1. Viruses and what they do - An overview Wednesday, August 25, 2010

2. Viruses (Encyclopedia Britannica)..infectious agents of small size and simple composition that can multiply only in living cells of animals, plants and bacteria. Viruses are obligate parasites that are metabolically inert when they are outside their hosts. They all rely, to varying extents, on the metabolic processes of their hosts to reproduce themselves. The viral diseases we see are due to the effects of this interaction between the virus and its host cell (and/or the host’s response to this interaction).

3. Viral Genomes Nucleic Acid DNA RNA Double Stranded Positive Negative RNADNA Single Stranded Double Stranded Single Stranded

4. SS RNA genomes Positive (sense) Negative (anti-sense) AUG GCA CGA UAC CGU GCU met ala arg +ve (sense) and -ve (anti-sense) RNA genomes

5. Virion capsomeres Capsid enveloped Virus or Virion envelope capsid “naked” virus particle or Virion herpes adeno

6. Proteins produced by viruses Structural proteins Non-structural proteins

7. Some viral shapes adenovirus parvovirus papillomavirus 100 nm 1 nm = 1 millionth of a mm 100 nm = 1 ten thousandth of a mm “ naked ” viruses

8. Some viral shapes herpesvirus parainfluenza virus influenzavirus poxvirus 1 nm = 1 millionth of a mm 100 nm = 1 ten thousandth of a mm 100 nm Enveloped viruses

9. Taxonomy What is it? On what is it based? Is it important? Do I need to remember all the details? International Committee on Taxonomy of Viruses

10. Viruses with ss DNA genomes Circoviridae Parvoviridae porcine circovirus canine parvovirus-2 feline panleukopenia virus porcine parvovirus (SMEDI)

11. Viruses with ds DNA genomes Poxviridae African swine fever virus Herpesviridae Adenoviridae Papovaviridae papillomaviruses adenoviruses bovine herpesvirus-1,2 porcine cytomegalovirus equine herpesviruses -1,4 african swine fever virus poxviruses malignant catarrhal fever virus

12. Viruses with ds RNA genomes Reoviridae Birnaviridae rotaviruses bluetongue virus african horse sickness infectious bursal disease (chickens) infectious pancreatic necrosis (salmonid fish)

13. Viruses with +ve RNA genomes Picornaviridae Caliciviridae Coronaviridae Arteriviridae Flaviviridae Togaviridae foot and mouth disease virus porcine enteroviruses feline calicivirus equine arterivirus, PRRS pestiviruses (BVD) coronaviruses equine encephalitis viruses flaviviruses (WNV)

14. Viruses with -ve RNA genomes Paramyxoviridae Rhabdoviridae Orthomyxoviridae Filoviridae Bunyaviridae parainfluenza virus canine distemper virus respiratory syncytial virus rabies virus vesicular stomatitis virus influenzaviruses Ebola virus Haantan virus Hendra, Nipah viruses

15. Viruses with reverse transcriptase Retroviridae Hepadnaviridae feline leukemia virus feline, bovine immunodeficiency viruses bovine, avian leukosis viruses caprine arthritis-encephalitis virus

16. Isolates, strains, serotypes and groups

18. antibodies to all viral proteins selective pressure on external viral proteins antibodies to external proteins neutralize virus no selective pressure on internal proteins

19. antibodies to all viral proteins selective pressure on external viral proteins antibodies to external proteins neutralize virus selective pressure forces slight change in external proteins

20. antibodies to all viral proteins selective pressure on external viral proteins antibodies to external proteins neutralize virus selective pressure forces slight change in external proteins virus, including changed virus, passed on to new host

21. process repeated, over time….. serum from original cat neutralizes does not neutralize NOTE: Only external proteins change. Internal proteins do not change

22. process repeated, over time serum from original cat neutralizes does not neutralize new serotype NOTE: Only external proteins change. Internal proteins do not change same serotype

23. Serotype - all isolates of a virus that can be neutralized by a common antiserum are said to belong to the same serotype. …..because of changes in external protein (internal proteins do not change) external proteins are called TYPE SPECIFIC antigens internal proteins are called GROUP SPECIFIC antigens

24. process repeated, over time new serotype same serotype different serotypes same group

25. Group and type specific antigens group specific antigen type specific antigen “naked” virus (eg FMDV) enveloped virus (eg influenza, FeLV)

26. Groups, types (sero-types), isolates and ‘strains’ Group Type -A Type - B Type - C Group specific antigen Type -A specific antigen Type - C specific antigen isolate

27. Serotypes and neutralizing antibody (eg. FMDV) serotypes of FMD virus AOCSAT1SAT2SAT3Asia antibodies against receptor binding protein of serotype A will neutralize viruses of serotype A but not of serotype C receptor receptor binding protein on viral surface

28. example - influenza group specific antigen type specific antigen serotype H1 serotype H5 serotype H7 test based on group specific antigen will detect all three vaccination against one serotype will not protect against others

29. Infection of a cell attachment entry and uncoating viral gene expression genome replication – DNA viruses – RNA viruses assembly and release – naked viruses – enveloped viruses

30. Distribution of the CCR5  32 mutation in human populations from PLoS Biology, Nov 2005

31. Errors in replication lead to “quasispecies” persistent infection mixture of variant viruses (quasispecies)

32. inclusion bodies A B CD

33. Release of virus Release by lysis of cell (cytopathic) or by budding (without death of cell, non-cytopathic)

34. Infection of a cell StageBiological implications Host defenses Drug intervention

35. Infection of the animal Entry - the beginning of infection – Local replication vs systemic spread Consequences of infection – none to illness (signs, symptoms) Signs and symptoms Why some animals get sick while others do not Patterns of disease

36. Why do some infected animals get sick and others don’t? Viral factors - virulence Host Factors

37. Viral Factors : Why are some isolates of a virus more likely to cause severe disease than others? determinants of viral virulence

38. Host Factors: Genetic Resistance – loss of receptors CCR5-  32 mutation and resistance to HIV – variation in immune response genes – genetic defects in defenses

39. Host factors: age related susceptibility – greater susceptibility of new born animals – greater susceptibility of adults prior exposure, acquired resistance maternal protection concurrent infections, immuno-suppression, increase in susceptible cells

40. Incubation period incubation period - time between infection and the appearance of clinical signs infection

41. Patterns of disease acute recurrent chronic or persistent slow clinical signs virus shedding virus difficult to detect

42. Resistance and Recovery

43. Resistance and recovery innate resistance – genetic – serum, mucous factors (complement, defensins) induced resistance – Toll-like receptors (dendritic cells) Pathogen Associated Molecular Patterns – interferons and inflammatory cytokines acquired immunity – humoral – cell mediated

44. After the Toll Rush, LAJ O’Neill, Science 303:1481 2004

45. Interferons I I I I I E Induced interferon genes antiviral effects activated factor infected cell Immune Modulation

46. Acquired anti-viral immunity (antibody) B Virus neutralized Virus infected cell Antibody targets Fc receptor bearing cell To kill virus infected cell

47. Acquired immunity (CMI) CD8 CD4 perforins Apoptosis trigger cytokines necrosis apoptosis virus replication suppressed

48. Prevention of infection and/or disease

49. Protection of the new born animal antibodies (possible CMI as well) in colostrum – maternal immunization – colostrum replacers – implications for immunization of young animals caution when using modified-live vaccines interference by maternal antibodies

50. Interference by maternal antibodies 24681012 * * * * * * passive antibody weeks after birth window of susceptibility interferes with vaccination minimum amount needed for protection can vaccinate in this range

51. Prevention of virus infections/disease vaccination – inactivated vaccines – attenuated vaccines – subunit – vectored – DNA vaccines management

52. Why vaccination sometimes fails to protect improper use genetic differences between animals antigenic differences blocking by maternal antibodies administration following infection (exceptions - rabies)

53. Diagnosis of viral diseases Why??

54. Sensitivity and Specificity

55. Diagnosis of viral diseases clinical signs virus detection detection of exposure Laboratory }

56. Detection of virus

57. isolation (isolation +immunological detection) quantitation (plaque assay, TCID50) PCR and real-time PCR haemagglutination (or HAI) ELISA (in clinic or lab) immunological detection (IH or IF) electron microscopy

58. Virus isolation (tissue culture, experimental animals) cultured cells (two dimensional animals) cytopathic effect immunofluorescence (anti-herpesvirus antibody)

59. Virus quantitation (plaques) plaques count plaques (plaque forming unit/ml)

60. Virus quantitation (TCID50, LD50) 10 -2 10 -3 10 -4 10 -5 10 -6 TCID 50 = 10 4 50% Tissue culture infectious dose

61. PCR Viral DNA primers First cycle Second cycle Twenty cycles million BVDV-2 BVDV-1a BVDV-1b

62. PCR tests offered by PDS, 2009 ($35) Avian influenza Avian paramyxovirus Bovine viral diarrhoea virus Equine influenza Equine herpesvirus (1 vs 4, neurotropic) Influenza A – pan species Malignant catarrhal fever Porcine circovirus PRRS Rotavirus (genotyping) Swine influenza West Nile virus

63. Haemagglutination (HA) virus No virus

64. Haemagglutination Dilution 248163264128256512 prozone titre 1024 No virus

65. Enzyme linked immunabsorbant assay (ELISA) Sample to be tested virus capturing antibody Detecting antibody Enzyme -> colour

66. In-clinic ELISAs Feline leukemia And Immunodeficiency viruses Others: canine parvovirus bovine herpesvirus - 1 bovine viral diarrhoea virus equine infectious anemia virus influenza-A porcine respiratory reproductive syndrome virus

67. Immunological detection ImmunohistochemistryImmunofluorescence bovine herpesvirus antigens in endothelial cells BHV-1 antigens in neuron In trigeminal ganglion

68. Detection of exposure

69. Detection of exposure or measure or humoral immunity (serology) virus neutralization haemagglutination inhibition ELISA

70. Virus neutralization Serially dilute serum 1/2 1/4 1/8 1/16……….1/512 Add equal amount of virus (100 plaque forming units) to each tube Infect cultured cells Last dilution that can prevent plaque formation is titer No serum1/21/41/2561/5121/128 --

71. HAI Serum dilution 1/20 1/40 1/80 1/160 1/320 1/640 1/1280 -ve control 1 wk8 wks Virus HA

72. Limitations of serology detects exposure and not when exposure occurred for correlation with disease – Paired sera – IgM – CSF

73. Virology Diagnostic Laboratories PDS, Saskatoon Veterinary Services Branch, Manitoba Agriculture, Winnipeg Animal Health Monitoring Lab, Abbotsford Central Laboratories for Veterinarians Ltd. Calgary

74. Sample collection and submission Choosing samples - considerations: – alive or dead – suspected pathogen(s), tropism, pathogenesis – phase of disease – virus detection or exposure Live animals – nasal swabs, transtracheal aspirates, respiratory secretions, scrapings – vesicular fluid, covering epithelium, biopsy from margin of lesion – feces or fecal swabs – clotted and unclotted blood – samples from unaffected animals

75. dead animals: – collect samples as soon as possible after death – affected organs – gut loops

76. Shipping the samples transport medium (from lab or buffered saline with 50 µg/ml gentamycin) 10% buffered formalin - < 1cm thick plastic, sealed containers, labeled with water proof ink ice packs vs frozen samples For PCR – Regular (ice, frozen) – RNALater

77. Samples should be accompanied by case history and suspected pathogen(s) treatment, vaccinations, numbers involved list of specimens

78. Viruses can be useful too biological control of pests cancer therapy gene therapy nanotechnology symbiotic virus-host relationships