Genomic surveillance of Rift Valley fever virus: from sequencing to lineage assignment.

Genetic evolution of Rift Valley fever virus (RVFV) in Africa has been shaped mainly by environmental changes such as abnormal rainfall patterns and climate change that has occurred over the last few decades. These gradual environmental changes are believed to have effected gene migration from macro (geographical) to micro (reassortment) levels. Presently, 15 lineages of RVFV have been identified to be circulating within the Sub-Saharan Africa. International trade in livestock and movement of mosquitoes are thought to be responsible for the outbreaks occurring outside endemic or enzootic regions. Virus spillover events contribute to outbreaks as was demonstrated by the largest epidemic of 1977 in Egypt. Genomic surveillance of the virus evolution is crucial in developing intervention strategies. Therefore, we have developed a computational tool for rapidly classifying and assigning lineages of the RVFV isolates. The computational method is presented both as a command line tool and a web application hosted at https://www.genomedetective.com/app/typingtool/rvfv/ . Validation of the tool has been performed on a large dataset using glycoprotein gene (Gn) and whole genome sequences of the Large (L), Medium (M) and Small (S) segments of the RVFV retrieved from the National Center for Biotechnology Information (NCBI) GenBank database. Using the Gn nucleotide sequences, the RVFV typing tool was able to correctly classify all 234 RVFV sequences at species level with 100% specificity, sensitivity and accuracy. All the sequences in lineages A (n = 10), B (n = 1), C (n = 88), D (n = 1), E (n = 3), F (n = 2), G (n = 2), H (n = 105), I (n = 2), J (n = 1), K (n = 4), L (n = 8), M (n = 1), N (n = 5) and O (n = 1) were also correctly classified at phylogenetic level. Lineage assignment using whole RVFV genome sequences (L, M and S-segments) did not achieve 100% specificity, sensitivity and accuracy for all the sequences analyzed. We further tested our tool using genomic data that we generated by sequencing 5 samples collected following a recent RVF outbreak in Kenya. All the 5 samples were assigned lineage C by both the partial (Gn) and whole genome sequence classifiers. The tool is useful in tracing the origin of outbreaks and supporting surveillance efforts.Availability: https://github.com/ajodeh-juma/rvfvtyping.

Enhanced Seroconversion to West Nile Virus Proteins in Mice by West Nile Kunjin Replicon Virus-like Particles Expressing Glycoproteins from Crimean-Congo Hemorrhagic Fever Virus.

Removal of genes coding for major parts of capsid (C), premembrane (prM), and envelope (E) proteins on the flavivirus genome aborts the production of infectious virus particles where the remaining genome forms a replicon that retains replicability in host cells. The C-prM-E proteins can also be expressed in trans with the flavivirus replicons to generate single-round infectious replicon virus-like particles (RVPs). In this study, we characterized the use of RVPs based on the Kunjin strain of WNV (WNVKUN) as a putative WNV vaccine candidate. In addition, the WNVKUN C-prM-E genes were substituted with the Crimean-Congo hemorrhagic fever virus (CCHFV) genes encoding the glycoproteins Gn and Gc to generate a WNVKUN replicon expressing the CCHFV proteins. To generate RVPs, the WNVKUN replicon was transfected into a cell line expressing the WNVKUN C-prM-E. Using immunoblotting and immunofluorescence assays, we showed that the replicon can express the CCHFV Gn and Gc proteins and the RVPs can transduce cells to express WNVKUN proteins and the CCHFV Gn and Gc proteins. Our study also revealed that these RVPs have potential as a vaccine platform with low risk of recombination as it infects cells only in one cycle. The immunization of mice with the RVPs resulted in high seroconversion to both WNV E and NS1 but limited seroconversion to CCHFV Gn and Gc proteins. Interestingly, we found that there was enhanced production of WNV E, NS1 antibodies, and neutralizing antibodies by the inclusion of CCHFV Gc and Gn into WNVKUN RVPs. Thus, this study indicates a complementary effect of the CCHFV Gn and Gc proteins on the immunogenicity by WNVKUN RVPs, which may be applied to develop a future vaccine against the WNV.

Epstein-Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus.

The Epstein-Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of a virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein-Barr virus.

A novel indirect ELISA based on glycoprotein Gn for the detection of IgG antibodies against Rift Valley fever virus in small ruminants.

Rift Valley fever virus (RVFV) is an emerging zoonotic pathogen that causes high morbidity and mortality in humans and livestock. In this paper, we describe the cloning, expression and purification of RVFV glycoprotein Gn and its application as a diagnostic antigen in an indirect ELISA for the specific detection of RVF IgG antibodies in sheep and goats. The performance of this Gn based ELISA is validated using a panel of almost 2000 field samples from sheep and goats from Mozambique, Senegal, Uganda and Yemen. All serum samples were also tested by virus neutralization test (VNT), the gold standard method for RVFV serological testing. Compared to the VNT results the Gn based ELISA proved to have an excellent sensitivity (94.56%) and specificity (95.57%). Apart from establishing this new diagnostic assay, these results also demonstrate a close correlation between the presence of RVFV Gn and neutralizing antibodies.