In-vitro and in-vivo study of the interference between Rift Valley fever virus (clone 13) and Sheeppox/Limpy Skin disease viruses.

Viral interference is a common occurrence that has been reported in cell culture in many cases. In the present study, viral interference between two capripox viruses (sheeppox SPPV and lumpy skin disease virus LSDV in cattle) with Rift Valley fever virus (RVFV) was investigated in vitro and in their natural hosts, sheep and cattle. A combination of SPPV/RVFV and LSDV/RVFV was used to co-infect susceptible cells and animals to detect potential competition. In-vitro interference was evaluated by estimating viral infectivity and copies of viral RNA by a qPCR during three serial passages in cell cultures, whereas in-vivo interference was assessed through antibody responses to vaccination. When lamb testis primary cells were infected with the mixture of capripox and RVFV, the replication of both SPPV and LSDV was inhibited by RVFV. In animals, SPPV/RVFV or LSDV/RVFV combinations inhibited the replication SPPV and LSDV and the antibody response following vaccination. The combined SPPV/RVFV did not protect sheep after challenging with the virulent strain of SPPV and the LSDV/RVFV did not induce interferon Gamma to LSDV, while immunological response to RVFV remain unaffected. Our goal was to assess this interference response to RVFV/capripoxviruses' coinfection in order to develop effective combined live-attenuated vaccines as a control strategy for RVF and SPP/LSD diseases. Our findings indicated that this approach was not suitable for developing a combined SPPV/LSDV/RVFV vaccine candidate because of interference of replication and the immune response among these viruses.

Safety and immunogenecity of a live attenuated Rift Valley fever vaccine (CL13T) in camels.

BACKGROUND: Rift Valley fever is an emerging zoonotic viral disease, enzootic and endemic in Africa and the Arabian Peninsula, which poses a significant threat to both human and animal health. The disease is most severe in ruminants causing abortions in pregnant animals, especially sheep animals and high mortality in young populations. High mortality rates and severe clinical manifestation have also been reported among camel populations in Africa, to attend however none of the currently available live vaccines against RVF have been tested for safety and efficacy in this species. In this study, the safety and efficacy (through a neutralizing antibody response) of the thermostable live attenuated RVF CL13T vaccine were evaluated in camels in two different preliminary experiments involving 16 camels, (that 12 camels and 4 pregnant camels). RESULTS: The study revealed that the CL13T vaccine was safe to use in camels and no abortions or teratogenic effects were observed. The single dose of the vaccine stimulated a strong and long-lasting neutralizing antibody response for up to 12 months. CONCLUSION: The presence of neutralization antibodies is likely to correlate with protection; however protection would need to be confirmed by challenge experiments using the virulent RVF virus.

Evaluation of Humoral Response and Protective Efficacy of an Inactivated Vaccine Against Peste des Petits Ruminants Virus in Goats.

Four goats were inoculated with an inactivated peste des petits ruminants virus (PPRV) vaccine. Three unvaccinated goats were kept as controls. After 36 days, the four goats were revaccinated. The immune response was monitored by virus neutralization test showing that two doses of the vaccine were able to stimulate strong immune response in all the vaccinated animals. The vaccinated goat and the controls were challenged with virulent PPRV intranasally. After PPRV challenge, the three control goats showed fever, viremia and virus excretion through mucosal surfaces, whereas the vaccinated goats were fully protected against PPRV infection and replication.

West Nile Disease Epidemiology in North-West Africa: Bibliographical Review.

West Nile fever (WNF) or West Nile disease (WND) is a mosquito-borne viral disease that can affect birds, humans and horses. West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. WNV is maintained in a mosquito-bird-mosquito transmission cycle, whereas humans and horses are considered dead-end hosts. In human and horses, symptoms range from unapparent infection to mild febrile illness, meningitis, encephalitis or death. WNV has a wide geographical range that includes portions of Europe, Asia, Africa, Australia (Kunjin virus), and in North, Central and South America. Migratory birds are thought to be primarily responsible for virus dispersal, including reintroduction of WNV from endemic areas into regions that experience sporadic outbreaks (Fields Virology, 2001, Lippincott Williams and Wilkins, Philadelphia, Pennsylvania, USA, 1043-1125). The occurrence of disease in humans and animals along with birds and mosquitoes surveillance for WNV activity demonstrates that the virus range has dramatically expanded including North, Central and South America as well as Europe and countries facing the Mediterranean Basin. WND infection in humans has been reported in Morocco in 1996 (Virologie, 1, 1997, 248), in Tunisia in 2007 (Ann. N. Y. Acad., 951, 2001, 117) (Med. Trop., 61, 2001, 487) and 2003 (Epidémiologie de la fièvre West Nile, 2012, Thèse de doctorat, Université Montpellier II, Sciences et techniques du Langueduc, Montpellier, France), and in Algeria in 1994 (Rev. Sci. Tech., 31, 2012, 829). Outbreaks of equine encephalitis have been also reported in Morocco in 1996 (Bull. OIE, 11, 1996, 867), in 2003 (Emerg. Infect. Dis., 11, 2005, 306) and in 2010 (World Animal Health Information Database. WAHID, 2010). Serological evidence of WNV has been demonstrated in the three countries in many species. The aim of this review was to assess the epidemiological situation of WND in north-west Africa comprising Morocco, Algeria and Tunisia, with an updated literature review based on of human cases and equine outbreaks reports as well as serological studies in these countries.

Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome.

A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/µl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection.

No evidence for replication of a field strain of bluetongue virus serotype 1 in the blood of domestic dogs.

The potential role of domestic dogs in the long-distance transmission of bluetongue virus (BTV) is currently unproven. This study set out, through an experimental infection study, to investigate whether domestic dogs mount a viraemia post-infection with a field strain of BTV serotype 1. All six experimentally infected dogs seroconverted within 14 days and viral RNA was detected in the blood of the dogs, albeit at significantly lower levels than that seen in domestic ruminants. There was no clear evidence for viral replication in the dogs as no increase in viral RNA was observed in, and it was not possible isolate virus from, the blood of the dogs. There was however evidence for a persistence of viral RNA in the blood of the dogs, which may be evidence for a low level of replication or could be indicative of persistence of the viral inoculum.

[Seroprevalence of pandemic influenza A (H1N1)pdm09 in two regions in Morocco following the 2010-2011 influenza season]. / Séroprévalence de la grippe pandémique A(H1N1)pdm09 dans deux régions du Maroc après la saison grippale 2010-2011.

PURPOSES: To study and to compare the prevalence of infection with the virus A(H1N1)pdm 2009 in the population of two regions of Morocco compared to preexisting antibody levels. PATIENTS AND METHODS: A total of 300 and 200 serum samples were collected in the region of Rabat and Meknes respectively. Samples were collected during March to April 2011. One hundred and fifty sera, collected in 2007 from blood donors, were recovered from the blood center. The research for antibodies to influenza A(H1N1)pdm09 was performed by hemagglutination inhibition assay. RESULTS: The overall prevalence of antibodies inhibiting hemagglutination at the Rabat region (67%) is significantly higher than that of Meknes (53%) while the rate of cross-reactive antibodies was 7.3%. The subjects under 25 years from the Rabat region have infection rates as high with an odds ratio of 2.45. Individuals with comorbidities have the lowest prevalence with an odds ratio of 0.61. The rate of influenza A(H1N1)pdm09 vaccination in the Rabat region is 7%. CONCLUSIONS: Immunization rates of the Moroccan population will prevent the occurrence of large outbreaks in the year 2011 to 2012 but the persistence of a naive population justifies the continuation of vaccination against A(H1N1)pdm09.

[Epidemiological aspects of West Nile virus infection in Morocco]. / Aspects épidémiologiques de l'infection à virus West-Nile au Maroc.

Morocco has undergone three outbreaks of West Nile fever. The first, in 1996, began with a case in a horse herd in Benslimane (central region) and spread to neighboring cities (in the central and northwestern regions). The next two outbreaks appeared after identical 7-year epidemiological latency periods (in 2003 and 2010) in the same area and season. The only human case, which was fatal, occurred during the 1996 outbreak. The West Nile virus strains circulating in Morocco are closely related to other western Mediterranean strains and are characterized by high virulence in horses.

Emerging viral diseases in dromedary camels in the Southern Morocco.

During the last fifteen years, new viral diseases such Bluetongue (BT), West Nile (WN), African horse sickness (AHS), Epizootic hemorrhagic disease (EHD) and Peste des Petits Ruminants (PPR) have extended their geographic distribution and emerged in North Africa and in Europe. Camel (Camelus dromedarius) is considered as a potential vector in the transmission of some of these diseases while it is host-specific for Camelpox (CP). A serological survey was conducted on 1392 sera to estimate changes of these diseases prevalence in camels over two different time spans (2003 and 2009) and across different sites of South Morocco. Results indicate clearly that BT was circulating in camels before the first notified outbreak in Morocco (2004) with 42% positive sera in Guelmim in 2003. BT and WN prevalence's increased over time from 11 to 25% and from 10 to 13% respectively. Higher prevalence of both diseases was found in camels living in oases indicating an increased intensity of viral circulation in these sites. Camels have been tested negative for AHS, EHD and PPR viruses while higher CP prevalence has been detected in camels living in Smara, the most closed site to eastern borders (i.e. Mauritania). The seroprevalence of CP in camels at interval of 7 years increases from 23% to 37%. This survey could provide information on the possible use of camel as a sentinel animal for surveillance of emerging diseases such BT and WN.

Experimental infection of camels with bluetongue virus.

Three camels aged 4-5 years were experimentally infected with Bluetongue virus serotype 1 (BTV-1) and were observed for 75 days. No clinical signs of disease were observed throughout the experiment, however all three animals seroconverted and developed BTV-1 specific neutralising antibodies after challenge. All three camels developed a viraemia from 7 days post infection albeit at a lower level than that usually observed in experimental infections of sheep and cattle. Virus was isolated from the blood of all three animals suggesting that camels may act as a reservoir for BTV and play an important role in its transmission.

Midge-transmitted bluetongue in domestic dogs.

The role of domestic dogs in the long-distance spread of bluetongue virus (BTV) remains unproven. It is currently known that dogs are capable of being infected with BTV, can mount an antibody response to the virus and in some cases die showing severe clinical signs of disease. Infection of dogs is currently thought to be by oral ingestion of infected meat or meat products rather than through vector feeding. In this study we show that a high percentage of domestic dogs in Morocco (21%) were seropositive for BTV and, as these dogs were fed tinned commercial food only, and had no access to other meat products, the most likely source of infection was through Culicoides midges. This finding increases the chances of dogs being infected with BTV during an outbreak but their role in the onward transmission of BTV remains unproven.

[Use of the immunoenzyme test ELISA-NS3 to distinguish horses infected by African horsesickness virus from vaccinated horses]. / Utilisation de l'épreuve immuno-enzymatique ELISA-NS3 pour différencier les chevaux infectés par le virus de la peste équine des chevaux vaccinés.

A vaccination protocol involving three horses, with five repeated injections of inactivated serotype 4 African horse sickness virus, was undertaken to determine a possible threshold for the appearance of antibodies against the non-structural protein NS3. Using an indirect enzyme-linked immunosorbent assay, with the recombinant NS3 protein as an antigen, the authors detected a response to NS3 as of the second injection for the first horse and after four injections for the second horse. No response to NS3 was detected for the third horse. The results show that the inactivated vaccine is insufficiently purified to eliminate the non-structural protein NS3. Therefore using the NS3 protein as a marker did not enable differentiation between vaccinated and infected horses.

Serological and virological responses in mules and donkeys following inoculation with African horse sickness virus serotype 4.

Two groups, comprising 4 donkeys and 4 mules (group 1) and 4 donkeys and 3 mules (group 2), were used to determine the duration of viraemia and to monitor the development of antibodies following inoculation with African horse sickness virus (AHSV). One group of animals was given a single dose of attenuated AHSV serotype 4 (AHSV 4) vaccine. The second group was inoculated with a virulent field strain of AHSV 4. Both groups were subsequently challenged with the virulent field strain of AHSV 4, 51 and 58 days, respectively, after their primary inoculation. Blood and serum samples, collected on alternate days after the primary inoculations and also after subsequent challenge, were assayed for virus and antibodies. Seven of the 8 AHSV vaccinated (group 1) and 7 of the 7 AHSV inoculated (group 2) animals showed humoral antibody responses after primary inoculation. Although no infectious virus could be isolated from either group for the duration of the study, reverse transcription-PCR data obtained for the second group did show the presence of AHSV viral RNA from as early as day 5 in mules and day 9 in donkeys after the primary inoculation. Viral RNA was detected consistently up to day 47 in some animals and intermittently thereafter. There was no evidence of a second viraemia in any of the animals after challenge. The detection of specific antibodies, against AHSV 4 NS3 protein, in all animals confirmed that both donkeys and mules were infected and that the virus had replicated.

Clinical, virological and immune responses of normal and immunosuppressed donkeys (Equus asinus africanus) after inoculation with African horse sickness virus.

To elucidate the role that donkeys may play in African horse sickness virus (AHSV) persistence during inter-epizootic periods we looked for clinical signs of infection and studied the viraemia and neutralising antibody kinetics in 3 immunocompetent and 3 immunosuppressed donkeys inoculated with AHSV-4. None of the donkeys developed signs of AHS. However infectious AHSV was isolated from the blood of the immunocompetent donkeys for up to 17 days post infection (dpi) and viral antigens were detected for up to 28 dpi. Immune cells also increased significantly from 35 to 60 dpi. There was no evidence of a recrudescence of viraemia following immunosuppression of these donkeys at 90 dpi despite a decrease in the numbers of immune cells. Infectious virus was not isolated from the blood of donkeys that had been immunosuppressed, prior to AHSV inoculation. However viral antigens were detected for up to 35 dpi. The titres of AHSV-specific neutralising antibodies and the number of immune cells were also significantly lower than in immunocompetent animals. Our findings suggest that donkeys may be able to play a role in the epidemiology of AHS but the ability of vectors to become infected by feeding upon viraemic donkeys needs to be assessed before the significance of that role can be fully understood.

Application of an indirect fluorescent antibody assay for the detection of African horse sickness virus antibodies.

An indirect fluorescent antibody (IFA) technique was used to screen and quantify antibodies against African horse sickness virus (AHSV) in equine sera. Results obtained with the IFA assay were compared directly with those obtained with standard complement fixation (CF) and virus neutralisation (VN) tests using horse sera from experimental studies and samples from the field. Positive fluorescent antibody titres were detected from as early as 7 days after primary vaccination and persisted for at least six months. The IFA technique offers a clear advantage over CF tests, where the antibodies are often of shorter duration and where sera from donkeys and mules are frequently anticomplementary. The sensitivity and specificity of the IFA test compared with the VN test were 98% and 83.3%, respectively. The IFA test is rapid, relatively easy to perform and inexpensive, and can be recommended as an alternative assay for screening different species of equidae in AHSV control and surveillance programmes.

Use of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridisation for the detection and identification of African horse sickness virus nucleic acids.

A coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) for the detection of African horse sickness virus (AHSV) dsRNA, has been developed using genome segment 7 as the target template for primers. RNA from isolates of all nine AHSV serotypes were readily detected. The potential inhibitory effects of either ethylene diamine tetra acetic acid (EDTA) or heparin on the RT-PCR were eliminated by washing blood samples before lysis of the red blood cells and storage. There was a close agreement in the sensitivity and the specificity of the RT-PCR and an indirect sandwich ELISA. Confirmation of the presence of AHSV using RT-PCR and dot-blot hybridization on blood samples collected from horses experimentally infected with AHSV serotype 4 (AHSV 4) and AHSV serotype 9 (AHSV 9), was achieved within 24 hours, compared to the period of several days required for virus isolation. The RT-PCR and virus isolation methods showed similar levels of sensitivity when used for the detection of AHSV in 3 horses infected with AHSV 4, and in 2 out of 3 horses infected with a less virulent isolate of AHSV 9. Although viraemia was detected in the third horse by virus isolation, from 6 to 14 days after infection, this animal remained consistently negative by RT-PCR. Conversely, AHSV viral RNA was detected by RT-PCR in the blood of 4 donkeys and 4 mules up to 55 days after their experimental infection despite the absence of any detectable infectious virus. RT-PCR is a sensitive and rapid method for detecting AHSV nucleic acids during either the incubation period at the start of an African horse sickness (AHS) epizootic, or for epidemiological investigations in species where clinical signs may be inapparent.

[Isolation and identification of camel poxvirus in Morocco]. / Isolement et identification du virus de la variole du dromadaire au Maroc.

The virus isolated from young dromedaries during a poxvirus infection, was cultivated on Vero cells. The infection of egg chorio-allantoic membrane caused pustulous lesions (pocks). When inoculated in newborn mice, adult mice or guinea pigs the virus had no pathogenic effect. The virus presented all the characteristics of a poxvirus when observed under the electronic microscope.

[Experimental study of postvaccinal immunity to sheep-pox]. / Etude expérimentale de l'immunité anticlaveleuse post-vaccinale.

Two live virus sheep-pox vaccines have been prepared on lamb testicular cells from strains Perego and RM65. Inocuity and activity have been compared to those of a commercial vaccine on a small number of lambs. Immunogenicity has been studied through neutralizing and precipitating antibodies, and through challenge resistance (strain Held), using a protective index. Results show that Perego vaccine titrated at 10(4) DICT50 gives a reliable immunity and does not provoke severe reaction post-inoculation. RM65 vaccine of which protection index is lower, only avoid infection to generalize. Although some correlation exists between seroneutralizing titers and protective index, virulent challenge appears and seems to give better data with regards to the level of protection conferred.