Zoonotic cases of camelpox infection in India.

This study reports the first conclusive evidence of zoonotic camelpox virus (CMLV) infection in humans associated with outbreaks in dromedarian camels (Camelus dromedaries) in northwest region of India during 2009. CMLV infection is usually restricted to camels and causes localised skin lesions but occasionally leads to generalised form of disease. However, the present outbreak involved camel handlers and attendants with clinical manifestations such as papules, vesicles, ulceration and finally scabs over fingers and hands. In camels, the pock-like lesions were distributed over the hairless parts of the body. On the basis of clinical and epidemiological features coupled with serological tests and molecular characterization of the causative agent, CMLV zoonosis was confirmed in three human cases. Clinical samples such as skin scabs/swabs and blood collected from affected animals and humans were analysed initially, for the presence of CMLV-specific antigen and antibodies by counter immunoelectrophoresis (CIE); serum neutralization test (SNT); plaque-reduction neutralization test (PRNT) and indirect immunoperoxidase test which was later confirmed by amplification of CMLV-specific ankyrin repeat protein (C18L) gene. Virus isolation was successful only from samples collected from camels. Further, sequence analyses based on three full-length envelope protein genes (A27L, H3L and D8L) revealed 95.2-99.8% and 93.1-99.3% homology with other Orthopoxviruses at nucleotide and amino acid levels, respectively. Phylogram of the three genes revealed a close relationship of CMLV with Variola virus (VARV). Considering the emerging and re-emerging nature of the virus, its genetic relatedness to VARV, zoonotic potential and productivity losses in camels; the control measures are imperative in curtailing economic and public health impact of the disease. This is the first instance of laboratory confirmed camelpox zoonosis in India.

Non-structural protein 3A for differentiation of foot-and-mouth disease infected and vaccinated animals in Haryana (India).

There are severe international trade restrictions on foot-and-mouth disease (FMD) affected areas. Because of endemic nature of FMD, India started FMD control programme (FMD-CP) using mass vaccination in selected states including Haryana (year 2003). Although no significant incidence of the disease was reported after launching FMD-CP in the state but in order to participate in international trade of animal and animal products, veterinary authorities have to prove that there is no FMD virus (FMDV) circulation in the animal population, for which it is necessary to differentiate the FMD infected and vaccinated animals. For this purpose, an in-house indirect ELISA utilizing baculovirus-expressed FMDV non-structural protein (NSP) 3A was used to find evidence for virus circulation (prevalence of anti-NSP 3A-specific antibodies) by examining serum samples that were collected either before start of FMD-CP or after completion of third phase (Pre-4th) of vaccination in Haryana (India). A significant reduction (P < 0.01) in prevalence of anti-NSP 3A-specific antibodies (possibly carriers) was observed 2 years after launching FMD-CP in Haryana. However, in cattle the percentage of animals with anti-NSP 3A-specific antibodies was found to be significantly higher (P < 0.01) than buffalo, both before (P < 0.01) and after (P < 0.01) launching FMD-CP in the state. The findings of this study suggest that use of FMDV vaccine in cattle and buffaloes in endemic areas reduces virus circulation (carriers) in the vaccinated herds and that the current 3ANSP-ELISA can be successfully used to monitor the FMDV circulation in endemic areas.

Comparative immunogenecity of foot and mouth disease virus antigens in FMD-haemorrhagic septicaemia combined vaccine and FMD vaccine alone in buffalo calves.

Humoral immune response was evaluated by monitoring the serum antibody titres and virus specific IgM titres against Foot and Mouth Disease (FMD) virus antigens in serum samples obtained from different groups of calves inoculated with combined vaccine or FMD vaccine alone, on 0, 7, 14, 21, 28, 42 and 56 days post-vaccination (DPV). The cellular immune response was monitored by MTT based lymphoproliferation in peripheral blood mononuclear cell cultures. Higher liquid phase blocking (LPB) ELISA antibody titres were observed in calves receiving combined vaccine as compared to calves immunized with FMD vaccine alone with the peak titres in both the groups obtained on 21 days post-vaccination. However, the virus specific IgM titres were significantly higher in group of calves inoculated with combined vaccine than FMD vaccine alone. The lymphoproliferative responses against FMDV types O, A22 and Asia 1 in the groups receiving combined vaccine and FMD vaccine alone started increasing gradually after day 14 and reached peak levels on 28 DPV followed by a gradual decline subsequently. The group receiving combined vaccine showed higher proliferative responses on in vitro stimulation with FMD virus type O, whereas, with FMD virus type Asia 1, the responses were significantly higher on 14 and 21 DPV as compared to the group immunized with FMD vaccine alone. However, in the group receiving combined vaccine, the responses on in vitro stimulation with FMD virus type A22 were significantly higher than FMD vaccine alone group on all DPV except on 42 DPV.

Bovine leukemia virus Gag particle assembly in insect cells: formation of chimeric particles by domain-switched leukemia/lentivirus Gag polyprotein.

A key stage in the life cycle of C-type retroviruses is the assembly of Gag precursor protein at the plasma membrane of infected cells. Here we report the assembly of bovine leukemia virus (BLV) gag gene product into virus-like particles (VLPs) using the baculovirus expression system. Expression of BLV Pr44(Gag) resulted in the assembly and release of VLPs, thereby confirming the ability of retroviral Gag polyprotein to assemble and bud from insect cells. Efficient particle formation required a myristoylation signal at the N-terminus of BLV Pr44(Gag). Recombinant baculoviruses expressing matrix (MA) or capsid-nucleocapsid (CA-NC) proteins of BLV were generated but neither of these domains was capable of assembling into particulate structures. To assess the compatibility of Gag domains between leukemia and lentivirus groups three different recombinant chimeras each expressing MA of one virus (e.g., simian immunodeficiency or BLV) and CA-NC of another (e.g., BLV or human T-cell leukemia virus type-I) were constructed. Each of the chimeric proteins assembled efficiently and budded as VLPs, suggesting that the MA and CA domains of these two evolutionary divergent retrovirus groups can be functionally exchanged without perturbation of Gag VLP formation. The lenti-leukemia chimeric Gag approach has potential for studying protein-protein interactions in other retroviruses.

Isolation of bluetongue virus from sheep in Rajasthan State, India.

A cytopathic agent was isolated in a baby hamster kidney (BHK)-21 cell-line from blood samples of cross-bred sheep showing typical bluetongue symptoms at Avikanagar in Rajasthan State, India. The cytopathic agent was identified as a bluetongue virus (BTV) by immunofluorescence, the immunoperoxidase test and electron microscopy of BHK-21 cells infected with the new isolate. The new isolate was typed as BTV serotype 1.

Purification and characterization of IgG1 and IgG2 from buffalo (Bubalus bubalis) serum and colostrum.

Buffalo IgG1 and IgG2 were purified from serum and colostrum using salt precipitation, dialysis, gel filtration and ion-exchange chromatography. Their purity was monitored by immunodiffusion and immunoelectrophoresis using anti-heavy chain specific sera and SDS-PAGE. Selective binding of IgG2 to protein-A was used to remove IgG2 from IgG1 preparations. The IgG1 and IgG2 had a molecular mass (Mr) of 162.0 and 161.5 kD, respectively and were found to consist of heavy (H) and light (L) chains. The H and L chains had Mr of 58 and 24 kD, respectively. Reduction-alkylation followed by gel filtration was used for the isolation of H and L chains. While intact H chains were obtained, the L chains appeared to be cleaved into 14 kD molecules and smaller fragments. The mean hexoses content of the serum IgG1 and IgG2 was 1.81 +/- 0.02% and 0.70 +/- 0.02%, respectively. The corresponding values for colostral IgG1 and IgG2 were 1.76 +/- 0.01% and 0.78 +/- 0.08%. Both the IgG subclasses activated homologous complement. These results suggest that buffalo and cattle IgG subclasses have many common characteristics and minor differences.