Identification of a murine cell line that distinguishes virulent from attenuated isolates of the morbillivirus Peste des Petits Ruminants, a promising tool for virulence studies.

Comprehensive pathogenesis studies on Peste des Petits Ruminants virus (PPRV) have been delayed so far by the absence of a small animal model reproducing the disease or an in vitro biological system revealing virulence differences. In this study, a mouse 10T1/2 cell line has been identified as presenting different susceptibility to virulent and attenuated PPRV strains. As evidenced by immunofluorescence test and RT-PCR, both virulent and attenuated PPR viruses penetrated and initiated the replication cycle in 10T1/2 cells, independently of the presence of the SLAM goat receptor. However, only virulent strains successfully completed their replication cycle while the vaccine strains did not. Since 10T1/2 cells are interferon-producing cells, the role of the type I interferon (type I IFN) response on this differentiated replication between virulent and attenuated strains was verified by stimulation or repression. Modulation of the type I IFN response did not improve the replication of the vaccine strains, indicating that other cell factor(s) not yet established may hinder the replication of attenuated PPRV in 10T1/2. This 10T1/2 cell line can be proposed as a new in vitro tool for PPRV-host interaction and virulence studies.

Crimean-Congo Hemorrhagic Fever Virus Antibodies among Livestock on Corsica, France, 2014-2016.

We conducted a serologic survey for Crimean-Congo hemorrhagic fever virus antibodies in livestock (cattle, sheep, and goats; N = 3,890) on Corsica (island of France) during 2014-2016. Overall, 9.1% of animals were seropositive, suggesting this virus circulates on Corsica. However, virus identification is needed to confirm these results.

Evolution of Attenuation and Risk of Reversal in Peste des Petits Ruminants Vaccine Strain Nigeria 75/1.

Peste des Petits Ruminants (PPR) is a highly infectious disease caused by a virus of the Morbillivirus genus. The current PPR eradication effort relies mainly on the implementation of massive vaccination campaigns. One of the most widely used PPR vaccines is the Nigeria 75/1 strain obtained after attenuation by 75 serial passages of the wild type isolate in cell cultures. Here we use high throughput deep sequencing of the historical passages that led to the Nigeria 75/1 attenuated strain to understand the evolution of PPRV attenuation and to assess the risk of reversal in different cell types. Comparison of the consensus sequences of the wild type and vaccine strains showed that only 18 fixed mutations separate the two strains. At the earliest attenuation passage at our disposal (passage 47), 12 out of the 18 mutations were already present at a frequency of 100%. Low-frequency variants were identified along the genome in all passages. Sequencing of passages after the vaccine strain showed evidence of genetic drift during cell passages, especially in cells expressing the SLAM receptor targeted by PPRV. However, 15 out of the 18 mutations related to attenuation remained fixed in the population. In vitro experiments suggest that one mutation in the leader region of the PPRV genome affects virus replication. Our results suggest that only a few mutations can have a serious impact on the pathogenicity of PPRV. Risk of reversion to virulence of the attenuated PPRV strain Nigeria 75/1 during serial passages in cell cultures seems low but limiting the number of passages during vaccine production is recommended.

Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus.

Several Avian paramyxoviruses 1 (synonymous with Newcastle disease virus or NDV, used hereafter) classification systems have been proposed for strain identification and differentiation. These systems pioneered classification efforts; however, they were based on different approaches and lacked objective criteria for the differentiation of isolates. These differences have created discrepancies among systems, rendering discussions and comparisons across studies difficult. Although a system that used objective classification criteria was proposed by Diel and co-workers in 2012, the ample worldwide circulation and constant evolution of NDV, and utilization of only some of the criteria, led to identical naming and/or incorrect assigning of new sub/genotypes. To address these issues, an international consortium of experts was convened to undertake in-depth analyses of NDV genetic diversity. This consortium generated curated, up-to-date, complete fusion gene class I and class II datasets of all known NDV for public use, performed comprehensive phylogenetic neighbor-Joining, maximum-likelihood, Bayesian and nucleotide distance analyses, and compared these inference methods. An updated NDV classification and nomenclature system that incorporates phylogenetic topology, genetic distances, branch support, and epidemiological independence was developed. This new consensus system maintains two NDV classes and existing genotypes, identifies three new class II genotypes, and reduces the number of sub-genotypes. In order to track the ancestry of viruses, a dichotomous naming system for designating sub-genotypes was introduced. In addition, a pilot dataset and sub-trees rooting guidelines for rapid preliminary genotype identification of new isolates are provided. Guidelines for sequence dataset curation and phylogenetic inference, and a detailed comparison between the updated and previous systems are included. To increase the speed of phylogenetic inference and ensure consistency between laboratories, detailed guidelines for the use of a supercomputer are also provided. The proposed unified classification system will facilitate future studies of NDV evolution and epidemiology, and comparison of results obtained across the world.

Cleavage site of Newcastle disease virus determines viral fitness in persistent infection cells.

Newcastle disease, caused by infection with virulent strains of Newcastle disease virus (NDV), poses a risk for the poultry industry. The virulence of NDV is mainly determined by the cleavage site of F protein. Lentogenic NDV can become velogenic after passages in SPF chicken brain and air sac based on some strains isolated from water birds, because the proportion of virulent-related strains gradually increases. In contrast, this proportion remains unchanged if NDV is passaged via 10-day-old SPF chicken embryos. This information suggests that environmental conditions rather than mutation affect NDV fitness in quasispecies. However, it is unknown how the environment selects virulent-related strains from a viral population. In this study, velogenic and lentogenic NDV marked by green or red fluorescence were used to establish persistent infection (PI) in BHK-21 cells. Monitoring viruses by different methods, we found that, without competition, persistently infected cells harbored lentogenic and velogenic NDV strains similarly in terms of viral release, viral spread and the period of persistent viral infection. In contrast, under competitive co-infection, velogenic NDV became dominant in quasispecies from the fifth passage of PI cells, which resulted in the progressive disappearance of the lentogenic NDV strain. This domination was concomitant with a short-term reduction in the superinfection exclusion and supernatant interference in PI cells resulting in a velogenic virus rebound. We concluded that virulent-related F protein cleavage site facilitates the spread and replication of NDV in conditions under which cells do not secret trypsin-like proteases and do not inhibit free virus infection, resulting in a gradual increase in virulent strains in quasispecies with the number of passages.

Peste des Petits Ruminants, the next eradicated animal disease?

Peste des Petits Ruminants (PPR) is a widespread viral disease caused by a Morbillivirus (Paramyxoviridae). There is a single serotype of PPR virus, but four distinct genetic lineages. Morbidity and mortality are high when occurring in naive sheep and goats populations. Cattle and African buffaloes (Syncerus caffer) are asymptomatically infected. Other wild ruminants and camels may express clinical signs and mortality. PPR has recently spread in southern and northern Africa, and in central and far-east Asia. More than one billion sheep and goats worldwide are at risk. PPR is also present in Europe through western Turkey. Because of its clinical incidence and the restrictions on animal movements, PPR is a disease of major economic importance. A live attenuated vaccine was developed in the 1980s, and has been widely used in sheep and goats. Current researches aim (i) to make it more thermotolerant for use in countries with limited cold chain, and (ii) to add a DIVA mark to shorten and reduce the cost of final eradication. Rinderpest virus-another Morbillivirus-was the first animal virus to be eradicated from Earth. PPRV has been proposed as the next candidate. Considering its wide distribution and its multiple target host species which have an intense mobility, it will be a long process that cannot exclusively rely on mass vaccination. PPR specific epidemiological features and socio-economic considerations will also have to be taken into account, and sustained international, coordinated, and funded strategy based on a regional approach of PPR control will be the guarantee toward success.

Serological and molecular investigation of Newcastle disease in household chicken flocks and associated markets in Eastern Shewa zone, Ethiopia.

Cross-sectional survey for Newcastle disease (ND) were conducted in nonvaccinated household flocks of village chickens to assess serological and virological ND status in households and associated live bird markets. In total, 1,899 sera and 460 pools of cloacal and tracheal swabs were sampled and tested using a commercial enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase polymerase chain reaction (rRT-PCR), respectively. Additionally, paired cloacal and tracheal swabs from 1,269 individual chickens were collected from markets and tested using RT-PCR. The prevalence of households with at least one seropositive chicken was higher during the dry season (27.4 %) than during the wet season (17.4 %) (P = 0.003). Viral genome was detected in 14.2 % of households during the wet season using a fusion (F) gene assay and in 24.2 % of households during the dry season using a polymerase (L) gene assay that targets both class I and class II viruses. At the markets sampled, overall bird level prevalence was 4.9 % for period 1 (F gene assay), and 38.2 % and 27.6 % for periods 2 and 3, respectively (L gene assay). Partial sequencing of the F gene (239 bp) cleavage site indicated that the majority of the circulating strains exhibited motifs specific to virulent strains. Seroepidemiology coupled with molecular analysis can be a useful tool to assess the status of NDV infection. The village chicken population in Ethiopia is endemically infected with virulent NDV that pose a significant threat to emerging small- and medium-scale commercial poultry production.

Circulation of avian influenza viruses in wild birds in Inner Niger Delta, Mali.

BACKGROUND: Avian influenza viruses (AIV) have been detected in wild birds in West Africa during the northern winter, but no information is available on a potential year-round circulation of AIV in West Africa. Such year-round circulation would allow reassortment opportunities between strains circulating in Afro-tropical birds and strains imported by migratory birds wintering in West Africa. OBJECTIVE AND METHOD: A 2-year longitudinal survey was conducted in the largest continental wetland of West Africa, the Inner Niger Delta in Mali, to determine the year-round circulation of AIV in wild birds. RESULTS AND CONCLUSIONS: Avian influenza virus RNA was detected during all periods of the year. Very low prevalence was detected during the absence of the migratory wild birds. However, a year-round circulation of AIV seems possible in West Africa, as shown in other African regions. West Africa may hence be another potential site of reassortment between AIV strains originating from both Afro-tropical and Eurasian regions.

RNA interference against animal viruses: how morbilliviruses generate extended diversity to escape small interfering RNA control.

Viruses are serious threats to human and animal health. Vaccines can prevent viral diseases, but few antiviral treatments are available to control evolving infections. Among new antiviral therapies, RNA interference (RNAi) has been the focus of intensive research. However, along with the development of efficient RNAi-based therapeutics comes the risk of emergence of resistant viruses. In this study, we challenged the in vitro propensity of a morbillivirus (peste des petits ruminants virus), a stable RNA virus, to escape the inhibition conferred by single or multiple small interfering RNAs (siRNAs) against conserved regions of the N gene. Except with the combination of three different siRNAs, the virus systematically escaped RNAi after 3 to 20 consecutive passages. The genetic modifications involved consisted of single or multiple point nucleotide mutations and a deletion of a stretch of six nucleotides, illustrating that this virus has an unusual genomic malleability.

Potential of adenovirus and baculovirus vectors for the delivery of shRNA against morbilliviruses.

Morbilliviruses are important pathogens of humans, ruminants, carnivores and marine mammals. Although good vaccines inducing long-term immunity are available, recurrent outbreaks of measles, canine distemper and peste des petits ruminants (PPR) are observed. In control strategies, antivirals thus could be useful to confine virus spread and application of interfering RNAs is a promising approach, provided they can be delivered efficiently into the host cells. We have constructed recombinant adenovirus and baculovirus vectors expressing short hairpin RNAs (shRNAs) against the PPR virus (PPRV) and compared them in vitro. It was found that both recombinant viruses inhibited PPRV replication with the baculovirus vector, which inhibited generation of infectious progeny by more than 2 log10 and the nucleoprotein expression of PPRV by 73%, being the more efficient. The results show that baculoviral shRNA-expressing vectors have the potential for therapeutic use against morbillivirus infections.

Newcastle disease virus in Madagascar: identification of an original genotype possibly deriving from a died out ancestor of genotype IV.

In Madagascar, Newcastle disease (ND) has become enzootic after the first documented epizootics in 1946, with recurrent annual outbreaks causing mortality up to 40%. Four ND viruses recently isolated in Madagascar were genotypically and pathotypically characterised. By phylogenetic inference based on the F and HN genes, and also full-genome sequence analyses, the NDV Malagasy isolates form a cluster distant enough to constitute a new genotype hereby proposed as genotype XI. This new genotype is presumably deriving from an ancestor close to genotype IV introduced in the island probably more than 50 years ago. Our data show also that all the previously described neutralising epitopes are conserved between Malagasy and vaccine strains. However, the potential implication in vaccination failures of specific amino acid substitutions predominantly found on surface-exposed epitopes of F and HN proteins is discussed.

Avian influenza in backyard poultry of the Mopti region, Mali.

This study reports the first evidence of circulation of avian influenza viruses (AIV) in domestic poultry in Mali. In the Mopti region, where AIV have already been isolated in migratory water birds, we sampled 223 backyard domestic birds potentially in contact with wild birds and found that 3.6% had tracheal or cloacal swabs positive by real-time reverse transcription PCR (rRT-PCR) for type A influenza viruses (IVA) and that 13.7% had sera positive by commercial ELISA test detecting antibodies against IVA. None of the birds positive by rRT-PCR for IVA was positive by rRT-PCR for H5 and H7 subtypes, and none showed any clinical signs therefore indicating the circulation of low pathogenic avian influenza. Unfortunately, no virus isolation was possible. Further studies are needed to assess the temporal evolution of AIV circulation in the Mopti region and its possible correlation with the presence of wild birds.

Identification and mapping of a region on the mRNA of Morbillivirus nucleoprotein susceptible to RNA interference.

The morbillivirus genus includes important pathogens such as measles virus (MV), peste des petits ruminants virus (PPRV), and rinderpest virus (RPV) and forms a group of antigenically related viruses. The viral nucleoprotein (N) is a well-conserved protein among the genus and plays a central role in the replication of the virus. Using a comprehensive approach for siRNA screening of the conserved sequences of the N gene, including sequence analysis and functional in vitro tests, we have identified a region for the design of siRNA effective for the control of PPRV, RPV, and MV replication. Silencing of the N mRNA efficiently shuts down the production of N transcripts, the expression of N protein, and the indirect inhibition of matrix protein, resulting in the inhibition of PPRV progeny by 10,000-fold. These results suggest that siRNA against this region should be further explored as a therapeutic strategy for morbillivirus infections.

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.