Genetic variability in VP1 gene of infectious bursal disease virus from the field outbreaks of Kerala, India.

Infectious bursal disease (IBD) is considered as menace as it affects poultry industry globally causing immunosuppression, high mortality and heavy economic loss. Outbreaks of IBD were reported in many states of India including Kerala. VP1 gene acts as an important factor in the process of virus encapsidation and its involvement in viral virulence and viral replication indicates its importance in infectious bursal disease virus (IBDV). The present study was conducted to carry out the molecular characterization of VP1 gene of virulent IBDV in Kerala. A total of 42 samples were processed for the detection and analysis of VP1 gene of IBDV. Out of 42 samples, 21 samples were positive for VP1 gene of IBD. The phylogenetic analysis of the partial VP1 gene sequences reveals the clustering of IBDV isolates into very virulent IBDV (vvIBDV) and non-virulent IBDV (vIBDV). Eighteen isolates (11 isolates from vaccinated flock and 7 from non-vaccinated flocks) clustered with very virulent strains. Three isolates (2 isolates were from vaccinated flock and 1 from non-vaccinated flock) clustered with non-virulent IBDV strains, showing more evolutionarily similarity to south Indian strain VCN14/ABT/MVC/India. It is observed that vvIBDV isolates from this study have common ancestor with the south Indian strain PY12 but showed 9-10% divergence from this strains. The amino acid analysis of these 21 isolates revealed that 17 isolates possessed the characteristic vvIBDV TDN amino acid triplet, while the three isolates had non-vIBDV NEG amino acid triplet at 145/146/147 position. The remaining isolate 1/CVASP/IBDV/VP1 shows unique PDN triplet instead of TDN. Two vvIBDV isolates (15/CVASP/IBDV/VP1 and 18/CVASP/IBDV/VP1) showed 100% nucleotide and amino acid similarity with intermediate plus vaccine strain. Four vvIBDV isolates showed neutral amino acid substitution K251R which was earlier reported in Indian strains but first time in south Indian isolates. The most common unique amino acid substitution observed in our study was neutral E269D amino acid substitution in 12 isolates, neutral amino acid substitution T329S in five isolates, neutral T174N and non-polar to polar amino acid substitution A178T in isolate 10/CVASP/IBDV/VP1, non-polar to polar amino acid substitution P360R in isolate 17/CVASP/IBDV/VP1 and non-polar to polar amino acid substitution P188S in isolate 1/CVASP/IBDV/VP1. These novel mutations in our study reveal the role of genetic drift in the evolution of vvIBDV strains. The isolate 2/CVASP/IBDV/VP1 from non-vaccinated flock shows VP1 gene of non-vIBDV, but possessing VP2 of vvIBDV type indicates this is evolved by genetic shift of segments A and B. This is the first genetic characterization study of field VP1 gene of IBDV isolates in Kerala, India.

Detection and molecular characterization of rotavirus of pigs in Kerala, India.

Group A rotaviruses (GAR) are an important cause of diarrhoea in infants and newborn animals especially pigs. In this paper, we report the detection, G and P typing and phylogenetic analysis of GAR of pigs in Kerala. A total of 100 fecal samples from diarrhoeic piglets were collected from organized farms in Wayanad, Ernakulam, Thrissur, and Palakkad districts of Kerala. The samples were tested for the presence of GAR employing reverse transcriptase polymerase chain reaction (RT-PCR) targeting VP6 gene. Positive samples were tested by G and P genotyping primers and representative amplicons were sequenced. Of the 100 samples, 12 were positive for GAR. The G and P types detected were G2, G4, G5, G6, G9, P[6] and P[19]. An untypable P type (P21-5 like) was also detected. In some of the samples more than one G type was detected. The nucleotide sequences of G2, G4 and G5 types were similar to those seen in pigs and that of G6 was similar to bovine sequences. G9, P[6] and P[19] sequences showed similarity to human rotavirus sequences. The findings of this study provide the first information on the G and P genotypes of GAR of pigs in Kerala.

Molecular characterisation of porcine reproductive and respiratory syndrome virus from pigs in Kerala.

Porcine reproductive and respiratory syndrome (PRRS) caused by an arterivirus is characterised by reproductive disorders in sows, and post-weaning pneumonia and growth reduction in piglets. Though the virus has been detected in Kerala, no systematic study has been carried out to ascertain its genotype and molecular epidemiology. In the present study, 7 PRRS virus (PRRSV) positive samples collected from incidences of PRRS in Kerala during 2017-2019 were subjected to ORF5, ORF7 and Nsp2 gene based reverse transcription polymerase chain reaction and the specific amplicons generated were sequenced. On BLAST analysis it was revealed that all the sequences were of genotype 2 (North American genotype). Phylogenetic analysis of ORF5 sequences, grouped them under subgenotype 4 with close clustering with other isolates from Kerala, Mizoram and Assam. Nsp2 gene sequence based phylogenetic analysis grouped the isolates under subgenotype 3 with similarities to isolates from Mizoram. Phylogenetic analysis based on ORF7, clustered the isolates under study with PRRSV isolates from Mizoram and Meghalaya. In Nsp2 sequences, a 30 amino acid discontinuous deletion was observed. On analysis of amino acid sequences of ORF5 of Kerala isolates and those from India, it was seen that the Kerala isolates showed closer similarity to PRRSV isolates from Assam than to the other Indian isolates. The study reveals that PRRSV strains prevalent in Kerala share close relationship with other PRRSV isolates in India. This may be due to spread of the virus from these regions to Kerala due to animal movement. Concerted efforts should be undertaken to check unauthorized animal movement to control spread of this economically important disease.

Identification and genetic analysis of infectious bursal disease viruses from field outbreaks in Kerala, India.

Recurrent infectious bursal disease (IBD) outbreaks were reported in different regions of Kerala, India. This paper reports the comparative genetic analysis of the hypervariable region of the VP2 gene of IBD virus isolates from the field outbreaks in Kerala. In phylogenetic analysis, the obtained field isolates fall into genogroup 1 and 3. In genogroup 3, all vvIBDV isolates shared a common ancestor with other south Indian isolates but isolates 9/CVASP/IBDV, 10/CVASP/IBDV, 12/CVASP/IBDV, 14/CVASP/IBDV and 17/CVASP/IBDV are most recently evolved and are diverged from the south Indian isolates. The amino acid sequence of 22 isolates was analysed, out of which 18 had conserved amino acids which were characteristic of vvIBDV. All the vvIBDV isolates obtained in the study had phenylalanine and valine at the position 240 and 294, respectively, similar to recently evolved Indian IBDV isolate (MDI14). But we observed T269A and S299N mutations in the isolate 6/CVASP/IBDV, and it is the first report of such mutations at these positions in India IBDV isolates. The isolate 11/CVASP/IBDV had a unique mutation of V225A which is not yet reported in IBDV isolates. Two isolates (15/CVASP/IBDV and 18/CVASP/IBDV) were 100% amino acid similar to intermediate plus vaccine strain. The isolates 8/CVASP/IBDV/VP2 and 19/CVASP/IBDV had amino acids unique for the intermediate vaccine with mutations observed at H253Q and V256I in 19/CVASP/IBDV, T270A and novel mutation N279Y in isolate 8/CVASP/IBDV. These two isolates had non-virulent classical heptapeptide sequence 'SWSARGS'; nevertheless, they produce field outbreaks of IBD. This is the first report of genetic characterisation of IBDV in Kerala, India.