Sequestration and Destruction of Rinderpest Virus-Containing Material 10 Years after Eradication.

In 2021, the world marked 10 years free from rinderpest. The United Nations Food and Agriculture Organization and World Organisation for Animal Health have since made great strides in consolidating, sequencing, and destroying stocks of rinderpest virus-containing material, currently kept by only 14 known institutions. This progress must continue.

Measles virus and rinderpest virus divergence dated to the sixth century BCE.

Many infectious diseases are thought to have emerged in humans after the Neolithic revolution. Although it is broadly accepted that this also applies to measles, the exact date of emergence for this disease is controversial. We sequenced the genome of a 1912 measles virus and used selection-aware molecular clock modeling to determine the divergence date of measles virus and rinderpest virus. This divergence date represents the earliest possible date for the establishment of measles in human populations. Our analyses show that the measles virus potentially arose as early as the sixth century BCE, possibly coinciding with the rise of large cities.

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.

New world origin of canine distemper: Interdisciplinary insights.

OBJECTIVE: Canine distemper virus (CDV), human measles virus (HMV), and rinderpest virus (RPV) of cattle are morbilliviruses that have caused devastating outbreaks for centuries. This paper seeks to reconstruct the evolutionary history of CDV. MATERIALS AND METHODS: An interdisciplinary approach is adopted, synthesizing paleopathological analysis of 96 Pre-Columbian dogs (750-1470 CE) from the Weyanoke Old Town, Virginia site, with historical reports, molecular analysis and morbilliviral epidemiology. RESULTS: Both measles (c.900CE) and rinderpest (c. 376 BCE) were first reported in Eurasia, while canine distemper was initially described in South America much later (1735 CE); there are no paleopathological indications of CDV in Weyanoke Old Town dogs. Molecularly, CDV is closely related to HMV, while viral codon usage indicates CDV may have previously infected humans; South American measles epidemics occurred prior to the emergence of canine distemper and would have facilitated HMV transmission and adaptation to dogs. CONCLUSIONS: The measles epidemics that decimated indigenous South American populations in the 1500-1700 s likely facilitated the establishment of CDV as a canine pathogen, which eventually spread to Europe and beyond. SIGNIFICANCE: Understanding the historical and environmental conditions that have driven morbilliviral evolution provides important insights into potential future threats of animal/human cross-species infections. LIMITATIONS: Interpreting historical disease descriptions is difficult and the archaeological specimens are limited. Molecular sequence data and codon usage analyses rely on modern viruses. SUGGESTIONS FOR FURTHER RESEARCH: Interdisciplinary approaches are increasingly needed to understand diseases of the past and present, as critical information and knowledge is scattered in different disciplines.

Analyses of nucleotide, codon and amino acids usages between peste des petits ruminants virus and rinderpest virus.

Peste des petits ruminants virus (PPRV) and rinderpest virus (RPV) are two causative agents of an economically important disease for ruminants (i.e., sheep, cattle and goat). In this study, the nucleotide, codon and amino acid usages for PPRV and RPV have been analyzed by multivariate statistical methods. Relative synonymous codon usage (RSCU) analysis represents that ACG for Thr and GCG for Ala are selected with under-representation in both PPRV and RPV, and AGA for Arg in PPRV and AGG for Arg in RPV are used with over-representation. The usage of nucleotide pair (CpG) tends to be removed from viral genes of the two viruses, suggesting that other evolutionary forces take part in evolutionary processes for viral genes in addition to mutation pressure from nucleotide usage at the third codon position. The overall nucleotide usage of viral gene is not major factor in shaping synonymous codon usage patterns, while the nucleotide usages at the third codon position and the nucleotide pairs play important roles in shaping synonymous codon usage patterns. Although PPRV and RPV are closely related antigenically, the codon and amino acid usage patterns for viral genes represent a significant genetic diversity between PPRV and RPV. Moreover, the overall codon usage trends for viral genes between PPRV and RPV are mainly influenced by mutation pressure from nucleotide usage at the third codon position and translation selection from hosts. Taken together, this is first comprehensive analyses for nucleotide, codon and amino acid usages of viral genes of PPRV and RPV and the findings are expected to increase our understanding of evolutionary forces influencing viral evolutionary pathway and adaptation toward hosts.

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.

Evidence for N7 guanine methyl transferase activity encoded within the modular domain of RNA-dependent RNA polymerase L of a Morbillivirus.

Post-transcriptional modification of viral mRNA is essential for the translation of viral proteins by cellular translation machinery. Due to the cytoplasmic replication of Paramyxoviruses, the viral-encoded RNA-dependent RNA polymerase (RdRP) is thought to possess all activities required for mRNA capping and methylation. In the present work, using partially purified recombinant RNA polymerase complex of rinderpest virus expressed in insect cells, we demonstrate the in vitro methylation of capped mRNA. Further, we show that a recombinant C-terminal fragment (1717-2183 aa) of L protein is capable of methylating capped mRNA, suggesting that the various post-transcriptional activities of the L protein are located in independently folding domains.

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.

Complete genome analysis of three live attenuated Rinderpest virus vaccine strains derived through serial passages in different culture systems.

The genomes of three South Korean Rinderpest virus vaccine strains (L72, LA77, and LA96) were analyzed in order to investigate their genetic variability. These three vaccine strains were all derived from the same virus strain origin (Fusan) through repeated passages in different culture systems. The full genome length of the three strains was 15,882 nucleotides, and the sequence similarity between the three South Korean RPV strains at the nucleotide level was 98.1 to 98.9%. The genetic distance between Nakamura III, L72, LA77, LA96, and LATC06 and the Kabete strain was greater than that between the Fusan and Kabete strains for the P, V, and C genes. The difference in pathogenicity among these strains might be due to the V gene, which has a positive (>1) selection ratio based on the analysis of synonymous (dS) and nonsynonymous (dN) substitution rates (dN/dS ratio [ω]).

A novel approach to generating morbillivirus vaccines: negatively marking the rinderpest vaccine.

The eradication of rinderpest virus (RPV) from the globe was possible through the availability of a safe and effective live attenuated vaccine and a suitable companion diagnostic test. However, the inability to serologically 'Differentiate between naturally Infected and Vaccinated Animals' (DIVA) meant that both the time taken to complete the eradication programme and the economic burden on countries involved was significantly greater than if a vaccine and companion diagnostic test that fulfilled the DIVA concept had been available. During the RPV eradication campaign serosurveillance for RPV was primarily based on a competitive ELISA using a RPV specific (C1) monoclonal antibody (mAb) directed against the viral haemagglutinin (H) protein but this test was not able to meet DIVA requirements. To provide proof of concept for the generation of novel morbillivirus DIVA vaccines we have identified, by phage display, and mutated residues critical for C1 mAb binding and assessed the functionality of mutants in an in vitro fusion assay. Finally we have incorporated mutated epitopes into a full length clone and rescued recombinant RPV using reverse genetics techniques. Here we describe a novel mechanism of marking morbillivirus vaccines, using RPV as a proof of concept, and discuss the applicability of this method to the development of marked vaccines for peste des petits ruminants virus (PPRV).

Characterization of the complete genomic sequence of the rinderpest virus Fusan strain cattle type, which is the most classical isolate in Asia and comparison with its lapinized strain.

In this study, we characterized the rinderpest virus (RPV) Fusan strain cattle type (B), which is the most classical isolate in Asia, by complete genomic sequence analysis and compared it with its lapinized Nakamura III (L) strain. The transversion rates of the M, F, and H genes were higher than those of other genes. In contrast, the deduced amino acid (aa) substitution rates of the P, C, and V genes were higher than those of other genes, although their transversion rates were not higher. The characteristic nucleotide (nt) or aa residues of the cattle-virulent B and Kabete 'O' strains were observed in the P/C/V, M, and L genes. According to these results, we speculate that nt/aa substitution in the P/C/V genes is one of the key determinants for the difference in the pathogenicity to cattle of the B and L strains.

Purifying selection can obscure the ancient age of viral lineages.

Statistical methods for molecular dating of viral origins have been used extensively to infer the time of most common recent ancestor for many rapidly evolving pathogens. However, there are a number of cases, in which epidemiological, historical, or genomic evidence suggests much older viral origins than those obtained via molecular dating. We demonstrate how pervasive purifying selection can mask the ancient origins of recently sampled pathogens, in part due to the inability of nucleotide-based substitution models to properly account for complex patterns of spatial and temporal variability in selective pressures. We use codon-based substitution models to infer the length of branches in viral phylogenies; these models produce estimates that are often considerably longer than those obtained with traditional nucleotide-based substitution models. Correcting the apparent underestimation of branch lengths suggests substantially older origins for measles, Ebola, and avian influenza viruses. This work helps to reconcile some of the inconsistencies between molecular dating and other types of evidence concerning the age of viral lineages.

Differentiating pestes des petits ruminants and rinderpest viruses by a novel monoclonal antibody.

Peste des petits ruminant (PPR) is an acute, febrile, viral disease of small ruminants with great economic importance. PPR and rinderpest (RP) viruses are antigenically related and need to be differentiated serologically. The use of monoclonal antibodies (MAbs) in ELISA for specific diagnostics and separation of PPR and RPV is important. For this purpose, six Balb/c mice were immunized with inactivated antigen from the Nijeria strain. Fusion cloning was performed 3 months later by directly using cloning plates, selecting the hybridoma colonies at an early stage with an inverted microscope, and transferring the colonies into 96-well plates with a micropipette. From 300 wells, nearly 56 hybridoma clones were found, from which, after testing in ELISA, 11 with higher titer were selected. Among these, only two clones were placed for limiting dilution (1H1, 6A12). Only one clone (6A12L1F12) had no cross-reactivity with RP, reacted with the N protein, and was of IgG2 isotype.

Simultaneous detection of Rift Valley Fever, bluetongue, rinderpest, and Peste des petits ruminants viruses by a single-tube multiplex reverse transcriptase-PCR assay using a dual-priming oligonucleotide system.

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions.

Genetic characterization of the Korean LATC06 rinderpest vaccine strain.

We sequenced the genome of LATC06 generated by in vitro passage in Vero cells of the lapinized-avianized (LA) strain and compared its sequence to those of other rinderpest viruses. The LATC06 genome consists of 15882 nucleotides. Its transcriptional regulatory control sequences (TRSs) at gene boundaries are identical to those of the Kabete O strain. Cleavage sites for generating F1/F2 proteins were identified in the same amino acid position (aa 108) as F proteins in LATC06, L13, RBT1, Kabete O, and RBOK strains. There are three predicted N-glycosylation sites of H proteins in LA (Japan) and LATC06 strains. The six epitopes of H protein in the LA (Japan) strain that elicit immunodominant humoral responses are also found in the LATC06 strain.

Rinderpest virus expressing enhanced green fluorescent protein as a separate transcription unit retains pathogenicity for cattle.

A full-length DNA clone of a virulent strain of rinderpest virus was constructed with the gene for the enhanced green fluorescent protein (eGFP) inserted as a separate transcription unit between the P and M genes. Rescue of the virus from the modified clone using reverse genetics generated a virus that grew to the same levels as the virus rescued from the unmodified DNA clone in cell culture. The recombinant virus expressed eGFP to a high level and was used to follow virus replication in real-time using live-cell imaging. Cattle infected with both the recombinant wild-type virus and the recombinant eGFP expressing virus developed clinical disease similar to that of the wild-type natural virus isolate. Detection of virus in circulating peripheral blood leukocytes was equivalent to that of the animals infected with the wild-type virus. The high level of expression of soluble eGFP by this virus allowed us to detect viral replication in infected animals by confocal microscopy. Imaging vibrating microtome sections by confocal microscopy provided good preservation of tissue and cellular architecture as well as revealing the sites of replication of the virus in different tissues of infected animals.

Origin of measles virus: divergence from rinderpest virus between the 11th and 12th centuries.

Measles, caused by measles virus (MeV), is a common infection in children. MeV is a member of the genus Morbillivirus and is most closely related to rinderpest virus (RPV), which is a pathogen of cattle. MeV is thought to have evolved in an environment where cattle and humans lived in close proximity. Understanding the evolutionary history of MeV could answer questions related to divergence times of MeV and RPV. We investigated divergence times using relaxed clock Bayesian phylogenetics. Our estimates reveal that MeV had an evolutionary rate of 6.0-6.5 x 10(-4) substitutions/site/year. It was concluded that the divergence time of the most recent common ancestor of current MeV was the early 20th century. And, divergence between MeV and RPV occurred around the 11th to 12th centuries. The result was unexpected because emergence of MeV was previously considered to have occurred in the prehistoric age. MeV may have originated from virus of non-human species and caused emerging infectious diseases around the 11th to 12th centuries. In such cases, investigating measles would give important information about the course of emerging infectious diseases.

Comparative and mutational analyses of promoter regions of rinderpest virus.

Comparative and mutational analysis of promoter regions of rinderpest virus was conducted. Minigenomic RNAs harboring the genomic and antigenomic promoter of the lapinized virulent strain (Lv) or an attenuated vaccine strain (RBOK) were constructed, and the expression of the reporter gene was examined. The activities of the antigenomic promoters of these strains were similar, whereas the activity of the genomic promoter (GP) of the RBOK strain was significantly higher than that of the Lv strain, regardless of cell type and the source of the N, P and L proteins. Increased replication (and/or encapsidation) activities were observed in the minigenomes that contained RBOK GP. Mutational analysis revealed that the nucleotides specific to the RBOK strain are responsible for the strong GP activity of the strain. It was also demonstrated that other virulent strains of RPV (Kabete O, Saudi/81 and Kuwait 82/1) have weaker GPs than that of the RBOK strain.

Recombinant L and P protein complex of Rinderpest virus catalyses mRNA synthesis in vitro.

Rinderpest virus belongs to the family of Paramyxoviridae, consisting of non-segmented negative sense RNA viruses. Viral transcription and replication are carried out by the RNA dependent RNA polymerase L protein which functions together with P protein as L-P complex. The exact events triggering the polymerase complex from transcription to replication function is poorly understood. In the present work, an in vitro transcription system has been described with partially purified L-P complex expressed in insect cells and viral genomic RNA. The relative abundance of each species of mRNA synthesized in vitro decreased from the 3' end of the genome to the 5' end similar to their abundance in virus infected cells. Recombinant L-P complex was unable to synthesize leader RNA suggesting the initiation of transcription from gene start site and not at the 3' end of the genome.