Application of Artificial Neural Networks for Dengue Fever Outbreak Predictions in the Northwest Coast of Yucatan, Mexico and San Juan, Puerto Rico.

Modelling dengue fever in endemic areas is important to mitigate and improve vector-borne disease control to reduce outbreaks. This study applied artificial neural networks (ANNs) to predict dengue fever outbreak occurrences in San Juan, Puerto Rico (USA), and in several coastal municipalities of the state of Yucatan, Mexico, based on specific thresholds. The models were trained with 19 years of dengue fever data for Puerto Rico and six years for Mexico. Environmental and demographic data included in the predictive models were sea surface temperature (SST), precipitation, air temperature (i.e., minimum, maximum, and average), humidity, previous dengue cases, and population size. Two models were applied for each study area. One predicted dengue incidence rates based on population at risk (i.e., numbers of people younger than 24 years), and the other on the size of the vulnerable population (i.e., number of people younger than five years and older than 65 years). The predictive power was above 70% for all four model runs. The ANNs were able to successfully model dengue fever outbreak occurrences in both study areas. The variables with the most influence on predicting dengue fever outbreak occurrences for San Juan, Puerto Rico, included population size, previous dengue cases, maximum air temperature, and date. In Yucatan, Mexico, the most important variables were population size, previous dengue cases, minimum air temperature, and date. These models have predictive skills and should help dengue fever mitigation and management to aid specific population segments in the Caribbean region and around the Gulf of Mexico.

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.

Epidemiological trends of dengue disease in Mexico (2000-2011): a systematic literature search and analysis.

This systematic literature review describes the epidemiology of dengue disease in Mexico (2000-2011). The annual number of uncomplicated dengue cases reported increased from 1,714 in 2000 to 15,424 in 2011 (incidence rates of 1.72 and 14.12 per 100,000 population, respectively). Peaks were observed in 2002, 2007, and 2009. Coastal states were most affected by dengue disease. The age distribution pattern showed an increasing number of cases during childhood, a peak at 10-20 years, and a gradual decline during adulthood. All four dengue virus serotypes were detected. Although national surveillance is in place, there are knowledge gaps relating to asymptomatic cases, primary/secondary infections, and seroprevalence rates of infection in all age strata. Under-reporting of the clinical spectrum of the disease is also problematic. Dengue disease remains a serious public health problem in Mexico.

Detection of hepatitis C virus coinfection in patients with dengue diagnosis.

Coinfection produced by dengue virus (DENV) and hepatitis C virus (HCV) is a serious problem of public health in Mexico, as they both circulate in tropical zones and may lead to masking or complicating symptoms. In this research, we detected active coinfected patients by HCV residing in the endemic city of Mérida, Yucatán, Mexico, with positive diagnosis to dengue during the acute phase. We performed a retrospective analysis of 240 serum samples from dengue patients. The IgM-ELISA serological test was used for dengue diagnosis, as well as viral isolation to confirm infection. DENV and HCV were detected by RT-PCR. Thus, 31 (12.9%) samples showed DENV-HCV coinfection, but interestingly the highest frequency of coinfection cases was found in male patients presenting hemorrhagic dengue in 19/31 (61.29%), with a predominance of 12 : 7 in males. Firstly, coinfection of DENV-HCV in Mérida, Mexico, was detected in young dengue patients, between 11 and 20 years old (38.7%), followed by those between 21 and 30 years old (32%); only 16.13% were between 0 and 10 years of age. Diagnosis of HCV infection in patients with dengue is highly recommended in order to establish potential risk in clinical manifestations as well as dictate patients' special care.

Serologic evidence of flavivirus infection in bats in the Yucatan Peninsula of Mexico.

We captured 140 bats of seven species in Merida City in the Yucatan Peninsula of Mexico in 2010. Serum was collected from each bat and assayed by plaque reduction neutralization test (PRNT) using six flaviviruses: West Nile virus, St. Louis encephalitis virus, and dengue viruses 1-4. Flavivirus-specific antibodies were detected in 26 bats (19%). The antibody-positive bats belonged to three species: the Pallas's long-tongued bat (Glossophaga soricina), Jamaican fruit bat (Artibeus jamaicensis), and great fruit-eating bat (Artibeus lituratus), and their flavivirus antibody prevalences were 33%, 24%, and 9%, respectively. The PRNT titers were usually highest for dengue virus 2 or dengue virus 4, but none of the titers exceeded 80. These data could indicate that most of the antibody-positive bats had been infected with dengue virus. However, because all titers were low, it is possible that the bats had been infected with another (perhaps unrecognized) flavivirus not included in the PRNT analysis, possibly a virus more closely related to dengue virus than to other flaviviruses. Each serum sample was assayed for flavivirus RNA by reverse transcription PCR, but all were negative.

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.

Productive container types for Aedes aegypti immatures in Mérida, México.

During 2007-2010, we examined which container types in Mérida, México, are most productive for Aedes aegypti (L.) immatures. Surveys for mosquito immatures followed routine surveillance methodology and container type classifications used by Servicios de Salud de Yucatán. Our main findings were that (1) small and larger discarded containers that serve no particular purpose and therefore can be removed from the environment contribute strongly to larval and pupal production in Mérida, and (2) the importance of different container types can vary among sets of residential premises as well as between dry and wet periods. These results may help to guide future implementation in Mérida of control efforts that target the most productive container types for Ae. aegypti immatures. Furthermore, if the Patio Limpio cleanup campaign that currently is ongoing in Mérida proves successful in removing discarded containers as important immature development sites, then we should see dramatic changes in the most productive container types in the future as the mosquito is forced to switch to other container types, which perhaps also will be easier to include in highly targeted mosquito control interventions.

Mosquito infestation and dengue virus infection in Aedes aegypti females in schools in Merida, Mexico.

We determined abundance of Aedes aegypti mosquitoes and presence of dengue virus (DENV) in females collected from schools in Mérida, México, during 2008 and 2009. Backpack aspiration from 24 schools produced 468 females of Ae. aegypti and 1,676 females of another human biter, Culex quinquefasciatus. Ae. aegypti females were collected most commonly from classrooms followed by offices and bathrooms. Of these females, 24.7% were freshly fed. Examination of 118 pools of Ae. aegypti females (total of 415 females) for presence of DENV RNA produced 19 positive pools (16.1%). DENV-infected pools were detected from 11 (45.8%) of 24 schools and came from different room types, including classrooms, offices, and bathrooms. The overall rate of DENV infection per 100 Ae. aegypti females was 4.8. We conclude that schools in Mérida present a risk environment for students, teachers, and other personnel to be exposed to mosquitoes and bites of DENV-infected Ae. aegypti females.

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.