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

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

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

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

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

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.

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.

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

Papillomas Epidermal Oral Genital Corneal Laryngeal Genital

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.

Progression of Human Papillomavirus Infection

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

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

Differentiation of Cutaneous Squamous Epithelium and HPV Infection Cycle

Differentiation of Cutaneous Squamous Epithelium and HPV Infection Cycle

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

Differentiation of Cutaneous Squamous Epithelium and HPV Infection Cycle

Differentiation of Cutaneous Squamous Epithelium and HPV Infection Cycle

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

Mucosal Syndromes

HCV-induced Cancers

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

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 10,000 500,000 Cervical Cancer Deaths 4,000 200,000 Sources: Mayo Clinic: Women’s Health. “Pap smear: Screening test for cervical cancer.” Available at: http://www.mayoclinic.com/health/pap-smear/HQ01177.

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

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)

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 2006. [second bullet] Three shots over 6 months are required to boost ones immune system.9 [third bullet] Total cost will be about $360.11 [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, 2006. A16. Mayo Clinic: Women’s Health. “Cervical cancer vaccine OK’d.” Available at: http://www.mayoclinic.com/health/cervical-cancer-vaccine/WO00120. DeNoon D. Cervical Cancer Vaccine Approved. WebMD Medical News. June 8, 2006.

Current ACIP and AAP Recommendations Routine immunization of males and females aged 11-12 years Immunization may be initiated as young as age 9 years Catch-up immunization for females aged 13-26 years Catch-up immunization for males aged 13-21 years Immunization of males aged 22-26 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

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

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.

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

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)

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

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

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

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

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

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

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

Time Course of Adenovirus Respiratory Infection

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

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

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

Adenoviruses: Diagnosis Virus isolation and propagation in epithelial cell cultures; within 2-20 days the virus causes a lytic infection with characteristic inclusion bodies Electron Microscopy Immunoassays (fluorescent antibody and ELISA) PCR DNA probe analysis

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

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

Parvovirus B19: Genome

Spread of B19 within the Body

Time Course of B19 Infection

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

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”

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

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.

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

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

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.