1. Naked DNA viruses Important Pathogens:
Human papillomavirus- cervical cancer
JC virus - PML (progressive multifocal leukencephalopathy)
BK virus - renal disease
Adenovirus- conjunctivitis, pharyngitis, GI
B19- Fifth disease, anemic complications

2. Why should I care whether or not a virus has an envelope?

3. Unenveloped viruses are…
Stable in hostile environments
Not damaged by drying, acid, detergent, and heat
Can infect the GI tract and survive the acid and bile
Can dry and still retain infective
Can spread easily via hands, dust, fomites

4. Unenveloped viruses are…
Released by lysis of host cells
Neutralizing mucosal and systemic antibodies are needed to prevent infection

5. Papovaviruses Topics: non-enveloped DNA viruses Papillomavirus: HPV
- causes warts & genital warts (condylomas)
- tumor virus (cervical carcinoma)
- new vaccine available
Polyomavirus: BK and JC viruses
- usually asymptomatic
In immuno-compromised:
- BK causes renal disease
- JC causes PML (progressive multifocal
leukoencephalopathy

6. General Features of Papovaviruses
Small non-enveloped icosahedral viruses with circular double-stranded DNA genome
Replicate in nucleus
Viruses may cause lytic, chronic, latent, and transforming infections, depending on the host cell
Hallmark: papovaviruses encode proteins that promote cell proliferation; the promotion of cell growth facilitates lytic viral replication in a permissive cell type but may oncogenically transform a cell that is nonpermissive.

7. Human Papillomavirus (HPV)
• 100+ varieties
• Most are harmless • 35 are transmitted sexually
• HPV 16,18, 31 and 45 are considered “high risk”
- Can lead to cervical cancer
• HPV 6, 11, 42, 43 and 44 are considered “low risk” Often lead to the development of genital warts
[title appears] As is so often the case, lack of knowledge breeds confusion. What are the facts about Human Papilloma Virus, its sexual transmission and cancer?
[blue text appears] Let’s begin with HPV. [first bullet] There are more than 100 varieties of HPV, [second bullet] most of which are harmless. [third bullet] 35 of the 100 are transmitted sexually. [fourth bullet] Some of these, notably HPV 16,18, 31 and 45, are considered “high risk” and [subbullet appears] can lead to cervical cancer, that is, cancerous change in the thin flat squamous cells that cover the neck of the uterus. [fifth bullet] Others, such as HPV 6, 11, 42, 43 and 44, are considered “low risk” for cancer but [subbullet appears] often lead to the development of genital warts.

8. Classification of HPVs
All HPVs
Cutaneous
HPV types
Mucosal
HPV types
Low-risk
Mucosal HPV types
High-risk
Mucosal HPV types
Benign
neoplasias
Benign neoplasias, malignancies

9. Papillomas
Epidermal Oral Genital
Corneal Laryngeal Genital

10. Epidemiology of Papillomaviruses
HPVs persist, asymptomatic shedding is likely.
HPVs are transmitted via direct or sexual contacts for certain types.
HPV infections are as frequent as common cold
6 million HPV infections yearly in US
~50% of college age women get HPV infection within four years of becoming sexually active
men likely high also, but less accurate detection
Major cause of cervical cancer (almost all cases)
Other cancers: most ano-genital,also head & neck, and possibly non-melanoma skin cancers.

11. Progression of Human Papillomavirus Infection

12. HPV genome E1 DNA helicase/NTPase
E2 Transcriptional trans-modulator, replication control
E4 Cytokeratin disruption
E5 Cell proliferation (binds PDGF receptor)
E6 Transforming protein (binds p53)
E7 Transforming protein (binds pRb)
L1 Major capsid protein
L2 Minor capsid protein

13. Replication Cycle of HPVs
Viral replication depends on the epithelial cell differentiation stage: It is persistent in the basal layer and active in differentiated cells
DNA-dependent DNA Polymerase

14. Differentiation of Cutaneous Squamous Epithelium and HPV Infection Cycle

15. Differentiation of Cutaneous Squamous Epithelium and HPV Infection Cycle

16. Viral Transformation
How do some viruses grow in cells that are differentiated?
Deregulate the cell cycle.
Papillomavirus depends on cellular replication machinery, so cell must in S (DNA synthesis) phase

17. Differentiation of Cutaneous Squamous Epithelium and HPV Infection Cycle

18. Differentiation of Cutaneous Squamous Epithelium and HPV Infection Cycle

19. Integration of E6/E7 into host cell chromosomes
Host cell promoter
E6/E7 ORF
Frequently precedes oncogenesis

20. Mucosal Syndromes

21. HCV-induced Cancers

22. Laboratory Diagnosis of HPV Infections
Test Detects
Cytology (pap smear) Koilocytic cells
DNA-based immuno-assay Viral nucleic acid
Advantages: High risk-specific, sensitive
Disadvantages: higher false positive rate
Pap stain: blue/green = basal squamous cells
black = nuclei
yellow = keratin

23. Pap Smear Indications
Women begin tests within 3 years of first sexual intercourse or after age 21
Categories of Pap Smear Results
1. Normal (Out of 50 million, 45 million are normal)
2. Atypical (2 million) - Result of general infection, irritation or trauma, or HPV
3. Pre-cancerous or cancerous (1.5 million of each) - Requires follow-up - 90% cure rate with early diagnosis
[title appears] How does HPV cause cancer? To answer this, lets take a step back some 50 years to the creation of the PAP smear by Dr. George Papanicolaou. [blue text and first bullet] His test, familiar to all today, involves collecting cells from the cervix during a routine pelvic exam and [subbullet appears] sending them for microscopic examination. [second bullet] Most women begin PAP tests within 3 years of first sexual intercourse or after age 21. [blue text appears] The results can fall into several categories. [#1 appears] The surface cells, called squamous cells, or mucus-producing glandular cells originating from inside the uterus or cervical canal, are often normal. In fact, of the 50 million PAP smears in the US each year, about 45 million are normal. [#2 appears] In about 2 million cases, the cells are labeled atypical, which is to say they are slightly abnormal. [subbullet appears] This may be the result of general infection, irritation or trauma, or from Human Papilloma Virus. [#3 appears] Of the remaining 3 million, approximately half are pre-cancerous (destined to slowly become cancers) and the other half are already cancerous, [first subbullet] requiring further follow-up [second subbullet] which is curative in 90% of the cases, with early diagnosis.
U.S. Worldwide
Cervical Cancer Diagnoses
10, ,000
Cervical Cancer Deaths
4, ,000
Sources: Mayo Clinic: Women’s Health. “Pap smear: Screening test for cervical cancer.” Available at:

24. HPV Treatment: HPV Prevention:
Warts are removed by surgical cryotherapy, electrocautery, or chemical means, although recurrences are common
Injection of interferon is beneficial
Surgery may be necessary for the removal of laryngeal papillomas
HPV Prevention:
Avoid direct contacts with infected tissue
Proper sexual precautions
Vaccine

25. Vaccine Approach Subunit vaccine: HPV L1 Virus-like Particles
it forms the structure of the virion without containing any HPV genetic material
Anti-L1 antibodies block HPV infection by binding L1 protein and neutralizing infection
T cells clear evading HPV virus
GSK- types 16 & 18
Merck- types 16,18 and 6,11 (wart causing)

26. Details About the Vaccine
• Available as of 2006
• 3 shots over 6 months are required to boost immune system
• Total cost: $360
- Insurers are expected to cover expense
• FDA declared vaccine safe and effective
• CDC recommendation:
 Girls & boys age 9-26 (before sexually active)
• Each state must weigh-in to decide if vaccine will be required to enter public school
[title appears] Now, lets get back to the vaccine. [first bullet] It should be available in the late summer of [second bullet] Three shots over 6 months are required to boost ones immune system.9 [third bullet] Total cost will be about $ [subbullet] It is expected insurers will cover the expense. [fourth bullet] The FDA has declared the vaccine safe and effective. [fifth bullet] The CDC will soon advise who should receive it. [subbullet and first subsubbullet] It is expected they will recommend all girls receive the shots at age 11 and 12, well before most are sexually active and potentially exposed to HPV. [second subsubbullet] There is also a possibility that boys, who can carry the virus as well, could be included, though the FDA has asked for more testing in boys to ensure its safety. [blue box and bullet appear] Finally, each state must weigh-in to decide whether the tests will be required to enter public school. [subbullet appears] These requirements are what assure sufficient population numbers to allow vaccines to gain an upper hand on high incidence, transmissible diseases.
HPV vaccine, given by age 11, will eliminate 70% of cervical cancer and save lives.
Sources: Dooren JC. Merck Cervical-Cancer Vaccine Cleared for Use in Girls, Women. The Wall Street Journal. June 9, A16.
Mayo Clinic: Women’s Health. “Cervical cancer vaccine OK’d.” Available at:
DeNoon D. Cervical Cancer Vaccine Approved. WebMD Medical News. June 8, 2006.

27. Current ACIP and AAP Recommendations
Routine immunization of males and females aged years
Immunization may be initiated as young as age 9 years
Catch-up immunization for females aged years
Catch-up immunization for males aged years
Immunization of males aged if not previously immunized (MSMs)
Not a strong recommendation due to lack of cost-efficacy models
Previous sexual activity is not a contraindication to HPV immunization

28. Emerging Problem: HPV head & neck cancer Vaccine may help
~90% caused by HPV 16
Recent study suggested that vaccine protects against 93% of oralpharyngeal cancers in women

29. Polyomaviridae
There were two human polyomaviruses, BK and JC viruses, named after the initials of the patients they were isolated from.
BKV and JCV are ubiquitous and usually cause asymptomatic infection in immunocompetent people
BKV and JCV may cause renal disease and progressive multifocal leukoencephalopathy (PML), respectively, in immunocompromised people
Merkel Cell polyomavirus: discovered in 2008 is associated with rare Merkel cell carcinomas
In 2007, two new polyomaviruses were isolated from patients with respiratory illness: KI & WU. Disease association unknown.

30. Epidemiology of Polyomaviruses
Polyomaviruses are ubiquitous
Polyomaviruses are transmitted by inhalation of infectious aerosols
BKV and JCV establish persistent and latent infections in kidney and some other tissues, and asymptomatic shedding is likely
Immunocompromised people are at risk for: JCV - PML (progressive multi-focal leukemia) BKV- renal disease

31. Spread of Polyomaviruses within the Body
viruria = virus in urine
hemmorhagic cystitis = inflammation &
bleeding of bladder
PML = immune mediated demyelination
& destruction of white matter
-usually fatal (~1-5% of AIDS patients)

32. JC virus - diagnosis of PML
Test Detects
Histologic examination Demyelination foci
MRI Brain lesions
Computed tomography Brain lesions
PCR analysis of Viral nucleic acid
CSF and brain biopsy

33. Magnetic Resonance Image (MRI) of PML Brain Lesions in Subcortical White Matter

34. Treatment, Prevention, and Control of Polyomaviral Infections
No specific treatment for polyomavirus infection is available, other than to decrease the immunosuppression
The ubiquitous nature of polyomaviruses makes it unlikely that the primary infection can be prevented

35. More Naked DNA viruses Adenovirus - ds DNA non-enveloped viruses
- productive and latent infections
- diseases include pediatric pneumonia & diarrhea,
keratoconjunctivitis
Parvovirus
- ss DNA non-enveloped virus
- B19 diseases: erythema infectiousum (Fifth Disease),
aplastic crisis in sickle cell patients,
acute hemolytic anemia,
hydrops fetalis

36. Adenoviridae
First isolated in 1953 from human adenoid (tonsil) cells
Virion: Icosahedral, with attachment fibers at each vertex
Envelope: no
Genome: ds DNA
Replication: nucleus
Productive and latent infections
Human serotypes: 49
Important diseases: pediatric pneumonia & diarrhea,
keratoconjunctivitis

37. Adenovirus: Pathogenesis
Spread by aerosol, close contact, or fecal-oral route
Virus infects first mucoepithelial cells causing direct cell damage
Virus persists in lymphoid tissue
Disease is determined by the tissue tropism of a virus
Different strains have different transmission & disease
Most: eye or respiratory
Strains 40 & 41 - fecal/oral with GI disease

38. Adenovirus Infections Associated with Disease or Persistence* * * * *

39. Time Course of Adenovirus Respiratory Infection

40. Cytopathology in Human Tissue
A: Lung tissue from a fatal case of adenovirus pneumonia: a desquamated alveolar lining cell with a basophilic inclusion body filling the entire nucleus
B: An desquamated cell with less-advanced cytopathology manifested by a smaller nuclear inclusion body, highlighted by a clear halo within the nuclear membrane
C: The nuclear inclusion is full of virions, of which there may be as many as 10,000 particles per cell A B C

41. Adenovirus Symptoms Strep Throat (5-15% of cases) Viral pharyngitis
- oral inflammation
- nasal inflammation
Strep Throat (5-15% of cases)
Viral pharyngitis
Viral: no fever, but cough

42. Adenovirus Symptoms Conjunctivitis (pink eye)
Bacterial Viral
generally associated with cold-like symptoms, but little of the pus/discharge of bacterial conjunctivitis

43. Adenoviruses: Diagnosis
Virus isolation and propagation in epithelial cell cultures; within days the virus causes a lytic infection with characteristic inclusion bodies
Electron Microscopy
Immunoassays (fluorescent antibody and ELISA)
PCR
DNA probe analysis

44. Adenoviruses: Treatment, Prevention, and Control
No specific therapy is available
Live oral vaccines for Ad4 and Ad7 are available for military recruits but are not used for civil population

45. Parvoviridae (parvus=small)
Virion: Icosahedral
Envelope: no
Genome: ss DNA
Replication: nucleus
Human pathogen: B19
Important diseases: Fifth Disease (erythema infectiousum),
aplastic crisis in sickle cell patients, acute hemolytic anemia, hydrops fetalis

46. Parvovirus B19: Genome

47. Spread of B19 within the Body

48. Time Course of B19 Infection

49. Parvovirus B19 Diseases Children/adults
Aplastic crisis: B19 can arrest red blood cell production. Esp. dangerous in patients with increased red blood cell turnover/chronic anemia (e.g. sickle cell anemia). Transfusion recommended.
Red-cell aplasia: chronic anemia in immunodeficient patients
Hydrops fetalis: infection of pregnant women in 1st 20 weeks of term can lead to fetal anemia in 10% of cases(miscarriage, stillbirth). Transfusion rec.
Congenital red-cell anemia: chronic anemia can result in treated hydrops fetalis cases
Fetal infection

50. Fifth Disease So… Why is it called Fifth disease?
Fifth Childhood exanthem (viral rash)
Chicken Pox
Measles
German Measles (rubella)
Roseola
Fifth Disease
Lacy Rash on legs
“Slapped Cheeks”

51. Pathogenesis of Diseases after B19 Infection in Children and Adults
transient aplastic crisis
pure red-cell aplasia
Fifth
Disease

52. Fetal Infection with Parvovirus B19
The fetal erythron consists of the liver and bone marrow. Packed red blood cells (prbc) are used for intrauterine transfusion. Congenital pure red cell aplasia (PRCA) can be a consequence treating fetal infection with B19 by giving a transfusion.

53. Hydrops fetalis
Human hydropic fetus and placenta after intrauterine parvovirus B19 infection
Hydrops is an edema (swelling due to lymph fluid) in subcutaneous tissue

54. Parvovirus B19: Diagnosis
Diagnosis of Erythema Infectiosum is usually based on the clinical presentation
Diagnosis for B19 disease is based on (I) detection of B19-specific IgM and IgG by ELISAs, (II) PCR
During pregnancy, hydrops can be detected by ultrasound

55. Parvovirus B19: Treatment, Prevention, and Control
Antiviral therapy in the form of commercial immunoglobulin preparations is effective in PRCA associated with persistent infection, although not all persistent infections are terminated with this treatment.
Treatment of hydropic infants with intrauterine transfusions has been reported, although this procedure is fraught with risk.
Prospects for a parvovirus B19 vaccine are good. A vaccine derived from self-assembled capsids expressed in recombinant baculoviruses induces a robust neutralizing antibody response in experimental animals, particularly when used with adjuvant.