Properties of Poxviruses Structure and composition Largest viruses dsDNA, 130-375 kb Brick shaped “dumbbell” core (contains nucleic acid) Lateral bodies (unknown function) Enveloped Resistant to inactivation More than 100 polypeptides Many target the immune response Replicate in cytoplasm Tend to be highly species-specific Some are being explored as gene therapy vectors Core Lateral bodies Envelope
Classification Family Poxviridae SubfamiliesGeneraMembers ChordopoxvirinaeOrthopoxvirusVariola, vaccinia, cowpox, monkeypox, camelpox (vertebrates)ParapoxvirusSealpox, parapox of deer, pseudocowpox AvipoxvirusCanarypox, fowlpox, pigeonpox, turkeypox, penguinpox CapripoxvirusGoatpox, sheeppox LeporipoxvirusHare fibroma, myoma, rabbit fibroma, squirrel fibroma SuipoxvirusSwinepox MolluscipoxvirusMolluscum contagium YatapoxvirusTanapox, Yaba monkey tumor EntomopoxvirinaeCapripoxvirus A (insects)Capripoxvirus B Capripoxvirus C
Replication Attachment, penetration and uncoating Membrane fusion Viral core dissociates, releasing Viral DNA Viral enzymes Virally-encoded RNA polymerase (prepackaged) synthesizes early mRNA Early proteins DNA polymerase Thymidine kinase Recombinase? Maturation Structural proteins are synthesized Virus synthesizes its own membranes Some virus exits by budding, but most when the cell dies
Smallpox Named smallpox to discriminate it from largepox (syphilis) No animal reservoir Two species Variola major (20% fatality; Genbank #VVCGAA) Variola minor (1-2% fatality; Genbank #VMVY16780) Smallpox has shaped civilization Earliest evidence: Egyptian mummies Ramses V (1157 BC) Introduced to the Americas by European explorers British army used smallpox as a biological weapon against the Pontiac Indians About 40 million native Americans died from European diseases, including smallpox Native Americans have limited MHC polymorphisms Killed 300 million people in the 20th century alone Total fatalities probably near 1 billion
Control and Eradication Vaccine Edward Jenner observed that milkmaids rarely contracted smallpox All had recalled earlier cowpox infections, which were nearly universal in milkmaids Cowpox only causes a mild infection in humans Jenner hypothesized that the infectious agent of cowpox protected against smallpox He inoculated a nephew by scarification with cowpox crusts termed variolation Today’s vaccine is live attenuated vaccinia virus Vaccinia’s genome looks similar to cowpox, but it is not identical In the 1950s, the Soviets proposed a global eradication program to the United Nations Some have suggested that the Soviets had a vaccine-resistant strain of variola, which could be used as a bioweapon The World Health Organization (WHO) Intensified Smallpox Eradication Programme program began in 1967 Smallpox was declared eradicated in 1980 (last case in 1977) Only two nations are supposed to have smallpox viruses today United States (CDC-Atlanta) Russia (VECTOR, Moscow) Smallpox
Smallpox Pathogenesis Respiratory transmission Incubation period 10-14 days After day 7 or so, humans begin shedding virus asymptomatically Principal reason for rapid spread Outbreaks tended to be in clusters Allowed the ring immunization containment strategy during the eradication program Clinical symptoms Fever Malaise Centrifugally-distributed exanthems macules papules pustules
Smallpox Pathogenesis in an Animal Model Problem: Smallpox virus was only known to infect humans Can an animal model be developed to study pathogenesis? No cases of smallpox since the 1970s Modern science could learn much from studying the course of infection and immune response in an animal model Because of 9/11, CDC and USAMRIID Scientists were granted permission by the WHO to infect cynomolgus macaques with smallpox in an attempt to assess pathogenesis using contemporary laboratory techniques Jahrling et al. PNAS. 101:15196-15200 Rubins et al. PNAS. 101:15190-15195 Required highest containment: BSL-4 Required >10 9 pfu iv for infection Resulted in fulminant disease fatal in most monkeys
Fig. 2. Gross lesions associated with smallpox virus infection of monkeys. (a) Hemorrhage on the serosal surface of the distal colon, monkey I-7, 6 days after exposure. Note the diffuse petechial hemorrhages and hemorrhagic, colic lymph nodes. (b) Mucosal surface of the distal colon described in a. Note the severe congestion, hemorrhage, and hemorrhagic colic lymph nodes. (c) Medial surface of the right arm of monkey I-4, 9 days after exposure. Smallpox pustules are predominantly discrete with occasional coalescence. Pock lesions developed synchronously and were more numerous distally, which is consistent with centrifugal distribution. (d) Palmar surface of the left hand of a monkey 11 days after exposure. Discrete and coalescing pustules retained integrity due to heavily keratinized palmar and digital epidermis. (e) Upper lip and nostrils from a cynomolgus monkey 11 days after exposure. Note synchronous development of pustules, some of which have umbilicated. Pustules that formed on the lightly keratinized mucosal surfaces of the nostrils have already ulcerated, resulting in dried exudates. Jahrling et al.
Fig. 4. Immunofluorescence examination of tissues from nonhuman primates infected with variola. (a) Staining for monocytes/macrophages (green) and viral antigen (red) indicated the presence of infected monocytes/macrophages (gold) in the lymphoid tissues (lymph node shown) and in circulation. (b) In addition to monocytes/macrophages, virus-infected endothelial cells (green) were readily observed. (c) Staining for monocytes/macrophages (green) and apoptosis (red) revealed the presence of numerous tingible body macrophages in lymphoid tissues. The majority of apoptotic cells were not monocytes/macrophages but instead were lymphocytes. A rare apoptotic monocyte/macrophage (arrow) is shown. (d) A monocyte/macrophage with a clearly stained nonapoptotic nucleus (blue) is shown engulfing two separate apototic bodies. Jahrling et al.
Fig. 5. Concentrations of D dimers, cytokines, and chemokines sequentially obtained from variola- infected monkeys. (a) D dimers (µg/ml) in sera of monkeys that received graded doses of variola strain India 1724 i.v. (b) IL-8 concentrations (pg/ml). (c) Monocyte chemoattractant protein 1 concentrations (pg/ml). (d) Macrophage inflammatory protein 1 concentrations (pg/ml). (e) IL-6 concentrations (pg/ml) in the sera of three monkeys infected i.v. with Harper strain (109 pfu). (f) Mean (± 1 SD) IFN- concentrations (pg/ml) in sera of monkeys infected i.v. with India 7124 and Harper strains (10 9 pfu). Jahrling et al.
Variola viruses do not exist in nature Some nations are suspected of having active bioweapons programs North Korea Syria Iran Israel Russia Some may have kept smallpox stocks hidden The world is ill-prepared for smallpox Vaccination is no longer routine Thus, a large susceptible population Smallpox as a Biological Weapon
Monkeypox 2003 US Outbreak 72 Cases Wisconsin, Illinois, Indiana Kansas, Missouri, Ohio Introduced from Africa to US pet supplier Gambian giant rat Prairie dogs Infection was not fatal to humans In Africa, disease is fatal in 1-10% of cases Democratic Republic of Congo - 10% West Africa - 1-2% The US strain was from west Africa Genomic comparisons revealed 5 genes mutant or missing in West African strains compared to central African strains WA strains do not have D14L, a protein that disables complement enzyme activity, while central African do