Experimental inoculation of a crow derived influenza A (H5N1) virus in chickens and its pathological and genetic characterization

We report the infectivity of a crow derived influenza A (H5N1) virus (A/crow/India/01TR01/2012) in chickens and its pathological and genetic characterization. Histopathological changes and immunohistochemistry staining of internal organs and skeletal muscle were consistent with influenza A virus infection. Real time RT-PCR and virus isolation results demonstrated the systemic spread of the virus in chickens with 100% mortality. Comparatively higher level of virus shedding was detected in oropharyngeal swab (7.63×106 viral RNA copy) than in cloacal swab (6.66 × 106 viral RNA copy). Concentrations of viral antigen in kidney, lungs, brain, spleen and large intestine were higher compared to pancreas and skeletal muscle. No genetic change was observed on interspecies transmission of the virus. The study revealed that the crow derived H5N1 virus is able to kill the poultry, underlining the need for close monitoring of presence of virus in poultry near crow roosting areas so that further transmission to other avian and mammalian hosts can be prevented

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Not AvailableBovine leukaemia virus (BLV) causes enzootic leucosis in cattle and is prevalent worldwide. Although recent studies have shown that BLV strains can be classified into 10 distinct genotypes, no information is available regarding the BLV genotype prevalent in cattle in India. To determine the genetic variability in BLV, in this study, 118 adult dairy cows from three states of India were screened for BLV infection by env gp51-specific ELISA and nested PCR. Of the 33 cows found positive by both PCR and ELISA, 10 selected BLV strains were subjected to molecular characterization. Phylogenetic analyses of partial and full-length env gp51 gene sequences of Indian BLV strains and other geographical diverse BLV strains representing all the 10 genotypes revealed that Indian strains belonged to BLV genotype 6. Although Indian strains showed close genetic proximity with the strains circulating in South America, they were classified into a new subgenotype within genotype 6. Alignment of deduced amino acid sequences in gp51 demonstrated substitutions mainly in conformational epitope G, neutralizing domain 2 and linear epitope D, with a novel mutation (threonine to alanine at residue 252) found in D-epitope of all the Indian BLV strains. Although serological evidence of BLV infection in India has been reported earlier, this study on molecular characterization of BLV strains established the existence of BLV genotype 6 in India. Additionally, the results of this study highlight the importance of genetic analysis of geographically diverse BLV strains to understand BLV global genetic diversity and further studies are required to determine BLV genetic diversity and extent of BLV infection in cattle in India.Not Availabl

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Not AvailableBovine leukaemia virus (BLV) causes enzootic leucosis in cattle and is prevalentworldwide. Although recent studies have shown that BLV strains can be classifiedinto 10 distinct genotypes, no information is available regarding the BLV genotypeprevalent in cattle in India. To determine the genetic variability in BLV, in this study,118 adult dairy cows from three states of India were screened for BLV infection byenvgp51‐specific ELISA and nested PCR. Of the 33 cows found positive by bothPCR and ELISA, 10 selected BLV strains were subjected to molecular characteriza-tion. Phylogenetic analyses of partial and full‐lengthenvgp51 gene sequences ofIndian BLV strains and other geographical diverse BLV strains representing all the10 genotypes revealed that Indian strains belonged to BLV genotype 6. AlthoughIndian strains showed close genetic proximity with the strains circulating in SouthAmerica, they were classified into a new subgenotype within genotype 6. Alignmentof deduced amino acid sequences in gp51 demonstrated substitutions mainly inconformational epitope G, neutralizing domain 2 and linear epitope D, with a novelmutation (threonine to alanine at residue 252) found in D‐epitope of all the IndianBLV strains. Although serological evidence of BLV infection in India has beenreported earlier, this study on molecular characterization of BLV strains establishedthe existence of BLV genotype 6 in India. Additionally, the results of this studyhighlight the importance of genetic analysis of geographically diverse BLV strains tounderstand BLV global genetic diversity and further studies are required to deter-mine BLV genetic diversity and extent of BLV infection in cattle in India.Not Availabl

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Not AvailableBirds can act as reservoirs of West Nile virus (WNV) with a key role in its epidemiology. WNV lineage 1 associated fatal cases of human encephalitis in 2011 and acute flaccid paralysis in 2013 were reported in Alappuzha district, Kerala, India. But no information is available on WNV circulation in domestic ducks, which are abundant, cohabit with humans and occupy wetlands and water bodies in the region. To determine the extent of WNV infection, we investigated 209 sera, 250 oral and 350 cloacal swab samples from local Chara and Chemballi domestic ducks (Anas platyrhynchos var domesticus) in the districts of Alappuzha, Kottayam, Kollam and Pathanamthitta collected during January and March 2015. The serum samples were tested for WNV antibodies first by a competition ELISA and then by a micro virus neutralization test (micro-VNT), while oral and cloacal swabs were subjected to WNV real-time RT-PCR. Ninety five ducks showed evidence of flavivirus antibodies by ELISA. End point neutralizing antibody titre against WNV and Japanese encephalitis virus (JEV) revealed WNV specific antibodies in 24 (11.5%) ducks in 3 districts, JEV specific antibodies in 21 (10%) ducks in 2 districts and flavivirus specific antibodies in 19 (9%) ducks. However, no WNV genomic RNA could be detected. The results of this study demonstrate evidence of widespread WNV and JEV infection in domestic ducks in Kuttanad region, Kerala with a higher seroprevalence to WNV than JEV. Additionally, it highlights the utility of domestic ducks as a surveillance tool to detect WNV/JEV circulation in a region.Not Availabl

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Not AvailableThe emergence of novel and divergent HoBi-like pestivirus (HoBiPeV) strains in cattle in Asia recently has raised concerns with regard to their reliable and accurate diagnosis. Hence, the aim of this study was to evaluate currently available BVDV diagnostic tests and HoBiPeV-specific diagnostic tests in detection of genetically divergent strains of HoBiPeV. One strain each of HoBiPeV-c and d were subjected to two BVDV diagnostic RT-PCR tests, one HoBiPeV specific RT-PCR test, three BVDV diagnostic qRT-PCR tests, one HoBiPeV specific qRT-PCR test and two BVDV antigen capture ELISAs. Archived cattle sera (n = 41) from farms with reports of HoBiPeV natural infection were assessed for detection of HoBiPeV antibodies by VNT and two commercial BVD antibody ELISA kits. BVDV diagnostic qRT-PCR tests had better sensitivity than BVDV diagnostic RT-PCR tests, while majority of them except a commercial kit showed a lower sensitivity for HoBiPeV-d strain. The HoBiPeV specific qRT-PCR test was found more sensitive than HoBiPeV specific RT-PCR but both had lower sensitivity for HoBiPeV-d strain, as displayed by primer/probe sequence mismatches. The BVDV Erns antigen ELISA detected both the strains of HoBiPeV, but with a lower sensitivity for HoBiPeV-d strain, whereas BVDV NS3 antigen ELISA failed to detect them even at a high HoBiPeV titre. Compared to VNT, commercial BVDV antibody ELISA showed low to moderate sensitivity in detection of HoBiPeV antibodies, with a failure rate of 31.25% for the whole virus antigen based ELISA and a failure rate of 56.25% for NS3 antibody ELISA. The present study demonstrated new challenges in HoBiPeV diagnosis indicating a need in improvement of both HoBiPeV specific diagnostic RT-PCR and qRT-PCR for better utility in HoBiPeV epidemiology and biological product safety. Although more studies are required, this study reinforces that combined use of BVDV Erns and NS3 antigen ELISA may have some utility in preliminary differentiation between HoBiPeV and BVDV infection in PI cattle. Additionally, we show that the comparative VNT has a better sensitivity in detection of HoBiPeV exposure and there is a need of robust antibody ELISA for reliable detection of antibodies against this emerging bovine pestivirus.Not Availabl

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Not AvailableBovine pestiviruses, bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2 enter the bovine and ovine cells exclusively through chathrin-mediated endocytosis and not through non-classical endocytosis. However, no report is available regarding the mechanisms of entry of the emerging bovine pestivirus, HoBi-like pestivirus (HoBiPeV) that causes disease in cattle, buffaloes, sheep and goats. This study was aimed to unravel the possibility of HoBiPeV in utilizing the alternate endocytic mechanisms in establishing infection in ovine cells by inhibiting the caveolae-mediated endocytosis by a cholesterol sequestering drug, nystatin and macropinocytosis by an actin disrupting drug, cytochalasin-D. The reduction in infectivity of the HoBiPeV was found to be least (7.55% and 1.45% in nystatin and cytochalasin-D treated cells respectively) even at the highest concentration of the drugs (10 ig/mL and 0.5 ig/mL of nystatin and cytochalasin respectively) used for the pre-treatment of ovine cells before infection. The results of this study demonstrate that the HoBiPeV does not use the alternate endocytic pathways for entry into ovine cells similar to BVDV-1 and BVDV-2. Further studies may unravel the cellular receptors involved and main entry mechanisms of HoBiPeV in the future.Not Availabl

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Not AvailableThe aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.Not Availabl

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Not AvailableBovine viral diarrhea virus (BVDV) is a pestivirus which infects cattle worldwide causing substantial economic losses in cattle farming. BVDV is divided into two recognized species, BVDV-1 and BVDV-2 and one tentative species, BVDV-3. Since, complete genome sequence analysis can provide better insights into molecular epidemiology of BVD, we report here the first complete genome sequence analyses of an Indian BVDV-2 strain isolated from cattle. The full-genome of strain Ind 141353 contains 12285 nucleotides (nt) with a single large open reading frame which codes for 3898 amino acids. Phylogenetic analysis indicated that this strain belongs to the BVDV-2a subtype and has highest (93%) level of genetic identity with the Chinese cattle strain JZ05-1. It was inferred that although introduction from China is possible, introduction of BVDV-2 into Indian and Chinese cattle from a common trade source cannot be ruled out completely. The results in this study extend the spectrum of pestivirus molecular data and provide important insights into BVDV molecular epidemiology.Not Availabl

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Not AvailableThe current study aimed to develop broadly protective vaccines for avian influenza. In an earlier study, HA stalk (universal flu vaccine) was found to be broadly protective against different subtypes of influenza virus in mice. Hence, we were interested to know its breadth of protective efficacy either alone or combined with inactivated rgH5N2 (clade 2.3.2.1a) vaccine against challenge viruses of homologous H5N1, heterologous H5N8 (clade 2.3.4.4) and heterosubtypic H9N2 virus in specific pathogen-free chickens. The rgH5N2 vaccine alone or in combination with HA stalk elicited sufficient pre-challenge immunity in the form of haemagglutination inhibiting (HI) antibodies and neutralizing antibodies (MNT) against H5N1, H5N8, and H9N2 in chickens. The rgH5N2 vaccine alone or in combination with HA stalk also attenuated the shedding of H5N1, H5N8 and H9N2 in chickens and protected against the lethal challenge of H5N1 or H5N8. In contrast, all HA stalk immunised chickens died upon H5N1 or H5N8 challenge and H9N2 challenged chickens survived. Our study suggests that the rgH5N2 vaccine can provide clinical protection against H5N1, H5N8 and can attenuate the viral shedding of H9N2 in chickens.Not Availabl

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Not AvailableOne of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro-inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte-derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF-α, -β and -γ were significantly upregulated in the H5N1 group. Pro-inflammatory cytokines (IL-1β, TNF-α and IL-18) were highly upregulated in early mid (IL-1), and late (IL-6) phases of H5N1 virus infection. IL-8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6 hr of infection. TLR3, 7, 15 and 21 were upregulated 24 hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus-induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV-infected cells.Not Availabl