Complete genome sequence of T'Ho virus, a novel putative flavivirus from the Yucatan Peninsula of Mexico.

BACKGROUND: We previously reported the discovery of a novel, putative flavivirus designated T'Ho virus in Culex quinquefasciatus mosquitoes in the Yucatan Peninsula of Mexico. A 1358-nt region of the NS5 gene was amplified and sequenced but an isolate was not recovered. RESULTS: The complete genome of T'Ho virus was sequenced using a combination of unbiased high-throughput sequencing, 5' and 3' rapid amplification of cDNA ends, reverse transcription-polymerase chain reaction and Sanger sequencing. The genome contains a single open reading frame of 10,284 nt which is flanked by 5' and 3' untranslated regions of 97 and 556-nt, respectively. Genome sequence alignments revealed that T'Ho virus is most closely related to Rocio virus (67.4% nucleotide identity) and Ilheus virus (65.9%), both of which belong to the Ntaya group, followed by other Ntaya group viruses (58.8-63.3%) and Japanese encephalitis group viruses (62.0-63.7%). Phylogenetic inference is in agreement with these findings. CONCLUSIONS: This study furthers our understanding of flavivirus genetics, phylogeny and diagnostics. Because the two closest known relatives of T'Ho virus are human pathogens, T'Ho virus could be an unrecognized cause of human disease. It is therefore important that future studies investigate the public health significance of this virus.

Modelling dengue fever risk in the State of Yucatan, Mexico using regional-scale satellite-derived sea surface temperature.

Accurately predicting vector-borne diseases, such as dengue fever, is essential for communities worldwide. Changes in environmental parameters such as precipitation, air temperature, and humidity are known to influence dengue fever dynamics. Furthermore, previous studies have shown how oceanographic variables, such as El Niño Southern Oscillation (ENSO)-related sea surface temperature from the Pacific Ocean, influences dengue fever in the Americas. However, literature is lacking on the use of regional-scale satellite-derived sea surface temperature (SST) to assess its relationship with dengue fever in coastal areas. Data on confirmed dengue cases, demographics, precipitation, and air temperature were collected. Incidence of weekly dengue cases was examined. Stepwise multiple regression analyses (AIC model selection) were used to assess which environmental variables best explained increased dengue incidence rates. SST, minimum air temperature, precipitation, and humidity substantially explained 42% of the observed variation (r2=0.42). Infectious diseases are characterized by the influence of past cases on current cases and results show that previous dengue cases alone explained 89% of the variation. Ordinary least-squares analyses showed a positive trend of 0.20±0.03°C in SST from 2006 to 2015. An important element of this study is to help develop strategic recommendations for public health officials in Mexico by providing a simple early warning capability for dengue incidence.

Chikungunya Virus in Febrile Humans and Aedes aegypti Mosquitoes, Yucatan, Mexico.

Chikungunya virus (CHIKV) was isolated from 12 febrile humans in Yucatan, Mexico, in 2015. One patient was co-infected with dengue virus type 1. Two additional CHIKV isolates were obtained from Aedes aegypti mosquitoes collected in the homes of patients. Phylogenetic analysis showed that the CHIKV isolates belong to the Asian lineage.

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.

Orthobunyaviruses, a common cause of infection of livestock in the Yucatan peninsula of Mexico.

To determine the seroprevalence of selected orthobunyaviruses in livestock in the Yucatan Peninsula of Mexico, a serologic investigation was performed using serum samples from 256 domestic animals (182 horses, 31 sheep, 1 dog, 37 chickens, and 5 turkeys). All serum samples were examined by plaque reduction neutralization test using Cache Valley virus (CVV), Cholul virus (CHLV), South River virus (SOURV), Kairi virus, Maguari virus, and Wyeomyia virus. Of the 182 horses, 60 (33.0%) were seropositive for CHLV, 48 (26.4%) were seropositive for CVV, 1 (0.5%) was seropositive for SOURV, 60 (33.0%) had antibodies to an undetermined orthobunyavirus, and 13 (7.1%) were negative for orthobunyavirus-specific antibody. Of the 31 sheep, 6 (19.3%) were seropositive for CHLV, 3 (9.7%) were seropositive for CVV, 4 (12.9%) were seropositive for SOURV, 16 (51.6%) had antibodies to an undetermined orthobunyavirus, and 2 (6.5%) were negative for orthobunyavirus-specific antibody. The single dog was seropositive for SOURV. Four (11%) chickens had antibodies to an undetermined orthobunyavirus, and 1 (20%) turkey was seropositive for CHLV. These data indicate that orthobunyaviruses commonly infect livestock in the Yucatan Peninsula.

Orthobunyavirus antibodies in humans, Yucatan Peninsula, Mexico.

We performed a serologic investigation to determine whether orthobunyaviruses commonly infect humans in the Yucatan Peninsula of Mexico. Orthobunyavirus-specific antibodies were detected by plaque reduction neutralization test in 146 (18%) of 823 persons tested. Further studies are needed to determine health risks for humans from this potentially deadly group of viruses.

Nucleotide sequencing and serologic analysis of Cache Valley virus isolates from the Yucatan Peninsula of Mexico.

Nucleotide sequencing was performed on part of the medium and large genome segments of 17 Cache Valley virus (CVV) isolates from the Yucatan Peninsula of Mexico. Alignment of these sequences to all other sequences in the Genbank database revealed that they have greatest nucleotide identity (97-98 %) with the equivalent regions of Tlacotalpan virus (TLAV), which is considered to be a variety of CVV. Next, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice that had been inoculated with a representative isolate from the Yucatan Peninsula (CVV-478) or the prototype TLAV isolate (61-D-240). The PRNT titers exhibited a twofold difference in one direction and no difference in the other direction suggesting that CVV-478 and 61-D-240 belong to the same CVV subtype. In conclusion, we demonstrate that the CVV isolates from the Yucatan Peninsula of Mexico are genetically and antigenically similar to the prototype TLAV isolate.

Identification of a novel subtype of South River virus (family Bunyaviridae).

We previously reported the isolation of South River virus (SORV) from a pool of mosquitoes collected in the Yucatan Peninsula of Mexico (Farfan-Ale et al. in Vector Borne Zoonotic Dis 10:777-783, 5). The isolate (designated SORV-252) was identified as SORV after a 197-nucleotide region of its small RNA genome segment was sequenced. In the present study, the complete small and medium RNA genome segments and part of the large RNA genome segment of SORV-252 were sequenced and shown to have 92%, 85% and 90% nucleotide sequence identity, respectively, to the homologous regions of the prototype SORV isolate (NJO-94F). To determine the antigenic relationship between SORV-252 and NJO-94F, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice inoculated with these viruses. SORV-252 and NJO-94F were distinguishable in the cross-neutralization assays; there was a twofold difference in the PRNT titers in one direction and a fourfold difference in the other direction, suggesting that SORV-252 represents a novel subtype of SORV. Additionally, SORV-252 and NJO-94F have distinct plaque morphologies in African green monkey kidney (Vero) cells. In conclusion, we provide evidence that a novel subtype of SORV is present in the Yucatan Peninsula of Mexico.

Sequence and phylogenetic data indicate that an orthobunyavirus recently detected in the Yucatan Peninsula of Mexico is a novel reassortant of Potosi and Cache Valley viruses.

We determined the complete nucleotide sequences of the small (S) and medium (M) RNA segments of an orthobunyavirus isolated from mosquitoes in the Yucatan Peninsula of Mexico. A 528-nt region of the large (L) RNA segment was also sequenced. The S RNA segment has greatest nucleotide identity to the homologous region of Cache Valley virus (CVV; 98%) followed by Potosi virus (POTV; 89%) and Northway virus (86%). The M RNA segment has 96% nucleotide identity to the homologous region of POTV, and less than 74% nucleotide identity to the homologous regions of all other orthobunyaviruses for which M segment sequence data are available. The L RNA segment has greatest nucleotide identity to the homologous region of POTV (98%) followed by CVV (82%) and Tensaw virus (77%). These data indicate that the virus, tentatively named Cholul virus (CHLV), is a novel reassortant that acquired its S RNA segment from CVV and its M and L RNA segments from POTV. Phylogenetic data support this conclusion.

Universal primers for the amplification and sequence analysis of 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'-GATTCCATACCCAGGAAGGADGG-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.

Host-feeding preference of the mosquito, Culex quinquefasciatus, in Yucatan State, Mexico.

Studies were conducted to determine the host-feeding preference of Culex quinquefasciatus Say (Diptera: Culicidae) in relation to the availability of human and domestic animals in the city of Merida, Yucatan State, Mexico. Mosquitoes were collected in the backyards of houses using resting wooden boxes. Collections were made five times per week from January to December 2005. DNA was extracted from engorged females and tested by PCR using universal avian- and mammalian-specific primers. DNA extracted from avian-derived blood was further analyzed by PCR using primers that differentiate among the birds of three avian orders: Passeriformes, Columbiformes and Galliformes. PCR products obtained from mammalian-derived blood were subjected to restriction enzyme digestion to differentiate between human-, dog-, cat-, pig-, and horse-derived blood meals. Overall, 82% of engorged mosquitoes had fed on birds, and 18% had fed on mammals. The most frequent vertebrate hosts were Galliformes (47.1%), Passeriformes (23.8%), Columbiformes (11.2%) birds, and dogs (8.8%). The overall human blood index was 6.7%. The overall forage ratio for humans was 0.1, indicating that humans were not a preferred host for Cx. quinquefasciatus in Merida.

Isolation and sequence analysis of Culex flavivirus from Culex interrogator and Culex quinquefasciatus in the Yucatan Peninsula of Mexico.

Previously, we reported a high prevalence of Culex flavivirus (CxFV) in Culex quinquefasciatus (Say) in the Yucatan Peninsula of Mexico. To determine whether other Culex spp. mosquitoes in this region are susceptible to natural CxFV infection, Cx. bahamensis (Dyar and Knab), Cx. coronator (Dyar and Knab), Cx. interrogator (Dyar and Knab), Cx. nigripalpus (Theobald) and Cx. opisthopus (Komp) in the Yucatan Peninsula of Mexico were tested for CxFV. Two pools of Cx. interrogator were positive. The envelope protein genes of these isolates and 16 isolates from Cx. quinquefasciatus were sequenced and shown to have > or =99.2% nucleotide identity. These data suggest that there is limited genetic diversity among CxFV isolates in the Yucatan Peninsula of Mexico.

Detection of flaviviruses and orthobunyaviruses in mosquitoes in the Yucatan Peninsula of Mexico in 2008.

A total of 191,244 mosquitoes from 24 species were collected in the Yucatan Peninsula of Mexico from January to December 2008, and tested for the presence of cytopathic virus by virus isolation in Vero cells. Eighteen virus isolates were obtained, all of which were orthobunyaviruses. These were identified by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing as Cache Valley virus (n=17) and South River virus (n=1). A subset (n=20,124) of Culex quinquefasciatus collected throughout the year was further tested by RT-PCR using flavivirus-specific primers. Flavivirus RNA was present in this mosquito species year-round. The overall flavivirus minimal infection rate, expressed as the number of positive mosquito pools per 1000 mosquitoes tested, was 7.7 and the monthly flavivirus minimal infection rates ranged from 4.3 to 16.6. Approximately one-third of the RT-PCR products were sequenced and all corresponded to Culex flavivirus, a recently discovered insect-specific flavivirus.

Serologic surveillance for West Nile virus and other flaviviruses in febrile patients, encephalitic patients, and asymptomatic blood donors in northern Mexico.

A clinical and serological investigation was performed to determine the presence of West Nile virus (WNV) among febrile and encephalitic patients in northern Mexico. In addition, asymptomatic blood donors were serologically assayed for WNV to determine the seroprevalence of WNV in the general population. The study cohort consisted of 1432 individuals (588 febrile patients, 44 encephalitic patients, and 800 asymptomatic blood donors). All subjects were negative for WNV IgM. Sixty subjects were reactive for dengue virus (DENV) IgM (16 blood donors and 44 febrile patients). A subset (n = 425) of individuals was also screened by ELISA for flavivirus IgG. The prevalence of flavivirus IgG in febrile patients, encephalitic patients, and blood donors ranged from 40% to 59%. A subset (n = 147) of sera reactive for flavivirus IgG was further tested by plaque reduction neutralization test. Six individuals with no history of travel during the preceding 12 months were seropositive for WNV. Another 65 individuals were seropositive for DENV1 and 24 were seropositive for DENV2. The high prevalence of dengue antibodies in northern Mexico appears to limit the incidence of WNV infection in this region. Article Summary Line: Antibodies to WNV, DENV-1, and DENV-2 were identified in humans in northern Mexico.

Complete nucleotide sequences of the small and medium RNA genome segments of Kairi virus (family Bunyaviridae).

We determined the complete nucleotide sequences of the small (S) and medium (M) RNA genome segments of a Kairi virus (KRIV) isolate from the Yucatan Peninsula of Mexico. The S segment consists of 992 nucleotides, and the M segment consists of 4,619 nucleotides. Phylogenetic analyses were conducted on each genomic segment, and these data are discussed. A 526 nucleotide region of the large (L) segment was also sequenced. This is the first study to present sequence and phylogenetic data for a KRIV isolate from Latin America.

West Nile virus activity in mosquitoes and domestic animals in Chiapas, México.

Prior to 2006, West Nile virus (WNV) had not been definitively detected in Chiapas, the southernmost state of Mexico, although it circulates elsewhere in Mexico and Central America. We collected over 30,000 mosquitoes and blood-sampled 351 domestic animals in Chiapas in search for evidence of current or recent transmission of WNV. Two mosquito pools tested positive for WNV RNA and 17 domestic animals tested positive for specific WNV-neutralizing antibodies, including young animals (<1 year old) in four of five sampled locations. The two WNV-positive mosquito pools were collected on the Pacific coastal plain of Chiapas in June, 2006, and included a pool of Culex nigripalpus, a suspected vector of WNV, and a pool of Cx. interrogator. The sequence of a 537-nucleotide portion of a cDNA amplicon derived from the WNV NS5 gene from the Cx. interrogator pool contained a single silent nucleotide substitution when compared to WNV strain NY99.

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'Ho 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' untranslated region.

Gonotrophic cycle estimate for Culex quinquefasciatus in Mérida, Yucatán, México.

West Nile virus (WNV) has been present in the Yucatán State, México, since 2002. Culex quinquefasciatus, one of the main vectors of WNV transmission in the United States, is also common in Mexico and may be a key vector of WNV transmission t o humans in t he Yucatán. The aim of this study was to determine the length of the gonotrophic cycle and the survival rates of Cx. quinquefasciatus from Mérida, Yucatán, during the rainy versus the dry season. Mosquitoes were collected during 25-day periods in October (rainy season) and in April (dry season), and captured females were classified by abdominal appearance (freshly fed, late-stage fed, half gravid, and subgravid). To determine the age structure as nulliparous and parous females and to calculate the gonotrophic cycle through a time series and the mosquito survival, we used Davidson formulae. Also, vitellogenesis analysis to monitor egg maturity was conducted during both seasons. Cross-correlation data suggested a similar length of the gonotrophic cycle (4 days) in both seasons. Oogenic development required a minimum of 72 h in each season. However, survival of the mosquito population collected in the rainy season was significantly higher (0.91) with a mean temperature of 28 +/- 1.57 degrees C than was survival in the dry season (0.78) with a mean temperature of 29 +/- 1.10 degrees C. Survival, although higher during the rainy season, did not influence the length of the gonotrophic cycle of Cx. quinquefasciatus in Yucatán.

Dengue virus circulation and evolution in Mexico: a phylogenetic perspective.

BACKGROUND: Dengue is the most important arthropod-borne viral infection in the Americas. In the last decades a progressive increment in dengue severity has been observed in Mexico and other countries of the region. METHODS: Molecular epidemiological studies were conducted to investigate the viral determinants of the emergence of epidemic dengue, dengue hemorrhagic fever and dengue shock syndrome as major public health problems in Mexico. Bayesian phylogenetic analyses were conducted to determine the origin, persistence and geographical dispersion of the four serotypes of dengue virus (DENV) isolated in Mexico between 1980 and 2002. Tests for natural selection were also conducted. RESULTS: The origin of some, but not all, strains circulating in Mexico could be inferred. Frequent lineage replacements were observed and were likely due to stochastic events. In situ evolution was detected but not associated with natural selection. Recent changes in the incidence and severity of dengue were temporally associated with the introduction and circulation of different serotypes and genotypes of DENV. CONCLUSIONS: Introduction of new DENV genotypes and serotypes is a major risk factor for epidemic dengue and severe disease. Increased surveillance for such introductions is critical to allow public health authorities to intervene in impending epidemics.

Antibodies to West Nile virus in asymptomatic mammals, birds, and reptiles in the Yucatan Peninsula of Mexico.

Surveillance for evidence of West Nile virus (WNV) infection in taxonomically diverse vertebrates was conducted in the Yucatan Peninsula of Mexico in 2003 and 2004. Sera from 144 horses on Cozumel Island, Quintana Roo State, 415 vertebrates (257 birds, 52 mammals, and 106 reptiles) belonging to 61 species from the Merida Zoo, Yucatan State, and 7 farmed crocodiles in Ciudad del Carmen, Campeche State were assayed for antibodies to flaviviruses. Ninety (62%) horses on Cozumel Island had epitope-blocking enzyme-linked immunosorbent assay (ELISA) antibodies to flaviviruses, of which 75 (52%) were seropositive for WNV by plaque reduction neutralization test (PRNT). Blocking ELISA antibodies to flaviviruses also were detected in 13 (3%) animals in the Merida Zoo, including 7 birds and 2 mammals (a jaguar and coyote) seropositive for WNV by PRNT. Six (86%) crocodiles in Campeche State had PRNT-confirmed WNV infections. All animals were healthy at the time of serum collections and none had a history of WNV-like illness.