Genome Analysis of Rift Valley Fever Virus, Mayotte

As further confirmation of a first human case of Rift Valley fever in 2007 in Comoros, we isolated Rift Valley fever virus in suspected human cases. These viruses are genetically closely linked to the 2006–2007 isolates from Kenya.

I dentifi ed during the 1930s in Kenya, Rift Valley fever (RVF) is a zoonotic disease that circulates in many African countries and in the Arabian Peninsula (1,2). RVF virus (RVFV) epizootics are characterized by large sweeping abortion storms and substantial death rates in adult livestock (primarily sheep, goats, and cattle), with the death rate for newborn animals approaching 100% (3). Humans typically are infected by bites of infected mosquitoes or by percutaneous or aerosol exposure to contaminated fl uids from infected animals. In most human cases, RVF is characterized by a self-limiting febrile illness (2-5 days), which progresses to more serious complications (hepatitis, encephalitis, blindness, or hemorrhagic syndrome) in only 1%-2% of infected persons (4,5). A large epizootic-epidemic occurred during 2006-2007 on the eastern African mainland, predominantly in Kenya (6) and Madagascar, during 2 successive rainy seasons (7).
In July 2007, a 12-year-old boy with a 2-month history of severe encephalitis was transferred from the Grande Comore, Union of the Comoros, to Mayotte (8,9). RVF infection was confi rmed by IgM serologic analysis. Because of the proximity of Comoros and Mayotte, the RVF situation among humans in Mayotte was assessed. In serum samples from 7 humans with dengue-like syndromes, RVFV IgM or RVFV RNA was detected. We report the isolations and full sequence analysis of 2 RVF viral isolates from these serum specimens.

The Study
During January-April 2007, seven patients native to Mayotte were admitted to the hospital for severe denguelike syndromes. Two patients were RVF seropositive by IgM and IgG, and the other 5 were positive by RVFVspecifi c reverse transcription PCR (RT-PCR) as detailed in Sissoko et al. (9). As described for other viruses, we used in-house IgM-capture enzyme immunoassays and in-house direct detection for IgG by using microplates coated with RVFV antigen and specifi c binding by using a peroxidaselabeled goat anti-human IgG conjugate (10).
RVFV isolates were obtained on Vero E6 cells from the serum of 2 hospitalized patients (serum collected on February 21 and March 20, 2008). RNA extracted by using the RNaid Kit (Qbiogene, Carlsbad, CA, USA) was reverse transcribed by PCR and amplifi ed by using SuperScript One-Step RT-PCR with platinum Taq kit (Invitrogen, San Diego, CA, USA) with primers targeting the small, medium, and large segments (adapted from [11]). Overlapping RT-PCR fragments were purifi ed by ultrafi ltration. Sequencing reactions were performed by using the Big Dye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequence chromatograms from both strands were obtained on automated sequence analyzer ABI3730XL (Applied Biosystems). For sequence analysis, contig assemblies and sequence alignments were performed by using BioNumerics version 6.6 (Applied-Maths, Sint-Martens-Latem, Belgium).
We used 2 methods for phylogenetic reconstruction: maximum likelihood and the Bayesian inference. The best models of nucleotide substitution for each dataset were selected from the uncorrected and corrected Akaike Information Criterion, the Hannan and Quinn performancebased decision theory and Bayesian Information Criterion of Jmodeltest version 0.1 and TREEFINDER version October 2008 (Munich, Germany, distributed by its author at www.treefi nder.de). The consensus substitution models proposed by the different software packages were selected for further analyses. Comparison of the maximumlikelihood method implemented by the TREEFINDER program with others was performed on the small, medium, and large segments by using the neighbor-joining and maximum parsimony methods from Mega5 software and the Bayesian approach by using MrBayes version 3.0B4 for phylogenetic reconstruction with random starting trees and run for 2,000,000 generations, sampling the Markov chains at intervals of 100 generations (12,13). Branch support values were obtained by using nonparametric bootstrapping with 1,000 resampling for PhyML and TREEFINDER and the posterior probabilities for the Bayesian approach estimated on 10,000 samples (sample frequency set to every 100th generation by using the Markov Chain Monte Carlo sampling). We compared topologies of the Because maximum-likelihood and Bayesian tree topologies obtained for the 3 segments were similar, only the small segment is presented. The Figure shows the Bayesian tree topology based on all RVFV small segments, with the HB29 phlebovirus from the People's Republic of China as an outgroup. Tree topologies are consistent with those generated in previous work (7,11). The reliability of the phylogenetic trees was confi rmed by performing bootstrap analysis. The Kenya 1 and Kenya 2 lineages clustered together with an overall bootstrap value of 92% but with sublineage bootstrap values of 56%-100%.

Conclusions
The The recent data published on RVFV Malagasy strains (7,14) support an epidemic cycle with introduction of the virus from outbreaks on mainland eastern Africa rather than an enzootic cycle in Madagascar. RVFV has been isolated from at least 40 species of mosquitoes in 8 genera.  identify all potential vector species in the island and animal surveys need to be conducted to help detect RVF at early stages to gain a better understanding of the ecologic and climatic factors that favor RVFV dissemination. These assessments will help in the development of appropriate control measures to better predict and respond to potential RVF outbreaks.