First report of Duck Hepatitis A virus genotype 2 in India.

Sudden death of ducklings was reported in a duck farm located at Tiruvallur district in Tamil Nadu, India. Disease investigation began with post mortem findings of dead birds revealing enlarged pale-pink / pale-yellow liver with multifocal petechiae and ecchymosis. A positive amplification with duck hepatitis A virus specific primers by reverse transcription-polymerase chain reaction (RT-PCR) on the tissue samples collected from dead birds indicated infection by duck hepatitis A virus (DHAV), an avian picornavirus, known to cause acute and high-mortality in ducklings. The virus isolation was successful in 9-days old embryonated chicken eggs, in primary chicken embryo fibroblast (CEF) cells and from experimentally infected ducklings. The embryonic death on day 5 to 7 post inoculation in chicken embryos with signs of cutaneous hemorrhage, edema and greenish yellow liver together with histopathology of embryonic liver and kidney further confirmed DHAV infection. TEM analysis of the infected allantoic fluid and infected CEF cell culture supernatant showed the presence of spherical shaped, non-enveloped virion particles of ~ 20-38 nm diameter, typical for DHAV. Experimental infection of ducklings with RT-PCR positive tissue supernatant caused 40% to 50% mortality with typical petechial hemorrhages on the surface of liver. Further, histopathological analysis and RT-PCR of the inoculated duckling's tissues confirmed the presence of DHAV. Nucleotide sequencing of the 5'UTR region and VP1 region confirmed duck hepatitis A virus genotype 2 (DHAV-2). To the best of our knowledge, this is the first report of laboratory confirmation of DHAV-2 in India. This study warrants the need for the extensive epidemiological surveillance to understand the prevalence of DHAV-2 in India and to take appropriate control measures to curtail the disease spread.

Experimental Infection of Foot and Mouth Disease in Indian Sheep and Goats.

Foot-and-mouth disease (FMD) is an economically important contagious disease of livestock mainly cattle, buffalo, sheep, goats, and pig. There is limited data available on pathogenesis of foot and mouth disease in goats. In the study, the sheep and goats were infected experimentally with a serotype O foot-and-mouth disease virus by different challenge routes. The sheep and goats challenged by coronary band route and coronary band and intra-dermo-lingual route exhibited FMD clinical signs at 2-5 days post challenge. Whereas intra-dermo-lingual challenged sheep and goats did not exhibit FMD clinical signs. Live virus could be isolated from blood of infected sheep and goats at 2-5 days post challenge. Viral RNA could be detected from blood of infected sheep and goats at 1-10 days post challenge. The neutralizing antibody titre was detected at 10 days post challenge and maintained up to 35 days post challenge in all infected sheep and goats. Non structural protein (NSP) antibodies were detected as early as 5-10 days post challenge and remain positive up to 35 days post challenge in the infected sheep and goats. In conclusion, the pathogenesis of sheep and goats with serotype O foot and mouth disease virus by different challenge routes could be demonstrated.

Phylogenetic analysis of the leader proteinase (Lpro) region of Indian foot and mouth disease serotype O isolates.

In this study, the nucleotide sequences of the complete leader proteinase (Lpro) region of 21 isolates of foot-and-mouth disease virus (FMDV) serotype O collected during various outbreaks in India were sequenced and compared with vaccine strains. The phylogenetic analysis of these Lpro sequences showed a difference in the clustering of the isolates based on the VP1 capsid coding region sequences. The comparison of amino acid sequences at the N terminus end of the Lpro region showed very high variability, although 2 conserved start codons (AUG) at 1st and 29th sites. Furthermore, all the amino acid residues that formed the active cleft site of the Lpro sequences of this study were conserved. These results suggest that Lpro sequences could also be used for phylogenetic comparison of FMDV isolates.

Emergence of antigenic variants with in serotype A foot and mouth disease virus in India and evaluation of a new vaccine candidate panel.

Emergence of genetically and antigenically divergent lineages/genotypes and poor intergenotypic antigenic coverage is a major concern in serotype A foot-and-mouth-disease virus (FMDV) in India. In 2009, to cover antigenic diversity emerged in serotype A virus field isolates, IND40/2000 was selected as the new vaccine strain for incorporation in the trivalent FMD vaccine formulation used in India. Although current vaccine strain (IND40/2000) covers most isolates antigenically, a few VP3(59)-deletion group isolates showed low r-value in routine vaccine matching exercise. The VP3(59)-deletion group within genotype 18 emerged first in late part of 2002 and in 2007 causing outbreaks along with non-deletion isolates of the same genotype. In case of emergence or re-emergence of more antigenically divergent isolates in future, a need for a new vaccine candidate to cover maximum isolates of both deletion and non-deletion group may arise. Four alternate candidate vaccine strains (IND281/2003, IND195/2007, IND360/2007 and IND123/2008) were selected based on set criteria and antigenic relationships with field isolates sampled between 2002 and 2009 were analyzed using a micro-neutralization test. Phylogenetic analysis based on capsid region of serotype A isolates revealed existence of two broad distinct clusters (VP3(59)-deletion and non-deletion group) within genotype 18. The VP3(59)-deletion group has diversified genetically with time giving rise to three different sub-lineages (clade18a, 18b and 18c). The present study indicates that the virus candidates IND281/2003 (VP3(59)-deletion group) and IND195/2007 (non-deletion group) can be used as an adjunct or alternative strain to currently used vaccine strain IND40/2000 in case of emergence of more antigenically divergent isolates in future.

Evaluation of FTA cards as a laboratory and field sampling device for the detection of foot-and-mouth disease virus and serotyping by RT-PCR and real-time RT-PCR.

Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for serodiagnosis pose a major problem in a tropical country like India, where there is maximum temperature fluctuation. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. The present study evaluated the usefulness of FTA Classic Cards for the collection, shipment, storage and identification of the FMDV genome by RT-PCR and real-time RT-PCR. The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards a useful option for transport of FMDV genome for identification and serotyping. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular document transport methods. Live virus cannot be isolated from samples collected in FTA cards, which is a limitation. This property can be viewed as an advantage as it limits the risk of transmission of live virus.

Serotyping of foot-and-mouth disease virus by antigen capture-ELISA using monoclonal antibodies and chicken IgY.

Laboratory detection of specific foot-and-mouth disease virus (FMDV) is routinely carried out by ELISA and RT-PCR. Identification and serotyping of FMDV by ELISA requires polyclonal antibodies raised in rabbits and guinea pigs. The polyclonal antibodies have certain disadvantages such as batch to batch variation, inconsistent yields of antibodies and limited quantity of serum obtained from individual animals. This paper describes a method wherein monoclonal antibodies and chicken IgY were used in an antigen capture-ELISA for serotyping of thirty tongue epithelial samples and sixty tissue culture fluids. The results were compared with the routine antigen detection ELISA. The present study indicated that monoclonal antibodies and chicken IgY can substitute conventional polyclonal antibodies for routine serotyping of FMDV.