Poxviridae

1. Poxviridae Molecular Virology

2. Introduction • Poxciruses have been known - the characteristic "pocks" produced by variola virus (Smallpox) . • The Pharaoh Ramses V (left) died of smallpox in 1157 B.C • The disease reached Europe in 710 A.D. and was transferred to America. • In the cities of 18th century Europe, smallpox reached plague proportions and was a feared scourge - highly infectious

3. Introduction • In 1967, WHO initiated a program to eradicate small pox from the third world nations . • Smallpox has now been eradicated - the last naturally occurring outbreak of smallpox was in Somalia on 26th October 1977. • The last known person to have natural smallpox of any kind lived .

4. Taxonomy

5. Taxonomy Family Poxviridae

6. Poxviruses Pathogenic for Humans

7. Morphology Poxviruses are the largest regular-shaped viruses.Oval or "brick-shaped" particles 200-400nm long - can be visualized by the best light microscopes .

8. Morphology • The external surface is ridged in parallel rows, sometimes arranged helically.

9. Morphology • The particles are extremely complex, containing many proteins (more than 100) and detailed structure is not known. • Biconcave (dumbbell-shaped), with two "lateral bodies" (function unknown).

10. Properties Morphology The outer surface is composed of lipid and protein which surrounds the core The core is composed of a tightly compressed nucleoprotein. They have a double-stranded DNA (130–375 kb) genome contained in a helical nucleocapsid structure seen as a dumbbell shape. Outside of which are several virus-encoded proteins in structures referred to as 'tubules'( mesh-like coat. ) This particle is referred to as an intracellular infectious virion.

11. Morphology • IMV - intracellular mature virions intracellular particles only have an inner membrane • EEV - extracellular enveloped virions The extracellular forms contain 2 membranes

12. Genome • Linear, d/s DNA of 130-300kbp (cross-linked ends). • No know origins • Replication in cell cytoplasma • Encodes all transcription and replication enzymes needed for viral gemone Harrison S C et al. PNAS 2004;101:11178-11192

13. Genome • Ends of genome consist : 1. a terminal hairpin loop (no free ends) 2. with several tandem repeat sequences 3. this arrangement is found at the ends of chromosomes from a number of different organisms Harrison S C et al. PNAS 2004;101:11178-11192

14. Genome • The ends of the genome form direct repeats called inverted terminal repeats (ITRs). • Most of the essential genes are located in the central part of the genome • There are ~250 genes in the genome.

15. Genome • Strand displacement DNA synthesis. No RNA primer • Complete mRNA synthesis system (RNA polymerase/capping enzyme/polyadenylation system) • Encode functions proteins DNA synthesis/nucleotide scavenging/immune escape mechanisms • Encode immune modulation factors interferon/complement/inflammatory responses • Encode many proteins initially stimulate host cell growth lead to cell lysis viral spread (Epidermal growth factor homologue) Harrison S C et al. PNAS 2004;101:11178-11192

16. Replication 1.Entry Intracellular mature virion (IMV) particles bind to unknown receptor(s) and fuse with the cell membrane. Extracellular enveloped virion (EEV) particles bind to unknown receptor(s) and are endocytosed into the cell.

17. Replication 2. Initial Uncoating The viral core particle (CORE) is released into the cytoplasm It containing: 1.viral genome, • viral DNA-dependent RNA polymerase 3. other enzymes

18. Replication 3. Early Transcription Early genes are immediately transcribed. It including immunomodulatory proteins, enzymes, and replication and transcription factors

19. Replication 4.Translocation The viral core particle translocates to the outside of the cell nucleus.

20. Replication 5. Secondary Uncoating The viral nucleoprotein (NP) complex, which contains the viral genome, is released. The viral genome is replicated 、transcription and translation of intermediate genes .

21. Replication 6. Late Transcription The viral late genes (coding for structural proteins, enzymes, and transcription factors) are transcribed and translated.

22. Replication 7. AssemblyConcatemeric intermediates are resolved into linear double-stranded DNA And packaged with late viral proteins into immature virions (IV). IVs mature into IMVs via an undescribed mechanism which may include processing of the IV through the Golgi apparatus.

23. Replication 8. ReleaseThe IMVs are transported to the periphery of the cell where they are released in one of three ways. • IMVs released via cell lysis remain IMVs • IMVs can bud through to the cell surface, picking up a viral envelope from the cell plasma membrane. • IMV can bud through the plasma membrane picking up an envelope and becoming an EEV. intracellular enveloped virus (IEV). cell-associated enveloped virus (CEV) intracellular mature virus (IMV)

25. Variola (smallpox) Clinical Features of Human Poxviruses • Smallpox is caused by two strains of the same virus: • Variola major - more common, causes a severe form of the disease • Mortality rate of 15 – 30% • Variola minor - causes a mild form of disease • 1 - 2% mortality rate

26. Transmission • Person  Person • Primarily droplet, or aerosol • smallpox in droplet nuclei can live between a few hours and a few days in the environment • No animal reservoir or vector • Very contagious • Persons are very sick before contagious

27. Pathogenesis of Variola Virus entry & multiply in respiratory epithelium Fever, Headache, Backache, SICK! NOT Infectious (12-14 d ) Blood circulation primary viremia NOT Infectious (2-4 d) Liver, spleen 2nd viremia Capillary vessel VERY Infectious Dermis macules -> papules -> vesicles -> pustule-> crust Multiplies in mouth and Dermis (deep layer of skin)  Rash

28. Case Definition of Smallpox • Illness with acute onset of fever greater than or equal to 101oF. • Followed by rash of firm, deep seeded pustules. • Centrifugal pattern of lesions compared to chickenpox. Rash distribution of smallpox vs. chickenpox. Adapted from World Health Organization, Diagnosis of Smallpox Slide Set, http://www.who.int/emc/diseases/smallpox/slideset.

29. The stages of lesions in poxvirus infections

30. skin rash ofVariola papules vesicles pustules crusts (Emond RTD., A colour atlas of infectious disease 1974)

31. Smallpox Lesions: • Start on Face, Arms, Mouth • Palms and Soles • Deeply Embedded into Skin • Similar Stage of Development • Occur in Very Sick Persons

32. Laboratory Diagnosis • Biopsy Intracytoplasmic inclusion bodies (varying size and duration) • Direct examination Electron microscopy Giemsa staining of infected cell scrape • Virus isolation Embryonated egg: chorioallantoic membrane (Pock lesions) Cell culture : primary monkey kidney cells • Serology hemagglutination inhibition test (only the Orthopoxvirus) neutralization test ELISA IFA • PCR-RFLP analysis / Real-Time PCR / Sequencing The only way to accurately and rapid distinguish between Variola, monkeypox and vaccinia infections.

33. Poxvirus infected cells Cowpox in primary monkey kidney cells, cytoplasmic inclusions with halo ( Guaneiri body) (Versteeg J., A Color Atlas of Virology,1985)

34. Pocks on chorioallantoicnic membrane

35. Other Poxvirus Infections • Poxvirus infections also occur in animals • Transmission to humans requires contact with infected animals • Infections of humans are usually mild • Can result in pox and scars but little other damage • Edward Jenner used cowpox to immunize individuals against smallpox Edward Jenner (1749-1823)

36. Edward Jenner (1749-1823) • Physician in England, credited with cowpox vaccination • Experimented with the folklore that milkmaids who contracted cowpox “did not take the smallpox.” • 1796, Jenner variolated the 8-year-old son of a local farmer with fluid from the cowpox pustules from the hand of a local dairymaid. • A few months later, the boy was injected with smallpox and failed to develop the disease.

37. Smallpox Eradication • Why was smallpox (variola) a good candidate for eradication? • Variola has a narrow host range. • There are no carriers. • There are no animal reservoirs. • A highly effective an inexpensive freeze-dried vaccine was available. • Surveillance of the disease was easy (centrifugal rash). • The WHO created a program to eradicate it.

38. Bioterrorism or Re-emergence? • Although smallpox has been eradicated, there are concerns about the potential use of variola virus as a weapon of terror. • Use of variola as a biological weapon has a long history. • Variola as germ warfare against Native American Indians, French and Indian Wars (1754 - 763). • Consequently, destruction of the last (official) remaining smallpox stocks held in Russia and USA has now been postponed indefinitely.

39. Bioterrorism or Re-emergence? • The possibility of an orthopoxvirus such as variola, monkeypox, camelpox or taterapox virus emerging or re-emerging as a threat to human health increases . • Finally, it is unclear whether all, only a few, or just one of the differences between the genomes of viruses such as camelpox and smallpox. • Genetic modification of camelpox to delete genes that are present in camelpox but absent in smallpox might be highly dangerous.

40. ApplicationExpression Vectors • Use of homologous recombination in infected cells to introduce foreign DNA coupled to VV promoter into virus genome. • Indicator systems such as beta-galactosidase to give recombinant plaques a blue color. • Problems for human vaccines: The proportion of the human population which has already been vaccinated - lifelong protection may result in poor response to recombinant vaccines (?). Dangerous in immunocompromised hosts.

41. ApplicationExpression Vectors • The possible solution to this may be to use Avipoxvirus vectors, as 'suicide vectors' - undergo abortive replication in mammalian cells: • can express high levels of foreign proteins • no danger of pathogenesis • no natural immunity in humans

42. Expression Vectors

43. 敬請指教 歡迎討論 Thanks for Your Attention

44. Monkeypox • Monkeypox occurs mainly in the rain forests of central and west Africa. The disease was first discovered in laboratory monkeys in 1958, but the virus also occurs in a number of African rodent species. In 1970, monkeypox was reported in humans for the first time. • In humans, monkeypox is similar to smallpox, although it is often milder. Unlike smallpox, monkeypox causes lymph nodes to swell (lymphadenopathy). The incubation period for monkeypox is about 12 days (range 7 to 17 days). The illness begins with fever, headache, muscle aches, backache, swollen lymph nodes, a general feeling of discomfort, and exhaustion. Within 1 to 3 days (sometimes longer) after the appearance of fever, the patient develops a papular rash (i.e., raised bumps), often first on the face but sometimes initially on other parts of the body. The lesions usually develop through several stages before crusting and falling off. The illness typically lasts for 2 to 4 weeks. Human monkeypox is believed to have a fatality rate of 1% to 10%.

45. Monkeypox • In June 2003 there was an outbreak of monkeypox in the USA resulting from importation of Gambian giant rats into the U.S. as pets. Import restrictions were subsequenbtly imposed on six specific genera of African rodents to try to prevent a repeat of this outbreak. • Is human monkeypox infection emergent disease or has it always been there but masked by smallpox? And now that we have eradicated smallpox, will monkeypox evolve to take it's place?