Real-Time RT-PCR Assays for Detection and Genotyping of West Nile Virus Lineages Circulating in Africa

West Nile virus (WNV) is an emerging arbovirus, circulating worldwide between birds and mosquitoes, which impacts human and animal health. Since the mid-1990s, WNV outbreaks have emerged in Europe and America and represent currently the primary cause of encephalitis in the United States. WNV exhibits a great genetic diversity with at least eight different lineages circulating in the world, and four (1, 2, Koutango, and putative new) are present in Africa. These different WNV lineages are not readily differentiated by serology, and thus, rapid molecular tools are required for diagnostic. We developed here real-time RT-PCR assays for detection and genotyping of African WNV lineages. The specificity of the assays was tested using other flaviviruses circulating in Africa. The sensitivity was determined by testing serial 10-fold dilutions of viruses and RNA standards. The assays provided good specificity and sensitivity and the analytical detection limit was 10 copies/ reaction. The RT-PCR assays allowed the detection and genotyping of all WNV isolates in culture medium, human serum, and vertebrate tissues, as well as in field and experimental mosquito samples. Comparing the ratios of genome copy number/infectious virion (plaque-forming units), our study finally revealed new insight on the replication of these different WNV lineages in mosquito cells. Our RT-PCR assays are the first ones allowing the genotyping of all WNV African variants, and this may have important applications in surveillance and epidemiology in Africa and also for monitoring of their emergence in Europe and other continents

Comparative full length genome sequence analysis of usutu virus isolates from Africa

Background: Usutu virus (USUV), a flavivirus belonging to the Japanese encephalitis serocomplex, was identified in South Africa in 1959 and reported for the first time in Europe in 2001. To date, full length genome sequences have been available only for the reference strain from South Africa and a single isolate from each of Austria, Hungary, and Italy. Methods: We sequenced four USUV isolates from Senegal and the Central African Republic (CAR) between 1974 and 2007 and compared the sequence data to USUV strains from Austria, Hungary, Italy, and South Africa using a Bayesian Markov chain Monte Carlo method. We further clarified the taxonomic status of a USUV strain isolated in CAR in 1969 and proposed earlier as a subtype of USUV due to an asymetric serological cross-reactivity with USUV reference strain. Results: A comparison of the four newly obtained USUV sequences with those from SouthAfrica_1959, Vienna_2001, Budapest_2005, and Italy_2009 revealed that they are all 96-99% and 99% similar at the nucleotide and amino acid levels, respectively. The phylogenetic relationships between these sequences indicated that a strain isolated in Senegal in 1993 is most closely related to the USUV strains detected in Europe. Analysis of a strain isolated from a human in CAR in 1981 (CAR_1981) revealed the presence of specific amino acid substitutions and a deletion in the 3′ noncoding region. This is the first fully sequenced human USUV isolate. The putative USUV subtype, CAR_1969, was 81% and 94% identical at the nucleotide and amino acid levels, respectively, compared to the other USUV strains. Our phylogenetic analyses support the serological identification of CAR_1969 as a subtype of USUV. Conclusions: In this study, we investigate the genetic diversity of USUV in Africa and the phylogenetic relationship of isolates from Africa and Europe for the first time. The results suggest a low genetic diversity within USUV, the existence of a distinct USUV subtype strain, and support the hypothesis that USUV was introduced to Europe from Africa. Further sequencing and analysis of USUV isolates from other African countries would contribute to a better understanding of its genetic diversity and geographic distribution

Les syncytines

Bien que l’infection par un rétrovirus ait généralement un effet délétère sur l’individu, la découverte de gènes de rétrovirus intégrés au génome depuis des millions d’années, et remplissant des fonctions physiologiques essentielles, révèle que ces éléments peuvent également jouer un rôle fondamental dans la biologie de leur hôte. Nous présentons ici l’exemple des syncytines, glycoprotéines d’enveloppe codées par des rétrovirus endogènes, capables d’induire la fusion cellulaire et nécessaires à la formation de l’architecture placentaire. Nous montrons que la capture de ces gènes s’est produite plusieurs fois au cours de l’évolution chez les mammifères euthériens et émettons l’hypothèse selon laquelle de tels événements auraient permis l’évolution, et éventuellement l’émergence, de la structure placentaire

Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new "syncytin" in a third order of mammals

Abstract Background Syncytins are envelope genes of retroviral origin that have been co-opted by the host to mediate a specialized function in placentation. Two of these genes have already been identified in primates, as well as two distinct, non orthologous genes in rodents. Results Here we identified within the rabbit Oryctolagus cuniculus-which belongs to the lagomorpha order- an envelope (env) gene of retroviral origin with the characteristic features of a bona fide syncytin, that we named syncytin-Ory1. An in silico search for full-length env genes with an uninterrupted open reading frame within the rabbit genome first identified two candidate genes that were tested for their specific expression in the placenta by quantitative RT-PCR of RNA isolated from a large set of tissues. This resulted in the identification of an env gene with placenta-specific expression and belonging to a family of endogenous retroelements present at a limited copy number in the rabbit genome. Functional characterization of the identified placenta-expressed env gene after cloning in a CMV-driven expression vector and transient transfection experiments, demonstrated both fusogenic activity in an ex vivo cell-cell fusion assay and infectivity of pseudotypes. The receptor for the rabbit syncytin-Ory1 was found to be the same as that for human syncytin-1, i.e. the previously identified ASCT2 transporter. This was demonstrated by a co-culture fusion assay between hamster A23 cells transduced with an expression vector for ASCT2 and A23 cells transduced with syncytin-Ory1. Finally, in situ hybridization of rabbit placenta sections with a syncytin-Ory1 probe revealed specific expression at the level of the junctional zone between the placental lobe and the maternal decidua, where the invading syncytial fetal tissue contacts the maternal decidua to form the labyrinth, consistent with a role in the formation of the syncytiotrophoblast. The syncytin-Ory1 gene is found in Leporidae but not in Ochotonidae, and should therefore have entered the lagomorpha order 12-30 million years ago. Conclusion The identification of a novel syncytin gene within a third order of mammals displaying syncytiotrophoblast formation during placentation strongly supports the notion that on several occasions retroviral infections have resulted in the independent capture of genes that have been positively selected for a convergent physiological role.</p