Sequencing of ZIKV genomes directly from Ae. aegypti and Cx. quinquefasciatus mosquitoes collected during the 2015-16 epidemics in Recife.

Zika virus (ZIKV) is a negative sense RNA virus from the Flaviviridae family, which was relatively unknown until the first human epidemic in Micronesia, in 2007. Since then, it spread to French Polynesia and the Americas. Recife, the capital of Pernambuco state and epicenter of the Zika epidemic in Brazil, experienced a large number of microcephaly cases and other congenital abnormalities associated to the ZIKV infection from, 2015 to 16. Evidences suggest that both Aedes aegypti and Culex quinquefasciatus mosquitoes from Recife are capable of replicating and transmitting the virus. Here, we conducted high throughput sequencing of ZIKV genomes directly from Ae. aegypti and Cx. quinquefasciatus mosquitoes collected during the ZIKV epidemics in Recife, in order to investigate the variability and evolution of the virus. We obtained 11 draft ZIKV genomes derived from 5 pools from each Ae. aegypti and Cx. quinquefasciatus species. Genome coverage breadth ranged from 16 to 100% and average depth from 45 to 46,584×. Two of these genomes were obtained from pools of Cx. quinquefasciatus females with no sign of blood in the abdomen. Amino acid substitutions found here were not species-specific. In addition, molecular clock dating estimated that ZIKV draft genomes obtained here were co-circulating in other regions of the country during the epidemics. Overall results highlight that viral mutations and even minor variants can be detected in genomes directly sequenced from mosquito samples and insights about natural viral genomic variability and viral evolution can be useful when designing tools for mosquito control programs.

Persistence of chikungunya ECSA genotype and local outbreak in an upper medium class neighborhood in Northeast Brazil.

The chikungunya East/Central/South/Africa virus lineage (CHIKV-ECSA) was first detected in Brazil in the municipality of Feira de Santana (FS) by mid 2014. Following that, a large number of CHIKV cases have been notified in FS, which is the second-most populous city in Bahia state, northeastern Brazil, and plays an important role on the spread to other Brazilian states due to climate conditions and the abundance of competent vectors. To better understand CHIKV dynamics in Bahia state, we generated 5 complete genome sequences from a local outbreak raised in Serraria Brasil, a neighbourhood in FS, by next-generation sequencing using Illumina approach. Phylogenetic reconstructions revealed that the new FS genomes belongs to the ECSA genotype and falls within a single strongly supported monophyletic clade that includes other older CHIKV sequences from the same location, suggesting the persistence of the virus during distinct epidemic seasons. We also performed minor variants analysis and found a small number of SNPs per sample (b_29L and e_45SR = 16 SNPs, c_29SR = 29 and d_45PL and f_45FL = 21 SNPs). Out of the 93 SNPs found, 71 are synonymous, 21 are non-synonymous and one generated a stop codon. Although those mutations are not related to the increase of virus replication and/or infectivity, some SNPs were found in non-structural proteins which may have an effect on viral evasion from the mammal immunological system. These findings reinforce the needing of further studies on those variants and of continued genomic surveillance strategies to track viral adaptations and to monitor CHIKV epidemics for improved public health control.

Genome sequencing reveals coinfection by multiple chikungunya virus genotypes in a recent outbreak in Brazil.

Chikungunya virus (CHIKV) is an RNA virus from the Togaviridae family transmitted by mosquitoes in both sylvatic and urban cycles. In humans, CHIKV infection leads to a febrile illness, denominated Chikungunya fever (CHIKF), commonly associated with more intense and debilitating outcomes. CHIKV arrived in Brazil in 2014 through two independent introductions: the Asian/Caribbean genotype entered through the North region and the African ECSA genotype was imported through the Northeast region. Following their initial introduction, both genotypes established their urban cycle among large naive human populations causing several outbreaks in the Americas. Here, we sequenced CHIKV genomes from a recent outbreak in the Northeast region of Brazil, employing an in-house developed Next-Generation Sequencing (NGS) protocol capable of directly detecting multiple known CHIKV genotypes from clinical positive samples. Our results demonstrate that both Asian/Caribbean and ECSA genotypes expanded their ranges, reaching cocirculation in the Northeast region of Brazil. In addition, our NGS data supports the findings of simultaneous infection by these two genotypes, suggesting that coinfection might be more common than previously thought in highly endemic areas. Future efforts to understand CHIKV epidemiology should thus take into consideration the possibility of coinfection by different genotypes in the human population.

Zika virus detection, isolation and genome sequencing through Culicidae sampling during the epidemic in Vitória, Espírito Santo, Brazil.

BACKGROUND: Zika virus (ZIKV) has been isolated from many mosquito species in nature, but it is believed that the main vectors in urban environments are species of the genus Aedes. Here, we detected and isolated ZIKV in samples from Aedes aegypti, Aedes taeniorhynchus and Culex quinquefasciatus, collected during the Zika epidemic in Vitória, southeast Brazil. Using quantitative real-time polymerase chain reaction, ZIKV detection was performed in mosquito samples collected from February to April 2016. RESULTS: Overall, six pools of mosquitoes were positive for ZIKV: four of Cx. quinquefasciatus, one of Ae. aegypti and one of Ae. taeniorhynchus. Their genomes were sequenced. CONCLUSIONS: These results support and strengthen the hypothesis that other mosquito species can also be involved in ZIKV transmission.