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.

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.

Identification and molecular characterization of novel and divergent HoBi-like pestiviruses from naturally infected cattle in India.

HoBi-like pestiviruses have been sporadically reported from naturally infected cattle in South America, Asia and Europe. While the closely related bovine viral diarrhoea virus 1 (BVDV-1) and BVDV-2 have been reported from cattle in India, the prevalence and diversity of HoBi-like viruses have not yet been studied. Here we report the genetic diversity and molecular characteristics of HoBi-like viruses, through systematic surveillance in cattle (n=1049) from 21 dairy farms across India during 2012-2013. On the basis of real-time RT-PCR, virus isolation and nucleotide sequencing results, of the 20 pestivirus positive cattle, HoBi-like viruses were identified in 19 cattle from four farms in three states and BVDV-1b in one cattle. Phylogenetic analysis of 5'-UTR and N(pro) region identified the circulation of two lineages of HoBi-like viruses in India, that were distinct to those circulating globally, highlighting the independent evolution of at least three lineages of HoBi-like viruses globally. Antigenic differences were also evident between the two Indian lineages. In addition to revealing that HoBi-like virus may be more widespread in Indian cattle than previously reported, this study shows greater genetic divergence of HoBi-like viruses indicating a need for continued pestivirus surveillance in cattle.

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.

Electromagnetic transition from the 4+ to 2+ resonance in 8Be measured via the radiative capture in 4He + 4He.

An earlier measurement on the 4+ to 2+ radiative transition in 8Be provided the first electromagnetic signature of its dumbbell-like shape. However, the large uncertainty in the measured cross section does not allow a stringent test of nuclear structure models. This Letter reports a more elaborate and precise measurement for this transition, via the radiative capture in the 4He + 4He reaction, improving the accuracy by about a factor of 3. Ab initio calculations of the radiative transition strength with improved three-nucleon forces are also presented. The experimental results are compared with the predictions of the alpha cluster model and ab initio calculations.

Measurement of the damping of the nuclear shell effect in the doubly magic 208Pb region.

The damping of the nuclear shell effect with excitation energy has been measured through an analysis of the neutron spectra following the triton transfer in the (7)Li induced reaction on (205)Tl. The measured neutron spectra demonstrate the expected large shell correction energy for the nuclei in the vicinity of doubly magic (208)Pb and a small value around (184)W. A quantitative extraction of the allowed values of the damping parameter γ, along with those for the asymptotic nuclear level density parameter ã, has been made for the first time.

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.

Molecular characterization of RNA and protein synthesis during a one-step growth curve of bovine viral diarrhoea virus in ovine (SFT-R) cells.

The aim of this study was to determine the kinetics of noncytopathic bovine viral diarrhoea virus (BVDV) multiplication and synthesis of BVDV specific RNA and proteins in ovine cells (SFT-R) during a one-step growth curve. The virus titre and RNA level were determined by focus-forming assay and real time RT-PCR. The RNA synthesis was detected by Northern blot while synthesis of E2 and NS3 proteins was assayed by immunohistochemistry and Western blot. The results showed that synthesis of viral RNA is initiated at 4h, NS3 and E2 proteins are detectable at 6-7h and the replication cycle is complete at 10-12h. Additionally, we provide evidence that NS2-3 protein was cleaved in ovine cells early during infection and in proliferated leukocytes of acutely infected sheep. This study showed that synthesis of BVDV RNA and proteins in ovine cells occurs at similar times as found in bovine cells.

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.