UNIT 1 VIRAL INFECTIONS (microbiology and parasitology)

Learn how to classify the infections caused by some selected viruses.
And be able to explain for others and you may pass your examination about viral infections.

1. NUB1106: Microbiology and Parasitology A. CLINICAL VIROLOGY

2. INTRODUCTION A. General Characteristics of Viruses Viruses do not fall strictly into the category cellular microorganisms as they do not possess a cellular organisation . Viruses are obligate intracellular organisms unable to self-replicate. 3. They lack enzymes necessary for proteins and nucleic acids synthesis and are dependent for replication on the synthetic machinery of host cells 4. They multiply by a complex process and not by binary fission 5. They are unaffected by antibiotics. 6. The genome is either DNA or RNA but never both. 7. Viruses do not have a system to produce ATP. 8. Range in size from100 to 400 nm(not seen by light microscope) 9. The classification of viruses is based on nucleic acid type, size and shape of virion , and presence or absence of an envelope.

3. Morphology of viruses Do not possess cellular organization Contain one type of nucleic acid either RNA or DNA Lack enzymes necessary for protein and nucleic acid synthesis machinery of host cells They multiply by complex process and not by binary fission. They are unaffected by antibiotics. They are sensitive to interferon.

4. MODE OF VIRAL MULTIPLICATION Adsorption: the virus attaches to its host cell specifically through host’s cell receptors. Penetration: the virus is engulfed into host cell Uncoating : the envelope(for enveloped viruses) of the virus is removed. Synthesis: replication and protein production. Assembly: viral proteins are assembled to form virion (new virus) Release: Enveloped viruses bud off of the membrane. a. Cell lysis: Naked viruses lyse host cell and leave through a hole in the plasma membrane. b. Budding: Intact virion pushes outward from a host's membrane. The mne wraps around the virion ; thus becoming the viral envelope.

5. Viral penetration to host cell

6. Viral budding(released from infected host cell)

7. Interaction between viruses and infected cells. cell death( cytocidal effect ) or even lysis( cytolysis ). cellular proliferation or malignant transformation ( oncogenic viruses ). Cellular autolysis. Respiratory syncytial virus(RSV) cause fusion of adjacent cell membaranes leading to polykaryocytosis or syncytium formation. Virus coded antigens may appear on the surface of infected cells,viral hemagglutin appears on the surface of cells infected with the infuenza virus and cause adsorption of erythrocytes to the cell surface( hemadsorption ) Certain viruses such as measles, mumps, adenoviruses, cytomegaloviruses and varicella virus cause damage to the chromosome of host cell. The most characteristic histological feature in virus infected cell is the appearance of inclusion bodies. The inclusion bodies may be present with cytoplasm(poxviruses), nucleus(herpesviruses) or both( measles viruses)

8. Specimen Processing for Diagnosis of Viral Diseases 1. Samples should generally come from the infected site. Skin infections: Rash site Blood (serum/plasma), stool, urine b. Respiratory infections: Sputum or throat swabs c. Central nervous system: For diagnosis of meningitis, cerebrospinal fluid (CSF) and serum, as well as stool or throat swabs for polio, d. Urogenital infections: Needle aspirates and endocervical and urethral swabs e. Gastrointestinal tract: Stool samples and rectal swabs f. Eye infections: Eye swabs and corneal scrapings g. Throat, nasal swab, ex. Corona virus

9. FAMILIES OF RNA VIRUSES Family Important human viruses Picornaviridae Rhinovirus, poliovirus, hepatitis A virus Orthomyxoviridae Influenza A, B, and C viruses Coronaviridae Coronavirus Rhabdoviridae Rabies virus Filoviridae Marburg and Ebola viruses

10. FAMILIES OF RNA VIRUSES Family Important human viruses Retroviridae Human immunodeficiency viruses Reoviridae Rotavirus Paramyxoviridae Measles, mumps, respiratory syncytial, parainfluenza Togaviridae Rubella virus Flaviviridae Yellow fever, dengue, hepatitis C

11. Polioviruses Morphology of poliovirus Poliovirus Poliovirus is transmitted by the fecal -oral route. The virus initially infects the gastrointestinal tract but spreads to the CNS.

12. Laboratory diagnosis and prevention Laboratory diagnosis: Specimens: blood, CSF , throat swab, and faeces . Prevention: Polio vaccines: The Salk(killed virus) vaccine( injectable poliovaccine (IPV) The Sabin vaccine is an attenuated vaccine. Live polio vaccine is administered orally and is therefore known as the oral polio vaccine(OPV). Killed vaccine induces only systemic antibody(IgG antibody, there is no intestinal immunity. Live vaccine on the other hand, also induces local immunity(IgA) and systemic immunity(IgG)

13. Arboviruses Arboviruses are also called arthropod-borne viruses, these are viruses of vertebrates biologically transmitted by hematophagous insect vectors. They multiply in blood sucking insects and are transmitted by bites to vertebrates hosts. Arboviruses are worldwide in distribution but are more common in tropical than in temperate zones. Arboviruses have been placed in Toga-, Flavi -, Bunya-, reo- and Rhabdovirus families. The ability to multiply in arthropods is their special characteristic. The most important arbovirus vectors are mosquitoes , followed by ticks.

14. Taxonomy of important arboviruses Family Genus Importany species Togaviridae Alphavirus Chikungunya Flaviviridae Flavivirus Yellow fever,dengue types1-4 Bunyaviridae Bunyavirus California encephalitis Phlebovirus Rift valley fever virus. Reoviridae Orbivirus Colorado tick fever

15. Bunyaviridae Are RNA viruses The virus is about 100 nm in diameter. Most bunyaviruses are mosquito-borne viruses, some are transmitted by sandflies, example Phlebotomus fever, or ticks. Bunyaviruses are so named from the type species Bunyamwera virus isolated from mosquitoes in Uganda, in 1946. The family Bunyaviridae contains four genera of medical importance: Bunyavirus , Phlebovirus , Nairovirus and Hantavirus. The virus is transmitted by culicoides .

16. Bunyaviridae Genus Phlebovirus : The major members of this genus are the sandfly fever and rift valley fever viruses. Rift valley fever is a mosquito-borne virus Flaviviruses : The family Flaviviridae contains only one genus, Flavivirus They are grouped into two groups: 1. mosquito-borne viruses 2. Tick-borne viruses

17. Yellow fever After an incubation period of 3-6 days, the disease starts as a fever of acute onset with chills, headache, nausea and vomiting. Jaundice, albuminuria and hemorrhagic manifestation develop and the patient may die of hepatic and renal failure. The virus is transmitted by the domestic Aedes aegyptii mosquito. The control of urban yellow fever can be achieved by eradicating the vectors mosquitoes. The vaccine is administered by subcutaneous inoculation

18. Dengue virus , Chikungunya virus Dengue virus is widely distributed throughout tropics and subtropics. Four types of dengue fever exist DEN1-4 Lymphadenopathy and maculopapular rash. Incubation period 3-14 days, as fever of sudden onset with headache, pain in the back and limbs, Dengue virus is transmitted from person to person by Aedes aegyptii mosquitoes.\ Chikungunya virus , the virus was first isolated from humans and Aedes aegypti mosquitoes from Tanzania. The name”chikungunya”is derived from the native word for the disease in which the patient lies “doubled up” due to severe joints pain. Epidemics of chikungunya have occurred in many African countries.

19. Togaviruses Togaviruses are spherical enveloped viruses with 50-70 nm in diameter, with a single stranded RNA. The family Togaviridae contains besides arboviruses belonging to genus Alphavirus, genus Rubivirus (rubella virus) Alphaviruses are mosquito-borne viruses, they are transmitted by culex and anopheles mosquitoes Rubella is rare in developed countries because of an effective vaccine.

20. Classification and properties of paramyxoviridae Genus Property Parainfluenzavirus Mumps Morbillivirus Pneumovirus Human viruses Parainfluenza 1-4 Mumps Measles RSV Diameter of nucleocapsid 18 nm 18 nm 18 nm 13 nm Fusion(F) protein present present present present Hemolysin present present present absent Hemagglutin / hemadsorption present present present absent Neuraminidase present present absent absent Intracellular inclusions in cytoplasm(C)/nucleus(N) C C N, C C

21. Mumps virus Mumps is an acute infectious disease commonly affecting children and characterised by nonsuppurative enlargement of the parotid glands. Properties: Mumps virus is a typical paramyxovirus possessing both HN and F proteins

22. Laboratory diagnosis and Prevention Specimens: saliva(within 4-5 days), urine(up to two weeks), CSF(8-9 days) after the onset of illness. Prevention :The Jeryl -Lynn strain of mumps virus(live vaccine) The vaccine is given as a single subcutaneous injection, either alone or in combination(MMR vaccine). It provides protection at least for ten years.

23. Parainfluenza viruses There are four types of parainfluenza viruses(1-4) Clinicla features: Parainfuenza viruses are responsible of 10% of respiratory infections in children. The most serious clinical disease is croup, which is mostly due to types 1 and 2. Type 3 causes lower respiratory disease such as bronchitis, bronchiolitis and pneumonia. In adults parainfluenza viruses cause sore throat Parainfluenza viral infection are confined to respiratory tract unlike Mumps which is systemic disease

24. Respiratory Syncytial Virus(RSV) Properties of the virus: RSV is pleomorphic , it has a size ranging from 150-300 nm. It is an enveloped virus, the envelop has two glycoproteins, the G protein which the virus attaches to cell surface, and the fusion F protein. RSV differs from other paramyxoviruses: 1. In not possessing the hemagglutin activity. 2. Does not have neuraminidase or hemolytic properties. 3. It has a smaller nucleocapsid (13nm)

25. Measles Morphology: It is spherical, pleomorphic particle, 120-250 nm in diameter, tightly coiled helical nucleocapsid , surrounded by the lipoprotein envelope carrying on the surface hemagglutinin(H) spikes The envelope also has the F protein, which mediate cell fusion and hemolytic activities. Cytopathic effect consist of multinucleate syncytium formation with numerous acidophilic nuclear and cytoplasmic inclusions, multinucleate giant cells. A safe and effective attenuated live vaccine is available The vaccine is either given alone or in combination at the MMR vaccine .

26. IMPORTANT HUMAN HEPATITIS VIRUSES Hepatitis A Hepatitis B Hepatitis C Hepatitis D Hepatitis E Family Picornaviridae Hepadnaviridae Flaviviridae Genome RNA DNA RNA RNA RNA Transmission Fecal -oral Parenteral , blood, sexually, needles,perinatal Parenteral , blood,needles Parenteral,blood,sexually,needles Fecal -oral Comments No chronic liver disease 5-10% chronic hepatitis, hepatocellular cancer Chronic infections Coinfection in patients infected with HBV high mortality rate in pregnant women

27. HEPATITIS VIRUSES Hepatitis A virus Infections Infections are spread by the fecal -oral route and are generally due to poor sanitation and hygiene. Food handling transmission is common. Vaccines are available.

28. Hepatitis C virus(HCV) Morphological structure: HCV measure 50-60 nm in diameter, single stranded RNA genome. It has an envelope with glycoprotein spikes. HCV has been classified in the genus Hepacivirus , family flaviviridae . HCV shows considerable genetic and antigenic diversity, at least six different genotypes and has many subtypes

29. Clinical features and epidemiology Mode of transmission: Transfusion of blood and blood components Other modes of contact with blood or blood products Injectable drug abusers, Sexual transmission The incubation period is 2-25 weeks. The acute illness is mild or anecteric , Jaundice is seen in about 5% of patients only. About 50 -80% of patients progress to chronic hepatitis. And some patients develop cirrhosis and hepatocellular carcinoma

30. Laboratory diagnosis The standard method of diagnosis is antibody detection by ELISA. Molecular method like PCR( nucleic acid amplification) 4. HCV antigen detection 5. Elevated liver enzymes No vaccine.

31. Hepatitis E virus(HEV) Morphology: family caliciviridae Clinicla features: Clinical severity and high case fatality rate 20-40% are seen in pregnant woman in the last trimester of pregnancy. Mode of transmission Faecal -oral route, by ingestion of food, drinks contaminated by infected faeces . It has been reported to be prevalent in animal reservoirs such as pigs

32. MEDICALLY IMPORTANT RNA VIRUSES Human immunodeficiency virus(HIV) Brief history: The emergency and pandemic spread of the acquired immunodeficiency syndrome(AIDS) have posed greatest challenge to public health. The full consequences of this phenomenon may not be evident for several years due to the silent spread and slow evolution of HIV infection. The first indication of this new syndrome came in 1981, with reports from New York and Los Angeles(USA), of sudden unexplained outbreaks of two very diseases, kaposi’s sarcoma and pneumocystis carinii pneumonia in young adults who were homosexuals or addicted to injected narcotics.

33. Human immunodeficiency virus(HIV) Brief history: They appeared to have lost their immune competence, making them vulnerable to fatal infections with relatively avurulent microorganisms, and other malignancies. This condition was named acquired immunodeficiency syndrome(AIDS). In 1983 Luc montagnier and colleagues from Pasteur institute, Paris(France) isolated a retrovirus from a west African patient with persistent generalised lymphadenopathy , which is a manifestation of AIDS, and called it lymphadenopathy associated virus(LAV).

34. Human immunodeficiency virus(HIV) Brief history: In 1984, Robert Gallo and colleagues from National Institute of Heath, USA, reported the isolation of retrovirus from AIDS patients and called it human T cell lymphotropic virus-III(HTLV-3) HTLV1 and HTLV2 have already been descrided earlier in association with human T-cell leukemia. To resolve this nomenclature confusion the international community of virus nomenclature in 1986 decided on generic name of human immunodeficiency virus(HIV) for these viruses. In 1985, serological tests(ELISA) became available for detection of anti-HIV antibodies.

35. Human immunodeficiency virus(HIV) HIV, the causative agent of AIDS, belongs to the lentiviruses subgroup of the family retroviridae . Strucrure : HIV is spherical, enveloped virus, with 90-120 nm in diameter, the nucleocapsid has an outer icosaehedral shell and an inner cone-shaped core. The genome is composed of two identical single stranded positive sense RNA copies, reverse transcriptase enzyme, which a characteristic feature of retroviruses. When the virus infects a cell, the viral RNA is first transcribed by the enzyme into single stranded DNA and then double stranded DNA(provirus) which is integrated into the host cell chromosome.

36. Major antigens of HIV A. Envelope antigens: 1. Spike antigen-gp120 (principle envelope Ag) 2. transmembrane pedicle protein-gp41. B. shell antigen: 1.nucleocapsid protein-p18 C.core antigens: 1. principle core antigen-p24 2. other core antigens-p15, p55 D . polymerase antigen-p31,p51,p66

37. Antigenic variation and diversity of HIV The original isolates of HIV and related strains prevalent all over the world belong to HIV type1. HIV strains first isolated from West Africa in 1986, which react with HIV type1 antiserum very weakly have been termed HIV type2. The envelope antigens of the two types are different, though their core polypeptides show some cross –reactivity. HIV-2 has 40% genetic identity with HIV-1, and it is less much virulent than HIV-1.

38. Pathogenesis Mode of transmission: 1. Sexual intercourse 2. Transfusion of blood and blood components Needle prick injury. Congenital transmission HIV virus attack CD4 lymphocytes principally but can infect any cell with CD4 surface antigen. Thus about 5-10 % of B-lymphocytes 10-20 % of monocytes and macrophages are susceptible. Specific binding of the virus to the CD4 receptor is by the envelope glycoprotein gp120.

39. Clinical features of HIV infection 1. Asymptomatic or latent infection It is a phase of symptomless infection(clinical latency) which may last up to several years. They show positive HIV antibodies during this phase, hence are infectious. Patient show as minor opportunistic infection, persistent generalised lymphadenopathy. 2. PGL is the presence of enlarged lymph nodes, at least 1 cm in diameter that persists at least for 3 months, without any other cause of illness.

40. Clinical features of HIV infection 3. AIDS related complex(ARC): This group includes patients with considerable immunodeficiency and present symptoms or minor opportunistic infections Symptoms such as fatigue, unexplained fever, persistent diarrhoea , marked weight loss, common opportunistic infections like candidiasis , herpes zoster, salmonelosis , TB. Generalised lymphadenopathy and splenomegaly are usually present. 4. AIDS:this is the end stage disease representing the irreversible breakdown of immune defense mechanisms leaving the patient exposed to progressive opportunistic infections and malignancies

41. Laboratory diagnosis A) Immunological tests: The total leucocyte count show leucopenia. The differential count show lymphocyte usually below 2000/cube mm The T4:T8 ratio is reversed, B) Specific tests for HIV infection: Antigen detection, the major core antigen(P24), is the earliest virus marker to appear in blood. The appearance of P24 antigenemia followed by IgM antibodies

42. Laboratory diagnosis 3. polymerase chain reaction: It is the most sensitive and specific test, PCR has become the golden standard for diagnosis in all stages of HIV infection 4. Antibody detection: It is the simplest and most widely employed technique for the diagnosis of HIV infection. The most widely used screening test is ELISA. While ELISA is ideal for sceening several serum samples at a time therefore a number of RAPID tests have been introduced such as immunochromatographic .

43. Laboratory diagnosis Antibodies may take 2-8 weeks to months appear after the infection, during this period the individual may be highly infectious. The seronegative infective stage in known as window period IgM antibodies disappear 8-10 weeks while the IgG antibodies remain throughout. When immunodeficiency becomes severe following clinical AIDS some components of anti-P-24 may disappear. 5. confirmatory tests: Western blot test

44. Laboratory diagnosis Westernblot test: It is the confirmatory commonly used, HIV proteins separated based on their electrophoretic mobility and molecular weight by acrylamide gel electropheresis are brotted onto the strips of nitrocellulose paper. These strips are reacted with test sera and with the enzyme conjugated antihuman globulin. A suitable substrate is then added which produce a prominent colour band where the specific Abs has reated with the separated viral proteins. In a positive serum, bands will be seen with multiple proteins, typically with P24 (gag gene, core Ag), P31(pol gene, reverse transcriptase) and gp 41, gp 120 or 160.( env gene, surface Ag)

45. Laboratory diagnosis A positive reaction with proteins representing the three genes gag, pol , env . Is conclusive evidence of HIV infection. The test may be considered positive even if it shows bands against at least two of the following genes products: P24, gp41, gp120/160. Westernblot is very costly, the practice is to perform either two diffent types of ELISA or an ELISA with any rapid test. HIV-1 infection is indicated by presence of at least 2 of 3 bands for HIV-2, gp36 is produced.

46. Laboratory monitoring of HIV infection Detection of CD4+ T cell count, when the count falls below 500 CD4+ T cells/cube mm, it is an indication of disease progression. Detection of antigen P24 level.