Microbiology, virology, immunology department DNA-VIRUSES: HERPESVIRUSES. ADENOVIRUSES RETROVIRUSES. AIDS. ONCOGENIC VIRUSES. PRIONS. Family Herpesviridae Morphology # icosahedral capsid (100 nm) # 162 capsomers # envelope(150-250 nm) # genome:linear double stranded DNA Replication nucleus budding through nuclear membrane Herpesvirus Subfamily Alphaherpesvirinae Betaherpesvirinae Gammaherpesvirinae Herpesviridae Alphaherpesvirinae (ectodermal tissue tropism) Betaherpesvirinae Grow rapidly Latency in sensory neurons Cytomegaloviruses (large balloon-like cells) Grow slowly Latency in salivary glands, kidneys, lymphocytes Gammaherpesvirinae (malignant catarrhal fever virus) Lymphoproliferative diseases Latency in lymphoid cells Human Herpesviruses (HHV) HHV-1 - > Herpes simplex virus 1 HHV-2 - > Herpes simplex virus 2 HHV-3 - > Varicella-zoster virus HHV-4 - > Epstein-Barr virus HHV-5 - > Cytomegalovirus HHV-6 - >HumanB cell lymphotropic virus HHV-7 - > isolated from CD4+ cell HHV-8 - > Kaposi’sarcoma associated herpesvirus (KSHV) Herpes simplex virus (HSV) Properties ectodermal tissue tropism vesicle lesion infection - > multinucleated cell latency • HSV-1- > trigerminal nerve ganglia • HSV-2- > lumbosacral nerve ganglia recurrent infection stimuli: stress,fever,sun light,drugs Cytopathic effect (right) (infected cells develop intranuclear acidophilic inclusion and then undergo necrosis ) HERPES SIMPLEX: Human Herpesvirus 1 (Herpes Labialis) & Human Herpesvirus 2 (Herpes Genitalis). Infection with herpes simplex virus may take several clinical forms. The infection is most often inapparent. The usual clinical manifestation is a vesicular eruption of the skin or mucous membranes. Infection is sometimes seen as severe keratitis, meningoencephalitis, and a disseminated illness of the newborn. HSV-1 life cycle: Primary infection. 1. Productive Infection HSV life cycle: Establishment of latency. 1. Productive Infection 2. Establishment of latency HSV life cycle: reactivation of latent virus. Common triggers: - sunburn - fever - “exam stress” 3. Reactivation of latent virus HSV life cycle:recurrent disease and viral shedding. 4. Cold sores. Virus spread to new host. 3. Reactivation of latent virus Herpes labialis (cold sores, herpes febrilis). This is the most common recurrent disease produced by type 1. The vesicle ruptures, leaving a painful ulcer that heals without scarring. The lesions may recur, repeatedly and at various intervals of time, in the same location. The permanent site of latent herpes simplex virus is the trigeminal ganglion. Herpes labialis Usually cold sores occur along the mucocutaneous junction of the lips. They may be single or clustered. They may develop following stress or another trigger or without an apparent stimulus. The duration is usually 10-14 days. Treatment is usually necessary only in immune-compromised patients. Herpes labialis Recurrent herpes infections can affect other parts of the body. Keratoconjunctivitis. The initial infection with herpesvirus may be in the eye, producing severe keratoconjunctivitis. Recurrent lesions of the eye appear as dendritic keratitis or corneal ulcers or as vesicles on the eyelids. With recurrent keratitis, there may be progressive involvement of the corneal stroma, with permanent opacification and blindness. Genital herpes (herpes progenitalis). Genital herpes is characterized by vesiculoulcerative lesions of the penis of the male or the cervix, vulva, vagina, and perineum of the female. The lesions are more severe during primary infection and may be associated with fever, malaise, and inguinal lymphadenopathy. Type 2 virus remains latent in lumbar and sacral ganglia. Natural history 1. Start as vesicles 2. Ulcer formation Severe HSV Outbreaks Neonatal infections Usually these are due to HSV-2. Herpesvirus type 2 may be transmitted to the newborn during birth by contact with herpetic lesions in the birth canal. Approximately half of the children born to mothers with active lesions will be infected. Neonatal infections The spectrum of illness produced in the newborn appears to vary from subclinical or local to severe generalized disease with a fatal outcome. Clinical syndromes include: Encephalitis Disseminated multi-organ infection (liver, spleen, lungs) Diagnosis of Herpes simplex virus infections Clinical syndrome recognition Electron microscopy Virus isolation Viral serology is generally not useful Laboratory Diagnosis The virus may be isolated from herpetic lesions (skin, cornea, or brain). The appearance of typical cytopathic effects in cell culture suggests the presence of herpesvirus in 1836 hours. Scrapings or swabs from the base of early herpetic lesions contain multinucleated giant cells. Serology: The agent is then identified by neutralization test or immunofluorescence staining with specific antiserum. Antibodies appear in 4-7 days; can be measured by NT, IHT, CFT, RIA and reach a peak in 2-4 weeks. HV, immune fluorescence test Tzanck Test A smear of an opened skin vesicle to detect multinucleated giant cells. Used to assay for HSV-1, HSV-2, and VZV. Tzanck heavens I do not have herpes! Treatment of HSV infections In normal hosts treatment provides only a 1-2 day benefit for cold sores Acute primary genital infections may be treated with acyclovir or a “cousin”. Recurrent genital infection can be prevented with the use of long term prophylactic acyclovir. Acyclovir can also be used to treat serious infections in severely immuno-compromised patients and in brain or systemic infections. VARICELLA-ZOSTER VIRUS (VZV) (Human Herpesvirus 3 – HHV-3) Varicella (chickenpox) is a mild, highly infectious disease, chiefly of children, characterized clinically by a vesicular eruption of the skin and mucous membranes. The causative agent is indistinguishable from the virus of zoster. Zoster (shingles) is a sporadic, incapacitating disease of adults (rare in children) that is characterized by an inflammatory reaction of the posterior nerve roots and ganglia, accompanied by crops of vesicles (like those of varicella) over the skin supplied by the affected sensory nerves. VARICELLA-ZOSTER VIRUS (Human Herpesvirus 3) (Chickenpox, Herpes Zoster, Shingles, Zona) Both diseases are caused by the same virus. Varicella is the acute disease that follows primary contact with the virus, whereas zoster is the response of the partially immune host to a reactivation of varicella virus present in latent form in sensory ganglia. Chickenpox Transmission Persons to person contact or sneezing/coughing Kids around 6 or 7 will usually spread it between each other. Pathogenesis & Pathology of Varicella: The route of infection is probably the mucosa of the upper respiratory tract. The virus probably circulates in the blood and localizes in the skin. Swelling of epithelial cells, ballooning degeneration, and the accumulation of tissue fluids result in vesicle formation. In nuclei of infected cells, particularly in the early stages, eosinophilic inclusion bodies are found. Varicella virus, pathogenesis Characterized by: Fever Raw, open, itchy sores Varicella Chickenpox Complications Bacterial Infections Viral Pneumonia Bleeding Problems Infection of the brain Treatment Acyclovir Tylenol In medieval times and still today, oatmeal baths are used to relieve itching. Prevention Vaccination(Varicella) A live attenuated vaccine is available. • Quarantine Or…for the extremely paranoid • Kill everyone around you Pathogenesis & Pathology. In addition to skin lesions — histopathologically identical with those of varicella — there is an inflammatory reaction of the dorsal nerve roots and sensory ganglia. Often only a single ganglion may be involved. As a rule, the distribution of lesions in the skin corresponds closely to the areas of innervation from an individual dorsal root ganglion. There is cellular infiltration, necrosis of nerve cells, and inflammation of the ganglion sheath. Zoster: Shingles If you come in contact with someone with Shingles and you haven’t had Chicken Pox yet, you will get Chicken Pox and not Shingles There is no vaccine for Shingles. There is work being done to create one. It is expected to take about 5 years Zoster Shingles CYTOMECALOVIRUS (Human Herpesvirus 5) Asymptomatic in normal humans, acquired as adult; also produces cytomegalic inclusion disease in newborn; also non-heterophile transfusion mononucleosis; problem in immunocompromised high degree of shedding in pregnancy may be an STD Retinitis, and detached retina in 25% treated with ganciclovir in severe cases Clinical Manifestations. Cytomegalovirus infection can result in one of three distinct clinical syndromes. Congenital cytomegalovirus infection: hepatospleno-megaly, retinitis, a petechial/purpuric skin rash, and involvement of the central nervous system (ventriculo-megaly, intracranial calcifications, etc). Clinical Manifestations. Mononucleosis syndrome (fever, malaise, atypical lymphocytosis, pharyngitis and, rarely, cervical adenopathy or hepatitis) Third clinical entity is cytomegalovirus infection in severely immunocompromised individuals. In these patients, infection can involve the lungs, gastrointestinal tract, liver, retina, and central nervous system Persistence of CMV common; 10-15% in teens; 50% by age 35 part of the differential for hepatitis Virus isolated from urine, blood, throat washings, saliva, tears, breast milk, semen, stool, amniotic fluid, cervical secretions and tissue for transplantation (now tested) Cytopathic Effect of VZV Cytopathic Effect of VZV in cell culture: Note the ballooning of cells. (Coutesy of Linda Stannard, University of Cape Town, S.A.) Cytomegalovirus Inclusion Diseases. Electron micrograph of a single animal cell infected with the cytomegalovirus. The intranuclear inclusion body has a typical” owl-eyed” apearence. Pathogenesis. Cytomegalovirus can cause persistent infection in various tissues, including those of the salivary glands, breasts, kidneys, endocervix, seminal vesicles and peripheral blood leukocytes. This persistent infection leads to chronic viral excretion by the involved organ. Transmission of virus is through contact with infected secretions. The average incubation period is four to six weeks. It should also be noted that the kidneys of organ donors can be a source of cytomegalovirus for the recipient, and that peripheral blood leukocytes have been implicated in the transmission of cytomegalovirus via blood transfusion. Epstein-Barr virus The usual source of infection is the saliva of persons shedding the virus. The virus has an affinity for receptors on the surface of B cells. Viral infection results in local replication and a secondary viremia. There is proliferation of both B cells and the T8 (suppressor) subset of T cells. EB HERPESVIRUS (Human Herpesvirus 4). EB (Epstein-Barr) virus is the causative agent of infectious mononucleosis and has been associated with Burkitt's lymphoma and nasopharyngeal carcinoma. Epidemiology. Epstein-Barr virus is transmitted by intimate contact. Pathogenesis. Epstein-Barr virus is tropic for Blymphocytes. Mononucleosis Characterized by fever, hepatosplenomegaly, pharyngitis, and lymphadenopathy (especially posterior auricular nodes) Peak incidence 15-20 years old. Abnormal circulating cytotoxic T cells (atypical lymphocytes). Most common during peak kissing years (“kissing disease”) Clinical manifestations of EBV infections Infectious mononucleosis The incubation period is 4-6 weeks. The illness may last from 2-8 weeks. Fatigue may persist for months Clinical manifestations of EBV infections Infectious mononucleosis Symptoms and signs tonsilar enlargement pharyngeal redness with or without pus cervical adenopathy (80-90%) large liver and spleen skin rash, esp. after ampicillin Diagnosis of EBV infection Increased lymphocytes and monocytes Atypical lymphocytes (usually >30%) Neutropenia (low neutophils) and thrombocytopenia (low platelets) Most have elevated liver enzymes Other specific serologic tests directed against viral antigens Atypical lymphocytes Typical pharyngitis Treatment of EBV infections Treatment is usually supportive Infectious mononucleosis Nasopharyngeal carcinoma Burkitt’s Lymphoma (1) Burkitt's lymphoma (BL) occurs endemically in parts of Africa (where it is the commonest childhood tumour) and Papua New Guinea. It usually occurs in children aged 3-14 years. It respond favorably to chemotherapy. It is restricted to areas with holoendemic malaria. Therefore it appears that malaria infection is a cofactor. Multiple copies of EBV genome and some EBV antigens can be found in BL cells and patients with BL have high titres of antibodies against various EBV antigens. Burkitt's lymphoma Oncogenic Properties: Herpesviruses have been linked with malignant diseases in humans : herpes simplex virus type 2 with cervical and vulvar carcinoma; EB virus with Burkilt 's lymphoma of African children and with nasopharyngeal carcinoma. Other Herpesviruses HHV-6 - Roseola or exanthem subitum so-called “sixth” exanthem HHV-7 - T lymphotropic and produces rashlike illness HHV-8 - associated with Kaposi’s sarcoma in AIDS patients DREAMS CAME TRUE? Adenoviruses (ADV) a group of viruses that infect the membranes (tissue linings) of the respiratory tract, the eyes, the intestines, and the urinary tract. first isolated from adenoidal tissue in 1953. Family Genus Adenoviridae Mastadenovirus Adenoviruses are double-stranded DNA viruses. They have icosahedral capsids with twelve vertices and seven surface proteins. The virion is non-enveloped, spherical and about seventy to ninety nm in size. ADENOVIRUS - Classification Subgroups- 6 subgroups (A-F), based on hemagglutination Serotypes- 1-49 (human) Common serotypes:- 1-8, 11, 21, 35, 37, 40 Enteric Adenoviruses belong to subgroup F Transmission Ingestion/Fecal-Oral Route Respiration (through respiratory droplets) Contact/Hand-to-eye transfer Venereal The types of adenovirus that cause respiratory and intestinal infections spread from person to person through respiratory secretions (coughs or sneezes) or fecal contamination. Fecal material can be ingested through contamination of water supplies, eating food contaminated by houseflies, or poor hygiene after handling diapers. Indirect transmission can occur through exposure to the contaminated surfaces of furniture and other objects. The types of adenovirus causing conjunctivitis may be transmitted by water (in lakes and swimming pools), by sharing contaminated objects (such as towels or toys), or by touch. Adenoviral infections affect infants and young children much more frequently than adults. Signs and Symptoms Depending on which part of the body is affected, the signs and symptoms of adenoviral infections vary: Febrile respiratory disease: bronchiolitis, croup, pneumonia Gastroenteritis Urinary tract infection: cervicitis, urethritis, hemorrhagic cystitis Eye Infections: Conjunctivitis, Keratoconjunctivitis Adenoviral infections Most adenoviral infections last from a few days to a week. Severe respiratory infections may last longer and cause lingering symptoms, such as a cough. Pneumonia can last anywhere from 2 to 4 weeks. Laboratory Diagnosis. The viruses have been isolated → from throat swabs, → conjunctival swabs, → rectal swabs, stools of patients, → urine, → from the eye (from patients with conjunctivitis). Laboratory Diagnosis. The viruses are isolated by inoculation of tissue cultures of human cells in which characteristic cytopathic changes are produced. A new serotype that has not been isolated in cell cultures can be detected by direct examination of fecal extracts by electron microscopy or by enzymelinked immunosorbent assay. Serology. In most cases, the neutralizing antibody titer of infected persons shows a 4-fold or greater rise against the type recovered from the patient in NT. The CF test, using the common antigen, is an easily applied method for detecting infection by any member of the group. A sensitive radioimmunoassay can measure serum antibody to type 5 fiber antigen. Treatment Adenoviral illnesses often resemble certain bacterial infections, which can be treated with antibiotics. But antibiotics don't work against viruses. Adenoviral infections usually don't require hospitalization. Prevention There's no way to completely prevent adenoviral infections. To reduce the risk of transmission hand-washing, keep shared surfaces such as countertops and toys clean, remove human with infections from group settings until symptoms subside. Chlorination of swimming pools, drinking water, wastewater High hygiene standards in opthamology practice Measures to prevent nosocomial transmission Adenovirus vaccine Oral live attenuated vaccine Strains 4, 7 Used in military recruits Statistics Every day in the world infect with HIV 14.000 people, about 6.000 – young men and women 15 - 24 years old. Image from http://www.hivaidssearch.com/hiv-aids-links.asp?id=811 Adult Prevalence of AIDS Image from http://www.hivaidssearch.com/hiv-aids-links.asp?id=811 Human Immunodeficiency Virus HIV, the etiologjcal agent of AIDS, belongs to the lentivirus subgroup of the family Retroviridae. Structure. HIV is a spherical enveloped virus, about 90-120 nm in size. The nucleocapsid has an outer icosaedral shell and an inner coneshaped core, enclosing the ribonucleoproteins. The genome is diploid, composed of two identical single stranded, positive sense RNA copies. KEY VIRAL ENZYMES Reverse transcriptase Integrase Protease Retrovirus virion assembly and release by budding some viral proteins inserted in cell membrane host cell chromosome virus and cell proteins in envelope new viral genomes and proteins provirus transcription of the provirus RNA DNA integration into host cell chromosome Types of HIV Virus HIV 1 Most common in sub-Saharan Africa and throughout the world Groups M, N, and O Pandemic dominated by Group M HIV 2 Group M comprised of subtypes A - J Most often found in West Central Africa, parts of Europe and India Pathogenesis. The receptor for the virus is the CD4 antigen and therefore the virus may infect any cell bearing the CD4 antigen on the surface. This is primarily the T4 (helper/inducer) lymphocyte. Some other immune cells also possess the CD4 antigen on the surface and so are susceptible to infection. Pathogenesis. Thus about 3 – 10 per cent of B lymphocytes and 10-20 per cent of monocytes and macrophages, including specialised macrophages such as alveolar macrophages in the lungs and Langerhans cells in the dermis are susceptible. Glial cells and microglia in the central nervous system are also found infected. Folicular dendritic cells from tonsils can be infected by HIV without the involvemnent of CD4. Specific binding of the virus to CD4 is by the envelope glycoprotein gp120. In an infected individual, HIV can be isolated from the blood, lymphocytes, cell free plasma, semen, cervical secretions, saliva, tears, urine and breast milk. The primary pathogenic mechanism in HIV infection is the damage caused to the T4 lymphocyte. The T4 cells decrease in numbers and the T4:T8 (helper: killer) cell ratio is reversed. Viral infection can suppress the function of infected cells without causing structural damage. Infected T4 cells do not appear to release normal amounts of interieukin-2, gammainterferon and other lymphokines. This has a marked dampening effect on cell mediated immune response. AIDS is primarily a sexually transmitted infection. It was transmitted predominantly among male homosexuals. The danger of infection is more for the passive partner because mucosal tears are very frequent during anal intercourse and virus laden lymphocytes in the semen can directly enter through these. In homosexual men, the relative risk of infection in the various sexual practices has been estimated in the descending order as ano-receptive, oro-receptive, ano-insertive and oro-instertive. PREVENTION OF HIV INFECTION The second mode of transmission is through blood and blood products. Before the danger of HIV transmission was recognised, many persons had received blood and blood products containing the infectious virus. Screening of blood donors is now mandatory. Even screening may not completely eliminate the danger as the early infectious case may be missed but the risk is reduced considerably. Contaminated needles can transmit the infection. This is particularly relevant in drug addicts who share syringes and needles. The use of unsterile syringes and needles by qualified and unqualified health workers makes iatrogenic infection likely. Even in large hospitals, sterilisation and asepsis are often unsatisfactory. The use of disposable syringes, needles and other equipment should be obligatory. This restriction also applies to the donation of semen, cornea, bone marrow, kidney and other organs as infection can be transmitted through any of these. Transmission of infection from mother to baby can take place before, during or after birth. As infection occurs in about half such babies. HIV may be present in breast milk and may rarely be transmitted through breast feeding. How HIV can NOT be transmitted Through air or by coughing and sneezing Through food or water Through sweat and tears By sharing cups, plates, and utensils with an infected person By touching, hugging and kissing an infected person By sharing clothes or shaking hands with an infected person By sharing toilets and bathrooms with an infected person By living with an infected person By mosquitoes, fleas, or other insects 101 WHO guidelines for clinical diagnosis of AIDS in adults 2 major signs & 1 minor sign (in absence of other explanation) Major signs: 1. Weight loss > 10% bodyweight 2. Diarrhea > 1 month 3. Fever > 1 month 103 WHO guidelines for clinical diagnosis of AIDS in an adult Minor signs: 1. Widespread enlarged lymph glands 2. Persistent cough for more than 1 month 3. Thrush (candida) of mouth & throat 4. Widespread itchy skin rash 5. Recurring shingles (herpes zoster) 6. Chronic, severe, spreading cold sores (HSV1) 7. Loss of memory 8. Loss of intellectual capacity 9. Peripheral nerve damage 104 105 © Teaching-aids at low cost 106 © Teaching-aids at low c 107 © Teaching-aids at low cost 108 © Teachings-aids at low cosr 109 © Teaching-ai Opportunistic Infections HIV slowly destroys CD4 cells over years of infection As the CD4 count drops, infections take ‘opportunity’ of this weakened immune system, resulting in opportunistic infections Pneumocystis carinii Dermatomycosis Herpes zoster Dermatitis Herpes ACQUIRED DEFICIENCY SYNDROME (AIDS) This is the end stage disease representing the irreversible breakdown of immune defense mechanisms, leaving the patient a prey to progressive opportunistic infections and malignancies. The clinical severity of AIDS varies with the type of infection or malignancy present. In early AIDS, many patients are ill only during episodes of infection which may respond to treatment. Between episodes they may be relatively well and able to resume normal life. Kaposi's sarcoma Kaposi's sarcoma Herpetic infection Еczema Warts Sarcoma Laboratory diagnosis Laboratory procedures for the diagnosis of HIV infection include tests for immunodificiency as well as specific tests for HIV. A. Immunological tests. The following parameters help to establish the immunodeficiency in HIV infection: l . Total leucocyte and lymphocyte count to demonstrate leucopenia and a lymphocyte count usually below 2,000 /c.mm. 2. T cell subset assays. Absolute T4 cell count will be usually less than 200/c.mm. T4: T8 cell ratio is reversed. 3. Platelet count will show thrombocytopenia. 4. Raised IgG and IgA levels. 5. Lymph node biopsy showing profound abnormalities. CD4 counts Number of CD4 cells in blood provides a measure of immune system damage CD4 count reflects phase of disease CD4 count: 500 – 1200: 200 – 500: < 200: Normal Beginning of HIV illness AIDS 122 B. Specific tests for HIV infection. These inc1ude demonstration of HIV antigens and antibodies and isolation of the virus. I. Antigen detection. The time course of appearance of detectable antigens and antibodies after VIIV infection is generally as follows: Following a single massive infection, as by blood transfusion, the virus antigens (p24) may be detectable in blood after about two weeks. IgM antibodies appear in about 4-6 weeks, to be followed by IgG antibodies. If the infecting dose is small, as following a needlestick injury, the process may be considerably delayed. The appearance of p24 antigenemia and viremia followed by the early antibody response coincide with the acute or seroconversion illness. Afterwards, p24 antigen disappears from circulation and remains absent during the long asymptomatic phase, to reappear only when severe clinical disease sets in. Tests for antigen detection are available only in specialized laboratories and therefore not used routinely. 2. Virus isolation. Once infected with HIV, a person remains infected for life. The virus is present in circulation and body fluids, mostly within the lymphocytes but some are also cell free. Virus titres are high early in infection, about a week before antibodies start appearing. Antibodies do not neutralize the virus and the two can coexist in the body. During the phase of asymptomatic infection, the virus titre is low and may not be detectable but when clinical AIDS sets in, the titre rises once again. An infected person may therefore be infectious throughout but the infectivity is highest in the early phase of infection (when the antibody tests are negative and the case may not be detected in screening tests) and again when the person becomes clinically ill, 3. Antibody detection. Demonstration of antibodies is the simplest and most widely employed technique for the diagnosis of HIV infection. However, it needs to be emphasized that it may take several weeks to months for antibodies to appear after infection, and during the later part of this period, the individual may be infectious. This seronegative infective stage is known as the window period. For this reason, antibody screening is not totally dependable for spotting infectious persons, for example, from among blood donors. Once antibodies appear they increase in titre and broaden in spectrum for the next several months. IgM antibodies disappear in 8-10 weeks while IgG antibodies remain throughout. When immunodeficiency becomes severe following clinical AIDS, some components of anti HIV antibody (e.g., anti-p24) may disappear. Serological tests for anti HIV antibodies are of two types screening and confirmatory tests. Add 1 drop specimen to well OraQuick test HIV Screening Tests Confirmatory Testing For Western blot: Venipuncture for whole blood Oral fluid specimen Follow-up testing of persons with negative or indeterminate Western blot results after 4 weeks DIAGNOSIS OF HIV INFECTION EIA Negative WESTERN BLOT Not indicated Positive Negative Positive Indeterminate Positive Positive REMARKS Reassure patient Re-test in 3 months Re-test in 3 months or perform PCR Counsel and treat patient HIV Life Cycle reverse transcriptase inhibitors like: Stavudine Tenofovir protease inhibitors like: Saquinavir Darunavir PREVENTION OF INFECTION