Modelling the expected rate of laboratory biosafety breakdowns involving rinderpest virus in the post-eradication era.

Now that we are in the rinderpest post-eradication era, attention is focused on the risk of re-introduction. A semi-quantitative risk assessment identified accidental use of rinderpest virus in laboratories as the most likely cause of re-introduction. However there is little data available on the rates of laboratory biosafety breakdowns in general. In addition, any predictions based on past events are subject to various uncertainties. The aims of this study were therefore to investigate the potential usefulness of historical data for predicting the future risk of rinderpest release via laboratory biosafety breakdowns, and to investigate the impacts of the various uncertainties on these predictions. Data were collected using a worldwide online survey of laboratories, a structured search of ProMED reports and discussion with experts. A stochastic model was constructed to predict the number of laboratory biosafety breakdowns involving rinderpest that will occur over the next 10 years, based on: (1) the historical rate of biosafety breakdowns; and (2) the change in the number of laboratories that will have rinderpest virus in the next 10 years compared to historically. The search identified five breakdowns, all of which occurred during 1970-2000 and all of which were identified via discussions with experts. Assuming that our search for historical events had a sensitivity of over 60% and there has been at least a 40% reduction in the underlying risk (attributable to decreased laboratory activity post eradication) the most likely number of biosafety events worldwide was estimated to be zero over a 10 year period. However, the risk of at least one biosafety breakdown remains greater than 1 in 10,000 unless the sensitivity was at least 99% or the number of laboratories has decreased by at least 99% (based on 2000-2010 during which there were no biosafety breakdowns).

Disease properties, geography, and mitigation strategies in a simulation spread of rinderpest across the United States.

For the past decade, the Food and Agriculture Organization of the United Nations has been working toward eradicating rinderpest through vaccination and intense surveillance by 2012. Because of the potential severity of a rinderpest epidemic, it is prudent to prepare for an unexpected outbreak in animal populations. There is no immunity to the disease among the livestock or wildlife in the United States (US). If rinderpest were to emerge in the US, the loss in livestock could be devastating. We predict the potential spread of rinderpest using a two-stage model for the spread of a multi-host infectious disease among agricultural animals in the US. The model incorporates large-scale interactions among US counties and the small-scale dynamics of disease spread within a county. The model epidemic was seeded in 16 locations and there was a strong dependence of the overall epidemic size on the starting location. The epidemics were classified according to overall size into small epidemics of 100 to 300 animals (failed epidemics), epidemics infecting 3,000 to 30,000 animals (medium epidemics), and the large epidemics infecting around one million beef cattle. The size of the rinderpest epidemics were directly related to the origin of the disease and whether or not the disease moved into certain key counties in high-livestock-density areas of the US. The epidemic size also depended upon response time and effectiveness of movement controls.

Infection of bovine dendritic cells by rinderpest or measles viruses induces different changes in host transcription.

The morbilliviruses are a closely related genus which are very similar in their sequences and share a common receptor, but nevertheless show significant restriction in the host species in which they cause disease. One contribution to this restriction might be the nature of the hosts' responses to infection. We have used microarrays to study the changes in the transcriptome of bovine dendritic cells after infection with wild-type (pathogenic) and vaccine (apathogenic) strains of rinderpest virus (RPV), a bovine pathogen, and a wild-type isolate of measles virus (MV), a morbillivirus that causes disease only in humans and some other primates. We found that, as previously observed in human cells, MV induces a rapid interferon response, while that induced by RPV was delayed and much reduced in magnitude. Pathogenic and apathogenic RPV also showed significant differences, with the latter inducing a slightly higher interferon response as well as significant effects on transcription of genes involved in cell cycle regulation.

A disease-mediated trophic cascade in the Serengeti and its implications for ecosystem C.

Tree cover is a fundamental structural characteristic and driver of ecosystem processes in terrestrial ecosystems, and trees are a major global carbon (C) sink. Fire and herbivores have been hypothesized to play dominant roles in regulating trees in African savannas, but the evidence for this is conflicting. Moving up a trophic scale, the factors that regulate fire occurrence and herbivores, such as disease and predation, are poorly understood for any given ecosystem. We used a Bayesian state-space model to show that the wildebeest population eruption that followed disease (rinderpest) eradication in the Serengeti ecosystem of East Africa led to a widespread reduction in the extent of fire and an ongoing recovery of the tree population. This supports the hypothesis that disease has played a key role in the regulation of this ecosystem. We then link our state-space model with theoretical and empirical results quantifying the effects of grazing and fire on soil carbon to predict that this cascade may have led to important shifts in the size of pools of C stored in soil and biomass. Our results suggest that the dynamics of herbivores and fire are tightly coupled at landscape scales, that fire exerts clear top-down effects on tree density, and that disease outbreaks in dominant herbivores can lead to complex trophic cascades in savanna ecosystems. We propose that the long-term status of the Serengeti and other intensely grazed savannas as sources or sinks for C may be fundamentally linked to the control of disease outbreaks and poaching.

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.

Phosphorylation status of the phosphoprotein P of rinderpest virus modulates transcription and replication of the genome.

The phosphoprotein P of paramyxoviruses is known to play more than one role in genome transcription and replication. Phosphorylation of P at the NH(2) terminus by cellular casein kinase II has been shown to be necessary for transcription of the genome in some of the viruses, while it is dispensable for replication. The phosphorylation null mutant of rinderpest virus P protein, in which three serine residues have been mutated, has been shown earlier to be non-functional in an in vivo minigenome replication/transcription system. In this work, we have shown that the phosphorylation of P protein is essential for transcription, whereas the null mutant is active in replication of the genome in vivo. The null mutant P acts as a transdominant repressor of transcriptional activity of wild-type P and as an activator of replication carried out by wild-type P protein. These results suggest the phosphorylation status of P may act as a replication switch during virus replication. We also show that the phosphorylation null mutant P is capable of interacting with L and N proteins and is able to form a tripartite complex of L-(N-P) when expressed in insect cells, similar to wild-type P protein.

Control of ruminant morbillivirus replication by small interfering RNA.

Peste-des-petits-ruminants virus (PPRV) and rinderpest virus (RPV) are two morbilliviruses of economic relevance in African and Asian countries. Although efficient vaccines are available for both diseases, they cannot protect the animals before 14 days post-vaccination. In emergencies, it would be desirable to have efficient therapeutics for virus control. Here, two regions are described in the nucleocapsid genes of PPRV and RPV that can be targeted efficiently by synthetic short interfering RNAs (siRNAs), resulting in a >80 % reduction in virus replication. The effects of siRNAs on the production of viral RNA by real-time quantitative PCR, of viral proteins by flow cytometry and of virus particles by appreciation of the cytopathic effect and virus titration were monitored. The findings of this work highlight the potential for siRNA molecules to be developed as therapeutic agents for the treatment of PPRV and RPV infections.

Rinderpest virus blocks type I and type II interferon action: role of structural and nonstructural proteins.

Rinderpest virus (RPV) is a paramyxovirus closely related to the human pathogen Measles virus. It causes severe disease in cattle, buffalo, and some wild animals; although it can infect humans, it does not cause disease. Here, we demonstrate that RPV blocks the action of both type I (alpha) and type II (gamma) interferons (IFNs) by blocking the phosphorylation and nuclear translocation of STAT1 and STAT2 and that this block is not related to species specificity. In addition, both wild-type virulent and vaccine strains of the virus blocked IFN action. Unlike the case with some other paramyxoviruses, neither STAT1 nor STAT2 is degraded upon virus infection. STAT1 is bound by both the viral structural protein P, and thereby recruited to concentrations of viral protein in the cell, and the nonstructural protein V. Although both P and V proteins bind to STAT1 and can block IFN action when expressed in transfected cells, the IFN antagonist activity of the P protein is weaker than that of the V protein. The viral C protein also seems to weakly block IFN-induced activation of STAT1 in transfection experiments. However, studies with knockout viruses showed that the viral V protein appears to be the dominant inhibitor of IFN signaling in the context of virus infection, since prevention of viral V expression restored the IFN sensitivity of infected cells. Although a change in the distribution pattern of STAT2 was observed in virus-infected cells, STAT2 was not bound by any viral protein.

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.

Importance of the extracellular and cytoplasmic/transmembrane domains of the haemagglutinin protein of rinderpest virus for recovery of viable virus from cDNA copies.

A specific interaction between the F and H proteins is required to enable fusion of the virus and host cell membranes and in some cases these proteins are not interchangeable between related viruses of the family Paramyxoviridae. For example, the F and H proteins of two ruminant morbilliviruses, rinderpest virus (RPV) and Peste-des-petits-ruminants virus (PPRV), are not interchangeable since viable virus could not be rescued from cDNA constructs where an individual glycoprotein gene of RPV was replaced with that from PPRV. To investigate which domain of the H protein, extracellular or cytoplasmic/transmembrane, was most important for preventing this interaction, two chimeric H gene constructs were made where the normal H gene of RPV was substituted with variant H genes where the transmembrane/cytoplasmic tail region (pRPV2C-PPRTm) or the whole ectodomain (pRPV2C-PPRExt) were derived from PPRV. Chimeric viruses were rescued from both the constructs and, while RPV2C-PPRTm virus grew to as high titres as the parent virus, RPV2C-PPRExt virus was extremely debilitated with respect to growth in tissue culture. Thus the ectodomain of H is the most important region required for effective interactions of the two glycoproteins for the recovery of viable virus. Nevertheless, the transmembrane/cytoplasmic domain of RPV alone can allow a chimeric virus to be rescued, which was not possible when the complete H gene was derived from PPRV. Both versions of the H protein and also the F protein were found to be incorporated into the envelope of the budded virions.

Wild-type Rinderpest virus uses SLAM (CD150) as its receptor.

Rinderpest virus (RPV) is a morbillivirus, related closely to the human pathogen Measles virus (MV). Although cell culture-adapted strains of RPV can infect many kinds of cell from different hosts, one such strain has previously been shown to have a detectable preference for cells expressing the MV receptor CD150 (SLAM), a protein found only on certain types of activated T cells, B cells and dendritic cells. Here, it is shown that the wild-type, virulent parent of the most common vaccine strain of RPV requires CD150 as a receptor, whilst the cell culture-adapted vaccine strain has acquired the ability to use heparan sulphate as an alternative receptor.

'Rescue' of mini-genomic constructs and viruses by combinations of morbillivirus N, P and L proteins.

Chloramphenicol acetyltransferase (CAT)-expressing negative-sense mini-genomic constructs of measles virus (MV) and rinderpest virus (RPV) were rescued by standard technology with helper plasmids expressing the nucleocapsid (N), phospho- (P) and large (L) proteins of MV, canine distemper virus (CDV) or RPV in order to determine whether the proteins of different viruses can function together. Homogeneous sets consisting of N, P and L plasmids derived from one virus were able to generate reporter gene expression from either mini-genomic construct. Heterogeneous sets of proteins from different viruses were not functional, with the exception that a low level of activity was obtained when MV N and P protein were combined with RPV L protein in the rescue of the MV mini-genomic construct, or CDV N was combined with RPV P and L in the rescue of the RPV mini-genome. However, only homogeneous sets of plasmids were able to rescue infectious virus from full-length anti-genome-expressing plasmids.

A role for virus promoters in determining the pathogenesis of Rinderpest virus in cattle.

Rinderpest virus (RPV) is a morbillivirus that causes cattle plague, a disease of large ruminants. The viral genome is flanked at the 3' and 5' genome termini by the genome promoter (GP) and antigenome promoter (AGP), respectively. These promoters play essential roles in directing replication and transcription as well as RNA encapsidation and packaging. It has previously been shown that individual changes to the GP of RPV greatly affect promoter activity in a minigenome assay and it was therefore proposed that individual nucleotide changes in the GP and AGP might also have significant effects on the ability of the virus to replicate and cause disease in cattle. The Plowright vaccine strain of RPV has been derived by tissue-culture passage from the virulent Kabete 'O' isolate (KO) and is highly attenuated for all ruminant species in which it has been used. Here, it was shown that swapping the GP and the first 76 nt of the AGP between virulent and avirulent strains affected disease progression. In particular, it was shown that flanking the virulent strain with the vaccine GP and AGP sequences, while not appreciably affecting virus growth in vitro, led to attenuation in vivo. The reverse was not true, since the KO promoters did not alter the vaccine's attenuated nature. The GP/AGP therefore play a role in attenuation, but are not the only determinants of attenuation in this vaccine.

Leader RNA of Rinderpest virus binds specifically with cellular La protein: a possible role in virus replication.

Rinderpest virus (RPV) is an important member of the Morbillivirus genus in the family Paramyxoviridae and employs a similar strategy for transcription and replication of its genome as that of other negative sense RNA viruses. Cellular proteins have earlier been shown to stimulate viral RNA synthesis by isolated nucleocapsids from purified virus or from virus-infected cells. In the present work, we show that plus sense leader RNA of RPV, transcribed from 3' end of genomic RNA, specifically interacts with cellular La protein employing gel mobility shift assay as well as UV cross-linking of leader RNA with La protein. The leader RNA synthesized in virus-infected cells was shown to interact with La protein by immunoprecipitation of leader RNA bound to La protein and detecting the leader RNA in the immunoprecipitate by Northern hybridization with labeled antisense leader RNA. Employing a minireplicon system, we demonstrate that transiently expressed La protein enhances the replication/transcription of the RPV minigenome in cells. Sub-cellular immunolocalization shows that La protein is redistributed from nucleus to the cytoplasm upon infection. Our results strongly suggest that La protein may be involved in regulation of Rinderpest virus replication.

Phosphoprotein P of Rinderpest virus binds to plus sense leader RNA: regulation by phosphorylation.

The negative sense genome RNA of Rinderpest virus, a Paramyxoviridae, is encapsidated with the nucleocapsid protein N and serves as a template for the viral RNA dependent RNA polymerase for transcription and replication. The viral RNA polymerase consists of the large protein L and the phosphoprotein P functioning as the P-L complex. We provide in this report, evidences for specific binding of P protein of Rinderpest virus to the plus sense leader RNA depending on its phosphorylation status. We have also demonstrated that P protein is released from the le RNA:P protein complex upon phosphorylation in vitro. Finally, we have identified that the C-terminal 358-389 amino acid residues of P protein is involved in le RNA binding. The leader RNA binding may signify a hitherto unidentified role for P protein in the viral RNA synthesis. Moreover, our results indicate a possible role for P protein in the transcription-replication switch through leader RNA binding.

Development of a reconstitution system for Rinderpest virus RNA synthesis in vitro.

The RNA dependent RNA polymerase of Rinderpest virus consists of two subunits-the large protein (L) and the phosphoprotein (P), where L is thought to be responsible for the catalytic activities in association with P protein which plays multiple roles in transcription and replication. The nucleocapsid protein (N) is necessary for encapsidation of genomic RNA, which is required as N-P complex. To understand the different steps of transcription and replication as well as the roles played by the three proteins, an in vitro reconstitution system for RNA synthesis is necessary which is not available for any morbillivirus. We describe here, an in vitro reconstitution system for transcription and replication of Rinderpest virus utilizing a synthetic, positive sense N-RNA minigenome template, free of endogenous viral polymerase proteins and recombinant viral proteins (P+L and P+N) expressed in insect cells by recombinant baculoviruses. We show that although L-P complex is sufficient to synthesize negative sense minigenome RNA, soluble N protein is necessary for encapsidation of RNA as well as synthesis of (+) sense leader RNA and (+) sense minigenome RNA.

Effect of single amino acid mutations in the conserved GDNQ motif of L protein of Rinderpest virus on RNA synthesis in vitro and in vivo.

The paramyxovirus RNA-dependent RNA polymerase consists of two subunits, the transcription co-factor phosphoprotein P and the large protein L, which possesses all the catalytic functions such as RNA synthesis (both transcription replication), methylation, capping and polyadenylation. The L protein has high sequence homology among the negative sense RNA viruses. The domains and residues on the L protein involved in the above-mentioned activities are not well defined, although the role of conserved GDNQ motif of the putative catalytic centre of L protein of few related viruses have been examined. In order to gain insight into the role played by the GDNQ motif of the L protein of Rinderpest virus (RPV), we have examined mutations at each amino acid in this motif of the L protein of Rinderpest virus and tested the biological activity in vivo and in vitro. Site directed mutants were generated and transiently expressed in mammalian cells and were shown to interact with P protein similar to wild type L. The biological activity of mutant L proteins has been tested in an in vitro reconstituted system capable of carrying out cell-free RNA synthesis on synthetic Rinderpest N-RNA template. Further, the role played by individual amino acids has also been defined in vivo using an in vivo minigenome replication/transcription system which indicated the importance of this conserved sequence in viral RNA synthesis.

Induction of apoptotic cellular death in lymphatic tissues of cattle experimentally infected with different strains of rinderpest virus.

The presence, type, and extent of cellular death in lymphatic tissues of cattle experimentally infected with rinderpest virus strains of different virulence was investigated morphologically. Cells with DNA strand breaks were identified in histological sections of palatine tonsil, spleen, and mesenteric and mandibular lymph nodes by the TUNEL (terminal desoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. In addition, representative samples of lymphatic tissues were examined by transmission electron microscopy. The results indicated that cellular disassembly in lymphatic tissues was caused by both apoptosis and oncosis. Cells with DNA strand breaks were observed in follicular and parafollicular areas of lymphatic tissues and their numbers were determined. A significant correlation was found between the number of TUNEL-positive cells and viral virulence. These results suggest that, in addition to oncosis, apoptotic cellular death in lymphatic tissues contributes substantially to the pathogenesis of rinderpest.

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.

Morbilliviruses use signaling lymphocyte activation molecules (CD150) as cellular receptors.

Morbilliviruses comprise measles virus, canine distemper virus, rinderpest virus, and several other viruses that cause devastating human and animal diseases accompanied by severe immunosuppression and lymphopenia. Recently, we have shown that human signaling lymphocyte activation molecule (SLAM) is a cellular receptor for measles virus. In this study, we examined whether canine distemper and rinderpest viruses also use canine and bovine SLAMs, respectively, as cellular receptors. The Onderstepoort vaccine strain and two B95a (marmoset B cell line)-isolated strains of canine distemper virus caused extensive cytopathic effects in normally resistant CHO (Chinese hamster ovary) cells after expression of canine SLAM. The Ako vaccine strain of rinderpest virus produced strong cytopathic effects in bovine SLAM-expressing CHO cells. The data on entry with vesicular stomatitis virus pseudotypes bearing measles, canine distemper, or rinderpest virus envelope proteins were consistent with development of cytopathic effects in SLAM-expressing CHO cell clones after infection with the respective viruses, confirming that SLAM acts at the virus entry step (as a cellular receptor). Furthermore, most measles, canine distemper, and rinderpest virus strains examined could any use of the human, canine, and bovine SLAMs to infect cells. Our findings suggest that the use of SLAM as a cellular receptor may be a property common to most, if not all, morbilliviruses and explain the lymphotropism and immunosuppressive nature of morbilliviruses.