R.P. Singh

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.

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.

A vero cell derived combined vaccine against sheep pox and Peste des Petits ruminants for sheep.

The combined sheep pox and Peste des Petits ruminants (PPR) vaccine was prepared in lyophilized form containing recommended doses of both vaccine viruses. Safety and immunogenicity of this combined vaccine was evaluated in sheep. Sheep immunized subcutaneously with 1ml of live attenuated vaccine consisting of 10(3)TCID(50) each of sheep pox virus (SPV) Romanian Fanar (RF) strain and Peste des Petits ruminants virus (PPRV-Sungri/96 strain) were monitored for clinical and serological responses for a period of four weeks post immunization (pi) and two week post challenge (pc). Specific antibodies directed to sheep pox virus could be demonstrated by indirect ELISA and serum neutralization test (SNT). Competitive ELISA and SNT were used for demonstration of antibodies to PPR virus. All the immunized animals resisted challenge with virulent SPV or PPRV on day 30pi, while control animals developed characteristic signs of disease. Specific virus could be detected in the unvaccinated control animals after challenge but not from any of the immunized sheep. Combined vaccine was found to be safe and potent as evident from sero conversion as well as challenge studies in sheep. This indicates that component vaccines did not interfere each other and can be used in target population for economic vaccination strategies.

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.

Comparative and temporal transcriptome analysis of peste des petits ruminants virus infected goat peripheral blood mononuclear cells

Peste des petits ruminanats virus (PPRV), a morbillivirus causes an acute, highly contagious disease - peste des petits ruminants (PPR), affecting goats and sheep. Sungri/96 vaccine strain is widely used for mass vaccination programs in India against PPR and is considered the most potent vaccine providing long-term immunity. However, occurrence of outbreaks due to emerging PPR viruses may be a challenge. In this study, the temporal dynamics of immune response in goat peripheral blood mononuclear cells (PBMCs) infected with Sungri/96 vaccine virus was investigated by transcriptome analysis. Infected goat PBMCs at 48h and 120h post infection revealed 2540 and 2000 differentially expressed genes (DEGs), respectively, on comparison with respective controls. Comparison of the infected samples revealed 1416 DEGs to be altered across time points. Functional analysis of DEGs reflected enrichment of TLR signaling pathways, innate immune response, inflammatory response, positive regulation of signal transduction and cytokine production. The upregulation of innate immune genes during early phase (between 2-5 days) viz. interferon regulatory factors (IRFs), tripartite motifs (TRIM) and several interferon stimulated genes (ISGs) in infected PBMCs and interactome analysis indicated induction of broad-spectrum anti-viral state. Several Transcription factors - IRF3, FOXO3 and SP1 that govern immune regulatory pathways were identified to co-regulate the DEGs. The results from this study, highlighted the involvement of both innate and adaptive immune systems with the enrichment of complement cascade observed at 120h p.i., suggestive of a link between innate and adaptive immune response. Based on the transcriptome analysis and qRT-PCR validation, an in vitro mechanism for the induction of ISGs by IRFs in an interferon independent manner to trigger a robust immune response was predicted in PPRV infection.