Recombinant adenovirus expressing the haemagglutinin of Peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR.

Peste des petits ruminants virus (PPRV) is a morbillivirus that can cause severe disease in sheep and goats, characterised by pyrexia, pneumo-enteritis, and gastritis. The socio-economic burden of the disease is increasing in underdeveloped countries, with poor livestock keepers being affected the most. Current vaccines consist of cell-culture attenuated strains of PPRV, which induce a similar antibody profile to that induced by natural infection. Generation of a vaccine that enables differentiation of infected from vaccinated animals (DIVA) would benefit PPR control and eradication programmes, particularly in the later stages of an eradication campaign and for countries where the disease is not endemic. In order to create a vaccine that would enable infected animals to be distinguished from vaccinated ones (DIVA vaccine), we have evaluated the immunogenicity of recombinant fowlpox (FP) and replication-defective recombinant human adenovirus 5 (Ad), expressing PPRV F and H proteins, in goats. The Ad constructs induced higher levels of virus-specific and neutralising antibodies, and primed greater numbers of CD8+ T cells than the FP-vectored vaccines. Importantly, a single dose of Ad-H, with or without the addition of Ad expressing ovine granulocyte macrophage colony-stimulating factor and/or ovine interleukin-2, not only induced strong antibody and cell-mediated immunity but also completely protected goats against challenge with virulent PPRV, 4 months after vaccination. Replication-defective Ad-H therefore offers the possibility of an effective DIVA vaccine.

Peste des petits ruminants virus tissue tropism and pathogenesis in sheep and goats following experimental infection.

Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions.

Early changes in cytokine expression in peste des petits ruminants disease.

Peste des petits ruminants is a viral disease of sheep and goats that has spread through most of Africa as well as the Middle East and the Indian subcontinent. Although, the spread of the disease and its economic impact has made it a focus of international concern, relatively little is known about the nature of the disease itself. We have studied the early stages of pathogenesis in goats infected with six different isolates of Peste des petits ruminants virus representing all four known lineages of the virus. No lineage-specific difference in the pathogenicity of the virus isolates was observed, although there was evidence that even small numbers of cell culture passages could affect the degree of pathogenicity of an isolate. A consistent reduction in CD4+ T cells was observed at 4 days post infection (dpi). Measurement of the expression of various cytokines showed elements of a classic inflammatory response but also a relatively early induction of interleukin 10, which may be contributing to the observed disease.

Peste des Petits Ruminants virus: an emerging threat to goat farming in Pakistan.

Pakistan at present is having more than 60 million head of goats, which consist of about 37 well-recognized breeds found in different regions of the country. Although the goat farming on commercial level is escalating in Pakistan, there are threats, which result this initiative into a loss. Among these threats, Peste des Petits Ruminants (PPR) outbreaks are causing huge economic damages. In this study, three outbreaks of PPR were confirmed at three different commercial farms in various regions of Punjab province and their economic impact on small ruminants farming was calculated. The disease started after 1-2 months of the establishment of these farms as the animals were purchased from different livestock markets. Disease started with sudden onset of respiratory and enteric clinical signs and spreads quickly. Disease caused mortality and morbidity of 10-15% and 20-40%, respectively, within a time period of 01-03 weeks. At these three farms, 116 of 365 animals exhibited the clinical disease, with an overall morbidity rate of 31.78%. A total of 43 animals died with mortality rate of 11.78% (43/365) causing a direct financial loss of $4300 (Pakistan Rupees 430,000/-), while the indirect cost due to treatment, loss of animal body condition, reduction in market value, increase veterinary services and labour was $7911 (Pak Rs. 791,100/-). Taken together, the results demonstrate that there is an urgent need to assess the economic impact of the disease throughout the country and to give proper emphasis for controlling PPR in sensitive regions where it is discouraging the investment in goat farming.

Two replication-defective adenoviral vaccine vectors for the induction of immune responses to PPRV.

Peste des petits ruminants is a highly contagious disease of small ruminants caused by a Morbillivirus, peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenovirus serotype 5 (Ad5) containing the highly immunogenic fusion protein (F) and hemaglutinine protein (H) genes from PPRV were constructed. HEK293A cells infected with either virus (Ad5-PPRV-F or -H) express F and H proteins respectively. These viruses were used to vaccinate mice by intramuscular inoculation. Both viruses elicited PPRV-specific B- and T-cell responses. Thus, after two immunizations, sera from immunized mice elicited neutralizing antibody response, indicating that this approach has the potential to confer protective immunity. In addition, we detected a significant antigen specific CD4(+) and CD8(+) T-cell response in mice vaccinated with either virus. These results indicate that these adenovirus constructs offer a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.

Peste des petits ruminants infection among cattle and wildlife in northern Tanzania.

We investigated peste des petits ruminants (PPR) infection in cattle and wildlife in northern Tanzania. No wildlife from protected ecosystems were seropositive. However, cattle from villages where an outbreak had occurred among small ruminants showed high PPR seropositivity, indicating that spillover infection affects cattle. Thus, cattle could be of value for PPR serosurveillance.

Early events following experimental infection with Peste-Des-Petits ruminants virus suggest immune cell targeting.

Peste-des-petits ruminants virus (PPRV) is a viral pathogen that causes a devastating plague of small ruminants. PPRV is an economically significant disease that continues to be a major obstacle to the development of sustainable agriculture across the developing world. The current understanding of PPRV pathogenesis has been heavily assumed from the closely related rinderpest virus (RPV) and other morbillivirus infections alongside data derived from field outbreaks. There have been few studies reported that have focused on the pathogenesis of PPRV and very little is known about the processes underlying the early stages of infection. In the present study, 15 goats were challenged by the intranasal route with a virulent PPRV isolate, Côte d'Ivoire '89 (CI/89) and sacrificed at strategically defined time-points post infection to enable pre- and post-mortem sampling. This approach enabled precise monitoring of the progress and distribution of virus throughout the infection from the time of challenge, through peak viraemia and into a period of convalescence. Observations were then related to findings of previous field studies and experimental models of PPRV to develop a clinical scoring system for PPRV. Importantly, histopathological investigations demonstrated that the initial site for virus replication is not within the epithelial cells of the respiratory mucosa, as has been previously reported, but is within the tonsillar tissue and lymph nodes draining the site of inoculation. We propose that virus is taken up by immune cells within the respiratory mucosa which then transport virus to lymphoid tissues where primary virus replication occurs, and from where virus enters circulation. Based on these findings we propose a novel clinical scoring methodology for PPRV pathogenesis and suggest a fundamental shift away from the conventional model of PPRV pathogenesis.

Virus excretion and antibody dynamics in goats inoculated with a field isolate of peste des petits ruminants virus.

A field isolate of peste des petits ruminants virus (PPRV) from an outbreak in Tibet, China, was inoculated into goats to investigate the dynamics of virus excretion and antibody production. Further, animals received PPRV vaccine strain Nigeria 75/1. Ocular, nasal and oral samples were tested for the presence of virus antigen by one-step real-time qualitative RT-PCR (qRT-PCR); competitive ELISA (c-ELISA) was used for the measurement of specific antibodies against PPRV. Virus particles could be detected as early as day 3 post-inoculation (pi) and virus excretion lasted for up to day 26 pi. All four goats inoculated with the PPRV field isolate were seropositive as early as day 10 pi. In animals inoculated with the vaccine strain, antibody was detected at day 14 pi, and levels of neutralizing antibodies remained above the protection threshold level (1 : 8) for 8 months. Both virus particles and neutralizing antibodies were detected earlier in goats challenged with the field isolate than in those receiving the vaccine strain.

Development of an epitope-based competitive ELISA for the detection of antibodies against Tibetan peste des petits ruminants virus.

AIMS: To develop an effective diagnostic kit, based on a competitive ELISA-based system (cELISA), for detecting serum antibody against peste des petits ruminants virus (PPRV). METHODS: Epitope peptides of the nucleocapsid (N) protein of Tibetan PPRV were synthesized chemically and injected into rabbits to prepare hyperimmune antisera. Test sera were incubated simultaneously with hyperimmune antisera and added to the wells of ELISA plates coated previously with recombinant N protein. Horseradish peroxidase-conjugated goat anti-rabbit antibody was employed to detect the quantity of hyperimmune antisera combined with recombinant N protein. RESULTS: A cELISA has been developed for monitoring PPRV infections with a cutoff value of 35. Relative sensitivity and specificity values of the epitope-based cELISA were 96.18 and 91.29%, respectively, when compared with a commercial cELISA kit in a test involving 1,039 serum samples. CONCLUSION: We report an efficient method for preparing antibody suitable for incorporation into a cELISA that can be used routinely for the detection of PPRV antibodies in serum samples. The method eliminated the requirement for virus culture and monoclonal antibody preparation, reduced the biorisk posed by virus-dependent manipulations, and the performance of the resultant cELISA compared favorably with a commercially available cELISA kit.

Prevalence of peste des petits ruminants among sheep and goats in India.

This study measured the clinical prevalence of peste des petits ruminants (PPR) among sheep and goats in India between 2003 and 2009 by analyzing clinical samples from suspected cases of PPR that were submitted to the Rinderpest and Allied Disease Laboratory, Division of Virology, IVRI, Mukteswar for PPR diagnosis. PPR outbreaks were confirmed by detecting PPR virus (PPRV)-specific antigen in the clinical samples. Clinical samples (blood, nasal swabs, spleen, lymph node, kidney, liver, intestine, and pooled tissue materials) were taken from a total of 592 sheep and 912 goats in different states of India and screened for the presence of PPRV antigen using a monoclonal antibody based sandwich ELISA kit. A total of 20, 38, and 11 laboratory-confirmed PPR outbreaks occurred among sheep, goat, and combined sheep and goat populations, respectively. Our findings provide evidence of widespread PPR endemicity in India. The underlying reasons could be variations in husbandry practices in different geographical regions, agro-climatic conditions, and livestock migration. Furthermore, decrease in the number of PPR outbreaks over time might be due to the effectiveness of current live PPR vaccines and timely vaccination of target species. Vaccination against PPR has been practiced in India since 2002 to control this disease.

Rescue of recombinant peste des petits ruminants virus: creation of a GFP-expressing virus and application in rapid virus neutralization test.

Peste des petits ruminants virus (PPRV) causes high mortality in goats and sheep and the disease has shown a greatly increased geographic distribution over the last 15 years. It is responsible for serious socioeconomic problems in some of the poorest developing countries. The ability to create recombinant PPRV would provide a useful tool for investigating the biology of the virus and the pathology of disease, as well as for developing new vaccines and diagnostic methods. Here we report the first successful rescue of recombinant PPRV from a full-length cDNA clone of the virus genome. Successful recovery of PPRV was achieved by using a RNA polymerase II promoter to drive transcription of the full-length virus antigenome. We have used this technique to construct a virus expressing a tracer protein (green fluorescent protein, GFP). The recombinant virus replicated as well as the parental virus and could stably express GFP during at least 10 passages. The newly established reverse genetics system for PPRV provides a novel method for constructing a vaccine using PPRV as a vector, and will also prove valuable for fundamental research on the biology of the virus. We found that our recombinant virus allowed more rapid and higher throughput assessment of PPRV neutralization antibody titer via the virus neutralization test (VNT) compared with the traditional method.

A novel recombinant Peste des petits ruminants-canine adenovirus vaccine elicits long-lasting neutralizing antibody response against PPR in goats.

BACKGROUND: Peste des petits ruminants (PPR) is a highly contagious infectious disease of goats, sheep and small wild ruminant species with high morbidity and mortality rates. The Peste des petits ruminants virus (PPRV) expresses a hemagglutinin (H) glycoprotein on its outer envelope that is crucial for viral attachment to host cells and represents a key antigen for inducing the host immune response. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether H can be exploited to generate an effective PPRV vaccine, a replication-competent recombinant canine adenovirus type-2 (CAV-2) expressing the H gene of PPRV (China/Tibet strain) was constructed by the in vitro ligation method. The H expression cassette, including the human cytomegalovirus (hCMV) promoter/enhancer and the BGH early mRNA polyadenylation signal, was inserted into the SspI site of the E3 region, which is not essential for proliferation of CAV-2. Infectious recombinant rCAV-2-PPRV-H virus was generated in transfected MDCK cells and used to immunize goats. All vaccinated animals produced antibodies upon primary injection that were effective in neutralizing PPRV in vitro. Higher antibody titer was obtained following booster inoculation, and the antibody was detectable in goats for at least seven months. No serious recombinant virus-related adverse effect was observed in immunized animals and no adenovirus could be isolated from the urine or feces of vaccinated animals. Results showed that the recombinant virus was safe and could stimulate a long-lasting immune response in goats. CONCLUSIONS/SIGNIFICANCE: This strategy not only provides an effective PPR vaccine candidate for goats but may be a valuable mean by which to differentiate infected from vaccinated animals (the so-called DIVA approach).

Experimental infection of alpine goats with a Moroccan strain of peste des petits ruminants virus (PPRV).

Peste des petits ruminants virus (PPRV) recently caused a serious outbreak of disease in Moroccan sheep and goats. Alpine goats were highly susceptible to PPRV with mortality rates approaching 100%, as opposed to local breeds of sheep which were less susceptible to the disease. The relative susceptibility of alpine goats was investigated through an experimental infection study with the Moroccan strain of PPRV. Severe clinical signs were observed in the alpine goats with virus being excreted through ocular, nasal and oral routes. No difference in the severity of the disease in goats was observed with different inoculation routes and transmission of the virus by direct contact was confirmed. This study confirmed the susceptibility of the alpine goat to PPRV infection and describes a challenge protocol that effectively and consistently reproduced severe clinical signs of PPR in experimentally infected goats.

Natural infection with peste des petits ruminants virus: a pre and post vaccinal assessment following an outbreak scenario.

Peste des petits ruminants virus (PPRV) infection was confirmed in a herd of goats (n = 55) at an organised farm in Islamabad, Pakistan. PPRV infection was confirmed using both antigen- and antibody-based detection methods, haemagglutination (HA) tests and molecular methods. Animals that survived natural infection developed a typical serological response and virus antigen was detected in fecal matter. Following determination of serological response to infection animals were grouped and either vaccinated or left unvaccinated: group 1 animals succumbed to infection (n = 5) and samples were analysed for PPRV antigen; group 2 animals developed clinical disease (n = 10) and were divided into 2 groups, half being vaccinated (group 2a) whilst the remainder were unvaccinated (group 2b); group 3 (n = 15) animals included those that developed only very mild clinical disease or no clinical disease; group 4 animals (n = 5) were negative for clinical disease and were housed as a negative control group. A variable antibody response was detected following resolution of the initial outbreak. Excretion of virus antigen was assessed at different time points following vaccination. Importantly, animals that were vaccinated (group 2a) excreted antigen in fecal matter for 1 month following vaccination whilst unvaccinated animals (group 2b) continued to shed virus antigen for 2 months. The potential for virus excretion in fecal matter and effects of vaccination upon virus infection are discussed. We postulate that excretion in fecal material may represent a mechanism of virus transmission following natural infection and that this mechanism may demonstrate a potential method by which PPRV outbreaks occur spontaneously in areas not previously known to have circulating virus.

Prevalence of antibodies to peste des petits ruminants virus before and during outbreaks of the disease in Awash Fentale district, Afar, Ethiopia.

The present study was conducted to assess the seroprevalence of peste des petits ruminants (PPR) in sheep and goats in Awash Fentale district, Afar, Ethiopia. Small ruminants in the district had poor herd immunity at the first visit and succumb to the disease then after. The seroprevalence during the time of an outbreak was much higher compared with the initial levels: 7.3% and 42.6% in sheep and goats, respectively. The higher seroprevalence figure in goats was suggestive of their relative susceptibility to PPR compared with sheep.

Effect of immunosuppression on pathogenesis of peste des petits ruminants (PPR) virus infection in goats.

In this study an attempt to address the effects of immunosuppression on pathogenesis of peste des petits ruminants (PPR) virus infection was undertaken. Cyclophosphamide and dexamethasone were used to immunosuppress the animals. The drug treated animals exhibited severe leukopaenia and lymphopaenia; one of the indicators of immunosuppression. Experimental peste des petits ruminants virus (PPRV) infection was then given to both drug-induced immunosuppressed and non-immunosuppressed goats and observed their effects. Findings indicated that, the immunosuppressed goats had a short period of viremia, more extensive and severe disease advancement and higher mortality rate than the non-immunosuppressed goats. PPRV antigen distribution in both ante-mortem and post-mortem materials was extensive and diffused in immunosuppressed animals than that of non-immunosuppressed. Some of the atypical organ(s)/tissues like liver, kidney, heart etc showed more antigen load than non-immunosuppressed group. Histopathological and immunohistochemical studies of tissues from the two groups showed that pathological changes in the non-immunosuppressed animals were confined only to gastrointestinal tract, whereas in the immunosuppressed animals histopathological changes and PPRV antigen distribution were more extensive and diffused. The present study indicated that immunosuppression increased the extent and severity of the pathological lesions associated with peste des petits ruminants virus infection.