Genetic characterization and ex-vivo neutralization behavior of bluetongue virus serotype-16 recovered from apparently healthy goat.

Bluetongue virus (BTV) infects almost all the domestic and wild ruminants though the clinical disease is most commonly reported in sheep and some species of deer. Goat and cattle are the most common asymptomatic reservoir of the virus. Full genome sequencing and serological characterization of the virus isolates are emphasized for understanding the phylogenetic relationship and molecular epidemiology of bluetongue (BT). In this study, we report phylogenetic and phenotypic antigenic relationship of a BTV serotype-16 (PDP2/13/Ind) recovered from an apparently healthy goat from the state of Uttarakhand, a hilly terrain of sub-Himalayan India with four other BTV-16 isolates. The full genome sequence data was analyzed and the phylogenetic relationship of the goat isolate with other BTV-16 was established. Phylogenetic analysis revealed cluster of PDP2/13/Ind along with other Indian BTV-16 isolates indicating their close ancestral relationship. A cohesive ancestral relationship, irrespective of the genome segments analyzed, was also observed between Indian and Mediterranean BTV-16. The mean substitution rate of different segments of BTV-16 isolates varied from 3.231 × 10-5 (seg-2) to 1.129 × 10-3 (seg-6) substitutions per site per year. Timescale analysis indicated that all the segments had an older ancestor. No statistically significant geographic structuring of BTV-16 isolates was observed indicating frequent gene flow. The goat isolate shares highest identity (99.5%-99.8%) with G53/ABT/HSR, a BTV-16 recovered from the western part of the country whereas high level of divergence (11.9%-33.3%) at genomic segment level was observed with a Nigerian BTV-16 (NIG1982/10). Phenotypic antigenic relationship (r) of PDP2/13/Ind with other isolate-specific hyperimmune serum (HIS) determined from serum neutralization titer was 0.672 ± 0.058 to 0.948 ± 0.09. On other hand, the calculated 'r' score was 0.636 ± 0.063 to 0.814 ± 0.201 when HIS against PDP2/13/Ind was used to neutralize the other BTV-16 isolates. The percentage antigenic similarity (R) of the PDP2/13/Ind with other BTV-16 isolates was 65.39 ± 5.38-87.67 ± 14.86. Data suggests presence of subtype antigenic variation amongst the BTV-16 isolates recovered from the goats of a geographically restricted area of the state of Uttarakhand, India.

Genetic and phylogenetic analysis of the outer capsid protein genes of Indian isolates of bluetongue virus serotype-16.

AIM: The aim of the study was to characterize bluetongue virus serotype 16 (BTV-16), recently isolated from different states of India. The evolutionary relationship of newly isolated BTV-16 and previously reported Indian and global BTV-16 isolates were compared using molecular analysis. MATERIALS AND METHODS: In the present study, five (n=5) BTV-16 isolates were used to amplify gene segment-2 and segment-6 encoding the outer capsid proteins VP2 and VP5, respectively. The amplified products were purified and sequenced by the Sanger sequencing method. The phylogenetic relationship and nucleotide identity of all five BTV-16 isolates were compared with previously reported Indian and global BTV-16 isolates. Nucleotide sequence data were aligned using the CLUSTAL W algorithm implemented in the MegAlign of DNASTAR program package (MegAlign 5.00, DNASTAR Inc., Madison, USA). Phylogenetic analyses were carried out using MEGA version 6.0 software with the best nucleotide substitution model. RESULTS: Phylogenetic analysis based on the VP2 and VP5 encoding genes, segregates Indian BTV-16 isolates in a distinct cluster with proximity to the Eastern topotype. Indian isolates make a monophyletic cluster with Eastern topotypes with Western topotype BTV-16 (BTV-16/NIG/AJ586694) occupying a separate cluster. Indian isolates were found to share 91.5%-97.5% and 96.5%-98.9% identity at the nucleotide and deduced amino acid (aa) level, respectively, to the global BTV-16 isolates. There is a high degree of variation with the Nigerian isolate with 27.0-27.7% and 26.0-26.9% at the nucleotide and aa sequence level, respectively. These data suggest that Indian BTV-16 isolates might have evolved separately within the Eastern BTV topotype. CONCLUSION: Phylogenetic analyses and nucleotide identity of BTV-16 isolates at the VP2 and VP5 gene encoded level indicate that isolates used in the present study might have evolved from a common Eastern topotype ancestor. The data presented in this study will be helpful for future selection of reference strains in a serological and molecular epidemiology study.

Differentiation of bovine herpesvirus1 subtypes based on UL0.5 gene sequencing.

Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis is one of the high economic importance diseases of cattle and caused by bovine herpesvirus1 (BoHV1). Based on the restriction endonuclease fingerprinting of viral DNA, the BoHV1 can be divided into three subtypes viz., BoHV1.1, 1.2a, and 1.2b. Since this method requires a pure viral DNA, it is time-consuming and labour intense. In the current study, the UL0.5 gene based PCR sequencing has been used for the subtyping of BoHV1. Out of five isolates, four had BoHV1-like signatures and one isolate had BoHV1.2-like signatures. Further, these viruses phylogenetically clustered under the respective subtypes. These results indicate that the UL 0.5 gene based PCR sequencing could be used as an alternate method of subtyping of BoHV1.

Structural analysis and cross-protective efficacy of recombinant 87 kDa outer membrane protein (Omp87) of Pasteurella multocida serogroup B:2.

Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic septicaemia (HS) in cattle and buffalo especially in tropical regions of Asian and African countries, is known to possess several outer membrane proteins (OMPs) as immunogenic antigens. In the present study, omp87 gene encoding for 87 kDa OMP (Omp87) protein of P. multocida serogroup B:2 strain P52, has been amplified (∼2304 bp), cloned in to pET32a vector and over-expressed in recombinant Escherichia coli as fusion protein. The recombinant Omp87 protein (∼102 kDa) including N-terminus hexa-histidine tag was purified under denaturing condition. Immunization of mice with rOmp87 resulted in increased antigen specific IgG titres in serum and provided protection of 66.6 and 83.3% following homologous (B:2) and heterologous (A:1) challenge, respectively. A homology model of Omp87 revealed the presence of two distinct domains; N-terminal domain with four POTRA repeats in the periplasmic space and a pore forming C-terminal ß-barrel domain (ß1- ß16) in the outer membrane of P. multocida, which belong to Omp85-TpsB transporter superfamily of OMPs. The study indicated the potential possibilities to use rOmp87 protein along with suitable adjuvant in developing subunit vaccine for haemorrhagic septicaemia and pasteurellosis in livestock.

Reassortant influenza A viruses in wild duck populations: effects on viral shedding and persistence in water.

Wild ducks of the genus Anas represent the natural hosts for a large genetic diversity of influenza A viruses. In these hosts, co-infections with different virus genotypes are frequent and result in high rates of genetic reassortment. Recent genomic data have provided information regarding the pattern and frequency of these reassortant viruses in duck populations; however, potential consequences on viral shedding and maintenance in the environment have not been investigated. On the basis of full-genome sequencing, we identified five virus genotypes, in a wild duck population in northwestern Minnesota (USA), that naturally arose from genetic reassortments. We investigated the effects of influenza A virus genotype on the viral shedding pattern in Mallards (Anas platyrhynchos) and the duration of infectivity in water, under different temperature regimens. Overall, we found that variation in the viral genome composition of these isolates had limited effects on duration, extent and pattern of viral shedding, as well as on the reduction of infectivity in water over time. These results support that, in wild ducks, functionally equivalent gene segments could be maintained in virus populations with no fitness costs when genetic reassortments occur.

Viral replication, persistence in water and genetic characterization of two influenza A viruses isolated from surface lake water.

Water-borne transmission has been suggested as an important transmission mechanism for Influenza A (IA) viruses in wild duck populations; however, relatively few studies have attempted to detect IA viruses from aquatic habitats. Water-isolated viruses have rarely been genetically characterized and evaluation for persistence in water and infectivity in natural hosts has never been documented. In this study, we focused on two IA viruses (H3N8 and H4N6 subtypes) isolated from surface lake water in Minnesota, USA. We investigated the relative prevalence of the two virus subtypes in wild duck populations at the sampling site and their genetic relatedness to IA viruses isolated in wild waterbirds in North America. Viral persistence under different laboratory conditions (temperature and pH) and replication in experimentally infected Mallards (Anas platyrhynchos) were also characterized. Both viruses were the most prevalent subtype one year following their isolation in lake water. The viruses persisted in water for an extended time period at constant temperature (several weeks) but infectivity rapidly reduced under multiple freeze-thaw cycles. Furthermore, the two isolates efficiently replicated in Mallards. The complete genome characterization supported that these isolates originated from genetic reassortments with other IA viruses circulating in wild duck populations during the year of sampling. Based on phylogenetic analyses, we couldn't identify genetically similar viruses in duck populations in the years following their isolation from lake water. Our study supports the role for water-borne transmission for IA viruses but also highlights that additional field and experimental studies are required to support inter-annual persistence in aquatic habitats.

Influenza A viruses in American White Pelican (Pelecanus erythrorhynchos).

The role of many wild waterbird species in the ecology and epidemiology of avian influenza viruses (AIV) remains unclear. We report the first isolation of AIV from American White Pelicans (Pelecanus erythrorhynchos; Pelecaniformes) in North America. Two H13N9 AIVs were isolated from hatchling birds in breeding colonies in Minnesota, USA, during 2007 and 2008. Based on molecular sequencing of the hemagglutinin and neuraminidase genes, the 2008 virus was genetically related to AIVs previously isolated from gulls and shorebirds in North America. The 2007 isolate was most related to AIVs from Eurasian gulls and North American ducks, reflecting both global movement of these viruses and reassortment between viruses associated with duck and gull reservoirs.

A Serine12Stop mutation in PB1-F2 of the 2009 pandemic (H1N1) influenza A: a possible reason for its enhanced transmission and pathogenicity to humans.

As the scientific community scrambles to define the ancestry and lineages of the eight segments of new pandemic H1N1 strain, we looked for unique genetic events in this virus's genome to explain the newly found enhanced virulence and transmissibility among humans. Genome annotations of this virus identified a stop mutation replacing serine at codon 12 (S12Stop) of the PB1-F2 protein, a virulence factor in influenza A viruses. Here, we discuss the significance of this finding and how it may contribute to host specialization, explaining the virtual absence of the H1N1 influenza A virus strain in pig populations. This finding is expected to lead to a better understanding of the transmission and pathogenesis of the 2009 pandemic strain.

The feasibility of using high resolution genome sequencing of influenza A viruses to detect mixed infections and quasispecies.

BACKGROUND: The rapidly expanding availability of de novo sequencing technologies can greatly facilitate efforts to monitor the relatively high mutation rates of influenza A viruses and the detection of quasispecies. Both the mutation rates and the lineages of influenza A viruses are likely to play an important role in the natural history of these viruses and the emergence of phenotypically and antigenically distinct strains. METHODOLOGY AND PRINCIPAL FINDINGS: We evaluated quasispecies and mixed infections by de novo sequencing the whole genomes of 10 virus isolates, including eight avian influenza viruses grown in embryonated chicken eggs (six waterfowl isolates - five H3N2 and one H4N6; an H7N3 turkey isolate; and a bald eagle isolate with H1N1/H2N1 mixed infection), and two tissue cultured H3N2 swine influenza viruses. Two waterfowl cloacal swabs were included in the analysis. Full-length sequences of all segments were obtained with 20 to 787-X coverage for the ten viruses and one cloacal swab. The second cloacal swab yielded 15 influenza reads of approximately 230 bases, sufficient for bioinformatic inference of mixed infections or quasispecies. Genomic subpopulations or quasispecies of viruses were identified in four egg grown avian influenza isolates and one cell cultured swine virus. A bald eagle isolate and the second cloacal swab showed evidence of mixed infections with two (H1 and H2) and three (H1, H3, and H4) HA subtypes, respectively. Multiple sequence differences were identified between cloacal swab and the virus recovered using embryonated chicken eggs. CONCLUSIONS: We describe a new approach to comprehensively identify mixed infections and quasispecies in low passage influenza A isolates and cloacal swabs and add to the understanding of the ecology of influenza A virus populations.