Evaluation of currently available bovine viral diarrhoea virus (BVDV) and HoBi-like pestivirus (HoBiPeV) specific diagnostic tests in detection of highly divergent HoBiPeVs in cattle.

The 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.

Porcine reproductive and respiratory syndrome virus induces concurrent elevation of High Mobility Group Box-1 protein and pro-inflammatory cytokines in experimentally infected piglets.

Porcine Reproductive and Respiratory Syndrome (PRRS), caused by PRRS virus (PRRSV), is one of the most important devastating diseases of pigs, characterized by reproductive failure in sows, and respiratory disease with heavy mortality in piglets. PRRS virus has been reported to elevate the levels of proinflammatory cytokines in the serum of infected pigs. High Mobility Group Box-1 (HMGB-1) protein is a cellular biomolecule belonging to the Danger Associated Molecular Patterns (DAMP) family, which stimulates immune cells to release pro-inflammatory cytokines upon release out of cells. The role of HMGB-1 in the pathogenesis of PRRSV remains largely unknown. In the present study, HMGB-1 levels in serum samples collected from six-week-old piglets infected intra-nasally with 2 × 105.75 TCID50/mL of Indian PRRSV (Ind-297221/2013) was estimated by ELISA up to 21 days post infection (dpi). Pro-inflammatory cytokine mRNA (IL-1ß, IL-6 and TNF- α) expression in PBL was estimated by SYBR green based real time PCR. Mean HMGB-1 concentration in serum was found to be significantly elevated in PRRSV infected piglets on 6 dpi as compared to uninfected control piglets. At mRNA level, significant increase in expression of HMGB-1 was observed from 4 to 5 dpi and from 11 to 13 dpi. IL-1ß and IL-6 mRNA were significantly upregulated between 4 and 6 dpi. Significant increase in TNF-α gene expression was seen only on 7 and 9 dpi. Higher levels of pro-inflammatory cytokines and HMGB-1 could be correlated with fever which was observed within 7 dpi in all the infected piglets and additionally around 13 dpi in the animal that died on 17 dpi. Thus, elevated HMGB-1 level in PRRSV infected piglets could be correlated with concurrent increase in pro-inflammatory cytokine (IL-6) mRNA. In-vitro studies were conducted in PRRSV infected Porcine Pulmonary Alveolar Macrophages (PAM) to ascertain HMGB-1 role in PRRS pathogenesis. The results of both in-vivo and in-vitro studies showed that HMGB-1 plays an important role in mediating the pro-inflammatory cytokine responses in PRRS pathogenesis.

Pathogenic characterization of porcine reproductive and respiratory syndrome virus of Indian origin in experimentally infected piglets.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important transboundary viral disease of pigs confronting the swine industry worldwide. This study was aimed to assess the pathogenic potential of PRRS virus belonging to genotype 2 that emerged in India in 2013. Nine 6-week-old piglets were inoculated intranasally with 2 × 105.75  TCID50 /ml of PRRSV (Ind-297221/2013). Three piglets were kept as uninfected controls. Blood and nasal swabs were collected daily up to 7 days post-infection (dpi) and on alternate days subsequently. Piglets were necropsied for tissue sample collection either on death or after euthanasia on 7, 14 or 21 dpi (one uninfected control and three PRRSV-infected piglets per interval). The virus caused high fever, typical blue ear, weight loss, respiratory distress, diarrhoea and leucopenia between 2 and 8 dpi. Two infected piglets died (on 3 and 17 dpi) during the course of study. The presence of virus in serum and nasal secretion was observed up to 19 and 17 dpi, respectively, with the maximum load between 4 and 7 dpi. Seroconversion started 6 dpi and the mean PRRSV antibody titre reached up to 640 by 21 dpi. Virus load was highest in tonsils at all the intervals, whereas in spleen and lymph nodes load was higher in later intervals. Major microscopic lesions in PRRSV-infected piglets included moderate to severe interstitial pneumonia, lymphoid depletion in tonsils and lymph nodes (cystic), thymic atrophy, reactive hyperplasia followed by lymphoid depletion in spleen. PRRSV antigen was consistently demonstrated by immunoperoxidase test in the lungs, spleen, tonsils and lymph nodes. Antigen distribution was more widespread on 7 and 14 dpi than on 21 dpi. The findings establish that the Indian PRRSV is highly pathogenic to piglets.

Induction profiles of mRNA of toll like receptors and cytokines in chickens pre-exposed to low pathogenic avian influenza H9N2 virus followed by challenge with highly pathogenic avian influenza H5N1 virus.

Herein, the induction of TLRs and cytokines in chickens pre-exposed to low pathogenic avian influenza H9N2 virus followed by challenge with highly pathogenic avian influenza (HPAI) H5N1 virus was studied. Four groups (1-4) of chickens inoculated with 106 EID50 of H9N2 virus were challenged with 106 EID50 of H5N1 virus on days 1, 3, 7 and 14 post H9N2 inoculation, respectively. In groups (1-4) TLRs and cytokines induction was studied in chicken PBMCs on day 3 post H5N1 challenge. In H5N1 control group TLRs (1, 2, 5 and 7) cytokines (IFNα, IFNß, IFNγ, IL1ß, IL2, IL4, IL8 and TGF ß3) were down regulated. In group 1 down regulation of cytokines and TLRs was similar to H5N1 control birds. Down regulation of TLRs and cytokines in H5N1 control and group 1 resulted death of all the chickens. In group 2, up-regulation of TLRs (3, 7 and 15) and induction of TNFα, IFNα, IFNß, IFNγ aided virus clearance leading to survival of all the chickens. In group 3 significant up-regulation of TLRs (3, 4 and 15) and significant induction of cytokines (IFNγ, TNFα, IL1ß, IL4, IL6, IL8, IL10 and TGF ß3) was detected. In group 4 significant up-regulation of TLRs (2, 3, 7 and 15) and significant induction of cytokines (IFNγ, TNFα, IL1ß, IL2, IL6, IL8 and IL10) was detected. In groups 3 and 4 simultaneous and significant induction of pro-inflammatory, antiviral and anti-inflammatory cytokine resulted cytokine dysregulation leading to death of (2/6) and (3/6) chickens respectively. Hence, the study revealed TLRs and cytokines role in modulating the H5N1 infection outcome in chickens pre-exposed to H9N2 virus.

First report on serological evidence of bovine viral diarrhea virus (BVDV) infection in farmed and free ranging mithuns (Bos frontalis).

Despite reports of BVDV infection in several domestic and wild ruminants, no information exists for mithun (Bos frontalis) species. Hence, this study was undertaken to determine prevalence of BVDV infection in mithuns, which contribute significantly to local economy in the North Eastern region of India. Blood and serum samples were collected between 2013 and 2016 from mithuns (n = 466) belonging to the states of Nagaland, Mizoram, and Arunachal Pradesh. Serum samples were tested for BVDV antibodies by a commercial ELISA and leukocytes were tested for BVDV by real-time RT-PCR. The overall true seroprevalence rate was 13.1% (95% confidence interval, CI: 6.9-17.8%) with higher prevalence in mithuns reared under semi-intensive system (27.5%) than in free-ranging mithuns (7.6%). Among the three states, seroprevalence (16.2%) was highest in Nagaland, while prevalence rates varied markedly among geographical locations. Age-wise data showed highest seroprevalence rate in >6-year-old animals (20.6%) than 2-6 years old (16.9%), 6 months-2 years old (8.5%), and <6-month-old animals (11.3%). The seroprevalence was higher in males (20.9%) than in females (12.1%). Among the four mithun strains, higher prevalence was evident in Manipur (30.3%) than Arunachal (21.3%), Nagaland (11.7%), and Mizoram strain (10.2%). However, no BVDV genomic RNA could be detected. The results provide first serological evidence of BVDV infection in mithun species and extend the knowledge on BVDV host range. The baseline data will help further investigations on epidemiology of BVD in mithun and its impact on mithun production.

Distribution pattern of bovine viral diarrhoea virus type 1 genome in lymphoid tissues of experimentally infected sheep.

In this study, cellular localization and the distribution pattern of BVDV genome in lymphoid tissues during the course of experimental acute BVDV-1 infection of sheep was investigated. Tonsils, mesenteric lymph nodes (MLN) and spleen were collected on 3, 6, 9, 12 and 15 days post infection (dpi) from twenty 4-month-old lambs, experimentally inoculated intra-nasally with 5 × 10(5) TCID50 of a non-cytopathic (ncp) BVDV-1 isolate, Ind-17555. Tissues collected from ten mock-infected lambs served as controls. In situ hybridization (ISH) was carried out in paraformaldehyde fixed paraffin embedded tissue sections using digoxigenin labelled riboprobe targeting 5'-UTR of BVDV-1. BVDV genome was detected at all the intervals from 3 dpi to 15 dpi in the lymphoid tissues with variations between the intervals and also amongst the infected sheep. During the early phase of acute infection, presence of viral genome was more in tonsils than MLN and spleen, whereas the distribution was higher in MLN during later stages. BVDV-1 genome positive cells included lymphocytes, macrophages, plasma cells, reticular cells and sometimes crypt epithelial cells. Genome distribution was frequently observed in the lymphoid follicles of tonsils, MLN and spleen, besides the crypt epithelium in tonsils, paracortex and medullary sinus and cords of MLN. Most abundant and widespread distribution of BVDV-1 genome was observed on 6 dpi while there was a reduction in number and intensity of positive signals by 15 dpi in most of the infected animals. This is the first attempt made to study the localisation of BVDV-1 in lymphoid tissues of acutely infected sheep by in situ hybridization. The results show that the kinetics of BVDV-1 distribution in lymphoid tissues of experimentally infected non-pregnant sheep follows almost a similar pattern to that demonstrated in BVDV infected cattle.

Expression and purification of recombinant H5HA1 protein of H5N1 avian influenza virus in E. coli and its application in indirect ELISA.

The PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) hemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.

Development of a RT-PCR ELISA for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants and its evaluation on clinical samples.

The aim of this study was to develop a reverse transcription polymerase chain reaction ELISA (RT-PCR ELISA) for detection of ruminant pestiviruses and to evaluate its diagnostic performance on clinical samples obtained from cattle, sheep and goats. Optimization was carried out by serial dilution of home-made digoxygenin-labelled RT-PCR product standards obtained from pestivirus isolates and pestivirus infected animals. The detection limit of the assay was 10TCID50/ml, similar to virus isolation and real-time RT-PCR but 10-fold higher than RT-PCR. The assay had high analytical specificity along with a good reproducibility. When the assay was evaluated on the samples obtained from animals infected experimentally with BVDV and from the field using virus isolation as standard, it showed a high diagnostic sensitivity (95.9%) and specificity (98.6%) and there was strong agreement (97.5% concordance) between the two tests. However, it displayed an increased diagnostic specificity and sensitivity over RT-PCR. Additionally, when a few samples (n=26) were tested by RT-PCR ELISA and real-time RT-PCR, 100% concordance was obtained between them. Our results showed that RT-PCR ELISA can be a rapid, cost effective and alternative molecular diagnostic test for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants in ordinary laboratory settings.

Serological evidence of West Nile virus infection in wild migratory and resident water birds in Eastern and Northern India.

To assess West Nile virus (WNV) infection in wild resident and migratory birds, we tested 3887 samples from 1784 birds belonging to 119 identified species within 30 families collected during 2008-10 from 13 states in India. The serum samples were tested for WNV antibodies initially by a competition ELISA and subsequently by a micro-plaque reduction neutralization test (Micro-PRNT), whereas tracheal and cloacal swabs were subjected to real-time RT-PCR for the detection of the WNV RNA. Twenty six birds (2.46%) out of 1058 tested showed evidence of flavivirus antibodies by ELISA. End point neutralization antibody determinations for WNV and Japanese encephalitis virus (JEV) showed that of the 22 ELISA positive sera, WNV-specific neutralizing antibodies were detected in 17 samples representing nine species of wild birds (residents: Purple swamphen, Little cormorant, Little egret, Black ibis and Spot-billed duck; residents with winter influx: Common coot and Mallard; migratory birds: Ruff and Purple heron), and two samples were positive for both WNV and JEV antibodies. The WNV-specific antibodies were most commonly detected in Mallards and Common coots. WNV genomic RNA was not detected by real-time RT-PCR. The results in this study suggest that wild resident birds are infected occasionally and wild migratory birds rarely with WNV. Additionally, our study provides evidence of WNV infection in eastern and northern India for the first time.

Small interfering RNAs targeting viral structural envelope protein genes and the 5ʹ-UTR inhibit replication of bovine viral diarrhea virus in MDBK cells.

Bovine viral diarrhea viruses (BVDVs) are important pathogens of cattle that occur worldwide, and for which no antiviral therapy is available. In the present study, the inhibitory effect of small interfering (si) RNAs on bovine viral diarrhea virus 1 (BVDV-1) replication in cultured bovine cells was explored. Four synthetic siRNAs were designed to target structural envelope region genes (Erns, E1, and E2) and one cocktail of siRNA was generated to target the 5ʹ-UTR of the BVDV-1 genome. The inhibitory effects of siRNAs were assessed by determination of infectious viral titer, viral antigen and viral RNA. The siRNA cocktail and three of the synthetic siRNAs produced moderate anti-BVDV-1 effect in vitro as shown by 25%-40% reduction in BVDV-1 antigen production, 7.9-19.9-fold reduction in viral titer and 21-48-fold reduction in BVDV-1 RNA copy number. Our findings suggest that siRNA cocktail targeted at the 5ʹ-UTR is a stronger inhibitor of BVDV-1 replication and the targets for siRNA inhibition can be extended to BVDV-1 structural envelope protein genes.

Genetic and antigenic characterization of bovine viral diarrhoea virus type 2 isolated from cattle in India.

Previous studies have shown that bovine viral diarrhoea virus type 1 (BVDV-1) subtype b is predominantly circulating in Indian cattle. During testing for exotic pestiviruses between 2007 and 2010, BVDV-2 was identified by real time RT-PCR in two of 1446 cattle blood samples originating from thirteen states of India. The genetic analysis of the isolated virus in 5' UTR, N(pro), entire structural genes (C, E(rns), E1 and E2), nonstructural genes NS2-3 besides 3' UTR demonstrated that the nucleotide and amino acid sequences showed highest similarity with BVDV-2. The entire 5' and 3' UTR consisted of 387 and 204 nucleotides, respectively, and an eight nucleotide repeat motif was found twice within the variable part of 3' UTR that may be considered as a characteristic of BVDV-2. The phylogenetic analysis revealed that the cattle isolate and earlier reported goat BVDV-2 isolate fall into separate clades within BVDV-2a subtype. Antigenic typing with monoclonal antibodies verified the cattle isolate also as BVDV-2. In addition, cross-neutralization tests using antisera raised against Indian BVDV strains circulating in ruminants (cattle, sheep, goat and yak) displayed significant antigenic differences only between BVDV-1 and BVDV-2 strains. This is the first identification of BVDV-2 in Indian cattle that may have important implications for immunization strategies and molecular epidemiology of BVD.