Rhabdovirus lecture

Morphology pathogenicity,laboratory diagnosis

1. RHABDOVIRUSES A lecture By – Dr.D.W.Deshkar Department of Microbiology D.Y.PATIL EDUCATION SOCIETY INSTTUTION DEEMED TO BE UNIVERSITY

2. INTRODUCTION Rhabdoviruses are – Rod or bullet shaped, enveloped viruses. Have nucleocapsid with helical symmetry. Genome is single stranded, unsegmented, negative sense RNA. ( Rhabdos = rod). Included under family Rhabdoviridae & contain viruses infecting mammals, reptiles,fish, insects & plants. 2

3. CLASSIFICATION Rhabdoviruses infecting mammals grouped in family Rhabdoviridae belong to TWO Genera. Vesiculovirus - containing vesicular stomatitis virus & related viruses like Chandipura virus (Arbovirus). Lyssavirus ( lyssa = madness, rage, a synonym for rabies) containing Rabies virus & related viruses. 3

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5. MORPHOLOGY Bullet shaped. 180 x 75 nm in size with one end rounded or conical & the other end planar or concave. Outer lipoprotein envelop carrying knob like spikes composed of glycoprotein G 5

6. MORPHOLOGY Spikes do not cover the planar end of virion. Beneath envelop is the membrane or matrix (M) protein layer which may be invaginated at the planar end. Membrane may project outwards from the planar end. 6

7. MORPHOLOGY Core of virion contains helically arranged nucleoprotein. Genome – unsegmented, linear, negative sense, single stranded RNA. Present in the nucleocapsid is RNA dependent RNA polymerase ( transcriptase) & some structural proteins. 7

8. RABIES VIRUS PROTEINS 8 Protein L -RNA dependent RNA polymerase Protein G- surface antigen Protein N -RNA binding protein Protein NS- phosphoprotein Protein M-membrane/matrix protein

9. RESISTANCE Sensitive to ethanol, iodine preparations, quaternary ammonium compounds, soap, detergents & lipid solvents such as ether, chloroform & acetone. Is inactivated by phenol, formalin, beta propiolactone, ultraviolet irradiation & sunlight. Thermal inactivation in 1 hr at 50 C & 5 min at 60 C. 9

10. RESISTANCE Dies at room temp, can survive for weeks when stabilised by 50% glycerol. Survive at 4 C. Can be preserved at - 70 C by lyophilisation. For storage in dry ice, the virus has to be sealed in vials as it is inactivated on exposure to CO 2 10

11. ANTIGENIC PROPERTIES 11 GLYCOPROTEIN – Surface spikes – composed of glycoprotein – imp. In pathogenesis, virulence & immunity. Imp. Properties as follows – Mediates binding of virus to acetylcholine receptors in neural tissue Induces haemagglutination inhibiting (HI) antibodies. Rabies virus has haemagglutinating activity, optimally seen at 0 - 4 C & pH 6.2. It is inactivated by heat 56 C for 30 – 60 min, ether, trypsin etc. Induces neutralising antibodies ( protective). Stimulates cytotoxic T cell immunity. Purified glycoprotein may act as safe, effective subunit vaccine.

12. ANTIGENIC PROPERTIES 12 NUCLEOPROTEIN – Is a nucleocapsid protein Imp. Having following properties – Induces complement fixing antibodies. Antibodies are not protective. Antigen is group specific & cross reactions do occur with rabies – related viruses. Antiserum prepared against the nucleocapsid antigen is used in diagnostic immunofluorescence tests.

13. ANTIGENIC PROPERTIES 13 OTHER ANTIGENS – Include - Two membrane proteins. Glycolipid. RNA dependent RNA polymerase.

14. HOST RANGE & CULTIVATION 14 ANIMALS – All mammals are susceptible to Rabies Cattle, cats & foxes – Highly susceptible. Skunks, opossums & fowl – relatively resistent. Humans & dogs occupy an intermediate position. Pups more susceptible than adults. Experimental infection can be produced in any lab. Animal but mice is choice.

15. HOST RANGE & CULTIVATION STREET VIRUS Definition : the virus recovered from naturally occurring cases of rabies is called “street virus” Sources : it is naturally occurring virus. It is found in saliva of infected animal. (continue) FIXED VIRUS Definition : the virus which has a short, fixed and reproducible incubation period is called “fixed virus S ources : it is prepared by repeated culture in brain of rabbit such that its I.P. is reduced & fixed 15 TYPES OF RABIES VIRUS -

16. HOST RANGE & CULTIVATION STREET VIRUS It produces Negri bodies Can be demo. In the brain of dying animal. Incubation period is long i.e. 20 to 60 days It is pathogenic for all mammals Cannot be used for preparation of vaccine FIXED VIRUS It does not form Negri bodies Incubation period is constant between 4-6 days It can be pathogenic for humans under certain conditions Is used for preparation of antirabies vaccine 16 TYPES OF RABIES VIRUS -

17. HOST RANGE & CULTIVATION CHICK EMBRYO Rabies virus can be grown in chick embryo. Mode of inoculation – Yolk sac. Serial propagation in chick embryo – development of attenuated vaccine strains like Flury & Kelev. Strains adapted to duck egg – give high yields – used for preparation of inactivated vaccine. TISSUE CULTURE 17 Rabies virus can grow in several primary & continuous cell cultures – chick embryo fibroblast, porcine or hamster kidney Fixed virus strains adapted for growth in human diploid cell, chick embryo & Vero cell cultures – used for the production of vaccine.

18. RABIES Primarily a Zoonotic disease of warm blooded animal such as :- Dogs, wild cats, Jackals, wolves etc. 18

19. RABIES COMMON FACTS 19 Mad Dog biting Humans lead to Rabies. Latin Rabhas means Frenzy. Hydrophobia Fear of Water, Saliva of Rabid dogs Pasture’s success – Vaccination Fixed virus from Rabbit injected into Joseph Meister Injected 13 injection of the cord vaccine.

20. RESERVOIR OF INFECTION 20 1 ) URBAN RABIES : From Dogs and cats.

21. RESERVOIR OF INFECTION 21 2) WILD LIFE RABIES : From jackals and foxes.

22. RESERVOIR OF INFECTION 22 3) BAT RABIES : Vampire bats which live on the blood of animals and men. These are one of the main causes of the death of bovine, around 0.5 to 1 million per year.

23. SOURCE OF INFECTION 23 Saliva of Rabid animal

24. RABIES HOST FACTORS All warm blooded animals including man. Rabies in man is a dead-end infection. MODE OF TRANSMISSION 24 ANIMAL BITES LICKS AEROSOL PERSON TO PERSON

25. INCUBATION PERIOD Normally it is 3 - 8 wks . May be short that is 4 days. Or may be prolonged for years. 25

26. PATHOGENESIS 26 Bite by Rabid dog or other animals Virus are carried in saliva virus deposited on the wound site. If untreated 50% will Develop rabies. Rabies can be produced by licks and corneal transplantation. Virus multiply in the muscle ,connective tissue, nerves after 48 – 72 hours. Penetrated nerve endings.

27. PATHOGENESIS 27 Live virus  Epidermis, Mucus membrane Peripheral nerve Centripetally CNS ( gray matter ) Centrifugally Other tissue (salivary glands,…)

28. PATHOGENESIS 28

29. PATHOGENESIS 29

30. SPREAD OF VIRUS 30 From Brain virus spread to Salivary glands, Conjunctival cell released into tears Kidney Lactating glands and Milk after pregnancy

31. PATHOGENESIS 31

32. PATHOGENESIS 32 Virus travels through axoplasam toward the spinal cord, at the rate of 3 mm/hour, Towards the brain Spread from brain centrifugally to various parts of the body. Multiplies in the salivary glands and shed in the saliva. Cornea, facial tissues skin. Incubation 1 – 3 months. May be average from 7 days to 3 years. Stages of the disease. Prodrome Acute encephalitis. Coma / Death.

33. BAD CATEGORY PRESENTATION 33 Furious Rabies Dumb ( Rage tranquille ) (Landry/ Guillain-Barre Syndrome

34. CLINICAL PICTURE CLINICAL FINDINGS SYMPTOMS 34 Bizarre behavior. Agitation Seizures. Difficulty in drinking. Patients will be able to eat solids Afraid of water - Hydrophobia. Even sight or sound of water disturbs the patient . But suffer with intense thirst. Spasms of Pharynx produces choking Death in 1 -6 days. Respiratory arrest / Death / Some may survive. Headache, fever, sore throat Nervousness, confusion Pain or tingling at the site of the bite Hallucinations Seeing things that are not really there Hydrophobia “Fear of water" due to spasms in the throat Paralysis Unable to move parts of the body Coma and death

35. CLINICAL MANIFESTATION 1 – Non specific prodrome 2 – Acute neurologic encephalitis Acute encephalitis Profound dysfunction of brainstem 3 – Coma 4 - Death ( Rare cases  recovery ) Non specific prodrome 1 - 2 days  1 week Fever, headache, sore throat Anorexia, nausea, vomiting, Agitation, depression Parenthesis or fasciculation's at or Around the site of inoculation of virus. 35

36. ACUTE NEUROLOGIC ENCEPHELITIS 36 1 – 2 days to < 1 week Excessive motor activity, Excitation, Agitation Confusion, Hallucinations, Delirium, Bizarre aberrations of thought, Seizures , Muscle spasms, Meningismus, Opisthotonic posturing Mental aberration ( Lucid period  coma ) Hypersalivation, Aphasia, Pharyngeal spasms Incordination, Hyperactivity Fever T > 40.6 Dilated irregular pupils Lacrimation , Salivation & Perspiration Upper motor neuron paralysis Deep tendon reflexes Extensor plantar responses ( as a rule ) Hydrophobia or Aerophobia (50 -70% )

37. LABORATORY DIAGNOSIS 1. SPECIMENS - Specimens tested are corneal smears, skin biopsy ( from face & neck), or saliva antemortem, & brain postmortem. 2. DIRECT MICROSCOPY – Antemortem – Demo. Of Rabies virus antigens by immunofluorescence. Direct immunofluorescence is done using monoclonal antibodies tagged with fluorescein isothiocynate. Immunoperoxidase staining used for Ag in tissues. Postmortem - Diagnosis on postmortem by demo. of Negri bodies in the brain. May be absent in 20%. 37

38. LABORATORY DIAGNOSIS Postmortem - Diagnosis on postmortem by demo. of Negri bodies in the brain. May be absent in 20%. 38 Negri bodies   round or oval inclusion bodies seen in the cytoplasm and sometimes in the processes of neurons of rabid animals after death. Negri bodies are Eosinophilic, sharply outlined, pathognomonic inclusion bodies (2-10 µm in diameter) found in the cytoplasm of certain nerve ..

39. LABORATORY DIAGNOSIS 3. ISOLATION- Animal inoculation - Isolation of virus by intracerebral inoculation in mice attempted from brain, CSF, saliva & urine Tissue culture - Isolation of virus in tissue culture cell lines ( WI 38, BHK 21, CER ). CPE minimal so identified by immunofluorescence. Antibody demo. - Demo. Of antibodies by ELISA. Molecular methods - Detection of rabies virus RNA by reverse transcriptase PCR is a sensitive method. 39

40. LABORATORY DIAGNOSIS IN DOGS & OTHER ANIMALS – Demonstration of Negri bodies in brain postmortem Impression smears stained by Seller’s technique (basic fuchsin + methylene blue in methanol). Demonstration of rabies virus antigen by immunofluorescence in salivary glands. Isolation of rabies virus by animal inoculation. 40

41. Prevention of human rabies post Exposure prophylaxis 41 General consideration:- Aim is to neutralize virus before entering CNS LOCAL WOUND TREATMENT a, Cleansing of wound(soap & water) b, Chemical treatment: Either Alcohol 400-700 ml /liter Tincture Iodine No more treatment with Ammonium compound No Carbolic acid and Nitric acid as it leave very bad scar c, Suturing avoided. d, Anti Rabies Serum e, Antibiotic and ATS f, Observe the animal for 10 days 3, Immunization 1,NERVOUS TISSUE VACCINE (NTV 2, Human diploid cell vaccine (HDCV)

42. ANTIRABIC VACCINES 42 Definition: it is fluid or dried preparation of Rabies “Fixed” virus grown in the Neural tissue of Rabbits, Sheep, Goats, Mice or Rats OR in embryonated duck eggs OR in cell culture

43. 43 Nervous Tissue vaccine Duck embryo vaccine Cell culture vaccine preparation From fixed virus grown in brain of sheep or other animals potency Low or variable Eliminate Neuroparalytic factors More potent more safer Doses Large nos: are required Fewer doses of small volume Side effects Severe & fatal reactions Allergic risks Fewer Uses Exposed subjects Used in UK,USA in past 1, (HDC) safe, potent Pre & post expos:Immunization Suckling mouse brain V Devoid of Neuroparalytic effect Used in Latin America Improvement over adult animal nervous tissue V Now purified DEV developed Improvement over adult animal nervous tissue V Not available in India & Pakistan 2Tissue culture 2 nd G (Non-human) Potent, low cost WHO recommendatio

44. ANTIRABIC VACCINES 44 NEURAL VACCINE – Semple vaccine Contain 5 % suspension. Of infected Sheep brain, ( Infected with fixed virus )Inactivated with Phenol at 37 c. Vaccines available after inactivation with Beta propiolactone. Used in India. Developed by Semple in 1911 at Central Research Institute Kasauli India. Derived from suckling mouse brain Type: Killed viral vaccine Dose: 2.5 ml S/C (Ant. Abdominal wall) Schedule: 14 doses

45. ANTIRABIC VACCINES 45 NON NEURAL VACCINES – E gg Vaccines – D uck Egg Vaccine – Prepared from a fixed virus adapted for growth in duck eggs & inactivated by betapropiolactone – Discontinued. L ive Attenuated Chick Embryo Vaccine – Two types of Flurry vaccines – Low egg passagge (LEP) – 40 – 50 egg passage for dogs & High egg passage (HEP) – 180 passage for cattle & cats.

46. ANTIRABIC VACCINES 46 TISSUE CULTURE VACCINES – Human diploid cell vaccine (HDCV) Developed by Koprowsky,Wiktor,and Plotkin Purified chick embryo cell vaccine (PCEC) Purified Vero cell vaccine (PVRV) Purified duck embryo vaccine (PDEV)

47. POST EXPOSURE PROPHYLAXIS 47 The vaccination is given on 0, 3, 7, 14, 30, and 90 th day Immunity lasts for 5 years Injected on deltoid region IM/SC Not to be given in the gluteal region

48. 48 HUMAN DIPLOID CELL VACCINES Dosage Pre- exposure prophylaxis 0 – 7 – 21 – or 28 – 56 days A booster after 1 year, Repeat once in 5 days, Post exposure Prophylaxis Sex doses 0 -3 -7-14 – 30 - 90 days Given IM or SC in the Deltoid region Don't inject in Gluteal region.

49. 49 PREEXPOSURE PROPHYLASIXIS DOSES Given on the following days 0, 7, 21,or 28 and 56 th day Generally given to Vet nary personal

50. 50 PASSIVE IMMUNISATION Human Rabies Immunoglobulin HRIG High Risk bitten on face and neck Given a dose of 20 IU /Kg wt Half at the site of bite and rest IM route. Active immunization should be initiated with passive immunization.

51. 51 FUTURE OF RABIES VACCINES A number of experimental vaccines are under development that may provide alternative safe and potent but less expensive vaccine options. These include DNA vaccines , recombinant viral vaccines, and recombinant protein vaccines. Further testing is needed to determine if and which one of these novel vaccines will make their way into mass production and application in the future.

52. 52 SUBUNIT OR GENETICALLY ENGINEERED VACCINES FOR RABIES A viral immunizing agent that has been treated to remove traces of viral nucleic acid so that only protein subunits remain. The subunits have less risk of causing adverse reactions. Several trails in progress

53. 53 CLASSIFICATION OF EXPOSURES Class I (slight Risk) 07 injection Licks on healthy unbroken skin. Scratches without oozing of blood. Class II (Moderate Risk) according to the Schedule plus one booster dose after 3 week Licks on fresh cuts. Scratches with oozing of blood. All bites except those on head, neck, face, palms and fingers. Minor wounds less than 5 in number. Class III (Severe Risk) according to the Schedule plus Two booster dose one after one week and another 2 week all bites or scratches with oozing of blood on neck, head, face, palms and fingers. Lacerated wounds on any part of the body. Multiple wounds 5 or more in number . Bites from wild animals.

54. THANKS 54