Department of Microbiology, Islamic Azad University, Falavarjan Branch Advanced Virology

1. IN THE NAME OF GOD Department of Microbiology, Islamic Azad University, Falavarjan Branch Advanced Virology dsDNA Viruses By: KeivanBeheshtiMaal

2. Large Enveloped dsDNA Viruses POXVIRUSES

3. 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} • UasinGishu disease {Central African horses} • variola virus {human; eradicated from nature} • volepox virus {California pinon mouse and voles}

4. 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

5. Virus Structure • most complex of the viruses that infect animals • dry weight vaccinia contains • 90% protein • 100 proteins • functional enzymes • polymerases, kinases, • ligasesetc., • structural proteins • 5% lipid • 3.2% DNA

6. 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 chlamydiaunder light microscope • complex internal structure

7. Virus Structure • "core" • biconcave = dumb bell shaped • tightly compressed nucleoprotein

8. 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).

9. Virus Structure • Core DNA • most essential genes located in the central part of the genome (highly consereved) • e.g assembly and replication • non‑essentialgenes 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

10. Virus Structure • CORE • Enzymes • Host RNA polymerase is in the cell nucleus • Pox replicates in cytoplasm (poxviruses use a virally-coded DNA -dependent RNA polymeraseneeded immediately upon infection) • in virions • flanked by 2 "lateral bodies" • function unknown

11. Virus Structure • surface of virus covered with filamentous protein • 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

12. Proteomics of Poxvirus • Proteomics: The protein composition of virion Aims: • important prerequisite for functional studies • Important prerequisite for studying pathogenicity responsible proteins

13. 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

14. 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 (ExtacellularVirions)

15. 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)

16. 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)

17. Proteomics of Poxvirus • 2) High Performance liquid Chromatography or SDS-PAGE 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)

18. 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

19. 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

20. Replication • attachment - vaccinia • host cell receptors for epidermal growth factor (EGF) • VGF for vaccinia growth factor.

21. Replication • 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

22. Replication • penetration • endosomesbecome acidified, • latent fusion activity of the virus proteins becomes activated • virionmembrane fuses with the endosomemembrane • delivery of the internal components of the virus to the cytoplasm

23. 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

24. 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]

25. 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

26. 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

27. Nucleic Acid synthesis • formation of high m.w. concatemers • cleaved and repaired to make virus genomes.

28. 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

29. 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

30. 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

31. VACV Life Cycle

32. AGENTS OF DISEASEinPoxviruses

33. 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

34. 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.

35. 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 • Vacciniavirus is an effective immunogen.

36. History • 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.

37. Vaccination/ Variolization • 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

38. 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

39. 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.

40. . • inoculation at other sites autoinoculation • face, eyelid, or other persons • (~ 6/10,000) • Erythematousor urticarial rashes

41. . • 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

42. Moderate to Severe Adverse Reactions • eczema vaccinatum • localized or systemic dissemination of vaccinia virus • generalized vaccinia • vesicular rash • ~3/10,000 vaccinations

43. vaccinianecrosum • severe, potentially fatal illness • progressive necrosis in the area of vaccination • postvaccinialencephalitis • 15%-25% die • 25% have permanent neurological disorders

44. 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).

45. 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 • Vacciniavirus is an effective immunogen

46. 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