Experimental infection and pathology of two highly pathogenic avian influenza H5N1 viruses isolated from crow and chicken in house crows (Corvus splendens).

We investigated the experimental infection of two highly pathogenic avian influenza H5N1 viruses isolated from crow (A/crow/Assam/142119/2008) and chicken (A/chicken/Sikkim/151466/2009) in house crows (Corvus splendens). Both viruses caused infection in crows, where four out of six and three out of six crows succumbed to H5N1 infection within 11 days post challenge by crow and chicken viruses, respectively. The major clinical signs in crows were wing paralysis, circling and torticollis. The virus shedding detected from swabs was not persistent in both crow nor chicken viruses. Both viruses were isolated more frequently from oral swabs than from cloacal swabs. Both virus strains were isolated from brain, lungs, heart, liver, pancreas, spleen, large intestines of crows that succumbed to H5N1 infection. The surviving birds seroconverted in response to H5N1 virus infection. Microscopically, both viruses caused coagulative necrosis in pancreas and kidneys. Brain showed gliosis and neuronal degeneration. This experimental study highlights that crows could be infected with H5N1 viruses from different hosts with minor differences in pathogenicity. Therefore, it is imperative to carry out surveillance of highly pathogenic avian influenza H5N1 virus in synanthropic birds along with biosecurity measures to mitigate the H5N1 spread in poultry population. Keywords: chicken virus; crow virus; highly pathogenic avian influenza; house crows.

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.

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.

Isolation of a lytic bacteriophage against virulent Aeromonas hydrophila from an organized equine farm.

A bacteriophage (VTCCBPA6) against a pathogenic strain of Aeromonas hydrophila was isolated from the sewage of an organized equine breeding farm. On the basis of TEM analysis, phage belonged to family Myoviridae. PCR amplification and sequence analysis of gp23 gene (encoding for major capsid protein) revealed phylogenetic resemblance to T4 like virus genus. Protein profiling by SDS-PAGE also indicated its resemblance to T4 like phage group. However, the comparison of its gp23 gene sequence with previously reported phages showed similarity with T4-like phages infecting Enterobacteriaceae instead of Aeromonas spp. Thus, to our knowledge, this report points toward the fact that a novel/evolved phage might exist in equine environment against A. hydrophila, which can be potentially used as a biocontrol agent.

Pathology of a H5N1, highly pathogenic avian influenza virus, in two Indian native chicken breeds and a synthetic broiler line.

In this study, susceptibility to H5N1 virus infection was studied in two Indian native chicken breeds viz. Kadaknath and Aseel (Peela) and an Indian synthetic broiler strain (Synthetic dam line (SDL-IC). Fifty birds from each genetic group were infected intra-nasally with 1000 EID50 of a highly pathogenic avian influenza virus (HPAIV) strain A/chicken/Navapur/India/7972/ 06 (H5N1) and observed for a period of 10 days. Significant differences in severity of clinical signs, gross lesions and time for onset of symptoms were observed. The overall severity of clinical signs and gross lesions was less in SDL-IC broilers as compared to the other two genetic groups. The mortality percentages were 100, 98 and 92% with Mean Death Time (MDT) of 3.12, 5.92 and 6.96 days, respectively for the two native breeds Kadaknath and Aseel (Peela), the and SDL-IC broiler strain. Comparison of histological lesions revealed differences in disease progression among the genetic groups. Vascular lesions such as disseminated intravascular coagulopathy (DIC) were predominant on 3 days post infection (dpi) in Kadaknath, and on 5 and 6 dpi in Aseel (Peela) and SDL-IC broiler. The mean log2 HA titres of the re-isolated virus from various organs of H5N1 AIV infected birds of the three genetic groups ranged from 2.32 (lung, trachea and bursa) to 5.04 (spleen) in Kadaknath; 2.32 (lung) to 6.68 (brain) in Aseel (Peela); and 2.06 (liver) to 7.01 (lungs and kidney) in SDL-IC broiler. These results suggest that the susceptibility to H5N1 highly pathogenic avian influenza virus infection differed among the three breeds; Kadaknath being highest followed by Aseel (Peela) and synthetic SDL-IC broiler. This is possibly the first report on the differences in the susceptibility of the India native breeds to H5N1 virus infection and its severity.

Peripheral blood mononuclear cells from field cattle immune to bovine viral diarrhea virus (BVDV) are permissive in vitro to BVDV.

The aim of this study was to determine the in vitro permissivity of peripheral blood mononuclear cells (PBMCs) from bovine viral diarrhea virus (BVDV)-immune field cattle to homologous and heterologous BVDVs. PBMCs from seventeen BVDV-naïve and sixteen BVDV-immune animals were infected with noncytopathic BVDV-1 or BVDV-2. The immune status of cattle was indicated by the presence of virus neutralizing antibodies, while viral load of PBMCs was determined by real-time RT-PCR. The results revealed that the PBMCs from naïve or immune animals were permissive to either BVDV-1 or BVDV-2, but the viral load was significantly higher for the naïve than for the immune animals. Furthermore, the load of homologous virus in PBMCs from immune animals was lower than that of heterologous virus. Our results provide evidence that the PBMCs from BVDV-immune cattle in field are susceptible to reinfection with homologous or heterologous BVDV, albeit to a lower extent in the former case.

Genetic variety of bovine viral diarrhea virus 1 strains isolated from sheep and goats in India.

UNLABELLED: Antigenic and genetic typing of pestiviruses isolated from Indian sheep and goats was carried out. Testing of 1777 sheep and 1026 goat blood samples collected between 2004 and 2008 resulted in isolation of twelve pestiviruses, seven from sheep and five from goats. All of them were antigenically typed as bovine viral diarrhea virus 1 (BVDV-1). Both the partial 5ʹ-UTR and entire non-structural autoprotease (Npro) gene of the pestiviruses were amplified by RT-PCR and sequenced. The phylogenetic analysis confirmed all twelve sheep and goat pestiviruses as BVDV-1 and they were further classified into two subtypes, BVDV-1b (seven) and BVDV-1c (five). This is for the first time that BVDV-1c was detected in sheep and goats. However, no association between the subtype and geographic area of origin was observed. Although closely related, BVDV-1b and BVDV-1c isolates of sheep and goats were placed in a different clade than previously reported Indian BVDV-1b/BVDV-1c isolates. This study confirmed widespread prevalence of BVDV-1 in Indian sheep and goats that has significance in the epidemiology of bovine viral diarrhea. KEYWORDS: bovine viral diarrhea virus; BVDV-1; goat; Npro; genetic typing; sheep; 5ʹ-UTR.

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.

Identification of other Cellular Receptors for Edema Factor of Bacillus anthracis by Independent Inhibition of Protective Antigen Evidenced by Inhibition of Embryo Growth and Angiogenesis.

Edema factor (EF) is one of the major secretory proteins of anthrax bacteria along with protective antigen (PA) and lethal factor (LF). Edema factor is a calmodulin-and calcium-dependent adenylate cyclase that increases intracellular levels of cAMP. Intracellular trafficking of EF occurs through PA by binding to ATR/CMG2 receptors, which are also involved in other physiological functions of cells. cAMP is a secondary messenger which activates multiple signaling cascades involved in the cytokinetics of actin molecules and cell junction formation. The present study evaluated the effect of EF on growth and angiogenesis patterns in chicken embryos in the in ovo model. Angiogenesis in the chorioallantoic membrane (CAM) of an embryonated chicken egg was decreased and embryo growth was delayed by EF despite the absence of trafficking moiety PA, which is required for transferring the EF molecule inside the cell. Angiogenesis inhibition and embryo growth retardation indicate the use of an alternative receptor by EF to modulate these cellular functions. Additionally, docking was performed between EF as a ligand and hepatocyte growth factor receptor (cMET) and vascular endothelial growth factor (VEGF) receptors, which are mainly involved in growth and angiogenesis. The analysis revealed a very strong binding of EF to cMET receptor (in terms of the number of hydrogen bonds and energy) compared to its ligand hepatocyte growth factor (HGF), which indicates the use of cMET receptor by EF and induction of angiogenesis and embryo growth retardation possibly by competitive inhibition of HGF ligand or receptor-mediated endocytosis.

Toxicopathological studies on the effects of T-2 mycotoxin and their interaction in juvenile goats.

Food and feeds contaminated with mycotoxins have been a threat to the rearing industry by causing some of the most fatal toxic reactions not only in the farm animals but also in humans who consume them. Toxicity to juvenile goats was induced by feed contamination with T-2 toxin (at 10 and 20 ppm dosage; group I and II, respectively). The toxicity impact was assessed on days 15 and 30 post treatment with respect to growth performance, oxidative stress, apoptotic studies and detailed pathomorphology. The study revealed that apart from the obvious clinical toxicosis (weakness, lethargy, and retardation in growth), the toxin fed groups also exhibited significant haematological (reduced hemoglobin, total leukocyte and thrombocyte counts) and biochemical changes (increased levels of oxidative stress markers with concomitant decrease in levels of serum and tissue catalase and superoxide dismutase). The pathomorphological and histological alterations suggested that the liver and intestine were the most affected organs. Ultra-structurally, varying degrees of degeneration, cytoplasmic vacuolations and pleomorphic mitochondria were observed in the hepatocytes and the enterocytes of the intestine. Kidney also revealed extensive degeneration of the cytoplasmic organelles with similar condensation of the heterochromatin whereas the neuronal degeneration was characterized by circular, whirling structures. In addition, the central vein and portal triad of the hepatocytes, cryptic epithelial cells of the intestine, MLNs in the lymphoid follicles, PCT and DCT of the nephronal tissues and the white pulp of the spleen exhibited extensive apoptosis. In this study, it was also observed that the expression of HSPs, pro-apoptotic proteins and pro-inflammatory cytokines were significantly upregulated in response to the toxin treatment. These results suggest that the pathogenesis of T-2 toxicosis in goats employs oxidative, apoptotic and inflammatory mechanisms.

Isolation and pathotyping of H9N2 avian influenza viruses in Indian poultry.

A total of 1246 faecal and tissue samples collected/received from 119 farms located in various states of India were processed for isolation of avian influenza viruses (AIV) during 2003-2004 as part of a program to monitor AIV infection in Indian poultry population. Avian influenza virus was isolated for the first time in India from poultry farms with history of drop in egg production, respiratory illness and increased mortality in Haryana state. A total of 29 H9N2 AIV isolates were obtained from the states of Punjab, Haryana, Uttar Pradesh, Gujarat, and Orissa and Union Territory Delhi. Subtyping was done by HI, RT-PCR and neuraminidase inhibition assay. Pathotyping of six representative isolates by intravenous pathogenicity index (0.0/3.0) in 6-8 weeks old chicken, trypsin dependency in cell culture and HA cleavage site analysis (335RSSR*GLF341) confirmed that these isolates are low pathogenic. Nucleotide sequence analysis of the HA gene showed that the Indian isolates are very closely related (95.0-99.6%) and shared a homology of 92-96% with H9N2 isolates from Germany and Asian regions other than that of mainland China. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) which indicates a preference to binding of alpha (2-6) sialic acid receptors. Two of the six isolates had 7 glycosylation sites in the HA1 cleaved protein and the remaining four had 5 sites. Phylogenetic analysis showed that they share a common ancestor Qa/HK/G1/97 isolate which had contributed internal genes of H5N1 virus circulating in Vietnam. Further characterization of Indian H9N2 isolates is required to understand their nature and evolution.

Prevalence of Bovine viral diarrhoea virus (BVDV) antibodies among sheep and goats in India.

The aim of this study was to determine the prevalence of pestivirus antibodies in sheep and goats in India. A total of 2803 serum samples collected between 2004 and 2008 from 1777 sheep in 92 flocks and 1026 goats in 63 flocks belonging to 13 states were tested by competition ELISA for detection of pestivirus antibodies. In sheep, the true prevalence rate was 23.4% (95% confidence interval: 22.9%-27.0%) and in goats it was 16.9% (95% CI: 16.4%-21.3%). The flock level seroprevalence was 66.3% for sheep and 54.0% for goats. Geographical variation in individual and flock prevalence was highly significant. A significant association (p < 0.05) was found between sheep and goat flocks having cattle contact and the flock level seroprevalence. The seroprevalence was lower in 6 months-1 year age group compared to the 1-2 year and >2 year age groups in both sheep and goats. Cross neutralization studies on 61 seropositive sheep and 34 seropositive goat samples representing all positive flocks, exhibited > four fold higher titre to bovine viral diarrhoea virus type 1 (BVDV-1) in 41 sheep and 23 goat samples and to BVDV-2 in one sheep and goat each. This study for the first time showed serological evidence of wide spread BVDV infections in Indian sheep and goats, with BVDV-1 predominating and BVDV-2 occasionally besides highlighting the potential risk of infection to other species, which needs to be considered whenever BVD control measures are initiated.

Molecular characterization of bovine viral diarrhea virus type 2 isolate originating from a native Indian sheep (Ovies aries).

The wide spread prevalence of bovine viral diarrhea virus type 1 (BVDV-1) in cattle and recent identification of BVDV-2 in goats in India warranted pestivirus surveillance in sheep. Nested reverse transcription-polymerase chain reaction (RT-PCR) was used to detect BVDV-2 in one of 1561 blood samples collected randomly from 78 sheep flocks in 11 states of India. Antigenic characterization of the isolated pestivirus using polyclonal and monoclonal antibodies typed the isolate as BVDV-2. When analyzed at genetic level in N(pro) (N-terminal autoprotease) and entire gene region coding structural proteins, namely capsid (C) protein and envelope proteins E(rns) (ribonuclease secreted), E1 and E2 genomic organization was the same for all pestiviruses, the nucleic acid and amino acid sequences showed highest similarity with those of BVDV-2. When compared with BVDV-2 isolate 890, the sequence homology was 83.7% for C, 84.6% for E(rns) and E1, and 81.5% for E2. The cleavage site C/E(rns) was found totally conserved while N(pro)/C was conserved only from C-terminus of N(pro), E(rns)/E1 site was conserved only from C-terminus of E(rns) and E1/E2 site was conserved only from C-terminus of E1. Phylogenetic analysis of nucleotide sequences in 5' untranslated regions (UTR), N(pro), E2, NS3 and NS5B regions placed the sheep isolate in a separate clade within BVDV-2 subtype b. This was supported by the presence of unique mutations in the structural protein coding regions beside NS3 and NS5B. To our knowledge this is the first report on the sequence analysis of the entire structural gene coding region of a BVDV-2b isolate. This is also the first occurrence of BVDV-2 subtype b in sheep, providing the evidence that this subtype can also occur in species other than cattle.

Identification of bovine viral diarrhea virus type 1 in yaks (Bos poephagus grunniens) in the Himalayan region.

Since cattle are widely infected by bovine viral diarrhea virus (BVDV) in India, we searched for pestivirus infection in yaks. Of 71 pure and crossbred yaks from Himalayan region, pestivirus antigen was detected by Ag-ELISA in three animals. Pestivirus in leukocyte and cell culture isolated virus samples originating from positive yaks was also confirmed by RT-PCR using panpestivirus specific primers selected from 5'-untranslated region (5' UTR). The 5' UTR, N(pro) and E2 regions were sequenced and used for genetic typing. Phylogenetic analysis revealed that pestiviruses detected in three Himalayan yaks were similar genetically, belonging to BVDV-1. Antigenic characterisation of yak pestivirus also confirmed the typing as BVDV-1. This is the first report on the identification of BVDV type 1 in yaks.

Infectious dose-dependent accumulation of live highly pathogenic avian influenza H5N1 virus in chicken skeletal muscle-implications for public health.

Highly pathogenic avian influenza viruses (HPAIV) of H5N1 subtype are a major global threat to poultry and public health. Export of poultry products, such as chicken and duck meat, is a known source for the cross-boundary spread of HPAI H5N1 viruses. Humans get infected with HPAI H5N1 viruses either by close contact with infected poultry or through consumption of fresh/undercooked poultry meat. Skeletal muscle is the largest soft tissue in chicken that has been shown to contain virus during systemic HPAIV infection and supports productive virus infection. However, the time between infection of a chicken with H5N1 virus and presence of virus in muscle tissue is not yet known. Further, it is also not clear whether chicken infected with low doses of H5N1 virus that cause non-fatal subclinical infections continue to accumulate virus in skeletal muscle. We investigated the amount and duration of virus detection in skeletal muscle of chicken experimentally infected with different doses (102 , 103 and 104 EID50 ) of a HPAI H5N1 virus. Influenza viral antigen could be detected as early as 6 hr after infection and live virus was recovered from 48 hr after infection. Notably, chicken infected with lower levels of HPAI H5N1 virus (i.e., 102 EID50 ) did not die acutely, but continued to accumulate high levels of H5N1 virus in skeletal muscle until 6 days post-infection. Our data suggest that there is a potential risk of human exposure to H5N1 virus through meat from clinically healthy chicken infected with a low dose of virus. Our results highlight the need to implement rigorous monitoring systems to screen poultry meat from H5N1 endemic countries to limit the global spread of H5N1 viruses.

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.

Genetic and antigenic characterization of bovine viral diarrhea virus type 2 isolated from Indian goats (Capra hircus).

Recent studies have shown that bovine viral diarrhea virus (BVDV) type 1 is widely prevalent in Indian cattle. In a surveillance of randomly collected 562 blood samples from seven states during 2004-2006, BVDV type 2 was detected in two native Indian goats by nested reverse transcription polymerase chain reaction (nRT-PCR). The virus isolated from them was classified antigenically as BVDV 2 on the basis of virus neutralization test and reactivity with monoclonal antibodies. Phylogenetic analysis of three different genomic regions, 5' un-translated region (5' UTR), E(rns) structural coding region and NS5B nonstructural coding region typed Indian goat isolate as BVDV 2a having close similarity with strains from North America and Europe suggesting its probable introduction through trade. It was placed in a separate clade within the 2a branch having unique mutations in E(rns) and NS5B region. This is the first report of BVDV 2 in India and only second time recorded in goat species. The isolation of BVDV 2 from goat warrants intensive surveillance in cattle and sheep.

Pathogenicity of an Indian isolate of bovine viral diarrhea virus 1b in experimentally infected calves.

The aim of this study was to determine the pathogenicity of an Indian bovine viral diarrhea virus (BVDV) 1b isolate in 7-9-months-old male calves. Infected (four) and control (two) calves were bled at three days interval for hematological, virological and serological studies until day 27. All infected calves developed respiratory illness, biphasic pyrexia, mild diarrhea, leucopenia and mild thrombocytopenia. Viraemia was demonstrated between 3 and 15dpi and the infected calves seroconverted by 15dpi. Prominent kidney lesions were endothelial cell swelling, proliferation of mesangial cells and podocytes leading to glomerular space obliteration. Degeneration and desquamation of cells lining seminiferous tubules were observed in two infected calves. Consolidation of lungs with interstitial pneumonia, mild gastroenteritis and systemic spread were also evident. It was concluded that Indian BVDV isolate induced moderate clinical disease in calves and glomerulonephritis resulting from acute BVDV infection was observed for the first time.

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.