Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico.

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.The authors thank Valeria Bussetti for expert technical assistance. This study was supported by the National Institutes of Health (awards 5R21AI067281, AI057158, 5R21AI067281 and AI088647), the United States Department of Defense and an intramural grant from Iowa State University. AEF is supported by a grant from the Wellcome Trust (award 106207).This is the final version of the article. It first appeared from the Microbiology Society via http://dx.doi.org/10.1099/jgv.0.00042

Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.The authors thank Valeria Bussetti for expert technical assistance. This study was supported by the National Institutes of Health (awards 5R21AI067281, AI057158, 5R21AI067281 and AI088647), the United States Department of Defense and an intramural grant from Iowa State University. AEF is supported by a grant from the Wellcome Trust (award 106207).This is the final version of the article. It first appeared from the Microbiology Society via http://dx.doi.org/10.1099/jgv.0.00042

Detection of RNA from a Novel West Nile-like Virus and High Prevalence of an Insect-specific Flavivirus in Mosquitoes in the Yucatan Peninsula of Mexico

As part of our ongoing surveillance efforts for West Nile virus (WNV) in the Yucatan Peninsula of Mexico, 96,687 mosquitoes collected from January through December 2007 were assayed by virus isolation in mammalian cells. Three mosquito pools caused cytopathic effect. Two isolates were orthobunyaviruses (Cache Valley virus and Kairi virus) and the identity of the third infectious agent was not determined. A subset of mosquitoes was also tested by reverse transcription-polymerase chain reaction (RT-PCR) using WNV-, flavivirus-, alphavirus-, and orthobunyavirus-specific primers. A total of 7,009 Culex quinquefasciatus in 210 pools were analyzed. Flavivirus RNA was detected in 146 (70%) pools, and all PCR products were sequenced. The nucleotide sequence of one PCR product was most closely related (71-73% identity) with homologous regions of several other flaviviruses, including WNV, St. Louis encephalitis virus, and Ilheus virus. These data suggest that a novel flavivirus (tentatively named T\u27Ho virus) is present in Mexico. The other 145 PCR products correspond to Culex flavivirus, an insect-specific flavivirus first isolated in Japan in 2003. Culex flavivirus was isolated in mosquito cells from approximately one in four homogenates tested. The genomic sequence of one isolate was determined. Surprisingly, heterogeneous sequences were identified at the distal end of the 5\u27 untranslated region

Universal primers for the amplification and sequence analysis pf actin-1 from diverse mosquito species

We report the development of universal primers for the reverse-transcription polymerase chain reaction (RT-PCR) amplification and nucleotide sequence analysis of actin cDNAs from taxonomically diverse mosquito species. Primers specific to conserved regions of the invertebrate actin-1 gene were designed after actin cDNA sequences of Anopheles gambiae, Bombyx mori, Drosophila melanogaster, and Caenorhabditis elegans. The efficacy of these primers was determined by RT-PCR with the use of total RNA from mosquitoes belonging to 30 species and 8 genera (Aedes, Anopheles, Culex, Deinocerites, Mansonia, Psorophora, Toxorhynchites, and Wyeomyia). The RT-PCR products were sequenced, and sequence data were used to design additional primers. One primer pair, denoted as Act-2F (5′-ATGGTCGGYATGGGNCAGAAGGACTC-3′) and Act-8R (5′-GATTCCATACCCAGGAAG-GADGG-3′), successfully amplified an RT-PCR product of the expected size (683-nt) in all mosquito spp. tested. We propose that this primer pair can be used as an internal control to test the quality of RNA from mosquitoes collected in vector surveillance studies. These primers can also be used in molecular experiments in which the detection, amplification or silencing of a ubiquitously expressed mosquito housekeeping gene is necessary. Sequence and phylogenetic data are also presented in this report

An Updated Review of the Invasive Aedes albopictus in the Americas; Geographical Distribution, Host Feeding Patterns, Arbovirus Infection, and the Potential for Vertical Transmission of Dengue Virus

Aedes (Stegomyia) albopictus is a mosquito native to Southeast Asia. Currently, it has a wide distribution in America, where natural infection with arboviruses of medical and veterinary importance has been reported. In spite of their importance in the transmission of endemic arbovirus, the basic information of parameters affecting their vectorial capacity is poorly investigated. The aim of the work was to update the distribution range of Ae. albopictus in the Americas, review the blood-feeding patterns, and compare the minimum infection rate (MIR) of the Dengue virus (DENV) between studies of vertical and horizontal transmission. The current distribution of Ae. albopictus encompasses 21 countries in the Americas. An extensive review has been conducted for the blood-feeding patterns of Ae. albopictus. The results suggest that the mosquito is capable of feeding on 16 species of mammals and five species of avian. Humans, dogs, and rats are the most common hosts. Eight arboviruses with the potential to infect humans and animals have been isolated in Ae. albopictus. In the United States of America (USA), Eastern equine encephalitis virus, Keystone virus, La Crosse Virus, West Nile virus, and Cache Valley virus were isolated in the Asian mosquito. In Brazil, Mexico, Colombia, and Costa Rica, DENV (all serotypes) has been frequently identified in field-caught Ae. albopictus. Overall, the estimated MIR in Ae. albopictus infected with DENV is similar between horizontal (10.95) and vertical transmission (8.28). However, in vertical transmission, there is a difference in the MIR values if the DENV is identified from larvae or adults (males and females emerged from a collection of eggs or larvae). MIR estimated from larvae is 14.04 and MIR estimated in adults is 4.04. In conclusion, it has to be highlighted that Ae. albopictus is an invasive mosquito with wide phenotypic plasticity to adapt to broad and new areas, it is highly efficient to transmit the DENV horizontally and vertically, it can participate in the inter-endemic transmission of the dengue disease, and it can spread zoonotic arboviruses across forest and urban settings

Study of Aedes aegypti population with emphasis on the gonotrophic cycle length and identification of arboviruses: implications for vector management in cemeteries

Aedes aegypti is the vector of the arboviruses causing dengue, chikungunya and zika infections in Mexico. However, its presence in public places has not been fully evaluated. In a cemetery from Merida, Yucatan, Mexico, the productivity of Ae. aegypti, the gonotrophic cycle, and the presence of Ae. aegypti females infected with arboviruses were evaluated. Immature and adult mosquitoes were inspected every two months between April 2016 to June 2017. For the gonotrophic cycle length, the daily pattern of total and parous female ratio was registered and was analyzed using time series analysis. Ae. aegypti females were sorted into pools and assayed for flavivirus RNA by RT-PCR and Sanger sequencing. Aedes aegypti immatures represented 82.86% (8,627/10,411) of the collection. In total, 1,648 Ae. aegypti females were sorted into 166 pools. Two pools were positive; one for dengue virus (DENV-1) and the other for zika virus (ZIKV). The phylogenetic analysis revealed that the DENV-1 is more closely related to isolates from Brazil. While ZIKV is more closely related to the Asian lineage, which were isolates from Guatemala and Mexico. We report some evidence of vertical transmission of DENV-1 in nulliparous females of Ae. aegypti. The gonotrophic cycle was four and three days in the rainy and dry season, respectively. The cemetery of Merida is an important focus of Ae. aegypti proliferation, and these environments may play a role in arboviruses transmission; probably limiting the efficacy of attempts to suppress the presence of mosquitoes in domestic environments

Detection of novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico using metagenomics and characterization of their in vitro host ranges.

A metagenomics approach was used to detect novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico. A total of 1359 mosquitoes of 7 species and 5 genera (Aedes, Anopheles, Culex, Mansonia and Psorophora) were sorted into 37 pools, homogenized and inoculated onto monolayers of Aedes albopictus (C6/36) cells. A second blind passage was performed and then total RNA was extracted and analysed by RNA-seq. Two novel viruses, designated Uxmal virus and Mayapan virus, were identified. Uxmal virus was isolated from three pools of Aedes (Ochlerotatus) taeniorhynchus and phylogenetic data indicate that it should be classified within the recently proposed taxon Negevirus. Mayapan virus was recovered from two pools of Psorophora ferox and is most closely related to unclassified Nodaviridae-like viruses. Two recognized viruses were also detected: Culex flavivirus (family Flaviviridae) and Houston virus (family Mesoniviridae), with one and two isolates being recovered, respectively. The in vitro host ranges of all four viruses were determined by assessing their replicative abilities in cell lines of avian, human, monkey, hamster, murine, lepidopteran and mosquito (Aedes, Anopheles and Culex) origin, revealing that all viruses possess vertebrate replication-incompetent phenotypes. In conclusion, we report the isolation of both novel and recognized RNA viruses from mosquitoes collected in Mexico, and add to the growing plethora of viruses discovered recently through the use of metagenomics

Discovery of a novel Tymoviridae-like virus in mosquitoes from Mexico.

A novel Tymoviridae-like virus, designated Ek Balam virus, was isolated from male Culex quinquefasciatus mosquitoes collected in Yucatan, Mexico. The genome was fully sequenced and shown to have no more than 69% nt sequence identity to its closest known relative. Mosquito cells were permissive to Ek Balam virus replication, but mammalian and avian cells were refractory, suggesting that vertebrates are not involved in the maintenance of the virus in nature