Long-term trial of protection provided by adenovirus-vectored vaccine expressing the PPRV H protein.

A recombinant, replication-defective, adenovirus-vectored vaccine expressing the H surface glycoprotein of peste des petits ruminants virus (PPRV) has previously been shown to protect goats from challenge with wild-type PPRV at up to 4 months post vaccination. Here, we present the results of a longer-term trial of the protection provided by such a vaccine, challenging animals at 6, 9, 12 and 15 months post vaccination. Vaccinated animals developed high levels of anti-PPRV H protein antibodies, which were virus-neutralising, and the level of these antibodies was maintained for the duration of the trial. The vaccinated animals were largely protected against overt clinical disease from the challenge virus. Although viral genome was intermittently detected in blood samples, nasal and/or ocular swabs of vaccinated goats post challenge, viral RNA levels were significantly lower compared to unvaccinated control animals and vaccinated goats did not appear to excrete live virus. This protection, like the antibody response, was maintained at the same level for at least 15 months after vaccination. In addition, we showed that animals that have been vaccinated with the adenovirus-based vaccine can be revaccinated with the same vaccine after 12 months and showed an increased anti-PPRV antibody response after this boost vaccination. Such vaccines, which provide a DIVA capability, would therefore be suitable for use when the current live attenuated PPRV vaccines are withdrawn at the end of the ongoing global PPR eradication campaign.

Complete genome of a 2014 isolate of peste des petits ruminants virus from Ethiopia.

This report describes the complete genome sequence of a peste des petits ruminants virus (PPRV) isolate from Ethiopia in 2014. The strain (PPRV/Ethiopia/Habru/2014), which showed a normal virulence and relatively low morbidity in the field, belongs to the North African subclade of Lineage IV.

A curated dataset of peste des petits ruminants virus sequences for molecular epidemiological analyses.

Peste des petits ruminants (PPR) is a highly contagious and devastating viral disease infecting predominantly sheep and goats. Tracking outbreaks of disease and analysing the movement of the virus often involves sequencing part or all of the genome and comparing the sequence obtained with sequences from other outbreaks, obtained from the public databases. However, there are a very large number (>1800) of PPRV sequences in the databases, a large majority of them relatively short, and not always well-documented. There is also a strong bias in the composition of the dataset, with countries with good sequencing capabilities (e.g. China, India, Turkey) being overrepresented, and most sequences coming from isolates in the last 20 years. In order to facilitate future analyses, we have prepared sets of PPRV sequences, sets which have been filtered for sequencing errors and unnecessary duplicates, and for which date and location information has been obtained, either from the database entry or from other published sources. These sequence datasets are freely available for download, and include smaller datasets which maximise phylogenetic information from the minimum number of sequences, and which will be useful for simple lineage identification. Their utility is illustrated by uploading the data to the MicroReact platform to allow simultaneous viewing of lineage date and geographic information on all the viruses for which we have information. While preparing these datasets, we identified a significant number of public database entries which contain clear errors, and propose guidelines on checking new sequences and completing metadata before submission.

Depletion of CD8+ T cells from vaccinated goats does not affect protection from challenge with wild-type peste des petits ruminants virus.

Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East and Asia. The disease is caused by peste des petits ruminants virus (PPRV); cell culture-attenuated strains of PPRV have been shown, both experimentally and by extensive use in the field, to be effective vaccines and are widely used. We have previously demonstrated that these vaccines elicit both serological (PPRV-specific antibody) and cell-based (PPRV-specific CD4+ and CD8+ T cells) immune responses. However, it is not known which of these responses are required for protection from PPRV, information that would be useful in the evaluation of new vaccines that are being developed to provide the capability to differentiate infected and vaccinated animals (DIVA capability). To begin to address this issue, we have used a complement-fixing monoclonal antibody recognizing caprine CD8 to deplete >99.9% of circulating CD8+ T cells from vaccinated goats. Animals were then infected with wild-type PPRV. Despite the absence of the CD8+ T-cell component of the vaccine-induced immune response, the vaccinated animals were almost fully protected, showing no pyrexia or viraemia, and almost no clinical signs. These data suggest that a virus-specific CD8+ T-cell response is not critical for protection against PPRV and that virus-specific antibody and/or CD4+ T cells are the main mediators of protection. We have also shown that the leucopenia caused by infection with wild-type PPRV affects all major classes of circulating leucocytes.

Full genome sequencing of archived wild type and vaccine rinderpest virus isolates prior to their destruction.

When rinderpest virus (RPV) was declared eradicated in 2011, the only remaining samples of this once much-feared livestock virus were those held in various laboratories. In order to allow the destruction of our institute's stocks of RPV while maintaining the ability to recover the various viruses if ever required, we have determined the full genome sequence of all our distinct samples of RPV, including 51 wild type viruses and examples of three different types of vaccine strain. Examination of the sequences of these virus isolates has shown that the African isolates form a single disparate clade, rather than two separate clades, which is more in accord with the known history of the virus in Africa. We have also identified two groups of goat-passaged viruses which have acquired an extra 6 bases in the long untranslated region between the M and F protein coding sequences, and shown that, for more than half the genomes sequenced, translation of the F protein requires translational frameshift or non-standard translation initiation. Curiously, the clade containing the lapinised vaccine viruses that were developed originally in Korea appears to be more similar to the known African viruses than to any other Asian viruses.

Complete Genome Sequence of a Lineage IV Peste des Petits Ruminants Virus from Turkey, 2018.

We report the whole-genome sequence of a peste des petits ruminants virus (PPRV) from a lamb exhibiting clinical signs in Turkey in September 2018. The genome of PPRV/Turkey/Central_Anatolia/2018 shows the highest nucleotide sequence identity (97.63%) to PPRV isolated in Turkey in 2000.

Comparison of the Immunogenicities and Cross-Lineage Efficacies of Live Attenuated Peste des Petits Ruminants Virus Vaccines PPRV/Nigeria/75/1 and PPRV/Sungri/96.

Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East, and Asia. Several effective vaccines exist for the disease, based on attenuated strains of the virus (PPRV) that causes PPR. While the efficacy of these vaccines has been established by use in the field, the nature of the protective immune response has not been determined. In addition, while the vaccine derived from PPRV/Nigeria/75/1 (N75) is used in many countries, those developed in India have never been tested for their efficacy outside that country. We have studied the immune response in goats to vaccination with either N75 or the main Indian vaccine, which is based on isolate PPRV/India/Sungri/96 (S96). In addition, we compared the ability of these two vaccines, in parallel, to protect animals against challenge with pathogenic viruses from the four known genetic lineages of PPRV, representing viruses from different parts of Africa, as well as Asia. These studies showed that, while N75 elicited a stronger antibody response than S96, as measured by both enzyme-linked immunosorbent assay and virus neutralization, S96 resulted in more pronounced cellular immune responses, as measured by virus antigen-induced proliferation and interferon gamma production. While both vaccines induced comparable numbers of PPRV-specific CD8+ T cells, S96 induced a higher number of CD4+ T cells specifically responding to virus. Despite these quantitative and qualitative differences in the immune responses following vaccination, both vaccines gave complete clinical protection against challenge with all four lineages of PPRV.IMPORTANCE Despite the widespread use of live attenuated PPRV vaccines, this is the first systematic analysis of the immune response elicited in small ruminants. These data will help in the establishment of the immunological determinants of protection, an important step in the development of new vaccines, especially DIVA vaccines using alternative vaccination vectors. This study is also the first controlled test of the ability of the two major vaccines used against virulent PPRV strains from all genetic lineages of the virus, showing conclusively the complete cross-protective ability of these vaccines.

Recent advances in viral vectors in veterinary vaccinology.

Viral vectored vaccines, particularly using vectors such as adenovirus, herpesvirus and poxviruses, are used widely in veterinary medicine, where this technology has been adopted much more quickly than in human medicine. There are now a large number of programmes to develop viral vector vaccine platforms for humans and very similar or identical vectors are being developed for veterinary medicine. The shared experiences of developing these new vaccine platforms across the two disciplines is accelerating progress, a striking example of the value of a 'One Health' approach. In particular, there is growing use of adenoviruses, either replicating or replication-incompetent, to create new vaccines for use in livestock or companion animals. Live replicating avian herpesvirus vectors are increasingly used as vaccines against poultry diseases.

Future research to underpin successful peste des petits ruminants virus (PPRV) eradication.

Peste des petits ruminants virus (PPRV) is a significant pathogen of small ruminants and is prevalent in much of Africa, the Near and Middle East and Asia. Despite the availability of an efficacious and cheap live-attenuated vaccine, the virus has continued to spread, with its range stretching from Morocco in the west to China and Mongolia in the east. Some of the world's poorest communities rely on small ruminant farming for subsistence and the continued endemicity of PPRV is a constant threat to their livelihoods. Moreover, PPRV's effects on the world's population are felt broadly across many economic, agricultural and social situations. This far-reaching impact has prompted the Food and Agriculture Organization of the United Nations (FAO) and the World Organisation for Animal Health (OIE) to develop a global strategy for the eradication of this virus and its disease. PPRV is a morbillivirus and, given the experience of these organizations in eradicating the related rinderpest virus, the eradication of PPRV should be feasible. However, there are many critical areas where basic and applied virological research concerning PPRV is lacking. The purpose of this review is to highlight areas where new research could be performed in order to guide and facilitate the eradication programme. These areas include studies on disease transmission and epidemiology, the existence of wildlife reservoirs and the development of next-generation vaccines and diagnostics. With the support of the international virology community, the successful eradication of PPRV can be achieved.

Complete Genome Sequence of Peste des Petits Ruminants Virus from Georgia, 2016.

We report here the complete genome sequence of a peste des petits ruminants virus (PPRV) from the first outbreak of the disease in Georgia in January 2016. Genome sequencing was performed using Illumina next-generation sequencing technology in conjunction with Sanger sequencing. This PPRV/Georgia/Tbilisi/2016 genome sequence clustered within lineage IV PPRV viruses.

Control of the induction of type I interferon by Peste des petits ruminants virus.

Peste des petits ruminants virus (PPRV) is a morbillivirus that produces clinical disease in goats and sheep. We have studied the induction of interferon-ß (IFN-ß) following infection of cultured cells with wild-type and vaccine strains of PPRV, and the effects of such infection with PPRV on the induction of IFN-ß through both MDA-5 and RIG-I mediated pathways. Using both reporter assays and direct measurement of IFN-ß mRNA, we have found that PPRV infection induces IFN-ß only weakly and transiently, and the virus can actively block the induction of IFN-ß. We have also generated mutant PPRV that lack expression of either of the viral accessory proteins (V&C) to characterize the role of these proteins in IFN-ß induction during virus infection. Both PPRV_ΔV and PPRV_ΔC were defective in growth in cell culture, although in different ways. While the PPRV V protein bound to MDA-5 and, to a lesser extent, RIG-I, and over-expression of the V protein inhibited both IFN-ß induction pathways, PPRV lacking V protein expression can still block IFN-ß induction. In contrast, PPRV C bound to neither MDA-5 nor RIG-I, but PPRV lacking C protein expression lost the ability to block both MDA-5 and RIG-I mediated activation of IFN-ß. These results shed new light on the inhibition of the induction of IFN-ß by PPRV.

Enhanced immunosurveillance for animal morbilliviruses using vesicular stomatitis virus (VSV) pseudotypes.

The measurement of virus-specific neutralising antibodies represents the "gold-standard" for diagnostic serology. For animal morbilliviruses, such as peste des petits ruminants (PPRV) or rinderpest virus (RPV), live virus-based neutralisation tests require high-level biocontainment to prevent the accidental escape of the infectious agents. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of neutralising antibodies against animal morbilliviruses. By expressing the haemagglutinin (H) and fusion (F) proteins of PPRV on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Serological responses against the four distinct lineages of PPRV could be measured simultaneously and cross-neutralising responses against other morbilliviruses compared. Using this approach, we observed that titres of neutralising antibodies induced by vaccination with live attenuated PPRV were lower than those induced by wild type virus infection and the level of cross-lineage neutralisation varied between vaccinates. By comparing neutralising responses from animals infected with either PPRV or RPV, we found that responses were highest against the homologous virus, indicating that retrospective analyses of serum samples could be used to confirm the nature of the original pathogen to which an animal had been exposed. Accordingly, when screening sera from domestic livestock and wild ruminants in Tanzania, we detected evidence of cross-species infection with PPRV, canine distemper virus (CDV) and a RPV-related bovine morbillivirus, suggesting that exposure to animal morbilliviruses may be more widespread than indicated previously using existing diagnostic techniques.

Protection of Cattle against Rinderpest by Vaccination with Wild-Type but Not Attenuated Strains of Peste des Petits Ruminants Virus.

UNLABELLED: Although rinderpest virus (RPV) has been eradicated in the wild, efforts are still continuing to restrict the extent to which live virus is distributed in facilities around the world and to prepare for any reappearance of the disease, whether through deliberate or accidental release. In an effort to find an alternative vaccine which could be used in place of the traditional live attenuated RPV strains, we have determined whether cattle can be protected from rinderpest by inoculation with vaccine strains of the related morbillivirus, peste des petits ruminants virus (PPRV). Cattle were vaccinated with wild-type PPRV or either of two established PPRV vaccine strains, Nigeria/75/1 or Sungri/96. All animals developed antibody and T cell immune responses to the inoculated PPRV. However, only the animals given wild-type PPRV were protected from RPV challenge. Animals given PPRV/Sungri/96 were only partially protected, and animals given PPRV/Nigeria/75/1 showed no protection against RPV challenge. While sera from animals vaccinated with the vaccine strain of RPV showed cross-neutralizing ability against PPRV, none of the sera from animals vaccinated with any strain of PPRV was able to neutralize RPV although sera from animals inoculated with wild-type PPRV were able to neutralize RPV-pseudotyped vesicular stomatitis virus. IMPORTANCE: Rinderpest virus has been eradicated, and it is only the second virus for which this is so. Significant efforts are still required to ensure preparedness for a possible escape of RPV from a laboratory or its deliberate release. Since RPV vaccine protects sheep and goats from PPRV, it is important to determine if the reverse is true as this would provide a non-RPV vaccine for dealing with suspected RPV outbreaks. This is probably the last in vivo study with live RPV that will be approved.

Determination of the minimum fully protective dose of adenovirus-based DIVA vaccine against peste des petits ruminants virus challenge in East African goats.

Peste des petits ruminants virus (PPRV) causes an economically important disease of sheep and goats, primarily in developing countries. It is becoming the object of intensive international control efforts. Current vaccines do not allow vaccinated and infected animals to be distinguished (no DIVA capability). We have previously shown that recombinant, replication-defective, adenovirus expressing the PPRV H glycoprotein (AdH) gives full protection against wild type PPRV challenge. We have now tested lower doses of the vaccine, as well as AdH in combination with a similar construct expressing the PPRV F glycoprotein (AdF). We show here that, in a local breed of goat in a country where PPR disease is common (Kenya), as little as 10(7) pfu of AdH gives significant protection against PPRV challenge, while a vaccine consisting of 10(8) pfu of each of AdH and AdF gives apparently sterile protection. These findings underline the utility of these constructs as DIVA vaccines for use in PPR control.

Efficient generation of vesicular stomatitis virus (VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies.

Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both test formats provide a reliable assessment of the strength and specificity of the humoral response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of morbillivirus neutralising antibodies. By expressing the haemagglutinin (H) and fusion (F) proteins of canine distemper virus (CDV) on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Further, by exchanging the glycoprotein expression construct, responses against distinct viral strains or species may be measured. Using this technique, we demonstrate cross neutralisation between CDV and peste des petits ruminants virus (PPRV). As an example of the value of the technique, we demonstrate that UK dogs vary in the breadth of immunity induced by CDV vaccination; in some dogs the neutralising response is CDV-specific while, in others, the neutralising response extends to the ruminant morbillivirus PPRV. This technique will facilitate a comprehensive comparison of cross-neutralisation to be conducted across the morbilliviruses.

Development of a helper cell-dependent form of peste des petits ruminants virus: a system for making biosafe antigen.

Peste des petits ruminants (PPR) is a viral disease of sheep and goats that is spreading through many countries in the developing world. Work on the virus is often restricted to studies of attenuated vaccine strains or to work in laboratories that have high containment facilities. We have created a helper cell dependent form of PPR virus by removing the entire RNA polymerase gene and complementing it with polymerase made constitutively in a cell line. The resultant L-deleted virus grows efficiently in the L-expressing cell line but not in other cells. Virus made with this system is indistinguishable from normal virus when used in diagnostic assays, and can be grown in normal facilities without the need for high level biocontainment. The L-deleted virus will thus make a positive contribution to the control and study of this important disease.

Antibodies to the core proteins of Nairobi sheep disease virus/Ganjam virus reveal details of the distribution of the proteins in infected cells and tissues.

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.

The nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space.

Nairobi sheep disease virus (NSDV) of the genus Nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in East and Central Africa, and in India, where the virus is called Ganjam virus. NSDV is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus, which also causes a haemorrhagic disease. As with other nairoviruses, replication of NSDV takes place in the cytoplasm and the new virus particles bud into the Golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. We have found that the overall structure of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment and the Golgi were unaffected by infection with NSDV. However, we observed that NSDV infection led to the loss of protein disulphide isomerase (PDI), an oxidoreductase present in the lumen of the endoplasmic reticulum (ER) and which assists during protein folding, from the ER. Further investigation showed that NSDV-infected cells have high levels of PDI at their surface, and PDI is also secreted into the culture medium of infected cells. Another chaperone from the PDI family, ERp57, was found to be similarly affected. Analysis of infected cells and expression of individual viral glycoproteins indicated that the NSDV PreGn glycoprotein is involved in redistribution of these soluble ER oxidoreductases. It has been suggested that extracellular PDI can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. The discovery of enhanced PDI secretion from NSDV-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses.

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.

Different functions of the common P/V/W and V-specific domains of rinderpest virus V protein in blocking IFN signalling.

The V proteins of paramyxoviruses are composed of two evolutionarily distinct domains, the N-terminal 75 % being common to the viral P, V and W proteins, and not highly conserved between viruses, whilst the remaining 25 % consists of a cysteine-rich V-specific domain, which is conserved across almost all paramyxoviruses. There is evidence supporting a number of different functions of the V proteins of morbilliviruses in blocking the signalling pathways of type I and II IFNs, but it is not clear which domains of V are responsible for which activities and whether all these activities are required for effective blockade of IFN signalling. We have shown here that the two domains of rinderpest virus V protein have distinct functions: the N-terminal domain acted to bind STAT1, whilst the C-terminal V-specific domain interacted with the IFN receptor-associated kinases Jak1 and Tyk2. Effective blockade of IFN signalling required the intact V protein.