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.

Rinderpest virus sequestration and use in posteradication era.

After the 2011 declaration of rinderpest disease eradication, we surveyed 150 countries about rinderpest virus stocks. Forty-four laboratories in 35 countries held laboratory-attenuated strains, field strains, or diagnostic samples. Vaccine and reagent production and laboratory experiments continued. Rigorous standards are necessary to ensure that stocks are kept under safe conditions.

Rinderpest eradication: appropriate technology and social innovations.

Rinderpest is only the second infectious disease to have been globally eradicated. In the final stages of eradication, the virus was entrenched in pastoral areas of the Greater Horn of Africa, a region with weak governance, poor security, and little infrastructure that presented profound challenges to conventional control methods. Although the eradication process was a development activity rather than scientific research, its success owed much to several seminal research efforts in vaccine development and epidemiology and showed what scientific decision-making and management could accomplish with limited resources. The keys to success were the development of a thermostable vaccine and the application of participatory epidemiological techniques that allowed veterinary personnel to interact at a grassroots level with cattle herders to more effectively target control measures.

Scientific background to the global eradication of rinderpest.

The global eradication of rinderpest was declared in 2011. This is the second infectious disease to have been eradicated from the world after smallpox, for which eradication was declared in 1980. From a scientific aspect, smallpox eradication was achieved by improvements in the Jenner vaccine, originally developed in the 18th century. Developments in vaccine technology and virological techniques during the 20th century have contributed to the eradication of rinderpest. The scientific background to rinderpest eradication is briefly reviewed vis-à-vis that of smallpox eradication.

Recombinant vaccines against the mononegaviruses--what we have learned from animal disease controls.

The mononegaviruses include a number of highly contagious and severe disease-causing viruses of both animals and humans. For the control of these viral diseases, development of vaccines, either with classical methods or with recombinant DNA virus vectors, has been attempted over the years. Recently reverse genetics of mononegaviruses has been developed and used to generate infectious viruses possessing genomes derived from cloned cDNA in order to study the consequent effects of viral gene manipulations on phenotype. This technology allows us to develop novel candidate vaccines. In particular, a variety of different attenuation strategies to produce a range of attenuated mononegavirus vaccines have been studied. In addition, because of their ideal nature as live vaccines, recombinant mononegaviruses expressing foreign proteins have also been produced with the aim of developing multivalent vaccines against more than one pathogen. These recombinant mononegaviruses are currently under evaluation as new viral vectors for vaccination. Reverse genetics could have great potential for the preparation of vaccines against many mononegaviruses.

Improvement and development of rapid chromatographic strip-tests for the diagnosis of rinderpest and peste des petits ruminants viruses.

This paper describes the improvement of a rapid diagnostic test for the detection of rinderpest virus (RPV) at pen-side and the development of a similar test for the detection of another Morbillivirus, peste de petits ruminants virus (PPRV). Using the Svanova Biotech format, prototype chromatographic strip test devices were developed for RPV and PPRV detection. For the RP device, the incorporation of a monoclonal antibody (Mab), which recognises additional RPV strains of RPV lineage 2, enhanced the range of reactivity of the rapid diagnostic test. The device detected antigen in animals infected experimentally with different RPV strains. It also showed detection levels similar to the RP Clearview™ device reported previously. In addition, RPV was also detected under field conditions in Pakistan. A PPRV specific Mab (C77) was used for the development of the PPR test. This Mab recognised a wide range of PPRV isolates and did not show any cross-reactivity with any other virus tested. In animal experiments the device was able to detect viral antigen in eye swabs taken from the animals. The PPRV test should be invaluable for future PPR control eradication programs.

Immune protection in animals: the examples of rinderpest and foot-and-mouth disease.

Fading immune protection in farmed animals may present a problem, particularly in free-ranging animals in nomadic and transhumant pastoral systems, where animals are not readily available for large-scale blanket vaccination programmes. Two veterinary examples of fading immune protection are discussed: rinderpest and foot-and-mouth disease (FMD). Both are devastating viral diseases of cattle that have a huge impact on the farming economy. Both diseases can be controlled by vaccination, although the post-vaccination immunity afforded by the rinderpest vaccine is markedly different from that induced by FMD vaccines. These differences may in part explain the respective advancement of international eradication campaigns: while global eradication of rinderpest is imminent, FMD viruses are still actively circulating in many parts of the world.

A sero-survey of rinderpest in nomadic pastoral systems in central and southern Somalia from 2002 to 2003, using a spatially integrated random sampling approach.

A cross-sectional sero-survey, using a two-stage cluster sampling design, was conducted between 2002 and 2003 in ten administrative regions of central and southern Somalia, to estimate the seroprevalence and geographic distribution of rinderpest (RP) in the study area, as well as to identify potential risk factors for the observed seroprevalence distribution. The study was also used to test the feasibility of the spatially integrated investigation technique in nomadic and semi-nomadic pastoral systems. In the absence of a systematic list of livestock holdings, the primary sampling units were selected by generating random map coordinates. A total of 9,216 serum samples were collected from cattle aged 12 to 36 months at 562 sampling sites. Two apparent clusters of RP seroprevalence were detected. Four potential risk factors associated with the observed seroprevalence were identified: the mobility of cattle herds, the cattle population density, the proximity of cattle herds to cattle trade routes and cattle herd size. Risk maps were then generated to assist in designing more targeted surveillance strategies. The observed seroprevalence in these areas declined over time. In subsequent years, similar seroprevalence studies in neighbouring areas of Kenya and Ethiopia also showed a very low seroprevalence of RP or the absence of antibodies against RP. The progressive decline in RP antibody prevalence is consistent with virus extinction. Verification of freedom from RP infection in the Somali ecosystem is currently in progress.

Charting methods for internal quality control for competition ELISA.

This chapter deals with relatively simple ways to use control charts to monitor the performance of ELISAs. A rinderpest competition ELISA, for the estimation of antibodies in serum samples, is used to demonstrate the methods. This assay is available in a kit form. Constant evaluation of the use of the kit is part of what is called internal quality control (IQC). Figure 1 shows an overview of the ELISA scheme described in this chapter. The details of the procedure, which involves plotting the data graphically (charting methods), are explained herein. The objectives of charting data are as follows: 1. To keep a constant record of all data. 2. To monitor the assay from plate to plate in any one day's testing. 3. To monitor the tests made from day to day, week to week, year to year. 4. To allow rapid identification of unacceptable results. 5. To allow recognition of reagent problems. 6. To identify trends in results (increasingly poor performance). 7. To identify when a new set of kit reagents is necessary. 8. To allow identification of differences among operators of the assay. 9. To fulfill various criteria for good laboratory practice. 10. To fulfill necessary requirements for external recognition to prove that tests are being performed at an acceptable level (increasingly important when results are used for international trading purposes).

The rinderpest virus non-structural C protein blocks the induction of type 1 interferon.

The innate immune response, in particular the production of type 1 interferons, is an essential part of the mammalian host response to viral infection. We have previously shown that rinderpest virus, a morbillivirus closely related to the human pathogen measles virus, blocks the actions of type 1 and type 2 interferons. We show here that this virus can also block the induction of type 1 interferon. The viral non-structural C protein appears to be the active agent, since expressing this protein in cells makes them resistant to activation of the interferon-beta promoter while recombinant virus that does not express the C protein activates this promoter much more than virus expressing the C protein. In addition, differences in activation of the interferon-beta promoter by different strains of rinderpest virus are reflected in differing abilities of their respective C proteins to block activation of the promoter by dsRNA. The C protein blocks the activation of this promoter induced by either cytoplasmic dsRNA or by Newcastle disease virus (NDV) infection, as well as activation induced by overexpression of several elements of the signalling pathway, including mda-5, RIG-I and IRF-3. The RPV C protein also blocks transcription from promoters responsive individually to the three transcription factors that make up the interferon-beta promoter enhanceosome, although it does not appear to block the activation of IRF-3.

Rescue of a chimeric rinderpest virus with the nucleocapsid protein derived from peste-des-petits-ruminants virus: use as a marker vaccine.

The nucleocapsid (N) protein of all morbilliviruses has a highly conserved central region that is thought to interact with and encapsidate the viral RNA. The C-terminal third of the N protein is highly variable among morbilliviruses and is thought to be located on the outer surface and to be available to interact with other viral proteins such as the phosphoprotein, the polymerase protein and the matrix protein. Using reverse genetics, a chimeric rinderpest virus (RPV)/peste-des-petits-ruminants virus (PPRV) was rescued in which the RPV N gene open reading frame had been replaced with that of PPRV (RPV-PPRN). The chimeric virus maintained efficient replication in cell culture. Cattle vaccinated with this chimeric vaccine showed no adverse reaction and were protected from subsequent challenge with wild-type RPV, indicating it to be a safe and efficacious vaccine. The carboxyl-terminal variable region of the rinderpest N protein was cloned and expressed in Escherichia coli. The expressed protein was used to develop an indirect ELISA that could clearly differentiate between RPV- and PPRV-infected animals. The possibility of using this virus as a marker vaccine in association with a new diagnostic ELISA in the rinderpest eradication programme is discussed.

Matrix protein and glycoproteins F and H of Peste-des-petits-ruminants virus function better as a homologous complex.

The matrix (M) protein of paramyxoviruses forms an inner coat to the viral envelope and serves as a bridge between the surface glycoproteins (F and H) and the ribonucleoprotein core. Previously, a marker vaccine (RPV-PPRFH) was produced for the control of peste des petits ruminants (PPR) disease, where the F and H genes of Rinderpest virus (RPV) were replaced with the equivalent genes from Peste-des-petits-ruminants virus (PPRV); however, this virus grew poorly in tissue culture. The poor growth of the RPV-PPRFH chimeric virus was thought to be due to non-homologous interaction of the surface glycoproteins with the internal components of the virus, in particular with the M protein. In contrast, replacement of the M gene of RPV with that from PPRV did not have an effect on the viability or replication efficiency of the recombinant virus. Therefore, in an effort to improve the growth of the RPV-PPRFH virus, a triple chimera (RPV-PPRMFH) was made, where the M, F and H genes of RPV were replaced with those from PPRV. As expected, the growth of the triple chimera was improved; it grew to a titre as high as that of the unmodified PPRV, although comparatively lower than that of the parental RPV virus. Goats infected with the triple chimera showed no adverse reaction and were protected from subsequent challenge with wild-type PPRV. The neutralizing-antibody titre on the day of challenge was approximately 17 times higher than that in the RPV-PPRFH group, indicating RPV-PPRMFH as a promising marker-vaccine candidate.

Rinderpest seroprevalence in wildlife in Kenya and Tanzania, 1982-1993.

Eight hundred and thirty five serum samples collected from eight wild artiodactyl species in Kenya and Tanzania between 1982 and 1993 were tested for virus-neutralising (VN) antibodies to rinderpest (RP) virus. Antibodies were found in 116 of 344 buffaloes (Syncerus caffer) but not in the other species including 349 wildebeest (Connochaetes taurinus). Most of the antibody positive buffaloes were from the Maasai Mara-Serengeti ecosystem (MM-SE) and would have had opportunity for exposure to the virus during the epidemic of rinderpest in buffalo confirmed there in 1982. Buffalo born after 1985 did not have antibody indicating that virus stopped circulating in this population at or around that time. This second demonstration that RP virus disappears from the MM-SE is further evidence that these species are not permanent reservoirs of this virus. Re-infection of wildlife is transient and they remain valuable sentinels for infection in nearby domestic livestock.

Inhibition of host peripheral blood mononuclear cell proliferation ex vivo by Rinderpest virus.

Rinderpest, or cattle plague, is caused by Rinderpest virus (RPV), which is related most closely to human Measles virus (MV), both being members of the genus Morbillivirus, a group of viruses known to have strong immunosuppressive effects in vitro and in vivo. Here, it was shown that peripheral blood mononuclear cells (PBMCs) isolated from cattle experimentally infected with either wild-type or vaccine strains of RPV impaired the proliferation of PBMCs derived from uninfected animals; however, in contrast to either mild or virulent strains of wild-type virus, the inhibition induced by the vaccine was both weak and transient. Flow-cytometric analysis of PBMCs obtained from cattle infected with different strains of RPV showed that the proportion of infected cells was virus dose-dependent and correlated with lymphoproliferative suppression.

Development of new generation rinderpest vaccines.

Veterinary science has benefited much from the advances in biotechnology during the past 20 years. New and improved diagnostic techniques for infectious diseases have been developed and new and highly effective vaccines to prevent such diseases have been introduced and more have been, or are about to be, field-tested. The latest development in negative strand virology, reverse genetics, the ability to rescue live virus from a DNA copy of the RNA genome, is being used to address questions concerning virus pathogenicity at the molecular level and to produce "marker" vaccines, i.e. vaccines that allow serological identification of all vaccinated animals. Such a vaccine would greatly benefit the continuing campaign for the global eradication of rinderpest since it would then be possible, by serological means, to detect wild type virus circulating in local areas or regions where it is still necessary to vaccinate and where the vaccination levels are below those required to eliminate the virus. Here we describe different approaches we have taken to produce such a vaccine using reverse genetics to add a marker to the existing and widely used Plowright rinderpest vaccine.

Effect of immunization with plasmid DNA encoding for rinderpest virus matrix protein on systemic rinderpest virus infection in rabbits.

Plasmid vaccine pBK-CMVMPILC113 expressing the matrix (M) gene of rinderpest virus was assessed for its potential to protect rabbits against a lethal viral challenge. Rabbits immunized with plasmids expressing the M gene were not protected when challenged with lapinized rinderpest virus, despite the production of anti-M antibodies, while rabbits immunized with rinderpest tissue culture vaccine were completely protected from a lethal challenge with lapinized rinderpest virus. The plasmid vaccine also had no significant effect on the lymphopenia in challenged rabbits. The results indicate that rinderpest M protein does not have a protective role in rinderpest infection.

Rinderpest virus H protein: role in determining host range in rabbits.

A major molecular determinant of virus host-range is thought to be the viral protein required for cell attachment. We used a recombinant strain of Rinderpest virus (RPV) to examine the role of this protein in determining the ability of RPV to replicate in rabbits. The recombinant was based on the RBOK vaccine strain, which is avirulent in rabbits, carrying the haemagglutinin (H) protein gene from the lapinized RPV (RPV-L) strain, which is pathogenic in rabbits. The recombinant virus (rRPV-lapH) was rescued from a cDNA of the RBOK strain in which the H gene was replaced with that from the RPV-L strain. The recombinant grew at a rate equivalent to the RPV-RBOK parental virus in B95a cells but at a lower rate than RPV-L. The H gene swap did not affect the ability of the RBOK virus to act as a vaccine to protect cattle against virulent RPV challenge. Rabbits inoculated with RPV-L became feverish, showed a decrease in body weight gain and leukopenia. High virus titres and histopathological lesions in the lymphoid tissues were also observed. Clinical signs of infection were never observed in rabbits inoculated with either RPV-RBOK or with rRPV-lapH; however, unlike RPV-RBOK, both RPV-L and rRPV-lapH induced a marked antibody response in rabbits. Therefore, the H protein plays an important role in allowing infection to occur in rabbits but other viral proteins are clearly required for full RPV pathogenicity to be manifest in this species.

Long term immunity in African cattle vaccinated with a recombinant capripox-rinderpest virus vaccine.

Cattle were vaccinated with a recombinant capripox-rinderpest vaccine designed to protect cattle from infection with either rinderpest virus (RPV) or lumpy skin disease virus (LSDV). Vaccination did not induce any adverse clinical responses or show evidence of transmission of the vaccine virus to in-contact control animals. Approximately 50% of the cattle were solidly protected from challenge with a lethal dose of virulent RPV 2 years after vaccination while at 3 years approx. 30% were fully protected. In the case of LSDV, all of 4 vaccinated cattle challenged with virulent LSDV at 2 years were completely protected from clinical disease while 2 of 5 vaccinated cattle were completely protected at 3 years. The recombinant vaccine showed no loss of potency when stored lyophylized at 4 degrees C for up to 1 year. These results indicate that capripoxvirus is a suitable vector for the development of safe, effective and stable recombinant vaccines for cattle.

Long-term sterilizing immunity to rinderpest in cattle vaccinated with a recombinant vaccinia virus expressing high levels of the fusion and hemagglutinin glycoproteins.

Rinderpest is an acute and highly contagious viral disease of ruminants, often resulting in greater than 90% mortality. We have constructed a recombinant vaccinia virus vaccine (v2RVFH) that expresses both the fusion (F) and hemagglutinin (H) genes of rinderpest virus (RPV) under strong synthetic vaccinia virus promoters. v2RVFH-infected cells express high levels of the F and H glycoproteins and show extensive syncytium formation. Cattle vaccinated intramuscularly with as little as 10(3) PFU of v2RVFH and challenged 1 month later with a lethal dose of RPV were completely protected from clinical disease; the 50% protective dose was determined to be 10(2) PFU. Animals vaccinated with v2RVFH did not develop pock lesions and did not transmit the recombinant vaccinia virus to contact animals. Intramuscular vaccination of cattle with 10(8) PFU of v2RVFH provided long-term sterilizing immunity against rinderpest. In addition to being highly safe and efficacious, v2RVFH is a heat-stable, inexpensive, and easily administered vaccine that allows the serological differentiation between vaccinated and naturally infected animals. Consequently, mass vaccination of cattle with v2RVFH could eradicate rinderpest.