P. Dhar

Recent epidemiology of peste des petits ruminants virus (PPRV)

Peste des petits ruminants (PPR) is an economically important viral disease of goats and sheep first described in west Africa in the 1940s. The virus has been circulating in parts of sub-Saharan Africa for several decades and in the Middle East and southern Asia since 1993, although the first description of the virus in India dates to 1987. To study the genetic relationship between isolates of distinct geographical origin, a selected region of the fusion (F) protein gene of the viruses was amplified using RT/PCR and the resulting DNA product sequenced for phylogenetic analysis. Viruses from 27 outbreaks in Asian and Middle Eastern countries, reported between 1993 and 2000, and two recent outbreaks from the African continent were compared with the prototype African strain. Of the four known lineages of PPR virus, lineage 1 and 2 viruses have been found exclusively in west Africa. Virus from an outbreak in Burkina Faso in 1999 fell into the lineage 1 group. Viruses of lineage 3 have been found in east Africa, where an outbreak in Ethiopia in 1996 was of this type, and also in Arabia and in southern India. However, there have been no further isolations of lineage 3 virus from India since the one reported in 1992 from Tamil Nadu. A virus of this lineage was found circulating in Yemen in 2001. In the past 8 years virus exclusively of the fourth lineage has spread across the Middle East and the Asian sub-continent, reaching east as far as Nepal and Bangladesh. This virus lineage was also reported from Kuwait in 1999. The geographical source of the new lineage 4 virus is unknown although it is most closely related to African lineage 1. The possibility that its earlier presence in northern India was masked by the circulation of Rinderpest virus, a related virus of cattle, is considered unlikely.

Comparative efficacy of various chemical stabilizers on the thermostability of a live-attenuated peste des petits ruminants (PPR) vaccine

Thermostability of a live-attenuated peste des petits ruminants (PPR) vaccine recently developed at Indian Veterinary Research Institute was studied using conventional lyophilization conditions. A total of four stabilizers viz., lactalbumin hydrolysate-sucrose (LS), Weybridge medium (WBM), buffered gelatin-sorbitol (BUGS) and trehalose dihydrate (TD) were used to prepare the lyophilized vaccine. The study revealed that the PPR vaccine lyophilized with either LS or TD is more stable than rest of the stabilizers having an expiry period of at least 45 days (so far studied) at 4 degrees C, 15-19 days at 25 degrees C and 1-2 days at 37 degrees C. However, at a temperature of 45 degrees C, BUGS had a marginal superiority, although lasted for few hours, followed by TD and LS with respect to shelf-life, LS and TD with respect to half-life. On the basis of half-life also LS followed by TD appeared superior at a temperature of 4, 25 and 37 degrees C. Reconstitution of vaccine with distilled water or 1M MgSO(4) or 0.85% NaCl maintained the required virus titre (2.5log(10)TCID(50) per dose) up to 8h at 37 degrees C and 7h at 45 degrees C. Among the three diluents, 1M MgSO(4) appeared to be the better diluent for reconstitution of lyophilized PPR vaccine, as the loss on dilution was lowest and maintain the required virus titre for a longer period. Investigation suggests for using LS as stabilizer for lyophilization and 1M MgSO(4) as vaccine diluent for the newly developed PPR vaccine.

A sandwich-ELISA for the diagnosis of Peste des petits ruminants (PPR) infection in small ruminants using anti-nucleocapsid protein monoclonal antibody.

Summary.A sandwich ELISA test using PPR specific monoclonal antibody (clone 4G6) to an epitope of nucleocapsid protein has been developed. The test uses polyclonal sera to capture the antigen from clinical samples (swabs and tissues). Captured antigens from clinical samples are detected using PPR specific monoclonal antibody. The test is specific to PPR as it failed to detect rinderpest vaccine virus (RBOK strain). Varieties of clinical samples originating from laboratory experiments (n = 231) and from field (n = 259) were employed to test the efficacy of sandwich-ELISA test. The test compared very well with an internationally accepted commercial Immune-capture ELISA kit, which uses biotinylated monoclonal antibody against the nucleocapsid protein. On a parallel testing using 490 clinical samples, 4G6 MAb based sandwich ELISA had an overall relative diagnostic specificity of 92.8% and diagnostic sensitivity of 88.9% compared to the commercial kit. The newly developed test is free from prozone phenomenon. PPR outbreaks from various parts of India have been confirmed using the test. Findings suggested that the newly developed ELISA is suitable for PPR diagnosis under field conditions.

Production and characterization of monoclonal antibodies to peste des petits ruminants (PPR) virus.

Peste des petits ruminants (PPR) is an acute, febrile viral disease of small ruminants, caused by a virus of the genus Morbillivirus. PPR and rinderpest viruses are antigenically related and need to be differentiated serologically. In the present study, 23 mouse monoclonal antibodies were produced by polyethyleneglycol (PEG)-mediated fusion of sensitized lymphocytes and myeloma cells. Among these, two belong to the IgM class and the remaining 21 to various subclasses of IgG. The MAbs from the IgG class designated 4B6 and 4B11 neutralized PPR virus in vitro. In radioimmunoprecipitation assay, 10 MAbs recognized nucleoprotein, 4 recognized the matrix protein and one each haemagglutinin and phosphoprotein. The remaining 7 MAbs failed to precipitate any defined viral protein. The reactivity pattern of the monoclonal antibodies in indirect ELISA indicated a close antigenic relationship within three Indian PPR (lineage 4) virus isolates and also within two rinderpest vaccine strains. All PPR virus isolates could be distinguished from rinderpest vaccine viruses on the basis of the reactivity pattern of all MAbs and anti-N protein MAbs. A set of six monoclonal antibodies specific to PPR virus could also be identified from the panel. From the panel of MAbs available, two MAbs were selected for diagnostic applications, one each for the detection of antigens and antibodies to PPR virus.

Apoptosis induced by peste des petits ruminants virus in goat peripheral blood mononuclear cells.

The ability of peste des petits ruminants virus (PPRV) to induce apoptosis in goat peripheral blood mononuclear cell (PBMC) culture was investigated. Goat PBMC were infected with PPRV and the infectivity was confirmed by cytopathic effect, demonstration of presence of infectious viral progeny and expression of viral antigens in the lymphocytes, cultured in vitro. Infected PBMC showed morphological features of apoptosis. DNA extracted from PPRV-infected cells displayed laddering pattern in agarose gel electrophoresis. Infected cells also showed significantly higher apoptotic indices measured by bisbenzimide staining than control cells. Electronmicrographs of PPRV-infected PBMC revealed features typical of apoptosis such as peripheral condensation of chromatin, blebbing of plasma membrane, fragmentation of nucleus and cell leading to formation of apoptotic bodies. Our results suggest that PPRV can induce apoptosis, in vitro, in goat lymphocytes.

Sequence analysis of the nucleoprotein gene of Asian lineage peste des petits ruminants vaccine virus.

The complete nucleotide sequence of the nucleocapsid (N) protein of the peste-des-petits ruminants vaccine virus (PPRV Sungri/96) belonging to the Asian lineage was determined. The gene was 1692 nucleotides in length and encoded a polypeptide of 525 amino acids. The PPRV Sungri/96 N gene has a nucleotide homology of 92% for PPRV Nigeria 75/1 to 55.5% for canine distemper virus. At amino acid level the homology was 94.1% with PPRV Nigeria 75/1, while with other morbilliviruses, PPRV Sungri/96 had only 71.4–64.9% amino acid identity. The phosphorylation prediction reveals eight conserved sites across morbilliviruses, whereas in the C-terminal portion of the protein the sites are not conserved. Phylogenetic analysis of different N proteins of morbilliviruses revealed five well-defined clusters as observed previously. To the best of our knowledge this is the first report describing the nucleocapsid gene sequence of PPRV Indian isolate.

Biological and molecular characterization of classical swine fever challenge virus from India

Aim: The aim of this study was biological and molecular characterization of classical swine fever (CSF) challenge virus from India. Materials and Methods: CSF challenge virus maintained at Division of Biological standardization was experimentally infected to two seronegative piglets. The biological characterization was done by clinical sign and symptoms along with postmortem findings. For molecular characterization 5’-nontranslated region, E2 and NS5B regions were amplified by reverse transcription polymerase chain reaction and sequenced. The sequences were compared with that of reference strains and the local field isolates to establish a phylogenetic relation. Results: The virus produced symptoms of acute disease in the piglets with typical post-mortem lesions. Phylogenetic analysis of the three regions showed that the current Indian CSF Challenge virus is having maximum similarity with the BresciaX strain (USA) and Madhya Pradesh isolate (India) and is belonging to subgroup 1.2 under Group 1. Conclusion: Based on biological and molecular characterization of CSF challenge virus from India is described as a highly virulent virus belonging to subgroup 1.2 under Group 1 along with some field isolates from India and Brescia strain.

An overview of process intensification and thermo stabilization for upscaling of Peste des petits ruminants vaccines in view of global control and eradication

Peste des petits ruminants (PPR) has been recognized as a globally distributed disease affecting the small ruminant population. The disease results in severe economic losses mainly to small land holders and low input farming systems. The control of PPR is mainly achieved through vaccination with available live attenuated vaccines. The thermo labile nature of PPR virus poses a major constraint in production of quality vaccines which often results in vaccine failures. The lack of quality vaccine production jeopardize the wide vaccination coverage especially in countries with poor infrastructure due to which PPR persists endemically. The vaccine production system may require augmentation to attain consistent and quality vaccines through efforts of process intensification integrated with suitable stabilizer formulations with appropriate freeze drying cycles for improved thermo tolerance. Manufacturing of live attenuated PPR vaccines during batch cultures might introduce defective interfering particles (DIPs) as a result of high multiplicity of infection (MOI) of inoculums, which has a huge impact on virus dynamics and yield. Accumulation of DIPs adversely affects the quality of the manufactured vaccines which can be avoided through use of appropriate MOI of virus inoculums and quality control of working seed viruses. Therefore, adherence to critical manufacturing standard operating procedures in vaccine production and ongoing efforts on development of thermo tolerant vaccine will help a long way in PPR control and eradication programme globally. The present review focuses on the way forward to achieve the objectives of quality vaccine production and easy upscaling to help the global PPR control and eradication by mass vaccination as an important tool.

Replacement of Animal Model for Propagation of Classical Swine Fever Challenge Virus by Adaption in the PK-15 Cell Line

Classical swine fever (CSF) challenge virus has been adapted in PK-15 cell line from infected splenic suspension of the challenge virus maintained hitherto by pig to pig passages. Confirmation of viral presence was done by reverse transcription-polymerase chain reaction (RT-PCR) and Fluorescent Antibody Technique (FAT). A reasonably good titre of 106.5 TCID50/ml was obtained at 6th passage level. The cell culture adapted challenge virus at a dose of 105.0 TCID50 produced CSF symptoms in pigs from 2 nd days post infection (dpi) onwards and succumbed to the infection between 11-12 dpi. Cell culture adapted CSF challenge virus offers advantage to inoculate exact virus particles over the traditional tissue suspension (20% w/v) in potency testing. Adapted challenge virus will replace the use of pigs for propagation of challenge virus; hence follows 4 R’s (replacement, reduction, refinement and rehabilitation) principle. This challenge virus can be attenuated by further serial passages and can be used to develop indigenous live attenuated cell culture based vaccine.