Protection of rabbits against lapinized rinderpest virus with purified envelope glycoproteins of peste-des-petits-ruminants and rinderpest viruses

Haemagglutinin (HA) and fusion (F) proteins of peste-des-petits-ruminants virus (PPRV) and rinderpest virus (RPV) were purified by immunoaffinity chromatography. The purified proteins were characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Rabbit hyperimmune sera were raised against the purified HA and F proteins and assayed by enzyme-linked immunosorbent assay (ELISA), haemagglutination-inhibition (HAI) and virus neutralization (VN) tests. The immunized animals were challenged with a virulent lapinized (rabbit-adapted) strain of RPV: Both HA and F proteins of PPRV protected rabbits against a lethal challenge with lapinized RPV. As expected, RPV HA and F proteins also conferred a similar protection against the homologous challenge. The postchallenge antibody responses were of a true anamnestic type.

Who really ate the fruit? A novel approach to camera trapping for quantifying frugivory by ruminants

Tropical forest ruminants disperse several plants; yet, their effectiveness as seed dispersers is not systematically quantified. Information on frequency and extent of frugivory by ruminants is lacking. Techniques such as tree watches or fruit traps adapted from avian frugivore studies are not suitable to study terrestrial frugivores, and conventional camera traps provide little quantitative information. We used a novel time-delay camera-trap technique to assess the effectiveness of ruminants as seed dispersers for Phyllanthus emblica at Mudumalai, southern India. After being triggered by animal movement, cameras were programmed to take pictures every 2 min for the next 6 min, yielding a sequence of four pictures. Actual frugivores were differentiated from mere visitors, who did not consume fruit, by comparing the number of fruit remaining across the time-delay photograph sequence. During a 2-year study using this technique, we found that six terrestrial mammals consumed fallen P. emblica fruit. Additionally, seven mammals and one bird species visited fruiting trees but did not consume fallen fruit. Two ruminants, the Indian chevrotain Moschiola indica and chital Axis axis, were P. emblica's most frequent frugivores and they accounted for over 95% of fruit removal, while murid rodents accounted for less than 1%. Plants like P. emblica that are dispersed mainly by large mammalian frugivores are likely to have limited ability to migrate across fragmented landscapes in response to rapidly changing climates. We hope that more quantitative information on ruminant frugivory will become available with a wider application of our time-delay camera-trap technique.

Performance of RT-PCR-ELISA for the detection of peste des petits ruminants virus

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.

Differentiation of rinderpest and peste des petits ruminants viruses using specific cDNA clones

The morbilliviruses which infect ruminants, rinderpest (RPV) and peste des petits ruminants (PPRV), are difficult to distinguish serologically. They can be distinguished by differential neutralisation tests and by the migration of the major virus structural protein, the nucleocapsid protein, on polyacrylamide gels. Both these methods are time consuming and require the isolation of live virus for identification; they are not suitable for analysis of material directly from post-mortem specimens. We describe a rapid method for differential diagnosis of infections caused by RPV or PPRV, which uses specific cDNA probes, derived from the mRNAs for the nucleocapsid protein of each virus, which can be used to distinguish unequivocally the two virus types rapidly.

Immune responses to hemagglutinin-neuraminidase protein of peste des petits ruminants virus expressed in transgenic peanut plants in sheep

Peste des petits ruminants (PPR) is an acute, highly contagious disease of small ruminants caused by a morbillivirus, Peste des petits ruminants virus (PPRV). The disease is prevalent in equatorial Africa, the Middle East, and the Indian subcontinent. A live attenuated vaccine is in use in some of the countries and has been shown to provide protection for at least three years against PPR. However, the live attenuated vaccine is not robust in terms of thermotolerance. As a step towards development of a heat stable subunit vaccine, we have expressed a hemagglutinin-neuraminidase (HN) protein of PPRV in peanut plants (Arachis hypogea) in a biologically active form, possessing neuraminidase activity. Importantly. HN protein expressed in peanut plants retained its immunodominant epitopes in their natural conformation. The immunogenicity of the plant derived HN protein was analyzed in sheep upon oral immunization. Virus neutralizing antibody responses were elicited upon oral immunization of sheep in the absence of any mucosal adjuvant. In addition, anti-PPRV-HN specific cell-mediated immune responses were also detected in mucosally immunized sheep. (C) 2010 Elsevier B.V. All rights reserved.

Context-dependency of a complex fruit-frugivore mutualism: temporal variation in crop size and neighborhood effects

The quantity of fruit consumed by dispersers is highly variable among individuals within plant populations. The outcome Of Such selection operated by firugivores has been examined mostly with respect to changing spatial contexts. The influence of varying temporal contexts on frugivore choice, and their possible demographic and evolutionary consequences is poorly understood. We examined if temporal variation in fruit availability across a hierarchy of nested temporal levels (interannual, intraseasonal, 120 h, 24 h) altered frugivore choice for a complex seed dispersal system in dry tropical forests of southern India. The interactions between Phyllanthus emblica and its primary disperser (ruminants) was mediated by another frugivore (a primate),which made large quantities of fruit available on the ground to ruminants. The direction and strength of crop size and neighborhood effects on this interaction varied with changing temporal contexts.Fruit availability was higher in the first of the two study years, and at the start of the season in both years. Fruit persistence on trees,determined by primate foraging, was influenced by crop size andconspecific neighborhood densities only in the high fruit availability year. Fruit removal by ruminants was influenced by crop size in both years and neighborhood densities only in the high availability year. In both years, these effects were stronger at the start of the season.Intraseasonal reduction in fruit availability diminished inequalities in fruit removal by ruminants and the influence of crop size and fruiting neighborhoods. All trees were not equally attractive to frugivores in a P. emblica population at all points of time. Temporal asymmetry in frugivore-mediated selection could reduce potential for co-evolution between firugivores and plants by diluting selective pressures. Inter-dependencies; formed between disparate animal consumers can add additional levels of complexity to plant-frugivore mutualistic networks and have potential reproductive consequences for specific individuals within populations.

Establishment of an in vitro transcription system for Peste des petits ruminant virus

Background: Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. Results: RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. Conclusion: This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.