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IN THE NAME OF GOD Department of Microbiology, Islamic Azad University, Falavarjan Branch Advanced Virology dsDNA Viruses By: Keivan Beheshti Maal 1 POXVIRUSES Large Enveloped dsDNA Viruses Genus Orthopoxvirus buffalopox virus {buffalo, cattle, human} camelpox virus {camel} (CMLV) cowpox virus {rodents, felines, bovines, human} ectromelia virus {mousepox} monkeypox virus {rodents, primates, human} rabbitpox virus {colonized rabbit only} raccoonpoxvirus {North America} skunkpox virus {North American striped skunk} taterapox virus {African gerbil} vaccinia virus {no natural reservoir} Uasin Gishu disease {Central African horses} variola virus {human; eradicated from nature} volepox virus {California pinon mouse and voles} Poxvirus Variola (smallpox) virus is most closely related to camelpox Both are believed to have evolved from a common ancestor probably a rodent poxvirus 5,000-10,000 years BC Virus Structure most complex of the viruses that infect animals dry weight vaccinia contains 90% protein 100 proteins functional enzymes polymerases, kinases, ligases etc., structural proteins 5% lipid 3.2% DNA Virus Structure Among largest, most complex animal viruses brick-shaped membrane bound viruses Non-obvious helical or icosahedral symmetry 360 x 270 x 250 nm Size of chlamydia under light microscope complex internal structure Virus Structure "core" biconcave = dumb bell shaped tightly compressed nucleoprotein Virus Structure Core Linear double-stranded DNA genome terminal hairpin loop advantage? several tandem (i.e. direct) repeat sequences ends of the genome form direct repeats inverted terminal repeats (ITRs). Virus Structure Core DNA most essential genes located in the central part of the genome (highly consereved) e.g assembly and replication - non-essential genes are located at the ends (much more diverse) e.g genes encode unique biological determinants: Host range Virulance factors Evasion from immune system General size: 130-300kbp Vaccinia 190,000 nucleotide base pairs completely sequenced Virus Structure CORE Enzymes Host RNA polymerase is in the cell nucleus Pox replicates in cytoplasm (poxviruses use a virally-coded DNA -dependent RNA polymerase needed immediately upon infection) in virions flanked by 2 "lateral bodies" function unknown surface of virus covered with filamentous protein Virus Structure envelope intracellular particles only have an inner membrane IMV - intracellular mature virions not host membrane extracellular forms contain 2 membranes EEV - extracellular enveloped virions second derived from Golgi or ER Proteomics of Poxvirus Proteomics: The protein composition of virion Aims: 1) important prerequisite for functional studies 2) Important prerequisite for studying pathogenicity responsible proteins Proteomics of Poxvirus Poxvirus gene classes: Early genes: 1) early and 2) immediate early (most of the core enzymes) Intermediate genes Late genes: 1) virion structural proteins 2) morphogenesis factors for assembly Proteomics of Poxvirus Infectious Forms: 1) Intracellular Mature Virions (IMV) 2) Intracellular Enveloped Virus (IEV) 3) Cell-Associated Extracellular Enveloped Virus (EEV) 4) Extracellular Enveloped Virus (EEV) Recent Classification: IMV = MVs (Mature Virions) IEV = WVs (Wrapped Virions) EEV = Evs (Extacellular Virions) Proteomics of Poxvirus Early Studies for Protein Analysis in VACV MVs SDS-PAGE Combination of SDS-PAGE and N-terminal a.a sequencing Identification of 12 unique virus encoded proteins (Takahashi et al., 1994) Two dimensional gel electrophoresis and a.a sequencing or immunoprecipitation Identification of 12 unique major membrane and core proteins (Jensen et al., 1996) Proteomics of Poxvirus 10 years later methods: 1) Gel-free liquid chromatography Identification of 75 viral proteins and their relative abundance (Chung et al., 2006) -enzymes -transcription factors -membrane proteins -core proteins - host interacting proteins A4: most abundant protein in MV particles (core protein complexes with core protein p4a/4a; morphogenesis) Proteomics of Poxvirus 2) High Performance liquid Chromatography or SDSPAGE in combination with electro-mass spectrometry (Yoder et al., 2006) Identificaton of 63 VACV virion proteins Confirmed the presence of most previously identified proteins Some previously identified proteins were not identified ( A2.5, A6, A9, A18, A21, A22, A25, A26, A28, A31, A45, C6, D7, D13, C5.5, G9, H2, H6, I2 and L5) Proteomics of Poxvirus 3) Gradient centrifugation with sucrose and cesium chloride (Resch et al., 2007) Identification of 80 proteins including 69 previously identified and 11 novel protein 15 previously reported proteins were not identified Determination of 10 most abundant MV proteins: [major proteins: F17, A3, A4, A10, A17 that perform 80% of MV protein mass)] All these studies highlight: 1)important role that different proteomics technologies have played in detection of proteins 2) importance of defining virion purity Replication Replication in cytoplasm of host cell. other DNA viruses in nucleus Use host enzymes for DNA synthesis Replication without any host cell enzymes for DNA synthesis. all of the enzymes necessary for DNA synthesis in virion can Replicate DNA but not mature in enucleated cells Replication attachment - vaccinia host cell receptors for epidermal growth factor (EGF) VGF for vaccinia growth factor. penetration direct penetration of the core enters cells via clathrin- coated pits require an acid pH for fusion to occur CAN’T fuse directly with the plasma membrane. taken up by invagination of clathrin coated pits into endosomes Replication penetration endosomes become acidified, latent fusion activity of the virus proteins becomes activated virion membrane fuses with the endosome membrane delivery of the internal components of the virus to the cytoplasm Replication uncoating two stages: Removal of the outer membrane as enters the cell particle (minus its outer membrane) is further uncoated Within minutes of entry: viral mRNA transcripts early' genes ~50% genome protein products complete the uncoating nucleocapsid released into the cytoplasm protein and NA synthesis viral factories bounded by virally synthesized membranes form the envelope of released mature virus proteins early VGF secreted causes non-infected cells to divide [proliferative disorders in some poxvirus infections] protein and NA synthesis proteins early VCP binds to C4b blocks the activation of classical complement pathway protein that binds to and neutralizes interferon gamma intermediate and late genes DNA synthesis post-translational processing of viral proteins structural proteins Nucleic Acid synthesis starts about 1-2 hrs makes - 10,000 copies/cell self-priming may nick at one or both ends from 3" end only - no Okazaki fragments Nucleic Acid synthesis formation of high m.w. concatemers cleaved and repaired to make virus genomes. Assembly some unknown contribution from the cell poxvirus gene expression and genome replication occur in enucleated cells maturation is blocked Assembly not understood probably involve interactions with the cytoskeleton e.g. actin-binding proteins Assembly Inclusions formed in cytoplasm mature into virus particles Actin 'comet tails' form shoot IEV through the cytoplasm to the cell surface possibly into adjacent cells an alternative mechanism for cell to cell spread? Highly processed and packaged genomic DNA accumulates mature viral particles within 24 hours of infection Release minority of mature enveloped virus fuse with the host cell plasma membrane released from the cell responsible for spread of the infection throughout body Most remains associated with the cell VACV Life Cycle AGENTS OF DISEASE IN POXVIRUSES Smallpox variola virus (VV) and vaccinia are the best known. VV strains are divided into variola major (25-30% fatalities) variola minor same symptoms but less than 1% death rate Incubation period is about 12 days Initial symptoms include high fever Fatigue head and back aches 2-3 days later lesions appear progress from macules to papules, and pustular vesicles. small blisters that itch and are extremely painful begin developing on the bodies extremities spread to the rest of body Twelfth day, the blisters scab over and leave permanent pitted scars. Death usually results if the virus reaches the brain, heart or lungs. Poxvirus Infection There is no other reservoir for VV but humans VV causes only acute infections, from which the infected person either: a) dies b) recovers with life-long immunity Vaccinia virus is an effective immunogen. first appeared in China and the Far East at least 2000 years ago. The Pharaoh Ramses V died of smallpox in 1157 B.C. skin lesions found on his mummy Marcus Aurelius Antonius, Roman philosopher-emperor, another victim; During his reign smallpox wiped out 2,000 people a day. History Chinese healers used variolation dried scabs from smallpox victims, ground to a powder blown up the nose. worked better if use variola minor widely practiced in the middle east for many centuries, Turkey fluid from smallpox vesicles scratched into the recipient's arm Vaccination/ Variolization POX Vaccinaion For more than 100 years, the "vaccine strains" were propagated from arm-to-arm Vaccination was almost universally adopted worldwide around 1800 for at least last 50 years, Vaccinia has been a distinct virus from Cowpox molecularly most similar to Buffalopox United States stopped vaccinating its military in 1989 civilians in the early 1980s Recently have started to vacccinate again Pox Vaccination current VACCINIA VACCINE Dryvax: the vaccinia (smallpox) vaccine currently licensed in the United States a lyophilized, live-virus preparation of infectious vaccinia virus (Wyeth Laboratories, Inc., Marietta, Pennsylvania). Previously: calf lymph with a seed virus derived from the New York City Board of Health (NYCBOH) strain of vaccinia virus and has a minimum concentration of 108 (PFU)/ml. inoculation at other sites . autoinoculation face, eyelid, or other persons (~ 6/10,000) Erythematous or urticarial rashes Side . Effects and Less Severe Adverse Reactions Reaction at site swelling and tenderness of regional lymph nodes, fever Approximately 70% of children experience >1 days of temperatures >100 F 15%-20% of children experience temperatures >102 Moderate to Severe Adverse Reactions eczema vaccinatum localized or systemic dissemination of vaccinia virus generalized vaccinia vesicular rash ~3/10,000 vaccinations vaccinia necrosum severe, potentially fatal illness progressive necrosis in the area of vaccination postvaccinial encephalitis 15%-25% die 25% have permanent neurological disorders ERRADICATION Less than 40 years ago, smallpox was endemic in 31 countries Yugoslavia as late as the early 1970s 1960's over 2 million people/year die WHO in 1965 decided to achieve eradication last naturally occurring outbreak was in Somalia on 26th October 1977 Endemic smallpox was declared eradicated in 1980 by the (WHO). ERRADICATION possible for 4 reasons: single stable serotype no other reservoir for variola virus than humans Infection spreads only from close contact with infected persons Vaccinia virus is an effective immunogen ERRADICATION variola virus causes only acute infections infected person either: dies recovers with life-long immunity most commonly from days 3-6 after onset of fever. Thus only infectious after show signs and symptoms know who exposed so can isolate Strict quarantine with respiratory isolation minimum of 16-17 days incubation : 10 - 12 days After eradication What to do with existing stocks consolidated into two collections 1976, WHO urged 75 labs in several countries that retained stocks of variola virus to destroy or transfer them to official WHO repositories in U.S. and Soviet Union. South Africa was last to destroy its virus stocks in 1983 CDC keeps about 400 different strains Moscow laboratory 200 strains in Novizbersk, Russia extent of stockpiles in other parts of the world unknown.