PLUS RNA to a complementary NEGATIVE strand that serves as a TEMPLATE and later is copied to many copies of DAUGHTER PLUS RNA. which is encapsidated to form the nucleocapsid. The PLUS STRANDED RNA viruses are: Astroviridae, Picornaviridae, Flaviviridae, Togaviridae, Coronaviridae, and Caliciviridae. 2. Negative-Strand Strategy: RNA viruses that replicate by the Negative-Strand strategy contain within the virion an RNA-dependent RNA polymerase. After Uncoating (which may be partial), the genomic RNA is copied to mRNAs, which are then translated to viral proteins (both structural proteins and nonstructural proteins). Thus, TRANSCRIPTION occurs BEFORE Translation. After Translation, the NEGATIVE genomic RNA is copied to a PLUS-STRANDED template, which is later copied to numerous copies of daughter Negative RNA. Some Negative RNA viruses have a fragmented genome and thus each fragment must be transcribed to mRNA and then the mRNA translated and then the fragment replicated via an complementary RNA template to daughter genomic RNA. Since Negative-Stranded RNA viruses must have the virion (endogenous) RNA polymerase, naked genomic RNA is NOT infectious (need the RNA polymerase for infectivity). The Negative-Stranded RNA viruses are the Rhabdoviridae, Paramyxoviridae, Orthomyxoviridae, Reoviridae, Arenaviridae, Bunyaviridae, and Filoviridae. Deltavirus is also Negative-Stranded but cannot replicate fully without Hepatitis B virus to provide HBsAg as its "envelope". 3. RETRO-STRATEGY: Only the Retroviridae employ the "retro" strategy. Viruses contain several enzymatic activities including REVERSE TRANSCRIPTASE, protease, RNase-H, and integrase. The genomic RNA is DIPLOID (2 copies of same 1X RNA; NOT 2X). Genomic RNA is copied to a DNA COPY called the PROVIRUS that integrates into host DNA. Later, the PROVIRUS is copied by cellular RNA PY II to LARGE RNA TRANSCRIPT that is both daughter genomic RNA, mRNA for GAG and GAG-POL, and spliced to mRNA for ENV. GAG and GAG-POL polyproteins are cleaved by viral protease to yield the viral proteins. ENV Polyprotein is glycosylated and cleaved by cellular protease. These viral proteins selfassembly and form the virion. Some of the Retroviridae are replication-defective and require a "helper virus" to replicate; many of these defective Retroviridae harbor a viral oncogene and cause transformation of the host cell. Some of the replication competent Retroviridae also cause transformation by a different mechanism. SYNTHESIS OF VIRAL GLYCOPROTEINS (PEPLOMER PROTEINS): Viral glycoproteins are synthesized by mechanisms identical to those for cellular glycoproteins. Briefly there are two types of glycosylation: 1. N-linked (N-glycosidic bond to side chain Nitrogen of Asparagine) oligosaccharide chains that are added by enzymatic reactions in the E.R. and then in the compartments of the Golgi. These oligosaccharides are added to Asparagine residues in the sequence of ASN-X (any amino acid)-THREONINE or SERINE. 2. O-Linked (O-glycosidic linkage to the Oxygen atom of either SERINE or THREONINE) oligosaccharide chains are added in the Golgi to serine residues. N-LINKED OLIGOSACCHARIDE GLYCOSYLATION OF PEPLOMER PROTEINS: The viral peplomer protein is synthesis by translation and has a hydrophobic sequence at its end (usually at NH2 end) called the SIGNAL SEQUENCE. This SIGNAL SEQUENCE (13 to 36 aa with highly hydrophobic stretch of 15aa) is recognized by the cellular SIGNAL RECOGNITION PARTICLE, which helps to transport the protein to the surface of the ENDOPLASMIC RETICULUM and to "dock" the protein to the E.R. As the protein is being synthesized on the polyribosomes, it is TRANSLOCATED across the E.R. membrane into the lumen of the E.R. In the case of "N-linked" glycoproteins, a large oligosaccharide chain comprised of relatively simple sugars (rich in mannose) is added " en bloc" (one step of adding whole chain) to specific Asparagine residues (ASN-X-THR or SER); the number of oligosaccharide chains varies for different glycoproteins. This oligosaccharide complex had been synthesized on an activated lipid carrier called DOLICHOL PHOSPHATE (a long lipid containing some twenty isoprene [C5] units). These "sugars" had been added to the carrier by a series of glycosyl transferase enzymes of the cell.

In the case of most (not all) glycoproteins, a cellular enzyme (signal peptidase) cleaves off the Signal Sequence. Usually 14 sugar residues (9 mannoses + 3 glucoses + 2 N-acetylglucosamines) are transferred en bloc to the ASP residue. Next, the N-linked "simple glycoprotein" glycosylated in the E.R. is transported, probably by a membrane vesicle, to the Golgi where much of the simple sugars are moved by cellular enzymes, and other sugars (fucose, sialic acid, N-acetyl-glucosamine) are added by cellular glysosyl transferase enzymes. the Golgi is a series of flattened membranes comprised of six membranous sacs (cisternae) organized as the CIS GOLGI, MEDIAL GOLGI, and TRANS GOLGI. Each Golgi compartment contains different cellular enzymes to remove certain sugars and to add other sugars to the simple oligosaccharide chain. Transport occurs in a unidirectional manner in vesicles of about 50nm. in the CIS Golgi, a mannose residue is removed; In the MEDIAL Golgi, two more mannoses are removed and an Nacetylglucosamine and a fucose are added. In the TRANS Golgi, galactose is added and then sialic acid is added to form the mature "complex". Thus CORE GLYCOSYLATION occurs in the E.R. while TERMINAL GLYCOSYLATION occurs within the Golgi organelle. Next, the processed viral glycoprotein (PEPLOMER) may be transported to the plasma membrane, to membrane vesicles, etc. and is inserted in the membrane. During the process of glycosylation, peplomer proteins may form DIMERS, or TRIMERS (HA peplomer of Influenzavirus) or TETRAMERS NA peplomer of Influenzavirus). Eventually, these glycoproteins are arranged as transmembrane proteins with a small portion remaining in the cytoplasm as a target for M protein or nucleocapsid to recognize. Thus, the glycoproteins are arranged as transmembrane proteins with a small portion remaining in cytoplasm as a target for M protein nucleocapsid to recognize. Thus, the glycoproteins become arranged as peplomers, and the interaction of the M protein or nucleocapsid with the glycoproteins in a region of cell membrane triggers the envelopment process. O-LINKED GLYCOSYLATION: Occurs within Golgi by poorly understood process in which sugars are added. The viral glycoprotein of some viruses have "O-linked" oligosaccharides which is linked to serine or Threonine. Some viral glycoproteins have BOTH N-linked and O-linked oligosaccharides on SAME protein. SIGNAL RECOGNITION PARTICLE: The mechanism by which secretory or membrane proteins are properly vectored to the membrane surface of the Endoplasmic Reticulum is that a cellular particle (composed of six proteins and a small RNA molecule of 300nt) called the "Signal Recognition Particle" (SRP) binds to the Signal Sequence of the nascent (growing) protein and stops protein synthesis. The Signal Sequence is a stretch of uncharged, primarily hydrophobic amino acids, 11 to 20 amino acids. The complex of ribosome-with the Signal Sequence of the nascent protein-SRP binds to the surface of the E.R. because its surface has Receptors for both the ribosome and SRP. Upon docking to the ER surface, the process of protein synthesis begins again and the Signal Sequence allows the protein to cross the ER membrane into the lumen. The process is called Translocation and the protein crosses as it is being made. The Signal Sequence is usually cleaved off by an enzyme (signal peptidase). Synthesis of the peplomer requires the host cell for cell organelles, molecules, and enzymes to make the "glyco" portion of the glycoprotein. The amino acid sequence of the protein is Viral-encoded. ADVANTAGES OF A GLYCOPROTEIN: FOR THE PEPLOMER: Carbohydrate groups are highly polar and hydrophilic, hence they increase water-solubility and assure protein can face the aqueous environment. For a virus this helps the peplomer interact with cell surface and may help to keep it in correct conformation. Carbohydrate stabilizes the protein and helps to protect from many proteases---important to keep peplomer intact for infectivity. Having carbohydrate assures that the peplomer will undergo a pathway to surface and promote virus egress in many cases, especially if peplomer is on plasma membrane e) CAPSID ASSEMBLY & MATURATION: Capsid assembly occurs by a self-assembly process in which the viral structural protein molecules interact with each other by non-covalent bonds to form either a helical or icosahedral or complex capsid. The site of capsid assembly varies. Several DNA viruses assemble their capsid in the nucleus (e. g. Herpes, Adeno, Papova, Parvo) while other viruses (Pox, Pico, Reo, Toga, Flavi, etc.) assemble the capsid within the cytoplasm. Some Retroviruses do not assemble their capsid until a very late stage when the virus is about to bud from the plasma membrane. Process of envelopment is complex and involves the alteration of areas of CELLULAR MEMBRANE in which cellular proteins are displaced and viral proteins associate with an area of the cell membrane. Viral PEPLOMER glycoproteins become INSERTED in the cell membrane as TRANSMEMBRANE proteins. If virus encodes a MATRIX Protein, the M protein associates with the cytophilic tail of the inserted peplomer protein. The viral nucleocapsid (either helical or icosahedral) then interacts with the Matrix Protein (if there is an M protein) or with the cytophilic tail of the peplomer. This PROTEIN-PROTEIN interaction triggers a BUDDING

PROCESS in which the nucleocapsid(s) become enveloped by this modified area of a cell membrane that harbors the inserted Peplomer Proteins and Matrix Protein (for some enveloped viruses).

Site of envelopment varies for different viruses. Herpesviruses obtain the envelope from the INNER NUCLEAR MEMBRANE into which a variety of PEPLOMER GLYCOPROTEINS have been inserted. in the case of the Herpesviridae, several TEGUMENT PROTEINS associate with the intra-nuclear portion of the peplomer glycoproteins. TEGUMENT of Herpesviridae lies between the ENVELOPE and ICOSADELTAHEDRAL CAPSID. The TEGUMENT contains several important proteins that the virion brings into the host cell to help turn on the Immediate Early genes and to shut off cellular protein synthesis. Many viruses obtain the ENVELOPE from the plasma membrane. Some viruses obtain their envelope from the Endoplasmic Reticulum membrane; other viruses obtain their envelope from the Golgi membrane. Poxviridae which are large DNA viruses that replicate only in the CYTOPLASM have a complex process t obtain their envelope, which may actually be several layers of double membranes. Briefly, the Poxviridae core structure obtains a double-layer of envelope from the Endoplasmic reticulum and later may obtain a second double-layer of membrane from the Golgi. During exit (egress) from the cell, the intracellular Poxviridae particle (called IEV = actually has four layers of four membranes) interacts by a fusion process with the cell plasma membrane and actually loses its most-outer membrane layer, thereby producing extracellular virions (EEV = Extracellular Enveloped Virions) that have a triple layer of membrane to form the envelope. Assembly And Maturation: Assembly of progeny nucleocapsids usually occurs at the site of genome replication - nucleus or cytoplasm - depending on the virus. Addition of an envelope may occur at the nuclear membrane (Herpesviridae), endoplasmic reticulum (some Togaviridae; Arenaviridae), Golgi (Coronaviridae, etc.), or plasma membrane (Orthomyxoviridae, Rhabdoviridae, Retroviridae, Paramyxoviridae, etc). The multi-layers of lipid-membrane structure of the Poxviridae apparently are added in the cytoplasmic "virus factories" from the E.R. and later from the Golgi. Release: This process differs greatly with different viruses. Some viruses lyse the infected cell and are released rapidly and in large quantities. Other viruses tend to remain cell-associated while some viruses are released slowly via vacuoles by a process that might be described as "reverse phagocytosis".

IX. NATURE AND TYPES OF FUNCTIONS MEDIATED BY VIRAL PROTEINS: AN OVERVIEW

This table is intended as general information. Do not attempt to memorize. The Table shows some of the many functions that viral proteins mediate: structural function, allow virus to attach and enter the cell, function in replication of viral genome, serve as accessory factors in viral genome replication, serve as trans-acting regulatory factors, serve to inhibit cellular macromolecular synthesis and alter the cell, function to help virus avoid/evade the immune response at several levels, and function in the transformation of the cell (tumor viruses) by several mechanisms. Viruses encode both STRUCTURAL and NON-STRUCTURAL proteins, and often a protein is multi-functional to help allow this small foreign package of genes (the virus) to compete against the large cell and to "train"" the cell to make viral macromolecules and more virus. VIRION PROTEINS STRUCTURAL PROTEINS (some examples; DO NOT MEMORIZE)) TYPE OF PROTEIN FUNCTIONS EXAMPLES Capsid Structure N capsid protein of Rhabdoviridae VP1, VP2 VP3 of poliovirus Peplomer Attachment glycoprotein HA protein of Influen; gp120 HIV Peplomer Penetration gp41 of HIV; F protein Paramyxo Peplomer Digests mucus NA of Influenzavirus E3 Corona(9-O-acetylneuraminic acid E3=esterase; Hemagg activity M (Matrix) Structure, Assembly M protein of Ortho, Paramyxo, Rhabdo Channel in ENV Forms channel for H+ ions M2 protein of Type A Influenzavir. Core Structure, Folds genome VP4 of poliovirus; P7/P9 of HIV alpha TIF ("Vp16") Transactivator in TEGUMENT alpha TIF = UL48 of Herpes simplex Regulatory Activate IE gene expression ICP4, ICP0, aTIF of Herpes Fiber of Adeno Attachment Penton Fiber at each Vertex

SPECIALIZED STRUCTURE: alpha TIF VP11/12 (UL46) VP13/14 (UL47) VHS Virion host shutoff UL11 protein UL45 protein UL37 Protein ICP0 ICP4 Protein Kinase US9 Protein Kinase UL13 VP1/2 = UL36

TEGUMENT PROTEINS OF HERPESVIRIDAE Transactivates IE genes UL48 protein 1,200 molecules Both associate with VP16 and Protein Complex in tegument modulates function of VP16 Destroys mRNAs how?? Associated with VP16 complex Myr tegument protein; viral egress? 18 K Protein that May link Tegument to inner envelope Function unknown Potent transactivator Turns on other IE genes ESSENTIAL IE gene Major IE protein PKs phosphorylate viral regulatory proteins? 300,000K interact w, 140K 150 molecules in tegument Helps vDNA to exit capsid ?

EXAMPLE OF CAPSID STRUCTURAL PROTEINS HERPESVIRUS CAPSID = FIVE PROTEINS T=16 (960 structural units in capsid) UL19 VP5 = 150K HEXONS + PENTONS 960 molecules UL38 VP19C = 50K TRIPLEX COMPONENT 375 molecules, connect HEX & PEN UL18 VP23 = 34K TRIPLEX COMPONENT 572 molecules, connect HEX & PEN UL35 VP26 = 12K Outer Surface Hexon/Penton 960 copies; joins capsid & tegument. UL26 VP24 = 26K PROTEASE, Internal POLIOVIRUS CAPSID: VP1, VP2 and VP3 make up the capsid; VP4 is internal core protein VP1-VP2-VP3 60 HeteroTrimers (Five per Vertex) Canyon at each vertex VP4 Internal: Associated with vRNA 60 molecules in core VPg One molecule Linked to vRNA Primer of vRNA synthesis SOME VIRAL PROTEINS WITH REPLICATION FUNCTIONS: SOME ARE ENZYMES DNA PY replicate viral DNA UL30 DNA PY of Herpes, Adeno, etc DNA PY Factor accessory factor UL42 processivity factor (DNA PY) RNA Replicase replicase of viral RNA "3D" RNA PY of polio, Primer of RNA synthesis primes genome synthesis VPg of polio Primer of DNA synthesis primers DNA synthesis 80K-dCTP complex of Adeno Ori bind Melt viral ORI, attract factors Ul9 protein of Herpesvirus DNA Binding Bind DNA; attract factors keep Replication fork open UL29=ICP8 of HSV Primase/Helicase prime DNA replication, uncoil UL5 HSV (UL8 + UL52 Helicase melt 2nd structure Coro, Flavi other RNA viruses RNA dep-DNA PY makes PROVIRUS Retroviruses RNA dep DNA PY makes RNA PREGENOME Hepadnaviridae VIRAL PROTEINS WITH ACCESSORY FUNCTIONS IN REPLICATION Uracil DNA glycosylase Removes Uracil in DNA (error) UL2 protein HSV Thymidine Kinase Phosphorylates TdR; scavenger TK of HSV =UL23 Ribonucleotide Reductase Converts diPO4 to deoxy di PO4 RR1 + RR2 of HSV UL39 + UL40 dUTPase Increase UTP-->dUTP pool UL50 HSV Alk Exonuclease Cleavage of DNA Concatemers? UL12 HSV DNA Package factor Functions in DNA process/Pkg UL6 of HSV Protease processes viral polyproteins Polio 2B, polio 3C Protease processes viral STR Protein Ul26 of HSV Protease many viruses encode protease Coro, Retro, Toga, Flavi, etc

VIRAL PROTEINS THAT SERVE IN TRANSCRIPTION AND AS TRANSCRIPTION FACTORS RNA Transcriptase copies RNA genome to mRNA P proteins of Orthomyxo all negative RNA viruses L protein of Rhabdo Poly A Polymerase Capping enzyme Methylase Trans-acting Factor Transacting factor Promotes egress Protein Kinase Adds Poly A tail adds 7methyl G to mRNA methylates viral mRNA Turns on viral promoter Anti-terminator prevents vRNA splicing PO4 viral proteins L protein of Rhabdo many Neg RNA, Reo, etc Reo, Rhabdo, Toga, etc Several RNA viruses, REO ICP4, ICP27,Herpesviruses TAT protein of HIV REV of HIV, RX HTLV PK of herpesviruses, etc

VIRAL PROTEINS THAT AFFECT HOST CELL MACROMOLECULAR PROCESSES/ORGANELLES Destroys Cap Binding Prevents cell mRNA translation 2A protein of Poliovirus Destroys cell mRNA Cleaves mRNA cap + 5' end PB2 Protein of Orthomyxo Inhibits Cell mRNA Splicing Blocks splicing of some mRNAs ICP27 protein HSV Inhibits Cellular RNA Synth. Blocks cell RNA Synthesis Sigma 3 Protein of Reovirus Inhibits cell DNA Synth. Blocks initiation of DNA synth Sigma 1 protein of Reovirus Destroys mRNA Degradation of mRNAs VHS protein of Herpes Blocks Host Translation Blocks Host mRNA NS1 protein of Influenzavirus egress from nucleus binds top Poly A tail of mRNA Damages cell membranes Viral Protein Inserted MANY viral Proteins inserted Alters cytoskeleton Alter cellular organization Viral proteins of many viruses VIRAL PROTEINS THAT RETARDS IMMUNE RESPONSE: SOME EXAMPLES Retards Immune System Inhibits MHC Presentation 19K of Adeno E3 gene Alters MHC transport IE protein ICP47 of HSV; Alters MHC protein UL18 of HCMV Retards antibody function Fc Receptor Binds Ig molecules gI/gE complex Herpesvirus Retard Immune response Block IFN function Small RNAs, Adeno, EBV block phosphor. of p!/eIF2 PK Retard immune response Bind to IL-1 crmA protein of cowpox virus Retard immune response Bind to C' receptor gC of HSV Retard immune response MANY LEVELS HIV PROTEINS (NEF, ENV, etc.) MECHANISMS OF VIRAL TRANSFORMS HOST CELL Human cancer Activates factors & cell genes Liver cancer activates cell genes ? Transformation Various means: signal pathway; mimic growth factor, protein kinase mimic receptors, mimic Trans factors Transformation LTR enhancer activates cONC LTR is not a protein Transformation Interacts with Tumor Suppressor Viral encoded anti-oncogene TAX protein HTLV X protein of HBV vONC of RETROVIRUSES DEFECTIVE RETRO Replication competent Retro T Ag SV40 rxs with Rb & p53 Adeno E1A binds to Rb Wart E7 protein binds to Rb

X. ONE STEP MULTIPLICATION CURVE: SEE POWERPOINT FIGURE. Extracellular virus refers to
virus made and released from the cell; total virus is extracellular + intracellular virus. The amount of virus produced per cell and the time required for production vary with virus and cell type. The curve usually plots infectious virus in plaque-forming units/ml versus time. Stages of a one-step curve are: ECLIPSE PERIOD: No virus is produced; only detectable virus is residual virus in the inoculum during this period, virus is penetrating, uncoating, and being replicated. LATENT PERIOD: Intracellular virus is being made, but no virus has been released. This period ends when virus release begins. RISE PERIOD: Period of maximal virus replication and release. Eventually, the curve levels off and maximal virus production has been reached. The virus yield (infectious particles per cell) varies from one virus-cell system to another. Poliovirus has replication cycle of only 6-8 hours; herpes simplex, have a cycle of 24-30 hours, some viruses such as cytomegaloviruses require 72 hours or longer to obtain maximal growth of virus.

Factors such as cell type, multiplicity of infection (virus added per cell at time of infection), temperature, etc. influence the kinetics of the replication curve. Much of our understanding of virus replication has come from the study of virus infected cells, using the techniques of modern molecular biology and biochemistry to analyze the time course of production of viral mRNAs, viral proteins, viral enzymes, virus maturation as well as the effect(s) infection has on cellular DNA, RNA and protein synthesis.

XI. ROLE OF THE HOST CELL IN VIRUS REPLICATION: Viruses are obligate intracellular parasites and
therefore require a living cell for their replication. Indeed, viruses are the ultimate obligate parasite since the HOST CELL supplies the requirements for the energy for replication, the building blocks (amino acids, oligosaccharides, ribonucleotides, deoxyribonucleotides, lipids, and membrane components) of their macromolecular components, and the site (organelles such as ribosomes, spliceosome, E.R., Golgi, membranes) and tools of assembly. Most of the enzymes required to generate viral genomic RNA or DNA and viral mRNAs are encoded by the cell, and virtually every factor required for protein synthesis and the transport of viral macromolecules within the cell is provided by the host cell. Of course, the viral genes encode the protein sequence (order of amino acids) and specify and retain the genetic code of the viral genome when the genomic sequences of DNA or RNA are replicated via a nucleic acid template of complementary sequence; also, most viruses encode for a limited number of enzymes (for some viruses, the enzyme is within the structure of the virus; most viral enzymes are nonstructural proteins made during infection). These viral-encoded enzymes usually play a role in the replication of the viral genome. Every cell type will not support the replication of every virus. Often, the failure of the virus to replicate is due to the lack or receptors for the virus. For example, poliovirus will NOT replicate in mouse cells, dog cells, cat cells, etc. because these cells lack the receptor for poliovirus attachment. In some cases, the failure of the virus to replicate is at the level of penetration; for example, the viral peplomer proteins of some viruses (such as the HA peplomer of Influenzavirus; or the F peplomer of measles virus, mumps virus, etc.) must be cleaved by a cellular protease to "activate" the peplomer for its role in penetration. Therefore, if the virus particle were replicated in a cell without this surface enzymatic activity, the, virus produced is not able to penetrate in the next cell to which it attaches. Therefore, part of the tropism of a virus for a certain tissue or cell type may be due to the presence of the receptor site on that cell type and/or to the ability of that cell type to produce virus with properly cleaved peplomer protein. A virus may enter a cell and still fail to be replicated because the proper expression of all viral genes in the correct sequence fails to occur in that cell type. Cells that have receptors for a virus and can support the replication of that virus are said to be Permissive. Cells that fail to support the replication of a given virus are said to be Nonpermissive. A nonpermissive infection in which the virus can enter the cell and undergo at least some events of replication in which the virus can enter the cell and undergo at least some events of replication (such as Immediate-Early gene expression) but cannot be fully replicated is referred to as an Abortive Infection. In the cases of some oncogenic viruses, the expression of some, but not all, viral genes may result in Transformation of the normal cell to a cell with altered properties. In some cases, the Transformation may be Oncogenic Transformation and the host cell becomes a cancer cell and may harbor viral genes without producing viral particles. Therefore the cell must provide a receptor site for the virus and the cell-virus interaction must trigger a penetration process. The cell then must function in the Uncoating of the virus. This may be by an enzymatic process or a process in which the virus interacts with cellular components [Endocytosis involves fusion of virus envelope (in a vesicle) with the endosome of the cell]. As the virus genome is copied to messenger RNA and mRNA synthesis occurs, the cell provides pool of ribonucleotides (ATP, UTP, CTP, GTP). Each of these required a long list of cellular enzymes for its synthesis. The cell also usually (not always) provides the enzymes for processing the messenger RNA (capping the 5' end and addition of Poly A tail at 3' end). If the viral mRNA is spliced, cell provides the enzymes and "spliceosome" to process the RNA. In the case of DNA viruses, mRNAs are made; also cell must supply dATP, dTTP, dGTP, & dCTP for viral DNA replication, which required many enzymes for their synthesis. Herpesviruses and Poxviruses encode for a FEW of these enzymes (such as thymidine kinase and ribonucleotide reductase), but virtually all of the DNA precursors are made by cellular enzyme systems.

When the viral messenger RNAs are translated to viral proteins, the cell must provide many factors, enzymes, and components. The pools of the many amino acids, the transfer RNA molecules, the ribosome subunits (each composed of ribosomal RNAs and numerous proteins), the initiation and elongation factors, the enzymes required for protein synthesis and activation of tRNAs, etc. etc. are provided by the host cell. In the case of the viral proteins destined to become glycoproteins, the cell provides the Signal Recognition Particle to transfer the nascent protein-ribosome complex to the E. R. membrane; it provides the ER structure, the energy, the oligosaccharide pool, dolichol-lipid intermediate, etc. that are required for the first step in glycosylation. The cell then provides energy and membranes to transport the "simple" glycoprotein, numerous enzymes to trim the "simple glycoprotein", the Golgi complex, and the numerous enzymes and complex sugars (fucose, N-acetylneuraminic acid, etc.) to synthesize the complex sugar. In the case of an enveloped virus, the cell provides a membrane structure that serves as the precursor to the viral envelope. Viral glycoproteins become inserted into this cell membrane and the virus uses the energy of the cell and cellular cytoskeletal elements to form the envelope during the envelopment process. Therefore, it is obvious that the virus employs a long list of metabolic systems and components of the cell for the synthesis of viral macromolecules and their assembly into viral macromolecules. Although the assembly of viral nucleocapsids is known to be a self-assembly process (viral polypeptides interact to form capsid precursor), viral envelopment and the vectoring of the virus within the cell appear to require the participation of cellular cytoskeletal elements (such as actin fibrils, myosin fibrils, tubulin in some cases).

XII. STRATEGIES TO GET MAXIMAL FUNCTIONS FROM LIMITED GENETIC INFORMATION

Viruses have a very limited amount of genetic information (genes) and must compete against a cell that has 10,000 fold to as much as 1 MILLION fold MORE genetic information. Thus, viruses have evolved to be fierce competitors and have employed various strategies to obtain maximal efficiency in their replication and have developed "molecular weapons" to compete with the host cell and its natural host. Some of the means and considerations that allow viruses to obtain maximal use of their limited genetic information and to compete with host are: 1. VIRUSES USE THE HOST CELL FOR ENERGY AND MOST FUNCTIONS: As discussed above, the cell provides the energy, organelles, building blocks, membranes etc. etc. for the virus to replicate. 2. STRUCTURE: VIRUSES USE REPEATED COPIES OF PROTEINS TO BUILD STRUCTURE Relatively little to a reasonable amount of the genetic information is used for structure. Many copies of the same few proteins build the structure. 3. SELF ASSEMBLY: The virus assembly is via self-assembly mechanism for capsids and uses cell membranes for envelopment. The virus did not waste genes to encode for enzyme to build its structure. 4. UNCOATING: DOES NOT REQUIRE ENZYMES FOR EARLY EVENTS: Again, the virus uses the cell and cell organelles (endosomes, etc.) to enter the cell and to be uncoated. 5. OVERLAP OF ORFs: SAME NUCLEOTIDES ENCODE MULTIPLE PROTEINS (BOTH STRANDS) Some viruses have overlapping Open Reading Frames such that the same stretch of its genome encodes for multiple proteins. Obvious examples are the Papovaviridae and Hepadnaviridae. 6. POLYPROTEINS MADE: USE ONLY ONE INITIATION SITE: Several viruses generate several proteins from a precursor protein and thus have only one set of cis elements for transcription and translation that generates several proteins. Also, in some cases, the cleavage of these polyproteins can occur in different ways to generate multiple proteins with different functions by use of parts of the same amino acid sequence. 7. SOME VIRAL PROTEINS ARE MULTIFUNCTIONAL: Many viral proteins are multi-functional to conserve genetic information. Some viral proteins have multiple enzymatic activities (RNA polymerase, Poly A polymerase, capping mRNA). The capsid protein of the Togaviridae is also the protease for processing itself. Tumor antigen protein of the Papovaviridae has multiple functions. 8. SPLICING: MAXIMAL USE OF ONE mRNA TO" ENCODE" MULTIPLE PROTEINS Several viruses encode an mRNA that can be spliced by the cell's spliceosome in several ways to generate multiple species of mRNA. This conserves use of regulatory elements and lets the cell do the work. 9. mRNA EDITING: Some viruses modify an mRNA such that the SAME RNA encodes different proteins by a simple mutation or addition of a nucleotide that changes the CODING FRAME. The Paramyxoviridae RNA Polymerase can edit certain viral mRNAs so that multiple proteins are made from same mRNA 10. GENE WITHIN A GENE: Some Herpesviruses have a "gene within a gene" such that two different proteins with different functions can be generated from same DNA sequence. 11. AUTOREGULATION: PRODUCE AMOUNT OF PROTEIN NEEDED: Many viral genes are tightly controlled and autoregulated so that the amount of gene product made is not excessive. This is efficient use of amino acid pools and nucleotide pools in the cell. Some cells such as the neuron have small pool size of these precursors and virus is wise to be efficient.

12. INHIBIT CELLULAR PROCESS: LESS COMPETITION FOR POOLS OF NTP & AA Viruses have a variety of means to block and reduce host macromolecular synthesis so that the cell cannot compete and cannot use pools of nucleotides, amino acids, ribonucleotides, etc. and its energy for itself. This small virus has shut off cellular processes so that it can use these pools of precursors, translation and transcription factors, spliceosomes, and organelles for its replication, not for the cell to use. 13. MANY MECHANISM TO EVADE THE IMMUNE SYSTEM: Viruses have many mechanisms to block and evade the immune system. Some are quite subtle (antigen drift HIV-1 and Influenzaviruses allows the virus to change its epitopes so that antibody will not neutralize the virus; Herpesvirus gI/gE proteins bind to Fc region of IgG to coat the virion with IgG to protect virion from anti-herpesvirus antibody) some are quite dramatic such as killing the key cell types that regulate the immune response. SELF EVALUATION QUIZ: 1. Define, describe or explain each of the following: a. capsid k. fusion t. Southern blot b. three types of capsid symmetry l. plaque u. Plus Stranded RNA virus c. promoter m. pock v. Trans-acting factor d. peplomer n. CPE w. icosadeltahedron e. envelope o. Prion x. tegument of a herpesvirus f. nucleocapsid p. Viroid y. capsomere g. origin of viruses o. Cell strain z. Matrix protein h. N linked oligosaccharide q. pitch aa. fiber of Adenoviridae i. quantal assay r. PCR bb. Cell Line j. viropexis s. Paired sera cc. Name the Lone Ranger's horse 2. Discuss the general approaches one can use to detect and quantitate animal viruses. What are the positive or negative features of each approach? Which methods require live, infectious virus. 3. Discuss the various functions that the host cell provides for the replication of a virus? 4. Draw a typical virus growth curve and label its "parts". 5. Name the families of DNA animal viruses. Which have a helical capsid? 6. Name the families of RNA viruses. Which have an envelope? Which have an icosahedral capsid. 7. Discuss the variation in the genomic structure (size, conformation, molecular features) of animal viruses. 8. Define peplomer and discuss its structure. Outline the steps in the glycosylation of a protein. 9. Discuss the means to cultivate animal viruses. What are the advantages of each? 10. Discuss the means to detect viral macromolecules in a clinical specimen. 11. Name five serological methods to detect antibody to a virus. Explain the principle of each method. 12. Discuss the general approaches used to make a CONFIRMED LAB Diagnosis of a viral disease. 13. Contrast key properties of being a PLUS-STRANDED RNA VIRUS vs being a NEGATIVE STRANDED RNA VIRUSES.