[show abstract][hide abstract] ABSTRACT: Soon aft er discovery of RNA interference (RNAi), its potential as eff ective antiviral therapy was recognized. Since then
RNAi has been variously exploited for antiviral purposes which could eff ectively block viral replication in vitro. For in
vivo use, however, delivery issue, toxicity, RNAi suppression and viral escape are still major hurdles. Here, we provide
an overview of the RNAi strategy and review the approaches that have been developed to surpass the obstacles and to
achieve targeted gene silencing for antiviral and other therapies.
[show abstract][hide abstract] ABSTRACT: Peste des petits ruminants virus, which causes a severe disease in sheep and goats, has only recently been officially declared to be present in Tanzania. An epidemiological study was carried out between September 2008 and October 2010 to investigate the incursion, persistence and spread of the virus in Tanzania. The investigation involved serosurveillance, outbreak investigation and computation of epidemiological indices such as the effective reproductive number, persistence and the threshold level for vaccination. Field and molecular epidemiological techniques were applied to isolate, characterise and trace the origin of the virus in Tanzania. A total of 2182 serum samples from goats and 1296 from sheep from 79 villages across 12 districts were investigated. Village-level prevalence of infection was variable (0.00% – 88.00%) and was higher in pastoral than in agro-pastoral villages. The overall antibody response to the virus was 22.10% (CI95% = 20.72% – 23.48%). About 68.00% and 73.00% of seropositive goats and sheep, respectively, did not show clinical signs. The proportion of seropositive animals differed significantly (p ≤ 0.001) between age groups, sex and farming practices. Real-time polymerase chain reaction results showed that the isolated strains belong to lineage III, whose origin is in East Africa and the Middle East. This indicates that one of the northern neighbouring countries is most likely the source of infection. The computed overall effective reproductive number, the threshold level of vaccination necessary to eradicate the disease and persistence were 4.75% and 98.00%, respectively. These estimates indicate that achieving elimination of the peste des petits ruminants virus from pastoral flocks will require significant effort and development of highly effective intervention tools.
The Onderstepoort journal of veterinary research. 01/2013; 80(1):593.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Virology 11/2011; 86(2):786-95. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interest in peste des petits ruminants virus (PPRV) has been stimulated by recent changes in its host and geographic distribution. For this study, biological specimens were collected from camels, sheep, and goats clinically suspected of having PPRV infection in Sudan during 2000-2009 and from sheep soon after the first reported outbreaks in Morocco in 2008. Reverse transcription PCR analysis confirmed the wide distribution of PPRV throughout Sudan and spread of the virus in Morocco. Molecular typing of 32 samples positive for PPRV provided strong evidence of the introduction and broad spread of Asian lineage IV. This lineage was defined further by 2 subclusters; one consisted of camel and goat isolates and some of the sheep isolates, while the other contained only sheep isolates, a finding with suggests a genetic bias according to the host. This study provides evidence of the recent spread of PPRV lineage IV in Africa.
[show abstract][hide abstract] ABSTRACT: 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.
Antiviral research 02/2011; 90(1):98-101. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Viral diseases of farm animals, rather than being a diminishing problem across the world, are now appearing with regularity in areas where they have never been seen before. Across the developing world, viral pathogens such as peste des petits ruminants virus (PPRV) place a huge disease burden on agriculture, in particular affecting small ruminant production and in turn increasing poverty in some of the poorest parts of the world. PPRV is currently considered as one of the main animal transboundary diseases that constitutes a threat to livestock production in many developing countries, particularly in western Africa and south Asia. Infection of small ruminants with PPRV causes a devastating plague and as well as being endemic across much of the developing world, in recent years outbreaks of PPRV have occurred in the European part of Turkey. Indeed, the relevance of many once considered 'exotic' viruses is now also high across the European Union and may threaten further regions across the globe in the future. Here, we review the spread of PPRV across Africa, Asia and into Europe through submissions made to the OIE Regional Reference Laboratories. Further, we discuss current control methods and the development of further tools to aid both diagnosis of the disease and prevention.
Journal of General Virology 12/2010; 91(Pt 12):2885-97. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: In mid-August 2004, an outbreak of a previously unknown fatal disease of camels was reported to Kassala State veterinary authorities. Several areas in the state were visited during August-October 2004 to collect epidemiological data and specimens for diagnosis. Clinically the disease was characterized by sudden death of apparently healthy animals and yellowish and later bloody diarrhea and abortion. The disease outbreaks coincided with the seasonal movement of animals towards autumn green pasture. Death was always sudden and proceeded with colic and difficulty in respiration. Mortality rate ranged between 0% and 50% and vary in accordance with the area with a mean of 7.4%. More than 80% of deaths were in pregnant and recently-delivered she-camels. All age, sex and breed groups were affected but more than 50% of deaths were reported in adult animals in comparison to calves and young camels. The main post-mortem findings include lung congestion and consolidation, paleness and fragility of liver, enlarged lymph nodes and congestion and hemorrhage of small intestine and stomach. Agar gel diffusion test (AGDT), RT-PCR and virus isolation in cell culture gave positive results for peste des petits ruminants virus (PPRV), a virus belonging to the Morbillivirus, Genus, member of the family Paramyxoviridae. The effect of this new devastating disease on camel production in the affected area was discussed as well as proposals for future research.
[show abstract][hide abstract] ABSTRACT: A one-step real-time Taqman RT-PCR assay (RRT-PCR) for peste des petits ruminants virus (PPRV) was developed to detect the four lineages of PPRV by targeting the nucleoprotein (N) gene of the virus. This new assay was compared to a conventional RT-PCR on reference strains and field materials. Quantitation was performed against a standard based on a synthetic transcript of the NPPR gene for which a minimum of 32 copies per reaction were detected with a corresponding C(t) value of 39. Depending on the lineage involved, the detection limit of RRT-PCR was decreased by one to three log copies relative to the conventional method. The lower stringency occurred with lineage III because of minor nucleotide mismatches within the probe region. The assay did not detect phylogenetically or symptomatically related viruses of ruminants (such as rinderpest, bluetongue, and bovine viral diarrhea viruses). However, it was capable of detecting 20% more positive field samples with low viral RNA loads compared to the conventional PCR method. When compared on a proficiency panel to the method developed by Bao et al. (2008), the sensitivity of the in-house assay was slightly improved on lineage II. It proved significantly faster to perform and hence better adapted for monitoring large numbers of at risk or diseased animals.
Journal of virological methods 05/2010; 165(2):168-77. · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study, carried out between September 2006 and January 2007, is the first cross-sectional serological investigation of peste-des-petits-ruminants (PPR) and Rift Valley fever (RVF) in Tunisia. The objective was to assess the potential need to develop a dual, recombinant PPR-RVF vaccine and how such a vaccine might be utilised in Tunisia. An overall PPR seroprevalence of 7.45% was determined, a finding supported by the high specificity (99.4%) and sensitivity (94.5%) of the ELISA used. On assessment of the diversity and density of mosquitoes in the sampling area, four species of RVF-vectors of the genus Aedes and Culex were identified. However, no serological evidence of RVF was found despite the use of a highly sensitive ELISA (99-100%). Larger scale investigations are underway to confirm these findings and the continuation of the emergency vaccination program against these two diseases remains valid.
The Veterinary Journal 02/2010; 187(3):402-4. · 2.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: The genus Capripoxvirus within the family Poxviridae comprises three closely related viruses, namely goat pox, sheep pox and lumpy skin disease viruses. This nomenclature is based on the animal species from which the virus was first isolated, respectively, goat, sheep and cattle. Since capripoxviruses are serologically identical, their specific identification relies exclusively on the use of molecular tools. We describe here the suitability of the G-protein-coupled chemokine receptor (GPCR) gene for use in host-range grouping of capripoxviruses. The analysis of 58 capripoxviruses showed three tight genetic clusters consisting of goat pox, sheep pox and lumpy skin disease viruses. However, a few discrepancies exist with the classical virus-host origin nomenclature: a virus isolated from sheep is grouped in the goat poxvirus clade and vice versa. Intra-group diversity was further observed for the goat pox and lumpy skin disease virus isolates. Despite the presence of nine vaccine strains, no genetic determinants of virulence were identified on the GPCR gene. For sheep poxviruses, the addition or deletion of 21 nucleic acids (7 aa) was consistently observed in the 5' terminal part of the gene. Specific signatures for each cluster were also identified. Prediction of the capripoxvirus GPCR topology, and its comparison with other known mammalian GPCRs and viral homologues, revealed not only a classical GPCR profile in the last three-quarters of the protein but also unique features such as a longer N-terminal end with a proximal hydrophobic alpha-helix and a shorter serine-rich C-tail.
Journal of General Virology 05/2009; 90(Pt 8):1967-77. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: The large (L) polymerase gene and the 5′-terminal UTR of the genome of peste des petits ruminants virus (PPRV), vaccine strain Nigeria 75/1, were cloned and sequenced. The L protein was also expressed in eukaryotic cells and its polymerase activity was quantitatively measured in a PPR reverse genetics assay using a reporter minigenome. Comparative sequence analysis of this functional L gene with corresponding genes of other morbilliviruses showed a degree of conservation exceeding 70%. The multiple sequence alignment and the phylogenetic study of L gene discriminated the morbilliviruses in 6 clusters, which are more closely related to Tupaia and Henipaviruses than to other paramyxoviruses. Important protein domains and functional motifs of the L polymerase of the PPRV Nigeria 75/1 vaccine were also identified by using different bioinformatics tools.
[show abstract][hide abstract] ABSTRACT: In Vietnam, for a number of specific geographical and historical reasons, the mountainous areas have preserved an exceptional diversity of wild and domestic animal species of high socioeconomic interest. This endemic genetic diversity fosters a rapid response to environmental change in mostly isolated local communities and, in particular, fosters the constant adaptation of ecosystems common to humans and farmed and wild animal populations and pathogens. During a 2-year study carried out in several mountainous regions of North Vietnam near the Chinese border, we surveyed 1697 breeders in 249 villages and gathered 5815 biological samples among the four main domesticated species of food animals: chickens, cattle, buffaloes, and goats. Serological analyses were carried out by ELISA on 726 sera in order to assess the prevalence of antibodies specific to two major diseases suspected to be present in the region: avian influenza (AI) and peste des petits ruminants (PPR). The results reported here reveal the presence of antibodies specific to AI, but not the H5N1 highly pathogenic strain, and the presence of antibodies specific to PPR, confirming that this disease, never previously described in Southeast Asia, is present in this region, with no mortality and little or no evidence of clinical cases. These are probably situations of co-evolutive epidemiological equilibrium between pathogen populations, which may have lost their virulence, and animal populations that have acquired genetic resistances over generations, either naturally or through genetic introgression from related wild species better adapted to such pathogens. These results suggest the need for more research, both short-term and, more globally, long-term.
Annals of the New York Academy of Sciences 01/2009; 1149:259-62. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Antiviral research 11/2008; 80(2):158-67. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Peste des petits ruminants (PPR) is a contagious viral disease of small ruminants in Africa and Asia. In 1999, probably the largest survey on PPR ever conducted in Africa was initiated in Ethiopia where 13 651 serum samples from 7 out of the 11 regions were collected and analyzed by competitive enzyme-linked immunosorbent assay (cELISA). The objective of this paper is to present the results of this survey and discuss their practical implications for PPR-endemic regions.
We explored the spatial distribution of PPR in Ethiopia and we investigated risk factors for positive serological status. Intracluster correlation coefficients (rho), were calculated for 43 wereda (administrative units).
Seroprevalence was very heterogeneous across regions and even more across wereda, with prevalence estimates ranging from 0% to 52.5%. Two groups of weredas could be distinguished on the basis of the estimated rho: a group with very low rho (rho < 0.12) and a group with very high rho (rho > 0.37).
The results indicate that PPRV circulation has been very heterogeneous, the values for the rho may reflect the endemic or epidemic presence of the virus or the various degrees of mixing of animals in the different areas and production systems. Age appears as a risk factor for seropositive status, the linear effect seeming to confirm in the field that PPRV is highly immunogenic. Our estimates of intracluster correlation may prove useful in the design of serosurveys in other countries where PPR is of importance.
BMC Veterinary Research 10/2008; 4:34. · 1.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: The most challenging task in RNA interference is the design of active small interfering RNA (siRNA) sequences. Numerous strategies have been published to select siRNA. They have proved effective in some applications but have failed in many others. Nonetheless, all existing guidelines have been devised to select effective siRNAs targeting human or murine genes. They may not be appropriate to select functional sequences that target genes from other organisms like viruses. In this study, we have analyzed 62 siRNA duplexes of 19 bases targeting three genes of three morbilliviruses. In those duplexes, we have checked which features are associated with siRNA functionality. Our results suggest that the intramolecular secondary structure of the targeted mRNA contributes to siRNA efficiency. We also confirm that the presence of at least the sequence motifs U13, A or U19, as well as the absence of G13, cooperate to increase siRNA knockdown rates. Additionally, we observe that G11 is linked with siRNA efficacy. We believe that an algorithm based on these findings may help in the selection of functional siRNA sequences directed against viral genes.
Antiviral Research 08/2008; 79(1):37-48. · 3.93 Impact Factor
[show abstract][hide abstract] ABSTRACT: For Mononegavirales, the template for transcription and replication is not the naked RNA but the nucleoprotein (N) encapsidated genomic and anti-genomic RNA. Because of this central role in the replication of these viruses, N has been the subject of numerous structural and functional mapping studies. Here, we report on the cloning of the Peste des Petits Ruminants virus (PPRV) N gene into the baculovirus vector and the expression of the protein in insect cells. By electron microscopy observation, we have shown that this recombinant PPRV N forms nucleocapsid-like particles in insect cells in the absence of other PPRV proteins, as reported for other paramyxoviruses. As it is known that the formation of these particles is first linked to the self-assembly of N, we have made several deletions in the PPRV N gene and expressed these mutants in insect cells. Analysis of these proteins by immunoprecipitation and electron microscopy observation enabled us to map the N-N interaction domains into two regions of PPRV N: aa 1-120 and 146-241. The fragment aa 121-145, which is not conserved within the morbillivirus group, is also required for the formation/stability of the nucleocapsid helical structure.
Virus Research 02/2008; 131(1):23-32. · 2.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: In tropical countries the diagnosis of viral infections of humans or animals is often hampered by the lack of suitable clinical material and the necessity to maintain a cold chain for sample preservation up to the laboratory. This study describes the use of filter papers for rapid sample collection, and the molecular detection and genotyping of viruses when stored over long periods at elevated temperatures. Infected blood was collected on filter papers, dried and stored at different temperatures (22, 32 and 37 degrees C) for various periods (up to 9 months). Two animal viruses, African swine fever, a large double-stranded DNA virus and Peste des Petits Ruminants, a negative single-stranded RNA virus, were used to validate the method. Filter papers with dried blood containing virus or control plasmid DNA were cut in small 5mm(2) pieces and added directly to the PCR tube for conventional PCR. Nucleic acid from both viruses could still be detected after 3 months at 32 degrees C. Moreover, the DNA virus could be detected at least 9 months after conservation at 37 degrees C. PCR products obtained from the filter papers were sequenced and phylogenetic analysis carried out. The results were consistent with published sequences, demonstrating that this method can be used for virus genotyping.
Journal of Virological Methods 01/2008; 146(1-2):257-65. · 1.90 Impact Factor