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Rabies is a fatal viral disease of serious public health implication. The disease is enzootic in India. In the present study, thirty six rabies virus isolates were obtained from terrestrial mammals of India during 2002–2012. Ecto-domain coding region of the glycoprotein gene from all the isolates were sequenced and the phylogenetic analysis was performed in relation to the global rabies and rabies related virus isolates. The Indian isolates grouped into two distinctly separate lineages with majority of the Indian isolates in Arctic like 1 lineage and the remaining isolates in sub-continental lineage. Isolates of the two distinct lineages were identified simultaneously from the same geographical region. Time scaled phylogenetic tree indicated that the sub-continental lineage of the virus is one of the earliest clade of rabies virus that diverged from bat rabies virus. On the contrary, the Arctic-like 1 lineage of India appeared to be a more recent divergence event. The amino acid sequence comparison revealed that all the major antigenic sites were almost conserved among the Indian isolates whereas few amino acid variations could be identified around site IIa, minor site I and IV. The d N / d S study based on G ecto-domain is in support of the earlier reports of strong purifying selection. In conclusion, it is evident that the Indian rabies virus isolates are of two major distinct lineages with distant phylogenetic and evolutionary relationship. [ABSTRACT FROM AUTHOR]

Abstract: The phylogenetic analysis of VP1 sequences of the 39 type O foot and mouth virus (FMDV) isolates collected from different regions of India during the year of 2001–12 revealed that all isolates belonged to the Middle East – South Asia (ME-SA) topotype. Based on the amount of divergence among the isolates, the viruses were further classified into three distinct lineages namely Ind 2001, PanAsia and PanAsia-2 as well as a minor, unnamed group. Ind 2001 lineage viruses accounted for most of the current type O outbreaks. At the nucleotide level these isolates showed a divergence of 2% to 14% with an average sequence variation of ∼9.9%. The serological spectrum of the current vaccine strain was studied by using bovine vaccinate serum (BVS) raised against O/IND/R2/75. All the current field isolates (n =24) were homologous (‘r’ value 0.4 to 1.0) to the vaccine strain. Examination of the amino acid sequences for selection pressure revealed the positive selection at amino acid sites 13 and 45. [Copyright &y& Elsevier]

Abstract: Small ruminants play an important role in the epidemiology of Foot-and-Mouth Disease (FMD). Small ruminants are vaccinated with one-half or one-third of cattle dose of oil-based or aqueous vaccines respectively. The extinction antigen payload in vaccine for protection in small ruminants is poorly studied. FMD seronegative Nellore sheep (n =30) and Osmanabadi goats (n =30) were vaccinated with different payloads of O1 Manisa vaccine (0.45–5μg). Vaccinated and sero-negative unvaccinated sheep (n =6) and goats (n =6) were challenged intradermally into the coronary band with O1 Manisa virus. The sheep and goats were monitored for signs of FMD and samples were collected for measuring viraemia and virus associated with nasal swabs and probang samples. Clotted blood was collected for serology. Vaccines containing antigen payload up to 0.94μg protected sheep and goats against challenge. Sheep and goats vaccinated with 0.45μg antigen payload were poorly protected against challenge. An antigen payload of 0.94μg was sufficient to offer complete protection and also absence of carrier status. Sheep and goats with no vaccination or with poor sero conversion to vaccination showed sub-clinical infection and became carriers. The results of the study suggest that vaccination offers protection from clinical disease even at a low payload of 0.94μg and hence one-half of cattle dose of the oil-based vaccine formulations is sufficient to induce protective immune response in sheep and goats. Since no live virus could be isolated after 5days post challenge from the nasal swab or probang samples even though viral RNA was detected, the risk of these animals transmitting disease was probably very low. [Copyright &y& Elsevier]

Abstract: Coccidiosis is a disease caused by intracellular parasites belonging to the genus Eimeria. In the present study, we transiently expressed two coccidial antigens EtMIC1 and EtMIC2 as poly histidine-tagged fusion proteins in tobacco. We have evaluated the protective efficacy of plant expressed EtMIC1 as monovalent and as well as bi-valent formulation where EtMIC1 and EtMIC2 were used in combination. The protective efficacy of these formulations was evaluated using homologous challenge in chickens. We observed better serum antibody response, weight gain and reduced oocyst shedding in birds immunized with EtMIC1 and EtMIC2 as bivalent formulation compared to monovalent formulation. However, IFN-γ response was not significant in birds immunized with EtMIC1 compared to the birds immunized with EtMIC2. Our results indicate the potential use of these antigens as vaccine candidates. [Copyright &y& Elsevier]

Abstract: Coccidiosis is an economically important disease affecting poultry industry and remains one of the major problems globally. Developing a cost effective sub-unit vaccine may help mitigate loss in the industry. Here, we report expressing one of the microneme proteins, EtMIC2 from Eimeria tenella in tobacco using Agrobacterium-mediated transient expression. The ability of plant expressed recombinant EtMIC2 in eliciting both humoral and cell-mediated immune responses were measured in the immunized birds. The protective efficacy in the vaccinated birds against a homologous challenge was also evaluated. Birds immunized with plant expressed EtMIC2 showed good sero-conversion, reduced oocyst output and increased weight gain when compared to control birds. Our data indicate that use of plant expressed recombinant EtMIC2 in birds was safe and had the potential in imparting partial protection in chickens against homologous challenge. [Copyright &y& Elsevier]

Abstract: The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P. pastoris were subjected to three step purification method involving density gradient centrifugations and size exclusion chromatography. The enriched VLPs were characterized using conformation-specific monoclonal antibodies in ELISA and by transmission electron microscopy. Mice immunized with a bivalent HPV16 and HPV18 VLPs developed high serum antibody titers to both HPV types that persisted for 190 days post vaccination. Serum of mice immunized with the HPV-VLP preparations could neutralize homologous pseudoviruses in an in vitro assays. Our results demonstrate that the L1 proteins expressed in P. pastoris fold properly as evidenced by assembly into VLPs and induction of type-specific neutralizing antibody response in mice. This work constitutes a step towards developing an alternate production platform for generating an affordable HPV vaccine to meet the needs of developing countries. [Copyright &y& Elsevier]

Abstract: The 3A region of foot-and-mouth disease virus has been implicated in host range and virulence. For example, amino acid deletions in the porcinophilic strain (O/TAW/97) at 93–102aa of the 153 codons long 3A protein have been recognized as the determinant of species specificity. In the present study, 18 type O FMDV isolates from India were adapted in different cell culture systems and the 3A sequence was analyzed. These isolates had complete 3A coding sequence (153aa) and did not exhibit growth restriction in cells based on species of origin. The 3A region was found to be highly conserved at N-terminal half (1–75aa) but exhibited variability or substitutions towards C-terminal region (80–153). Moreover the amino acid substitutions were more frequent in recent Indian buffalo isolates but none of the Indian isolates showed deletion in 3A protein, which may be the reason for the absence of host specificity in vitro. Further inclusive analysis of 3A region will reveal interesting facts about the variability of FMD virus 3A region in an endemic environment. [Copyright &y& Elsevier]

Abstract: Intestinal infection with Eimeria, the etiologic agent of avian coccidiosis, stimulates protective immunity to subsequent colonization by the homologous parasite, while cross-protection against heterologous species is poor. As a first step toward the development of a broad specificity Eimeria vaccine, this study was designed to assess a purified recombinant protein from Eimeria maxima gametocytes (Gam82) in stimulating immunity against experimental infection with live parasites. Following Gam82 intramuscular immunization and oral parasite challenge, body weight gain, fecal oocyst output, lesion scores, serum antibody response, and cytokine production were assessed to evaluate vaccination efficacy. Animals vaccinated with Gam82 and challenged with E. maxima showed lower oocyst shedding and reduced intestinal pathology compared with non-vaccinated and parasite-challenged animals. Gam82 vaccination also stimulated the production of antigen-specific serum antibodies and induced greater levels of IL-2 and IL-15 mRNAs compared with non-vaccinated controls. These results demonstrate that the Gam82 recombinant protein protects against E. maxima and augments humoral and cell-mediated immunity. [Copyright &y& Elsevier]

Abstract: India like several other South East Asian and African countries continues to face the public health and economic problems associated with the disease. Our objective was to perform a limited sequence analysis of a portion of nucleoprotein gene of 22 rabies virus isolates obtained from domestic animals in Southern India during 2004–2005. These isolates were compared with rabies virus isolates originating from Asia, Europe, Africa and North America. The phylogenetic analysis showed that RV isolates in Southern India belong to genotype 1. They were similar to one another forming a single major genetic cluster not ordered by geography or species of origin. However, they were dissimilar to RV isolates in Northern India and in other parts of the world. The data indicated that dog rabies virus variants are the major circulating viruses and control of dog rabies would result in overall reduction in the burden and incidence of rabies in India. [Copyright &y& Elsevier]

Abstract: Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18IU. The results indicated a good correlation with the NIH mouse potency assay (r =0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation. [Copyright &y& Elsevier]

Abstract: The rabies vaccine is produced by inactivation of rabies virus propagated on BHK21 cells. In the rabies inactivation process, BEI is added at a final concentration of 1.6mM to the viral harvest at 37°C, followed by a second dose of BEI at 24h post-inactivation. Inactivation was confirmed by the mice innocuity test and tissue culture amplification test as per B.P (Vet) 2004. Validation of test procedure is essential as per cGMP requirement. The dose of BEI was validated by using lower and higher concentrations of BEI in inactivation process. The study indicated that BEI at a lower concentration (0.4mM) was able to inactivate the rabies virus within 30h and the routine concentration (1.6mM) of BEI is effective in inactivating rabies virus within 18h. The amplification test used for confirming the inactivation of the live virus was validated by spiking the sample with different dilutions of pretitrated live rabies virus. The test revealed that the amplification method is sensitive to detect live rabies virus if present in the inactivated sample. The validation of BEI as an inactivant and the amplification test are discussed. [Copyright &y& Elsevier]

The complete inactivation of Foot and Mouth Disease (FMD) virus is a critical requirement in the production of FMD vaccine to ensure the safety of the product. Binary ethyleneimine (BEI) is an aziridine compound, produced from bromoethylamine hydrobromide (BEA) commonly used for the inactivation of FMD virus during vaccine manufacturing. The validation of BEI, when used as an inactivant, is essential to ensure the quality of the inactivating agent and the validity of the process. In the present study, the inactivation kinetics of Foot and Mouth Disease virus (O, A and Asia-1 serotypes) were determined for different concentrations of BEI (0.4mM, 0.8mM, 1.2mM, 1.6mM and 2.0mM). Statistically significant differences in the inactivation kinetics were observed between 0.4mM and 1.6mM of BEI. The results indicated that BEI at 1.6mM was able to inactivate the FMD virus within 8–10h. Increasing the concentration of BEI beyond 1.6mM did not appreciably improve the inactivation process. No differences in the inactivation kinetics were found between the various serotypes studied. This study can be used as a guideline for routine procedures for validating the quality of BEA and the inactivation process. [Copyright &y& Elsevier]