Complete nucleotide sequences of the large RNA genome segmentsof Main Drain and Northway viruses (family Peribunyaviridae)

AbstractThe large RNA genome segments of Main Drain virus (MDV) and Northway virus (NORV) were fully sequenced and shownto consist of 6860 and 6875 nucleotides, respectively. Sequence alignments revealed that the large RNA segment of MDVis most closely related to the corresponding region of NORV, with 76.8% nucleotide sequence identity, and the large RNAsegment of NORV is most closely related to the corresponding region of Maguari virus, with 79.1% identity

Evidence that Lokern virus (family Peribunyaviridae) is a reassortant that acquired its small and large genome segments from Main Drain virus and its medium genome segment from an undiscovered virus

Abstract Background Lokern virus (LOKV) is a poorly characterized arthropod-borne virus belonging to the genus Orthobunyavirus (family Peribunyaviridae). All viruses in this genus have tripartite, single-stranded, negative-sense RNA genomes, and the three RNA segments are designated as small, (S), medium (M) and large (L). A 559 nt. region of the M RNA segment of LOKV has been sequenced and there are no sequence data available for its S or L RNA segments. The purpose of this study was to sequence the genome of LOKV. Methods The genome of LOKV was fully sequenced by unbiased high-throughput sequencing, 5′ and 3′ rapid amplification of cDNA ends, reverse transcription-polymerase chain reaction and Sanger sequencing. Results The S and L RNA segments of LOKV consist of 952 and 6864 nt. respectively and both have 99.0% nucleotide identity with the corresponding regions of Main Drain virus (MDV). In contrast, the 4450-nt. M RNA segment has only 59.0% nucleotide identity with the corresponding region of MDV and no more than 72.7% nucleotide identity with all other M RNA segment sequences in the Genbank database. Phylogenetic data support these findings. Conclusions This study provides evidence that LOKV is a natural reassortant that acquired its S and L RNA segments from MDV and its M RNA segment from an undiscovered, and possibly extinct, virus. The availability of complete genome sequence data facilitates the accurate detection, identification and diagnosis of viruses and viral infections, and this is especially true for viruses with segmented genomes because it can be difficult or even impossible to differentiate between reassortants and their precursors when incomplete sequence data are available

Production of a chimeric flavivirus that contains the major structural glycoprotein genes of T’Ho virus in the genetic background of Zika virus

Abstract T’Ho virus is a poorly characterized orthoflavivirus most closely related to Rocio virus and Ilheus virus, two orthoflaviviruses associated with human disease, suggesting that T’Ho virus could also be a human pathogen. The genome of T’Ho virus has been sequenced but an isolate has never been recovered, impeding its phenotypic characterization. In an attempt to generate recombinant T’Ho virus, the entire viral genome was synthesized as three overlapping DNA fragments, joined by Gibson assembly, and transfected into mosquito cells. Several cell culture passages were performed, but virus was not recovered. Subsequent experiments focused on the development of a chimeric orthoflavivirus that contains the premembrane and envelope protein genes of T’Ho virus in the genetic background of Zika virus. The chimeric virus replicated in mosquito (C6/36) and vertebrate (Vero) cells, demonstrating that the major structural glycoproteins of T’Ho virus permit entry into both cell types. The chimeric virus produced plaques in Vero cells that were significantly smaller than those produced by Zika virus. The chimeric virus can potentially be used as a surrogate diagnostic reagent in place of T’Ho virus in plaque reduction neutralization tests, allowing T’Ho virus to be considered in the differential diagnosis

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

Arbovirus Surveillance near the Mexico–U.S. Border: Isolation and Sequence Analysis of Chikungunya Virus from Patients with Dengue-like Symptoms in Reynosa, Tamaulipas

A total of 1,090 residents of the city of Reynosa, Tamaulipas, on the Mexico–U.S. border presented at hospitals and clinics of the Secretariat of Health, Mexico, in 2015 with symptoms characteristic of dengue. Dengue virus (DENV) antigen was detected by enzyme-linked immunosorbent assay in acute sera from 134 (12.3%) patients. Sera from select patients (N = 34) were also tested for chikungunya virus (CHIKV) RNA by quantitative reverse transcription–polymerase chain reaction. Thirteen (38.2%) patients, including five DENV antigen-positive patients, were positive. Sera from three CHIKV RNA-positive patients were further assayed by virus isolation in cell culture and CHIKV was recovered on each occasion. The genome of one isolate and structural genes of the other two isolates were sequenced. In conclusion, we present evidence of CHIKV and DENV coinfections in patients who live near the Mexico–U.S. border and provide the first genome sequence of a CHIKV isolate from northern Mexico

Surveillance for Flaviviruses Near the Mexico-U.S. Border: Co-circulation of Dengue Virus Serotypes 1, 2, and 3 and West Nile Virus in Tamaulipas, Northern Mexico, 2014-2016

A clinical, serological, and molecular investigation was performed to determine the presence of dengue virus (DENV) and other flaviviruses among residents of the city of Reynosa, Tamaulipas, on the Mexico–U.S. border in 2014–2016. The sample population consisted of 2,355 patients with suspected dengue, in addition to 346 asymptomatic individuals recruited during a household-based epidemiological investigation designed to identify flavivirus seroconversions. Sera were collected from patients with suspected dengue in the acute phase of illness and from asymptomatic individuals at enrollment and every 5–7 months for 19 months. Sera from suspected dengue patients were tested for DENV antigen by enzyme-linked immunosorbent assay (ELISA), and select antigen-positive sera were further tested using a serotype-specific, quantitative reverse transcription–polymerase chain reaction. Sera from the household cohort were tested for flavivirus-reactive antibodies by immunoglobulin (Ig) M and IgG ELISAs using DENV antigen. A total of 418 (17.7%) patients with suspected dengue had laboratory-confirmed DENV infections, including 82 patients who were positive for DENV RNA. The most frequently detected serotype was DENV-1 (61 patients), followed by DENV-2 (16 patients) and DENV-3 (five patients). A total of 217 (62.7%) asymptomatic individuals had flavivirus-reactive antibodies at enrollment, and nine flavivirus-naïve individuals seroconverted. Sera from a subset of dengue patients and household participants, including all those who seroconverted, were further tested by plaque reduction neutralization test, resulting in the detection of antibodies to DENV-1, DENV-2, and West Nile virus. In summary, we provide evidence for the co-circulation of multiple flaviviruses in Reynosa, Tamaulipas, on the Mexico–U.S. border

Detection of Antibodies to Lokern, Main Drain, St. Louis Encephalitis, and West Nile Viruses in Vertebrate Animals in Chihuahua, Guerrero, and Michoacán, México

We conducted serologic surveillance for flaviviruses and orthobunyaviruses in vertebrate animals in Mexico in 2018–2019. Sera were collected from 856 vertebrate animals, including 323 dogs, 223 horses, and 121 cows, from 16 species. The animals were from 3 states: Chihuahua in northwest Mexico (704 animals) and Guerrero and Michoacan on the Pacific Coast (27 and 125 animals, respectively). Sera were assayed by plaque reduction neutralization test using four flaviviruses (dengue type 2, St. Louis encephalitis, West Nile, and Zika viruses) and six orthobunyaviruses from the Bunyamwera (BUN) serogroup (Cache Valley, Lokern, Main Drain, Northway, Potosi, and Tensaw viruses). Antibodies to West Nile virus (WNV) were detected in 154 animals of 9 species, including 89 (39.9%) horses, 3 (21.4%) Indian peafowl, and 41 (12.7%) dogs. Antibodies to St. Louis encephalitis virus (SLEV) were detected in seven animals, including three (0.9%) dogs. Antibodies to Lokern virus (LOKV) were detected in 22 animals: 19 (8.5%) horses, 2 (1.7%) cows, and a dog (0.3%). Antibodies to Main Drain virus (MDV) were detected in three (1.3%) horses. WNV and LOKV activity was detected in all three states, SLEV activity was detected in Chihuahua and Michoacan, and MDV activity was detected in Chihuahua. None of the animals was seropositive for Cache Valley virus, the most common and widely distributed BUN serogroup virus in North America. In conclusion, we provide serologic evidence that select flaviviruses and BUN serogroup viruses infect vertebrate animals in Chihuahua, Guerrero, and Michoacan. We also provide the first evidence of LOKV and MDV activity in Mexico