1. Adenoviruses and Poxviruses
18 Muharram 1428/ 6th Feb 2007
SBM 2044
Medical Microbiology

2. Adenoviruses
A valuable systems for biochemical and molecular studies of eukaryotic cell processes.
Icosahedral symmetry; Linear ds DNA; no envelope; capsids composed of 252 capsomeres. Hexon and penton capsomeres are 2 main components. Hexon is the major protein forming the 20 triangular faces of the viral capsid.
Human adenovirus are divided into 6 groups (AF) based on their physical, chemical, and biological properties.

3. Adenovirus
Penton base carries a toxin-like activity  cytopathic effects and rapid detachment of cells from the surface
Fibers contain type-specific antigens for serotyping and possess haemagglutinating activity.

4. Adenoviruses Exist in all parts of the world, all year round.
Different serotypes may infect different groups of people:
Eg. 1, 2, 5, 6 – 1st years of life;
4, 18 & 19 – not until adulthood;
3, 4 & 7 – common among military recruits.
Spread person-to-person by respiratory and ocular secretions

5. Pathogenesis

6. Adenovirus Replication
Replicate well only in cells of epithelial origin.
A) Virus attachment:
Adenovirus attaches to host cells via the fiber structures. The receptor is usually CAR (coxsackie-adenovirus receptor).
Internalisation of the virus takes place when the penton base interacts with cellular integrins.
Virus then internalised into endosomes, and by acidic pH into cytosol.
Next, uncoating commences in cytoplasm and finishes in the nucleus, and simultaneously ends the host-virus molecular interaction.

7. Adenovirus Replication
B) Early events:
Is the stage before the onset of viral DNA synthesis.
Aims: to induce the host cell to enter the S phase of the cell cycle; to express viral functions that protect the infected cell from host defense mechanisms; and to synthesise viral genes for replication.
>20 early proteins are synthesised, many of which are important for viral replication. Eg. E1A is vital before the synthesis of other early regions. The E1B encodes proteins that block apoptosis that occurs due to E1A functions.

8. Adenovirus Replication
C) Replication and Late Events:
Virus replicates in the nucleus.
Late genes mainly encode structural proteins.
D) Viral Assembly and Maturation:
Virion is made in the nucleus.
Capsomeres self-assemble into empty-shell capsids in the nucleus, then only DNA enters the capsids.

9. Adenovirus Replication
E) Virus Effects on Host Cells:
Adenoviruses encode several gene products to counteract antiviral agents from host. Virus prevent the activation of an interferon-inducible kinase that phosphorylates and inactivates eukaryotic initiation factor 2.
Adenovirus E3 inhibits cytolysis of infected cells by host responses. E3 gp19-kDa protein blocks movement of the MHC class I antigen to the cell surface, protecting the cell from cytotoxic T lymphocytes-mediated lysis. Also blocks induction of cytolysis by the cytokine TNF-α.
Cytopathology: marked rounding, enlargement, aggregation of affected cells into grape-like clusters. Infected cells do not lyse. Sometimes cells can be seen with rounded intranuclear inclusions containing DNA (≈ CMV, but without multinucleated giant cells or syncytia).

10. Gene Therapy Adenovirus as gene delivery vehicles or DNA vaccination.
Advantage: because replication-defective virus is able to lyse the endosome after internalisation and release DNA into the cytoplasm.
Prototype for adenoviral therapy is AV5

11. Adenovirus Infections
Adenoviruses infect and replicate in epithelial cells of resp T, eye, GIT, UT etc. Some may persists as latent infections for years in adenoids and tonsils, and are shed in the faeces several months after infection.
Adenoviruses 1-7 are the common types. A single serotype may produce more than one clinical illness.
Respiratory diseases with cough, nasal congestion and sore throat are usually associated with Group C viruses, commonly found in infants and children.

12. Adenovirus Infections
Adenoviral pneumonia – by adenovirus 3, 7 and 21 – are thought to be the cause of childhood pneumonia.
Acute respiratory disease syndrome among military recruits, which caused by types 4 and 7.
Others: keratoconjunctivitis by types 8, 19 & 37; and enteritis in children by types 40 & Types 11 and 21 may cause acute haemorrhagic cytitis in children esp boys. Patients receiving transplants are immunocompromised are susceptible to pneumonia usually caused by Types 1-7 eg. Children receiving liver tx → adenovirus hepatitis.

13. Laboratory diagnosis
Infected cells can be cultured, and tissues are looked for rounding and clustering of swollen cells. Adenovirus also cause increase glycolysis in cells, hence the growth medium becomes more acidic. Viruses can be detected by immunofluorescence against antihexon Ab.
PCR of samples using primers from a conserved viral sequence (eg. Hexon VA1) for all serotypes.
Examination of faecal extracts by electron M, ELISA or by latex agglutination. Since adenovirus can be excreted for prolonged periods, the presence of virus does not necessarily mean it is associated with disease.

14. Treatment and Prevention
Most infections are mild and require no therapy. But serious adenovirus illness can be managed by treating symptoms and complications.
Careful hand washing environment surface by sodium hypochlorite (bleach).
Vaccine: 1971 – US focuses on military with live adenovirus types 4 & 7, enclosed in gelatine-coated capsules which allows the virus to bypass resp T and be released in intestine instead.

16. Poxviruses

17. Poxviruses
Largest and most complex of viruses; characterised by extensive rash and high fever.
Include variola virus, etiologic agent of smallpox million deaths in 20th century, in 1967 alone, 15 million contracted with the disease, and 2 million deaths – apparently eradicated. But some strains are still kept for research purpose. Smallpox as biological weapon.
Complex structure, brick and oral-shaped. The extracellular forms contain 2 membranes (EEV - extracellular enveloped virions), intracellular particles only have an inner membrane (IMV - intracellular mature virions).
Ds DNA, linear, kbp. Virions contain >100 polypeptides.

18. Adenovirus vs. Host
Poxviruses are unique – replication in cytoplasm, suggest that poxviruses encode viral genes for many cellular proteins.
Viral-encoded host modifier genes:
1. polypeptide of early genes of vaccinia V is closely related to epidermal GF and to transform GF-α.
2. genes resemble mammalian genes for proteins that would inhibit host defense mechanism eg. TNF-Receptor, IFN-γ Receptor, IL-1-Receptor and complement binding protein. All this disrupts complement and ck network hence enhanced virus replication.

19. Poxvirus Infections in Humans
Control by variolation – inoculating someone with mild forms of smallpox to produce immunity.
Smallpox vaccination used vaccinia virus. Unlike variola, vaccinia has a broad host range including mice. Some strains cause serious disease in animals.

20. Pathogenesis
Variola virus entered the mucous membranes of the upper resp T. Followed by: 1. primary multiplication in lymphoid tissue; 2. transient viraemia; 3. secondary multiplication; 4. secondary and intense viraemia; 5. clinical disease.
6-9 days after infection, lesions in the mouth ulcerated and discharge virus (early).
Later, pustules broke down and discharged virus into the environment.

21. Smallpox

22. Pathogenesis
All the layers of the skin were involved, and there was actual necrosis of the dermis. Hence, scarring after variola infections.
Vaccinia virus causes localised pustular lesions only at the site of inoculation.
Incubation period is days. Pustules formed crusts that fell off after 2 weeks.

23. Replication I
Receptors are not known, but probably >1 on different cell types. For Vaccinia, probably the EGF receptor (epidermal growth factor).
Penetration is complex and may also involve >1 mechanism.
Uncoating occurs in two stages, removal of the outer membrane as the particle enters the cell and in the cytoplasm, the particle (minus its outer membrane) is further uncoated and the core passes into the cytoplasm.
Gene expression is carried out (exclusively?) by viral enzymes associated with the core and is divided into 2 phases:
Early genes: ~50% genome, expressed before genome replication
Late genes: expressed after genome replication; late promoters are dependent on DNA replication for activity.

24. Replication II
Poxvirus gene expression has been studied in detail because of the interest in the use of Vaccinia virus as a vector for expression of heterologous genes.
Genome replication is believed to involve self-priming, leading to the formation of high m.w. concatemers (isolated from infected cells) which are subsequently cleaved and repaired to make virus genomes. The many virus-encoded enzymes involved in replication (e.g. thymidine kinase - tk) offer potential targets for chemotheraputic agents.
Assembly occurs in the cytoskeleton; the events involved in putting together such a complex particle are not understood, but probably involve interactions with the cytoskeleton (e.g. actin-binding proteins). Inclusions are formed in the cytoplasm which mature into virus particles. Actin 'comet tails' form which shoot IEV through the cytoplasm to the cell surface, and possibly into adjacent cells - these have been timed moving at 3µm/min. This may provide an alternative mechanism for cell to cell spread (c.f. EEV).
Overall, replication of this large, complex virus is rather quick ~12h.

26. Laboratory diagnosis
1) Specimen lesions poxviruses are stable and remain viable for weeks without refrigeration.
-Direct examination – rapid (1hr) identification, large enough to be seen by light microscopy.
2) Differentiate among poxviruses by inoculation of vesicular fluid onto chorioallantoic membrane of chick embryos. Look for lesions on membrane – variola pocks are much smaller

27. Pock on chick chorion-allantois membrane induced by India-3a strain
Pock on chick chorion-allantois membrane induced by India-3a strain. 1- epithelium, 2 - connective tissue, 3 - blood vessel. Arrows show congested blood vessel near epithelium. Thick arrow shows border of the pock.

28. Laboratory diagnosis
- look for growth in cell cultures. Only orthopoxviruses grow well in human and non-human cells.
3) PCR test specific virus.
Differential diagnosis is important: smallpox may be confused with drug rashes, varicella.
Rx: vaccinia immunoglobulin vaccinia virus – limited to CDC; methisazone – chemotherapeutic agent and prophylaxis.

29. Transmission
Highly contagious, very stable in extracellular environ; by respiratory spread; dried virus in crusts from skin lesions could survive in clothes and other materials.
Highly contagious once fever had begun ~1st week rash. Resp droplets infections come earlier than skin lesions.

30. Smallpox amenable to total eradication
No known non-human reservoir
1 stable serotype
Had made 1 effective vaccine
Subclinical infection cases did not occur
No chronic asymptomatic
Severe patients get quick attention from medical authorities, hence interrupt cycle.
Vaccination: vaccinia virus infected on calves  vaccine prepared from vesicular lesions in skin  vaccine contains 40% glycerol, 0.4% phenol.

31. Reactions 3-4d: papule increases in size  5-6d: vesiculation
9d : maximum size  pustular. Dessication up to 2 wks, whereby depressed pink scar turns white.
Person is fully protected after vesicular or pustular surrounding a central lesion (scab or ulcer)~7d. If not, vaccination again.

32. Other Poxviruses
Monkeypox – also orthopoxvirus, rare zoonosis; acquired by direct contact with wild animals in Africa.
Pronounced lymphadenopathy-feature not seen in smallpox or chickenpox
Several outbreaks: Zaire, 2003 US.
Cowpox – also orthopoxvirus, less sever and milder lesions confined to teats and udders.
Direct milking with hands
Buffalopox
Orf virus – occupational disease, mainly seen in sheep and goats.

33. Tumours associated with poxviruses
Molluscum contagiosum:
Is a benign epidermal tumour
Had not transmitted to animals
Has not been grown in tissue cultures
Studied in human lesion by EM, Molluscipoxvirus causes small pink wart-like tumous, rarely on palms, soles or mucous membranes.
Tanapox and Yaba Monkey Tumour:
Tanapox is a fairly common skin infection in Africa, Kenya and Democratic Republic of Congo.
Yatapoxvirus – morphologically similar to orthopox