Domestic Dogs as Sentinels for West Nile Virus but not Aedes-borne Flaviviruses, Mexico.

We tested 294 domestic pet dogs in Mexico for neutralizing antibodies for mosquito-borne flaviviruses. We found high (42.6%) exposure to West Nile virus in Reynosa (northern Mexico) and low (1.2%) exposure in Tuxtla Gutierrez (southern Mexico) but very limited exposure to Aedes-borne flaviviruses. Domestic dogs may be useful sentinels for West Nile virus.

Biosafety at Home: How to Translate Biomedical Laboratory Safety Precautions for Everyday Use in the Context of COVID-19.

Population adoption of social distancing measures during the COVID-19 pandemic is at times deficient, increasing the risk of SARS-CoV-2 transmission. Healthcare workers and those living in areas of intense transmission may benefit from implementing biosafety measures in their daily lives. A mixed-methods approach, combining components of single negotiation text and the Delphi method, was used to create a COVID-19 biosafety-at-home protocol. A consensus building coordinator liaised with 12 experts to develop the protocol over 11 iterations. Experts had more than 200 years of combined experience in epidemiology, virology, infectious disease prevention, and public health. A flyer, created from the final protocol, was professionally designed and initially distributed via social media and institutional websites/emails in Ecuador beginning on May 2, 2020. Since then, it has been distributed in other countries, reaching ∼7,000 people. Translating research laboratory biosafety measures for the home/street environment might be challenging. The biosafety-at-home flyer addresses this challenge in a user-friendly format.

Complete genomic sequences of Venezuelan equine encephalitis virus subtype IIID isolates from mosquitoes.

Venezuelan equine encephalitis virus (VEEV) is an important pathogen of medical and veterinary importance in the Americas. In this report, we present the complete genome sequences of five VEEV isolates obtained from pools of Culex (Melanoconion) gnomatos (4) or Culex (Melanoconion) pedroi (1) from Iquitos, Peru. Genetic and phylogenetic analyses showed that all five isolates grouped within the VEEV complex sister to VEEV IIIC and are members of subtype IIID. This is the first report of full-length genomic sequences of VEEV IIID.

Lack of Evidence of Sylvatic Transmission of Dengue Viruses in the Amazon Rainforest Near Iquitos, Peru.

Dengue viruses (DENV) are currently responsible for more human morbidity and mortality than any other known arbovirus, and all four DENV are known to exist in sylvatic cycles that might allow these viruses to persist if the urban (Aedes aegypti) cycle could be controlled. To determine whether DENV were being maintained in a sylvatic cycle in a forested area about 14 km southwest of Iquitos, Peru, a city in which all 4 serotypes of DENV circulate, we placed 20 DENV seronegative Aotus monkeys in cages either in the canopy or near ground level for a total of 125.6 months. Despite capturing >66,000 mosquitoes in traps that collected some of the mosquitoes attracted to these monkeys, blood samples obtained once a month from each animal were tested and found to be negative by an enzyme-linked immunosorbent assay for IgM and IgG antibodies to dengue, yellow fever, Venezuelan equine encephalitis, Oropouche, and Mayaro viruses. Although all four DENV serotypes were endemic in nearby Iquitos, the findings of this study did not support a DENV sylvatic maintenance and transmission cycle in a selected area of the Amazon rainforest in northeastern Peru.

Effect of Holding Conditions on the Detection of Chikungunya and Venezuelan Equine Encephalitis Viruses in Mosquito Pools.

Emerging and re-emerging arboviruses continue to be a threat to global public health, and viral surveillance of mosquito populations is critical for mosquito control operations. Due to the tropical climate of many of the affected areas, it may be difficult to maintain a cold chain as the samples travel from collection sites to laboratories for testing. We determined how suboptimal holding temperatures affected the ability to detect viruses in pools of mosquitoes. Adult female Aedes albopictus and Ae. taeniorhynchus individuals were inoculated with chikungunya virus or Venezuelan equine encephalitis virus suspensions, respectively, and placed at 26°C for 8 days. One infected mosquito was then added to a vial of 24 negative mosquitoes and held at -80°C, -20°C, 4°C, 22°C, or 35°C for up to 14 days. Mosquito pools were analyzed for both infectious virus by plaque assay and for viral ribonucleic acid (RNA) with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). At higher temperatures, the amount of infectious virus decreased rapidly, but viruses in samples held at 4°C or lower remained relatively stable. In contrast, viral RNA was detectable from pools held at all temperatures and holding times by RT-qPCR. Cycle threshold (Ct) values increased as temperatures and holding times increased. These findings suggest that if viral RNA detection is the goal of surveillance efforts, then mosquito pools do not require storage at ≤4°C. This enhances the feasibility of field-based arbovirus surveillance programs in which maintaining a cold chain may not be a possibility.

Identification and Genomic Analysis of a Novel Group C Orthobunyavirus Isolated from a Mosquito Captured near Iquitos, Peru.

Group C orthobunyaviruses are single-stranded RNA viruses found in both South and North America. Until very recently, and despite their status as important vector-borne human pathogens, no Group C whole genome sequences containing all three segments were available in public databases. Here we report a Group C orthobunyavirus, named El Huayo virus, isolated from a pool of Culex portesi mosquitoes captured near Iquitos, Peru. Although initial metagenomic analysis yielded only a handful of reads belonging to the genus Orthobunyavirus, single contig assemblies were generated for L, M, and S segments totaling over 200,000 reads (~0.5% of sample). Given the moderately high viremia in hamsters (>107 plaque-forming units/ml) and the propensity for Cx. portesi to feed on rodents, it is possible that El Huayo virus is maintained in nature in a Culex portesi/rodent cycle. El Huayo virus was found to be most similar to Peruvian Caraparu virus isolates and constitutes a novel subclade within Group C.

Species Diversity, Seasonal, and Spatial Distribution of Mosquitoes (Diptera: Culicidae) Captured in Aotus Monkey-Baited Traps in a Forested Site Near Iquitos, Peru.

This study was conducted to determine the relative abundance, diversity, seasonal, and vertical distributions of potential mosquito vectors in the Amazon Basin, Peru. A total of 66,097 mosquitoes (50 mosquito species from 12 genera) were collected from May 2001 through March 2002 at a forested site near Iquitos, Peru. Mosquitoes were collected using Aotus nancymae Hershkovitz monkey-baited CDC light traps set for 12-h day and night periods at varying heights (e.g., ground and canopy) in the forest. Of the 12 genera, three accounted for 75% of all mosquitoes collected: Culex (33%), Aedes (23%), and Psorophora (18%). The most prevalent species collected were Aedes serratus (Theobald), Culex pedroi Sirivanakarn & Belkin, Psorophora albigenu (Peryassu), and a combination of Mansonia indubitans Dyar & Shannon and Mansonia titillans (Walker), which accounted for 56% of all mosquitoes captured. In general, mosquitoes were collected more often at night and on the ground. Exceptions include Coquillettidia venezuelensis (Theobald), which were collected in relatively even numbers at both day and night and most Mansonia and some species of Anopheles, which were collected more often in the canopy. Total mosquito populations had two peaks, June-July (Ma. indubitans/titillans and Cq. venezuelensis) and December-January (Ps. albigenu, Cx. pedroi, and Ae. serratus). Observations of the eight most collected mosquitoes indicated that behavioral shifts were not observed between collection months. These data provide a better understanding of the species diversity, population density, and seasonal distribution of potential mosquito vectors within the Amazon Basin region and allow for the development of appropriate vector and disease prevention strategies.

Determinants of Anopheles seasonal distribution patterns across a forest to periurban gradient near Iquitos, Peru.

As part of a field ecology study of arbovirus and malaria activity in the Amazon Basin, Loreto Department, Peru, we collected mosquitoes landing on humans at a forest site and inside and outside of residences and military barracks at periurban, rural, and village sites. We collected 11 Anopheles spp. from these four sites. An. darlingi, the principal malaria vector in the region, accounted for 98.7% of all Anopheles spp. collected at Puerto Almendra. Peaks in landing activity occurred during the December and April collection periods. However, the percent of sporozoite-positive Anopheles spp. was highest 1-2 months later, when landing activity decreased to approximately 10% of the peak activity periods. At all sites, peak landing activity occurred about 2 hours after sunset. These data provide a better understanding of the taxonomy, population density, and seasonal and habitat distribution of potential malaria vectors within the Amazon Basin region.

Phylogenetic relationships among orthobunyaviruses isolated from mosquitoes captured in Peru.

The Orthobunyavirus genus of the family Bunyaviridae is comprised of over 220 extremely diverse viral species. Members of this genus are often associated with acute febrile illness in animals and humans. As part of a longterm study of the ecology of arboviruses in the Amazon basin of Peru, we have isolated over 60 orthobunyaviruses from mosquitoes. The identification of many of these isolates by fluorescent antibody assay has been confounded by the lack of specificity of many available reagents. Therefore, we initiated genetic characterization, based on the S and M genomic segments, of selected viral isolates. Based on comparisons of the nucleotide sequences of the nucleocapsid gene, Wyeomyia, a virus in the Bunyamwera group, was the most related Orthobunyavirus species. Within the nonstructural S (NSs) open reading frame of the S segment, we found four conserved stop codons for the Peruvian isolates. Detailed comparisons of Bunyamwera, Simbu viruses, Group C viruses, and California viruses revealed all four of these NSs stop codons only appeared in Wyeomyia and the Peruvian isolates, and Guaroa conserved one of these stop codons. Such an apparent obliteration of the native NSs protein has not been described. Analysis of partial M segment amino acid sequence supports the conclusion that the viruses in this study are members of an uncharacterized orthobunyavirus group.

Seasonal distribution, biology, and human attraction patterns of mosquitoes (Diptera: Culicidae) in a rural village and adjacent forested site near Iquitos, Peru.

This study was conducted as part of a field-ecology study of arboviral and malarial activity in the Amazon Basin, Loreto Department, Peru, to determine the relative abundance, species diversity, and seasonal and vertical distributions of potential mosquito vectors. Mosquitoes were captured either by volunteers using mouth aspirators while mosquitoes attempted to land on the collectors or in dry ice-baited ABC light traps. Anopheles darlingi, the principal malaria vector in the region, was the most commonly captured anopheline mosquito in Puerto Almendra village (99%) while landing on humans, with a mean of 37.1 mosquitoes captured per 24-h period, representing nearly one half of all mosquitoes collected. An. darlingi human landing activity began shortly after sunset, peaked at 2000-2100 hours, and declined gradually until sunrise. This species readily entered houses, because 51% of the An. darlingi captured by paired collectors, stationed inside and outside houses, were captured indoors. Human landing collections provided a more accurate estimate of human attraction of An. darlingi, capturing 30 times as many as co-located dry ice-baited ABC light traps. In contrast, eight times as many Culex (Melanoconion) species, including known arbovirus vectors, were captured in light traps as by co-located human collectors. Despite being located within 300 m of the village collection site, only a few Anopheles species were captured at the forest collection site, including only 0.1 An darlingi/ 24 h, thus indicating that An. darlingi activity was directly associated with the rural village. These data provide a better understanding of the taxonomy, population density, and seasonal distribution of potential mosquito vectors of disease within the Amazon Basin region and allow for the development of appropriate vector and disease prevention strategies that target vector populations.

Protection of hamsters by Venezuelan equine encephalitis virus candidate vaccine V3526 against lethal challenge by mosquito bite and intraperitoneal injection.

In an attempt to improve the current live, attenuated vaccine (TC-83) for Venezuelan equine encephalitis virus (VEEV), specific mutations associated with attenuation of VEEV in rodent models were inserted into a full-length cDNA clone of the Trinidad donkey strain of VEEV by site-directed mutagenesis. Because some viruses have been reported to be more pathogenic when introduced by mosquito bite than the same virus introduced by needle inoculation, there were concerns that the presence of mosquito saliva, or changes in the virus caused by replication in a mosquito, might allow the virus to overcome the protective effects of prior vaccination with V3526. Therefore, we determined if hamsters vaccinated with V3526 were protected from challenge with the virulent Trinidad donkey strain of VEEV. All non-vaccinated hamsters died after intraperitoneal challenge or after being fed on by VEEV-inoculated Aedes taeniorhynchus. In contrast, hamsters vaccinated with V3526 were resistant to intraperitoneal challenge and infection by VEEV-infected Ae. taeniorhynchus. Therefore, the V3526 candidate vaccine elicits protection against VEEV infection by mosquito bite.

Protection of hamsters by Venezuelan equine encephalitis virus candidate vaccine V3526 against lethal challenge by mosquito bite and intraperitoneal injection.

In an attempt to improve the current live, attenuated vaccine (TC-83) for Venezuelan equine encephalitis virus (VEEV), specific mutations associated with attenuation of VEEV in rodent models were inserted into a full-length cDNA clone of the Trinidad donkey strain of VEEV by site-directed mutagenesis. Because some viruses have been reported to be more pathogenic when introduced by mosquito bite than the same virus introduced by needle inoculation, there were concerns that the presence of mosquito saliva, or changes in the virus caused by replication in a mosquito, might allow the virus to overcome the protective effects of prior vaccination with V3526. Therefore, we determined if hamsters vaccinated with V3526 were protected from challenge with the virulent Trinidad donkey strain of VEEV. All non-vaccinated hamsters died after intraperitoneal challenge or after being fed on by VEEV-inoculated Aedes taeniorhynchus. In contrast, hamsters vaccinated with V3526 were resistant to intraperitoneal challenge and infection by VEEV-infected Ae. taeniorhynchus. Therefore, the V3526 candidate vaccine elicits protection against VEEV infection by mosquito bite.

Experimental transmission of West Nile virus by Culex nigripalpus from Honduras.

As a result of concerns regarding the geographic spread of West Nile virus (WNV) to Central America, we evaluated the potential for Honduran Culex nigripalpus Theobald to transmit this virus. We tested individual mosquitoes captured in Olancho Province, Honduras, in September 2003. Mosquitoes were allowed to feed on 2- to 4- day-old chickens previously inoculated with a New York strain (Crow 397-99) of WNV. Infection rates in Cx. nigripalpus ranged from 81%-96% after feeding on chickens with viremias between 10(6.3) and 10(7.4) plaque-forming units per milliliter. Development of a disseminated infection was directly correlated with holding time after the infectious blood meal as 68% (19/28) of the mosquitoes tested 20 days after the infectious blood meal had a disseminated infection as compared to 38% (15/40) of the mosquitoes tested 14 days after feeding on the same viremic chickens (viremia = 10(6.97.4)). Nearly all (4/5) Cx. nigripalpus with a disseminated infection that fed on susceptible chickens transmitted virus by bite. In addition, 8 (57%) of 14 Cx. nigripalpus with a disseminated infection transmitted virus when tested by a capillary tube feeding assay. Based on its efficiency of viral transmission in this study and its role in the transmission of the closely related St. Louis encephalitis virus in the southeastern United States, Cx. nigripalpus should be considered a potentially important vector of WNV in Honduras and the rest of Central America.

Genetic analysis of South American eastern equine encephalomyelitis viruses isolated from mosquitoes collected in the Amazon Basin region of Peru.

Identifying viral isolates from field-collected mosquitoes can be difficult and time-consuming, particularly in regions of the world where numerous closely related viruses are co-circulating (e.g., the Amazon Basin region of Peru). The use of molecular techniques may provide rapid and efficient methods for identifying these viruses in the laboratory. Therefore, we determined the complete nucleotide sequence of two South American eastern equine encephalomyelitis viruses (EEEVs): one member from the Peru-Brazil (Lineage II) clade and one member from the Argentina-Panama (Lineage III) clade. In addition, we determined the nucleotide sequence for the nonstructural P3 protein (nsP3) and envelope 2 (E2) protein genes of 36 additional isolates of EEEV from mosquitoes captured in Peru between 1996 and 2001. The 38 isolates were evenly distributed between lineages II and III virus groupings. However, analysis of the nsP3 gene for lineage III strongly suggested that the 19 isolates from this lineage could be divided into two sub-clades, designated as lineages III and IIIA. Compared with North American EEEV (lineage I, GA97 strain), we found that the length of the nsP3 gene was shorter in the strains isolated from South America. A total of 60 nucleotides was deleted in lineage II, 69 in lineage III, and 72 in lineage IIIA. On the basis of the sequences we determined for South American EEEVs and those for other viruses detected in the same area, we developed a series of primers for characterizing these viruses.

Endemic eastern equine encephalitis in the Amazon region of Peru.

Eastern equine encephalitis virus (EEEV) causes severe neurologic disease in North America, but only two fatal human cases have been documented in South America. To test the hypothesis that alphavirus heterologous antibodies cross-protect, animals were vaccinated against other alphaviruses and challenged up to 3 months later with EEEV. Short-lived cross-protection was detected, even in the absence of cross-neutralizing antibodies. To assess exposure to EEEV in Peru, sera from acutely ill and healthy persons were tested for EEEV and other alphavirus antibodies, as well as for virus isolation. No EEEV was isolated from patients living in an EEEV-enzootic area, and only 2% of individuals with febrile illness had EEEV-reactive IgM. Only 3% of healthy persons from the enzootic region had EEEV-neutralizing antibodies. Our results suggest that humans are exposed but do not develop apparent infection with EEEV because of poor infectivity and/or avirulence of South American strains.

Endemic Venezuelan equine encephalitis in northern Peru.

Since Venezuelan equine encephalitis virus (VEEV) was isolated in Peru in 1942, >70 isolates have been obtained from mosquitoes, humans, and sylvatic mammals primarily in the Amazon region. To investigate genetic relationships among the Peru VEEV isolates and between the Peru isolates and other VEEV strains, a fragment of the PE2 gene was amplified and analyzed by single-stranded conformation polymorphism. Representatives of seven genotypes underwent sequencing and phylogenetic analysis. The results identified four VEE complex lineages that cocirculate in the Amazon region: subtypes ID (Panama and Colombia/Venezuela genotypes), IIIC, and a new, proposed subtype IIID, which was isolated from a febrile human, mosquitoes, and spiny rats. Both ID lineages and the IIID subtype are associated with febrile human illness. Most of the subtype ID isolates belonged to the Panama genotype, but the Colombia/Venezuela genotype, which is phylogenetically related to epizootic strains, also continues to circulate in the Amazon basin.

Seasonal distribution, biology, and human attraction patterns of culicine mosquitoes (Diptera: Culicidae) in a forest near Puerto Almendras, Iquitos, Peru.

This study was conducted as part of a field ecology study of arboviral activity in the Amazon Basin, Peru, to determine the taxonomy, frequency, seasonal, and vertical distributions of potential mosquito vectors. In addition, the relative efficiency of human-landing collections and dry ice-baited Centers for Disease Control (CDC)-type light traps was determined for collecting mosquitoes. A total of 70 species of mosquitoes from 14 genera were collected from June 1996 through December 1997 at a forested site near Puerto Almendras, approximately 20 km west-southwest of Iquitos, Peru. Three species [Psorophora (Janthinosoma) albigenu (Peryassu), Ochlerotatus (Ochlerotatus) fulvus (Wiedemann), and Ochlerotatus (Ochlerotatus) serratus (Theobald)] accounted for 70% of all mosquitoes captured in human-landing collections. Overall, biting activity occurred throughout the 24-h cycle but was higher during the daytime, primarily because of large populations of two day-biting species, Ps. albigenu and Oc. serratus. Oc. fulvus was active throughout the 24-h cycle but was more frequently collected during the evening. Oc. fulvus, Ps. albigenu, Culex (Melanoconion) pedroi Sirivanakarn & Belkin, and a mixture of Culex (Melaonoconion) vomerifer Komp, and Culex (Melanoconion) gnomatos Sallum, Huchings & Ferreira, accounted for 73% of the mosquitoes captured during darkness) by human collectors. In general, Ochlerotatus spp. and Psorophora spp. were more commonly captured in human-landing collections, whereas most Culex spp. were more frequently collected in the dry ice-baited CDC-type light traps. In general, mosquito populations were lowest from June through August when river levels were at their lowest. Two large population peaks occurred in November-December and in February-March as a result of "flood water" mosquito populations (e.g., Ps. albigenu). These data provide a better understanding of the taxonomy, population density, and seasonal distribution of potential mosquito vectors within the Amazon Basin region and allow for the development of appropriate vector and disease prevention strategies.

Vector competence of Mexican and Honduran mosquitoes (Diptera: Culicidae) for enzootic (IE) and epizootic (IC) strains of Venezuelan equine encephalomyelitis virus.

Experimental studies evaluated the vector competence of Ochlerotatus taeniorhynchus (Wiedemann), Culex cancer Theobald, Culex pseudes (Dyar and Knab), Culex taeniopus Dyar and Knab, and a Culex (Culex) species, probably Culex quinquefasciatus Say, and Culex nigripalpus Theobald from Chiapas, Mexico, and Tocoa, Honduras, for epizootic (IC) and enzootic (IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. Culex pseudes was highly susceptible to infection with both the IC and IE strains of VEE (infection rates >78%). Patterns of susceptibility to VEE were similar for Oc. taeniorhynchus collected in Mexico and Honduras. Although Oc. taeniorhynchus was highly susceptible to the epizootic IC strains (infection rates > or = 95%, n = 190), this species was less susceptible to the enzootic IE strain (infection rates < or = 35%, n = 233). The Culex (Culex) species were refractory to both subtypes of VEE, and none of 166 contained evidence of a disseminated infection. Virus-exposed Cx. pseudes that refed on susceptible hamsters readily transmitted virus, confirming that this species was an efficient vector of VEE. Although Oc. taeniorhynchus that fed on hamsters infected with the epizootic IC strain transmitted VEE efficiently, only one of six of those with a disseminated infection with the enzootic IE virus that fed on hamsters transmitted virus by bite. These data indicate that Cx. pseudes is an efficient laboratory vector of both epizootic and enzootic strains of VEE and that Oc. taeniorhynchus could be an important vector of epizootic subtypes of VEE.

Effect of triethylamine on the recovery of selected South American alphaviruses, flaviviruses, and bunyaviruses from mosquito (Diptera: Culicidae) pools.

We evaluated the effect of triethylamine (TEA) on the recovery of infectious virus from pools of mosquitoes for two South American alphaviruses (eastern equine encephalomyelitis and Venezuelan equine encephalomyelitis subtypes IIIC and ID), one flavivirus (Ilheus) and two bunyaviruses (Mirim [Guama group] and Itaqui [group C]). Mosquitoes were inoculated intrathoracically with virus, held for 7-10 d at 26 degrees C, and handled under one of four regimens before testing for the presence of virus by plaque assay. Mosquitoes were killed by freezing at - 70 degrees C for 3 min and tested immediately for the presence of virus; killed by freezing at -70 degrees C for 3 min and then held at room temperature for 1 h before testing for the presence of virus; anesthetized with TEA and assayed immediately for the presence of virus; or anesthetized with TEA and then held at room temperature for 1 h before being assayed for the presence of virus. For each of the viruses tested, viral titers in mosquitoes anesthetized with TEA were similar to those in mosquitoes killed by freezing at-70 degrees C. Likewise, there was no significant difference in viral titers in mosquitoes anesthetized with TEA and held at room temperature for 1 h or in mosquitoes frozen at -70 degrees C and held at room temperature for 1 h before being processed for virus by isolation. Triethylamine is advantageous for the handling of mosquitoes in a field environment. The elimination of the need for a cold chain, without compromising virus recovery, increases the feasibility of conducting research projects requiring the isolation of live virus from mosquitoes in remote tropical environments.