Genetic evolution of Newcastle Disease Virus sub-genotype VII.2 isolates, diagnosed from vaccinated poultry farms of Gujarat, India.

Newcastle disease was suspected in 37 commercial poultry farms, including 12 layer and 25 broiler farms in four districts of Gujarat, India. Vaccination had been done in 32 (20 broilers and 12 layers) farms. Tissue samples from each farm were pooled as one sample. In egg embryo inoculation, HA-HI and PCR, respectively, 32/37, 29/37, and 24/37 samples were found positive. Pathotyping by mean death time calculation and primer combination PCR revealed velogenic NDV, which was later confirmed with the presence of the 112-RRQKR*F-117 sequence at the F protein cleavage site. Phylogenetic analysis of full F gene sequences (N=10) confirmed the presence of sub-genotype VII.2 in 9/10 sequences, and genotype II in one sample. These 9 sequences were only 0.7 to 2.6 % divergent with two VII.2 (=VIIi) sequences (HQ697254.1 chicken/Banjarmas/Indonesia and KU862293.1 Parakeet/Karachi/Pakistan) but had 2.2 to 3.6 % diversion from two VII.2 sequences (OR185447 and MZ546197) from India. Then branching was found from sequences of VIIh, VIIk (VII.2), and VIIa (VII.1.2), and then from sub-genotypes VII.1.1 and VII.1.2. Due to less than 5 % diversion, these sequences could not be qualified as new sub-genotype in evolutionary distance analysis. At the amino acid level, our sequences had aa N-T-I-A-L-T at 24-79-125-385-445-482. Whereas at the same positions, in most of the retrieved VII.2 sequences and vaccines, the sequence was S-A-V-T-Q/I- E/A. Two sequences revealed additional six and four amino acid differences,respectively.This indicates rapid continuous genetic evolution of sub-genotype VII.2 and partially explains vaccinal immunity escape.

Doppler indices of uterine arteries and mRNA transcripts of hormone receptors in cervix vis-à-vis dilatation therapy in buffaloes affected with incomplete cervical dilatation associated with uterine torsion.

The present study aimed to investigate the Doppler indices and mRNA transcripts of hormone receptors in relation to the response of dilatation therapy in incomplete cervical dilatation (ICD) associated with uterine torsion in buffaloes. Out of 36 successfully detorted uterine torsion cases, eight buffaloes revealed a fully dilated cervix, while the remaining 28 had ICD, and subjected to dilatation therapy (500 µg cloprostenol + 2 mg estradiol benzoate + 80 mg valethamate bromide + 50 IU oxytocin + 250 mL calcium borogluconate). The responses of dilatation therapy were assessed in 26 buffaloes as one died, and one could not follow up. Doppler indices of middle uterine arteries on trans-rectal ultrasound were evaluated pre- and 30-60 min post-detorsion. Cervical tissue biopsies were collected from 16 buffaloes to study mRNA transcripts of hormone receptors. The duration, degree, location of uterine torsion, fetal viability, consistency of the cervix, relaxation of pelvic ligaments, udder engorgement, and gestation length were also recorded to evaluate the response of dilatation therapy. The 73.08% (19/26) buffaloes responded to the therapy with a duration ranging from 2 to 56 hrs (18.41 ± 4.11). The significantly increased blood flow volume (BFV) and time-average peak velocity (TAP) while the significantly reduced resistive index (RI) and pulsatility index (PI) in an ipsilateral middle uterine artery (MUA) at post-detorsion were observed in dilation therapy responded than the not-responded group. The mRNA transcripts of estradiol receptors-α (ESR1), prostaglandin receptors (PTGFR), and oxytocin receptors (OXTR) were upregulated by 7.47, 6.63, and 8.72-fold in the ICD group, respectively. The Doppler indices along with duration of illness, location of uterine torsion, consistency of the cervix, and udder engorgement can be used to predict the response of dilatation therapy in ICD associated with uterine torsion. The upregulated mRNA expression of ESR1, PTGFR and OXTR is mandatory for success of dilatation therapy.

Surveillance and Molecular Characterization of SARS-CoV-2 Infection in Non-Human Hosts in Gujarat, India.

Since December 2019, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been spreading worldwide, triggering one of the most challenging pandemics in the human population. In light of the reporting of this virus in domestic and wild animals from several parts of the world, a systematic surveillance study was conceptualized to detect SARS-CoV-2 among species of veterinary importance. Nasal and/or rectal samples of 413 animals (dogs n= 195, cattle n = 64, horses n = 42, goats n = 41, buffaloes n = 39, sheep n = 19, cats n = 6, camels n = 6, and a monkey n = 1) were collected from different places in the Gujarat state of India. RNA was extracted from the samples and subjected to RT-qPCR-based quantification of the target sequences in viral nucleoprotein (N), spike (S), and ORF1ab genes. A total of 95 (23.79%) animals were found positive, comprised of n = 67 (34.35%) dogs, n= 15 (23.43%) cattle, and n = 13 (33.33%) buffaloes. Whole SARS-CoV-2 genome sequencing was done from one sample (ID-A4N, from a dog), where 32 mutations, including 29 single-nucleotide variations (SNV) and 2 deletions, were detected. Among them, nine mutations were located in the receptor binding domain of the spike (S) protein. The consequent changes in the amino acid sequence revealed T19R, G142D, E156-, F157-, A222V, L452R, T478K, D614G, and P681R mutations in the S protein and D63G, R203M, and D377Y in the N protein. The lineage assigned to this SARS-CoV-2 sequence is B.1.617.2. Thus, the present study highlights the transmission of SARS-CoV-2 infection from human to animals and suggests being watchful for zoonosis.

Molecular and immunogenic characterization of BHK-21 cell line adapted CVS-11 strain of rabies virus and future prospect in vaccination strategy.

Development of a cost effective quality vaccine is a key issue in rabies control programme in developing countries. With this perspective, in the present study, challenge virus standard (CVS)-11 strain of rabies virus was adapted to grow in BHK-21 cells, characterized, compared with other viruses including global vaccine strains and field isolates from Indian subcontinent and China at molecular level. This cell adapted virus was evaluated for the production of cost effective veterinary vaccine. The maximum virus titre achieved was 10(7) fluorescent focus unit (FFU)/mL at 10th passage level. There was no nucleotide difference in the nucleoprotein (N) and glycoprotein (G) genes after adaptation in cell line. Phylogenetic analysis showed that adapted virus was grouped with global vaccine strains, closest being with other CVS strains but distinct from the Indian field isolates. Global vaccine strains including cell adapted CVS-11 virus have 83-87 % identity at nucleotide level of G gene with Indian field viruses. Growth kinetics of cell culture adapted virus showed that the optimum virus titer (around 10(7) FFU/mL) could be obtained at around 48 h post infection by co-cultivation method using 0.1 multiplicity of infection inoculums at 37 °C. These findings can be used for up scaling of vaccine production. The protective efficacy of test vaccine produced using 10(6.95) FFU/mL cell culture harvest showed 1.17 IU/mL relative potency by NIH test. Further, adapted virus was found to be suitable for use in rapid fluorescent focus inhibition test.