Brief description about Picornaviridae family

An insight into the RNA virus family Picornaviridae

1. PICORNAVIRIDAE Submitted by- Jagnoor Singh Sandhu L-2021-V-48-M

2. HISTORY In 1897, Loeffler and Frosch demonstrated 1 st animal virus – FMD virus . In 1909, Landsteiner, Popper identified the Poliovirus, the cause of human poliomyelitis .

3. CLASSIFICATION Picornaviruses are classes under Baltimore’s viral classification system as group IV viruses as they contain a single stranded, positive sense RNA genome. The family Picornaviridae originally comprises of atleast eight genera : Aphthovirus , Enterovirus, Teschovirus, Cardiovirus, Erbovirus, Kobuvirus, Hepatovirus and Parechovirus . Four new genera viz., Tremovirus , Sapelovirus, Senecavirus and Avihepatovirus , have recently been added. The former genus Rhinovirus was abolished in 2006, and the member viruses human rhinovirus ( cause of common colds) and two bovine rhinoviruses are included in the genus Enterovirus.

4. VIRION PROPERTIES Picornavirus virions are very small, smooth and round in outline, non-enveloped and have icosahedral symmetry. The genome consists of a single molecule of linear, positive sense, single stranded RNA. Both the 5’ and 3’ ends contain untranslated regulatory sequences and has a covalently linked protein, VPg at uncapped 5’ end and polyadenylated tail at its 3’ end. Genomic RNA is infectious. The capsid is constructed from 60 copies of identical subunit each containing 4 capsid proteins : VP1 (1D), VP2 (1B), VP3 (1C) and VP4 (1A) and a single copy of the genome linked protein, VPg (3B).

5. Radial depth cued images of picornavirus particles with a color gradient from innermost ( dark blue ) to outermost ( white ) surfaces. virus structure with different proteins VP1 is the most external and immunodominant of the picornavirus capsid proteins

6. VIRION STABILITY Most picornaviruses are relatively heat stable . Aerosols of aphthoviruses are less stable, but may remain viable for several hours under high humidity. They vary in their stability at low pH and such differences were previously utilised to classify them. Ex : Aphthoviruses are unstable below pH 7 whereas other picornaviruses are stable at acidic pH 3. Because of differences in their pH stability, only certain disinfectants are suitable for use against each virus. Ex : Sodium carbonate 10% (washing soda) is effective against FMD viruses.

7. VIRAL REPLICATION

8. Viral Proteins and their Functions

9. GENUS VIRUS DISEASE HOST Aphthovirus Foot and mouth disease Foot and mouth Cattle, sheep, swine virus disease ,goats, wild ruminants Equine rhinitis A virus Systemic respiratory Horses, camelids disease Bovine rhinitis B virus Mild respiratory Cattle disease Cardiovirus Encephalomyocarditis virus Encephalomyelitis and myocarditis Swine, elephants in contact with rodents Enterovirus Human enterviruses A, B, C Aseptic meningitis, Humans and D poliomyelitis, myocarditis Human enterovirus C Poliomyelitis Humans (Poliovirus) Human rhinoviruses A,B and Respiratory disease Humans C

10. GENUS VIRUS DISEASE HOST Enterovirus Swine vesicular disease virus Bovine enteroviruses (includes bovine rhinovirus 1,3) Simian enteroviruses Porcine enterovirus B Swine vesicular disease Mild enteric and respiratory disease Asymptomatic infection Asymptomatic infection Pigs Cattle Primates Swine Erbovirus Equine rhinitis B virus Mild rhinitis Horses Kobuvirus Bovine kobuvirus Enteritis Cattle Teschovirus Porcine teschovirus 1 : PTV 1 Polioencephalomyelitis Swine ( TESCHEN/TALFAN DISEASE) Porcine teschovirus 2-11 Usually asymptomatic , Swine mild diarrhoea, pericarditis

11. GENUS VIRUS DISEASE HOST Hepatovirus Hepatitis A virus Hepatitis A Humans Tremovirus Avian encephalomyelitis virus Avian encephalomyelitis Chickens Sapelovirus Porcine sapelovirus (PEV A & PEV 8) along with PTV 1,3,6 SMEDI (Stillbirth, mummification, embryonic death and infertility) Pigs Avihepatovirus Duck hepatitis A virus Duck viral hepatitis Ducks

12. FOOT AND MOUTH DISEASE It is a highly contagious disease of even-toed ungulates and is characterized by fever and vesicle formation on epithelial surfaces. It is listed under OIE list A disease and pose significant economic losses. SYNONYMS : Aphthous fever Afta epizootica

13. ETIOLOGY: FMD is caused by foot and mouth disease virus of the genus Aphthovirus of the family Picornaviridae . There are seven serotypes ( identified by cross protection and serologic test ) of FMDV : O,A,C,Asia1,SAT1,SAT2 and SAT3. Within the serotypes there are many subtypes. For epidemiological purposes,isolates of FMDV within a given serotype are classified ( based on VP1 gene) according to their topotype. Ex: For serotype O, there are atleast 7 topotypes, reflecting its wide geographic occurrence from South America across Africa to Southeast Asia.

14. HOST AND DISTRIBUTION FMD affects a wide variety of cloven – footed domestic and wild animal species. Cattle, water buffalo, swine, sheep, goats, camels and many wild ruminants are susceptible. Horses are refractory to the infection. Clinical signs are most severe in cattle and swine. FMD is widely distributed throughout the world and the infection is still enzootic in much of Africa, Asia and the Middle East. In India, O, A, and Asia 1 serotypes are prevalent . Serotype C was found in Philippines in 1976-1981, 1983-1990, & in 1994

15. DISTRIBUTION SEROTYPES GEOGRAPHIC DISTRIBUTION O, A, C South America, Eastern European countries O, A, C, SAT 1, 2 and 3 Africa O, A, C, Asia 1 Asia

16. TRANSMISSION FMD is spread rapidly by movement of infected animals. The main mode of transmission is through the inhalation of droplets. But, direct or indirect contact with affected animals Ingestion of infected food. Mechanical transmission by contaminated objects (clothing, hands, footwear, vehicles). Inoculated with contaminated vaccines and veterinary instruments. Insemination with contaminated semen and so on can all produce infection. Long distance spread is dependent on wind direction and speed and is favored by low temperature, high humudity and overcast skies.

17. TRANSMISSION Reasons for the rapidity of spread: The highly infectious nature of the virus. The rapid replication cycle with very high virus yields. Production of high-titer virus in respiratory secretions. Large volumes of droplets and aerosols of virus shed by infected animals. The stability of virus in such droplets. The short incubation period. The excretion of virus before 24 hours of onset of clinical signs results in rapid virus dissemination. The involvement of sheep or other animals that show minimal signs of infection may also contribute to rapid spread.

18. PATHOGENESIS

19. CLINICAL FEATURES CATTLE: Incubation period – 2 to 8 days. Mortality is low; morbidity – 100% Pyrexia (39.4 – 40.6 ⁰ C), anorexia and depression. Marked decrease in milk yield and abortion due to pyrexia. Drooling of saliva. Vesicle formation ( tongue, dental pads, gums, soft palate, nostrils, interdigital space, coronary bands and teats. Rupture of vesicles – crater like lesions producing characteristic smacking sound. Calves upto 6 months of age – die from acute myocarditis. Complications of FMD lesions – superinfection of lesions, hoof deformations, mastitis, permenant impairment of milk production, permenant weight loss and impairment of thermoregulatory mechanism (“PANTERS”).

20. LESIONS Vesicles in buccal and foot areas. Fluid filled vesicles – enlarge and coalesce with adjacent ones – rupture and slough leaving an eroded area which is covered with gray fibrinous coating. Degeneration and necrosis of myocardium – “tigeroid heart”.

21. A B C A, B – Drooling of saliva C – Foot lesions may cause shifting of weight to front legs; reluctant to move and have a hunched back

22. A B C A – Ruptured vesicles on tongue B – Ruptured vesicle on upper gums and muzzle C – Ruptured vesicles in the interdigital space

23. A C A – Vesicles on the teat B– Tigeroid heart appearance in heart C – “ Hairy panters ” (complications of FMD) B A

24. CLINICAL FEATURES PIGS: Lameness is often the first sign of FMD. Foot lesions may be more severe and painful. Vesicles within the mouth are not common, although common on the snout. No drooling of saliva . Abortion and suppuration of denuded areas resulting in loss of claws and prolonged lameness. Other signs: fever, anorexia, reluctance to move, vesicles on coronary band, interdigital space, and snout. LESIONS: Detached hoof, tigeroid appearance of myocardium.

25. A B A – Vesicles on the snout B – ruptured vesicles in the interdigital space and coronary band

26. DIAGNOSIS FIELD DIAGNOSIS : Based on characteristic clinical signs and lesions. Whenever salivation and lameness occurs simultaneously with vesicular lesions FMD should be suspected. VIRUS ISOLATION AND IDENTIFICATION: Clinical materials: In early infection : vesicular fluid, epithelial tissue from the edge of recently ruptured vesicles, blood (in anticoagulant), milk and serum samples. In more advanced cases : Oesophageal/pharyngeal fluids (from ruminants), pharyngeal swabs (from swine) are ideal from animals in convalescent stage. Dead animals : Tissue samples from lymph nodes, thyroid, adrenal gland, kidney and heart. Should be sent within 24 hrs to the laboratory or should be frozen -70 ⁰ C and sent.

27. DIAGNOSIS Virus isolation: Cell cultures : The virus grows well in cell cultures. Primary cultures of bovine, porcine or ovine kidney and primary bovine thyroid cells are more sensitive than established cell lines such as baby hamster kidney ( BHK-21 ) or pig kidney ( IB-RS-2 ) cells. CPE ( rounding and flattening of the cells, breaking down of the intracellular bridges and finally cell death) are produced typically in 24-48 hrs. Laboratory animals : Unweaned mice (2-7 days old) are commonly used for cultivation of FMDV. Virus identification: The isolated virus can be identified by ELISA, RT-PCR or neutralization test.

28. CPE in FMDV-infected cells. Still images from a live-cell experiment in which BHK-21 cells were infected with FMDV as shown up. Normal BHK-21 cell line

29. DIAGNOSIS IMMUNOLOGICAL METHODS OF VIRUS IDENTIFICATION : ELISA: Is preferred test for detection and identification of FMDV serotypes. It is more sensitive than CFT and is not affected by complementary factors. NUCLEIC ACID DETECTION METHODS: RT-PCR: Can be used to identify specific serotypes of FMDV. In situ hybridization SEROLOGICAL TESTS: VNT and ELISA are prescribed tests for international trade and are used for detection of specific antibody response against FMDV.

30. PREVENTION AND CONTROL Immunity is not considered life-long. Recovered animals can be infected immediately with other serotypes. Frenkel method : In this method of vaccine production, the normal tongue epithelium is removed, minced, placed in a nutrient broth and inoculated with FMDV. After replication, the virus is inactivated with formalin and adjuvanted with aluminium hydroxide. At present, instead of tongue epithelium, BHK-21 cell lines are used. Formaldehyde, Binary ethyleneimine (BEI) is used as inactivating agent and aluminium hydroxide-saponin or oil is used as adjuvant. Protection for 4-6 months. Though molecular vaccines are available, they are not much effective and not economical. PUBLIC HEALTH SIGNIFICANCE: - Rarely zoonotic with often subclinical infection. Clinical signs include pyrexia, anorexia and vesiculation.

31. Vaccination Schedule

32. SWINE VESICULAR DISEASE Swine Vesicular Disease (SVD) is mild vesicular disease of pigs. This was first described in Italy in 1966 and still occurs as endemic. This may occur sporadically in European and Asian countries. ETIOLOGY: It is caused by swine vesicular disease virus ( genus. Enterovirus ). The pig is the natural host for the virus. The virus is resistant to low pH and temperatures, hence transmitted easily between countries in affected meat. Pork products prepared without heat treatment (such as salami) can harbor virus for several months.

33. TRANSMISSION Direct or indirect contact: Infected animals or faeces Contaminated environment Ingestion: - Contaminated meat scraps Virus excretion: Nose, mouth, faeces Up to 48 hrs. before clinical signs

34. PATHOGENESIS Vesicles contain high titers of virus and virus in large quantities is excreted in the faeces (upto 2 months after infection). Carrier status is been detected rarely.

35. CLINICAL FEATURES IP: upto 7 days. Disease is often detected by the sudden appearance of lameness in several pigs in a herd. Affected pigs show transient fever, dullness, inappetance and vesicles appear at the junction between the heel and coronary band and then spread to encircle the digit. Less commonly lesions are found on the snout, lips and tongue. Occasionally, some infected swine develop signs of encephalomyelitis, such as ataxia, circling and convulsions. Subclinical disease is more common.

36. Vesicles on snout and heel

37. DIAGNOSIS Based on symptoms and lesions. But laboratory diagnosis is essential to distinguish the vesicular diseases. Disease/Virus Susceptible animals Resistant species FMD Cattle, sheep, goat and pigs Horse Swine vesicular disease Pigs Cattle, sheep, goat and horse Vesicular exanthema of pigs Pigs Cattle, sheep, goat and horse Vesicular stomatitis Cattle, pigs and horses Sheep and goat

38. DIAGNOSIS Clinical materials : Vesicular fluid or epithelium, blood and serum. Virus isolation: Cell culture: SVD virus grows well in pig kidney cells and produce CPE as early as 6 hrs. Laboratory animal: Isolated by the intracerebral inoculation of newborn mice, which develop paralysis and die. Virus identification: ELISA can be used to detect Ag. Nucleic acid detection methods: RT-PCR tests and real time RT-PCR. Serological tests: VNT and ELISA are commonly used test.

39. Prevention and Control Preventive measures include screening imported pigs, restricting the importation of pork products that may contain virus, restricting swill feeding, and regulating the disposal of garbage from international airplanes and ships. Some countries conduct routine surveillance and pre- and post-export testing, particularly in Europe. No commercial vaccines are available . Outbreaks are controlled by quarantining infected farms and regions, tracing pigs that may have been exposed, culling infected and in-contact pigs, and cleaning and disinfecting the affected premises.

40. AVIAN ENCEPHALOMYELITIS Synonym: Epidemic tremors. Avian encephalomyelitis (AE) is an economically important disease of young chicken. It is placed under the new genus, Tremovirus and is closely related to Hepatitis A virus. Transmission: Main mode transmission is by fecal-oral route. Vertical transmission via the egg may also occur. It produces enteric infection and the virus is shed in feces.

41. CLINICAL FEATURES Occurs in chickens – 1 to 21 days of age. IP: 1 to 2 days after vertical transmission and 11 days after horizontal transmission. Characterized by dullness, progressive ataxia, tremors (particularly of the head and neck), weight loss, blindness, paralysis and in severe cases, prostration, coma and death. Recovered birds show CNS disorders. Eggs from infected layers show a reduced hatchability and increased loss of hatched chicks. It causes relatively mild encephalomyelitis in quail, turkeys, pigeons and pheasants.

42. LESIONS No obvious macroscopic lesions. Lesions of viral encephalitis seen throughout the CNS, microscopically. Non-suppurative encephalomyelitis and lymphocytic accumulation are characteritic. Central chromatolysis of neurons in the medulla oblongata is strongly indicative of AE.

43. DIAGNOSIS Based on the clinical signs and histopathologic lesions. Clinical materials: Tissues such as brain, pancreas and paired sera. Virus isolation may be done in cell culture, embryonated eggs via yolk sac route (5 – 7 days old) and virus identification by immunofluorescence staining of tissues from affected chicks. Nucleic acid detection methods by RT-PCR. Serological tests by ELISA using purified or recombinant antigens are commonly used. CONTROL: Can be achieved by either depopulation or vaccination. Attenuated virus vaccines administered in the drinking water are available.( after 8 weeks of age, but at least 4 weeks before the onset of egg laying).

44. Precautions In accordance with the WOAH, all susceptible birds on site should be vaccinated concurrently with the dose recommended by the manufacturer. Administration of live AE vaccine to flocks may result in a transient decrease in egg production for up to 14 days. Do not administer live AE vaccine to breeders within 4 weeks of onset of lay or during egg production. The AE virus is shed in faeces and transmitted vertically in eggs for up to 4 weeks following vaccination. To avoid clinical AE in the progeny, eggs for hatching should not be taken from the flock until 4 weeks have elapsed following vaccination. Birds less than 8 weeks-of-age exposed to live AE vaccine may display clinical neurological disease.

45. Vaccination AE- Poxine  is recommended for the prevention of avian encephalomyelitis and fowl pox to healthy pullets between the ages of eight weeks and four weeks before the start of egg production. Store this vaccine at not over 45°F (7°C). Do not vaccinate within 21 days before slaughter. Vaccine administration Live AE vaccine - drinking water or wing web from 10 weeks-of-age until 4 weeks before onset of lay.

46. DUCK VIRAL HEPATITIS ETIOLOGY: Duck hepatitis virus 1 is the most common. Classified under the new genus, Avihepatovirus of the family Picornaviridae. Duck hepatitis viruses 2 and 3 are now classified as astroviruses (genus Avastrovirus of the family Astroviridae). TRANSMISSION: Highly contagious and the virus is excreted in feces. The infection is transmitted by either direct contact or through fomites. Rats have been described as reservoir. Introduced into susceptible population through duck meat products.

47. CLINICAL FEATURES AND LESIONS IP: 1-5 days. Common in ducklings less than 21 days. Clinical signs: stand still with partially closed eyes, loss balance so that fall to one side, paddle spasmodically and die. Mortality may reach 100%. Lesions : Hepatomegaly, edematous, mottled with punctate or ecchymotic hemorrhages. Extensive hepatic necrosis.

48. (A,C)  Signs of opisthotonos and spasmodic kicking.  (B,D)  Non-infected Pekin and Muscovy controls showed normal liver appearance.  (E,F)   Hemorrhagic spots on the liver surface of Pekin ducklings.  (G,H)  Muscovy livers showed severe congestion.

49. DIAGNOSIS Based on the history, clinical signs and characteristic necropsy findings. Clinical materials: Liver is the ideal material. Virus can be isolated in duck hepatocyte cultures/duck embryo liver. DH A V - 1 produces CPE characterized by cell rounding. Virus identification by immunofluorescence staining. Nucleic acid detection methods: RT-PCR Serological tests: VNT has been developed. Control: Recovered ducks are immune. Hyperimmune serum reduces losses during outbreaks. Attenuated virus vaccines are available commercially. Inactivated vaccines against DH A V - 1 can also be used to prime the ducks to produce high antibody titre.

50. Prevention & control DHAV-1 infections can be controlled by the use of live attenuated virus vaccines and an inactivated virus vaccine. They are administered to breeder ducks to confer passive immunity to ducklings. Ducklings susceptible to DHAV-1 may be passively protected with a chicken egg yolk antibody preparation. DAstV-2 infections can be controlled by the use of a live attenuated virus vaccine given to breeder ducks to confer passive immunity to ducklings. DVH type II is caused by duck astrovirus type 1 (DAstV-1) Control is based on vaccination and biosecurity. There is no specific treatment for duck viral hepatitis infection. Prevention and control is based on strict biosecurity and implementation of vaccination protocols. 

51. REFERENCES: Fenner’s veterinary virology – 5 th edition. Veterinary virology by Frederick A. Murphy – 3 rd edition.