Arbovirus investigations in Argentina, 1977-1980. II. Arthropod collections and virus isolations from Argentine mosquitoes

Prospective surveys for arboviruses were carried out in Santa Fe, Corrientes, and Chaco provinces, Argentina, aperiodically during 1977-1980. A total of 313,233 mosquitoes and 598 biting flies other than mosquitoes were collected and tested for virus in 5,197 and 45 pools, respectively. Forty virus strains were isolated, all from mosquitoes, as follows: Santa Fe Province: 4 Gamboa group viruses from Aedeomyia squamipennis, 1 strain each of St. Louis encephalitis virus from Culex pipiens quinquefasciatus and Culex (Culex) spp.; Corrientes Province: a single strain of a newly discovered Anopheles A serogroup virus, Las Maloyas, from Anopheles albitarsis; and Chaco Province: 4 Gamboa group viruses from Ad. squamipennis, 6 strains of new Bunyaviridae (1 Antequera, 1 Barranqueras, and 4 Resistencia) from Culex (Melanoconion) delpontei, 3 strains of a new subtype of western equine encephalitis virus and 1 strain of Para virus from the Cx. (Mel.) ocossa group, 12 strains of a newly discovered subtype (VI) of the Venezuelan equine encephalitis complex from Cx. (Mel.) delpontei, and 1 strain each from Ad. squamipennis, Aedes scapularis, Ae. spp., Cx. (Cux.) spp., Cx. (Mel.) ocossa group, Mansonia spp., and Psorophora spp. Bloodmeals from 265 engorged mosquitoes were identified by precipitin test. These data, coupled with data on engorgement rates for 25,995 mosquitoes from bait collections, provide information on the host feeding patterns of several mosquito species. This information is discussed, along with data on relative abundance of mosquito species, within the context of the vector relationships of the species from which viruses were isolated. The association of Cx. (Mel.) delpontei with 18 strains of 4 different viruses in Chaco Province, plus its catholic feeding habits, clearly indicate for the first time the importance of this species as an arbovirus vector.     

2019—2020年哈密市蚊虫种类分布及携带病毒调查

目的　了解新疆维吾尔自治区哈密市蚊虫种类及病毒携带情况,并鉴定该市尖音库蚊所属亚种。方法　分别于2019、2020年7月中旬,在新疆维吾尔自治区哈密市伊州区、伊吾县、巴里坤县采用诱蚊灯法捕捉蚊虫,鉴定所捕获蚊虫种属及尖音库蚊种属。采用逆转录PCR技术检测捕获蚊虫携带黄病毒属、甲病毒属、布尼亚病毒属、流行性乙型脑炎病毒、辽宁病毒、Tahyna病毒、蜱传脑炎病毒及西尼罗病毒情况。结果　在哈密市伊州区、伊吾县、巴里坤县共捕获蚊虫1 496只,分属于3属3种。尖音库蚊为优势蚊种,占所捕获蚊虫总数的65.91%（986只）;其次为里海伊蚊,占30.55%（457只）;阿拉斯加脉毛蚊最少,仅占3.54%（53只）。所捕获尖音库蚊经雄蚊尾器鉴定为尖音库蚊指名亚种。经逆转录PCR法检测,所捕获蚊虫黄病毒属、甲病毒属、布尼亚病毒属、流行性乙型脑炎病毒、辽宁病毒、Tahyna病毒、蜱传脑炎及西尼罗病毒均为阴性。结论　2019—2020年哈密市存在尖音库蚊、里海伊蚊、阿拉斯加脉毛蚊3种蚊虫,以尖音库蚊为优势蚊种;所捕获尖音库蚊均为指名亚种,未发现蚊类携带相关虫媒病毒。     

Ecological studies of enzootic Venezuelan equine encephalitis in north-central Venezuela, 1997-1998.

From 1997-1998, we investigated the possible continuous circulation of epizootic Venezuelan equine encephalitis (VEE) virus suggested by a 1983 subtype IC interepizootic mosquito isolate made in Panaquire, Miranda State, Venezuela. The study area was originally covered by lowland tropical rainforest but has been converted into cacao plantations. Sentinel hamsters, small mammal trapping, mosquito collections, and human serosurveys were used to detect active or recent virus circulation. Six strains of subtype ID VEE virus were isolated from hamsters that displayed no apparent disease. Four other arboviruses belonging to group A (Togaviridae: Alphavirus), two Bunyamwera group (Bunyaviridae), and three Gamboa group (Bunyaviridae) arboviruses were also isolated from hamsters, as well as 8 unidentified viruses. Venezuelan equine encephalitis-specific antibodies were detected in 5 small mammal species: Proechimys guairae, Marmosa spp., and Didelphis marsupialis. Mosquito collections comprised of 38 different species, including 8 members of the subgenus Culex (Melanoconion), did not yield any virus isolates. Sera from 195 humans, either workers in the cacao plantation or nearby residents, were all negative for VEE virus antibodies. Sequences of 1,677 nucleotides from the P62 gene of 2 virus isolates indicated that they represent a subtype ID lineage that is distinct from all others characterized previously, and are unrelated to epizootic VEE emergence.     

Evaluation of methods to assess transmission potential of Venezuelan equine encephalitis virus by mosquitoes and estimation of mosquito saliva titers

Determining the dose of an arbovirus transmitted by a mosquito is important to design transmission and pathogenesis studies simulating natural infection. Several different artificial infection and transmission methods used to assess vector competence and to estimate the dose injected during mosquito feeding have not been fully evaluated to determine whether they accurately reflect natural transmission. Additionally, it is not known whether different mosquito vectors transmit similar amounts of a given virus. Therefore, we compared three traditional artificial transmission methods using Venezuelan equine encephalitis virus (VEEV) and Aedes albopictus and Ochlerotatus taeniorhynchus mosquitoes. Both the mosquito species and the infection route used affected the amount of virus detected in the saliva after a 10-day extrinsic incubation period. Median titers of virus detected in saliva of Ae. albopictus and Oc. taeniorhynchus mosquitoes ranged from 0.2 to 1.1 log(10) (mean 0.7-1.4 log(10)) and 0.2 to 3.2 log(10) (mean 1.0-3.6 log(10)) plaque-forming units, respectively. The results of this study will aid in the design of transmission and pathogenesis studies involving arboviruses.     

The Role of Innate Immunity in Conditioning Mosquito Susceptibility to West Nile Virus

Arthropod-borne viruses (arboviruses) represent an emerging threat to human and livestock health globally. In particular, those transmitted by mosquitoes present the greatest challenges to disease control efforts. An understanding of the molecular basis for mosquito innate immunity to arbovirus infection is therefore critical to investigations regarding arbovirus evolution, virus-vector ecology, and mosquito vector competence. In this review, we discuss the current state of understanding regarding mosquito innate immunity to West Nile virus. We draw from the literature with respect to other virus-vector pairings to attempt to draw inferences to gaps in our knowledge about West Nile virus and relevant vectors.     

Distribution of mosquitoes and mosquito-borne arboviruses in Yunnan Province near the China-Myanmar-Laos border

Economic development and increased tourism in the southern region of Yunnan Province in China, adjacent to several countries in Southeast Asia, has increased the likelihood of import and export of vectors and vector-borne diseases. We report the results of surveillance of mosquitoes and mosquito-borne arboviruses along the border of China-Myanmar-Laos in 2005 and 2006, and information associating several arboviruses with infections and possibly disease in local human populations. Seventeen mosquito species representing four genera were obtained, and 14 strains of mosquito-borne viruses representing six viruses in five genera were isolated from Culex tritaeniorhynchus. In addition, IgM against Japanese encephalitis virus, Sindbis virus, Yunnan orbivirus and novel Banna virus was detected in acute-phase serum samples obtained from hospitalized patients with fever and encephalitis near the areas where the viruses were isolated. This investigation suggests that Japanese encephalitis virus, Sindbis virus, and lesser-known arboviruses circulate and may be infecting humans in the China-Myanmar-Laos border region.     

Transmission Potential of Two Chimeric Western Equine Encephalitis Vaccine Candidates in Culex tarsalis

Western equine encephalitis virus (WEEV) is a zoonotic alphavirus that circulates in western North America between passerine birds and mosquitoes, primarily Culex tarsalis. Since it was isolated in 1930, WEEV has caused tens of thousands of equine deaths in addition to thousands of human cases. In addition because WEEV is a virus of agricultural importance in addition to a public health threat, we developed two live-attenuated chimeric vaccine candidates that have been shown to be immunogenic and efficacious in mouse models. Vaccine candidate strains were developed by inserting the structural protein genes of WEEV strain McMillan (McM) or CO92-1356 into a Sindbis virus (SINV) strain AR339 backbone. The SIN/McM chimera also derived the N-terminal half of its capsid gene from a North American eastern equine encephalitis virus (EEEV) strain FL39-939 (henceforth referred to as SIN/EEE/McM). Although these vaccines do not generate viremia in mice, we further assessed their safety by exposing Cx. tarsalis to artificial blood meals containing high viral titers of each vaccine candidate. Both viruses exhibited a decreased rate of infection, dissemination, and transmission potential compared with the parental alphaviruses. Specifically, SIN/CO92 infected 37% of mosquitoes and disseminated in 8%, but failed to reach the saliva of the mosquitoes. In contrast, the SIN/EEE/McM virus was unable to infect, disseminate, or be transmitted in the saliva of any mosquitoes. These findings suggest that both vaccine candidates are less competent than the parental strains to be transmitted by the primary mosquito vector, Cx. tarsalis, and are unlikely to be reintroduced into a natural WEEV transmission cycle.     

Mosquito Immunity against Arboviruses

Arthropod-borne viruses (arboviruses) pose a significant threat to global health, causing human disease with increasing geographic range and severity. The recent availability of the genome sequences of medically important mosquito species has kick-started investigations into the molecular basis of how mosquito vectors control arbovirus infection. Here, we discuss recent findings concerning the role of the mosquito immune system in antiviral defense, interactions between arboviruses and fundamental cellular processes such as apoptosis and autophagy, and arboviral suppression of mosquito defense mechanisms. This knowledge provides insights into co-evolutionary processes between vector and virus and also lays the groundwork for the development of novel arbovirus control strategies that target the mosquito vector.     

Serologic survey of domestic animals for zoonotic arbovirus infections in the Lacandón Forest region of Chiapas,Mexico

A serologic survey in domestic animals (birds and mammals) was conducted in four communities located in the Lacandón Forest region of northeastern Chiapas, Mexico, during June 29 to July 1, 2001, with the objective to identify zoonotic arboviruses circulating in this area. We collected 202 serum samples from healthy domestic chickens, geese, ducks, turkeys, horses and cattle. The samples were tested by plaque-reduction neutralization test for antibodies to selected mosquito-borne flaviviruses (family Flaviviridae), including St. Louis encephalitis (SLE), Rocio (ROC), Ilheus (ILH), Bussuquara (BSQ), and West Nile (WN) viruses, and selected alphaviruses (family Togaviridae), including Western equine encephalitis (WEE), Eastern equine encephalomyelitis (EEE), and Venezuelan equine encephalitis (VEE) viruses. Neutralizing antibodies to SLE virus were detected in two (8%) of 26 turkeys, 15 (23%) of 66 cattle, and three (60%) of five horses. Antibodies to VEE virus were detected in 29 (45%) of 65 cattle. Because some of these animals were as young as 2 months old, we demonstrated recent activity of these two viruses. Sub-typing of the VEE antibody responses indicated that the etiologic agents of these infections belonged to the IE variety of VEE, which has been reported from other regions of Chiapas. WN virus-neutralizing antibodies were detected in a single cattle specimen (PRNT(90) = 1:80) that also circulated SLE virus-neutralizing antibodies (PRNT(90) = 1:20), suggesting that WN virus may have been introduced into the region. We also detected weak neutralizing activity to BSQ virus in four cattle and a chicken specimen, suggesting the presence of this or a closely related virus in Mexico. There was no evidence for transmission of the other viruses (ROC, ILH, EEE, WEE) in the study area.     

Growth characteristics of the chimeric Japanese encephalitis virus vaccine candidate, ChimeriVax-JE (YF/JE SA14--14--2), in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes

The Japanese encephalitis (JE) virus vaccine candidate, ChimeriVax-JE, which consists of a yellow fever (YF) 17D virus backbone containing the prM and E genes from the JE vaccine strain JE SA14--14--2, exhibits restricted replication in non-human primates, producing only a low-level viremia following peripheral inoculation. Although this reduces the likelihood that hematophagous insects could become infected by feeding on a vaccinated host, it is prudent to investigate the replication kinetics of the vaccine virus in mosquito species that are known to vector the viruses from which the chimera is derived. In this study ChimeriVax-JE virus was compared to its parent viruses, as well as to wild-type JE virus, for its ability to replicate in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes. Individual mosquitoes were exposed to the viruses by oral ingestion of a virus-laden blood meal or by intrathoracic (IT) virus inoculation. ChimeriVax-JE virus did not replicate following ingestion by any of the three mosquito species. Additionally, replication was not detected after IT inoculation of ChimeriVax-JE in the primary JE virus vector, Cx. tritaeniorhynchus. ChimeriVax-JE exhibited moderate growth following IT inoculation into Ae. aegypti and Ae. albopictus, reaching titers of 3.6-5.0 log(10) PFU/mosquito. There was no change in the virus genotype associated with replication in mosquitoes. Similar results were observed in mosquitoes of all three species that were IT inoculated or had orally ingested the YF 17D vaccine virus. In contrast, all mosquitoes either IT inoculated with or orally fed wild-type and vaccine JE viruses became infected, reaching maximum titers of 5.4-7.3 log(10) PFU/mosquito. These results indicate that ChimeriVax-JE virus is restricted in its ability to infect and replicate in these mosquito vectors. The low viremia caused by ChimeriVax-JE in primates and poor infectivity for mosquitoes are safeguards against secondary spread of the vaccine virus.     

Heparan sulfate binding by natural eastern equine encephalitis viruses promotes neurovirulence

The Alphavirus genus of the family Togaviridae contains mosquito-vectored viruses that primarily cause either arthritogenic disease or acute encephalitis. North American eastern equine encephalitis virus (NA-EEEV) is uniquely neurovirulent among encephalitic alphaviruses, causing mortality in a majority of symptomatic cases and neurological sequelae in many survivors. Unlike many alphaviruses, NA-EEEV infection of mice yields limited signs of febrile illness typically associated with lymphoid tissue replication. Rather, signs of brain infection, including seizures, are prominent. Use of heparan sulfate (HS) as an attachment receptor increases the neurovirulence of cell culture-adapted strains of Sindbis virus, an arthritogenic alphavirus. However, this receptor is not known to be used by naturally circulating alphaviruses. We demonstrate that wild-type NA-EEEV strain FL91-4679 uses HS as an attachment receptor and that the amino acid sequence of its E2 attachment protein is identical to those of natural isolates sequenced by RT-PCR amplification of field samples. This finding unequivocally confirms the use of HS receptors by naturally circulating NA-EEEV strains. Inactivation of the major HS binding domain in NA-EEEV E2 demonstrated that the HS binding increased brain replication and neurologic disease but reduced lymphoid tissue replication, febrile illness signs, and cytokine/chemokine induction in mice. We propose that HS binding by natural NA-EEEV strains alters tropism in vivo to antagonize/evade immune responses, and the extreme neurovirulence of wild-type NA-EEEV may be a consequence. Therefore, reinvestigation of HS binding by this and other arboviruses is warranted.     

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.     

Arbovirus surveillance in six states during 1972.

A virus surveillance project was established and maintained during 1972 along 10 major river drainages in six states. Mosquitoes, biting flies, and blood specimens from sentinel equines were collected during 83 field trip visits to 141 arthropod collecting sites and 22 sentinel locations from April into December 1972. There were 173,074 mosquitoes tested and 303 arboviruses isolated from 11 of 41 species. From 13,388 biting flies tested, 8 arbovirus isolations were obtained in 1 of 5 species. There was no isolation of Venezuelan equine encephalitis (VEE) virus. Western equine encephalitis (WEE) virus isolates were the most numerous and were followed by Turlock, St. Louis encephalitis, Hart Park, California encephalitis, and Bunyamwera (BUN) group viruses. The first isolation of WEE from the mosquito Cullex (Mel). erraticus is reported, as is the extension of the ranges for Buttonwillow virus from California to New Mexico and Texas. Also a single isolation of the BUN group from Culicoides variipennis extends the range of this virus-vector relationship from California to Texas. New distribution records for mosquito species previously unreported for Arizona, Louisana, New Mexico, and Oklahoma are reported. The sentinel burros detected WEE serologic conversions at two sites in New Mexico and at one in Texas. The surveillance project provided state and federal officials with current information on the status of arbovirus activity, including the absence of VEE activity during 1972, and it demonstrated the existence of the potential for WEE epizootics and epidemics throughout a wide geographic area of the Western United States.     

Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication

Most alphaviruses and many other arboviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans, and nonhuman primates. Consequently, they can be propagated in most vertebrate and insect cell cultures. This ability of arboviruses to infect arthropods and vertebrates is usually essential for their maintenance in nature. However, several flaviviruses have recently been described that infect mosquitoes but not vertebrates, although the mechanism of their host restriction has not been determined. Here we describe a unique alphavirus, Eilat virus (EILV), isolated from a pool of Anopheles coustani mosquitoes from the Negev desert of Israel. Phylogenetic analyses placed EILV as a sister to the Western equine encephalitis antigenic complex within the main clade of mosquito-borne alphaviruses. Electron microscopy revealed that, like other alphaviruses, EILV virions were spherical, 70 nm in diameter, and budded from the plasma membrane of mosquito cells in culture. EILV readily infected a variety of insect cells with little overt cytopathic effect. However, in contrast to typical mosquito-borne alphaviruses, EILV could not infect mammalian or avian cell lines, and viral as well as RNA replication could not be detected at 37 °C or 28 °C. Evolutionarily, these findings suggest that EILV lost its ability to infect vertebrate cells. Thus, EILV seems to be mosquito-specific and represents a previously undescribed complex within the genus Alphavirus. Reverse genetic studies of EILV may facilitate the discovery of determinants of alphavirus host range that mediate disease emergence.     

Larval nutritional stress does not affect vector competence for West Nile virus (WNV) in Culex tarsalis

In some mosquito species the conditions experienced by larvae during development have been shown to lead to changes in susceptibility to various arboviruses in the adult female. Since laboratory mosquitoes are generally reared under ideal conditions, mosquito vector competence experiments in the laboratory may not accurately reflect vector?virus relationships in nature. We examined the consequences of larval nutritional stress on Culex tarsalis vector competence for West Nile virus (WNV). Larval nutrition deprivation resulted in increased development time, decreased pupation and emergence rates, and smaller adult female body size. However, infection, dissemination, and transmission rates for WNV at 5, 7, and 14 days postfeeding were not consistently affected. These results suggest that larval nutritional rearing protocols are not a major factor in laboratory estimates of WNV vector competence in Cx. tarsalis.     

云南景东县乙型脑炎流行状况调查

目的调查云南省景东县流行性乙型脑炎的流行现状。方法媒介蚊虫调查采用诱蚊灯通宵捕蚊方法,蚊虫乙脑病毒检测采取RT-PCR方法,健康人群血清乙脑病毒抗体检测采用ELISA。结果共采集到蚊虫4属15种6653只,其中三带喙库蚊和中华按蚊属于当地的优势种群,分别占总捕获蚊数的82.07%和10.46%。RT-PCR检测三带喙库蚊和中华按蚊共16批1310只,各有1株感染乙脑病毒;ELISA检测当地健康人群血清乙脑病毒抗体阳性率为72.96%,不同年龄组人群乙脑病毒抗体水平差异有统计学意义(χ2=51.589,P0.01)。结论云南景东县蚊虫种类较多,三带喙库蚊和中华按蚊有传播乙型脑炎的作用。     

Vector competence of California mosquitoes for California encephalitis and California encephalitis-like viruses.

Mosquitoes collected from coastal, inland valley, and alpine locations in California were evaluated for their experimental vector competence for two viruses in the California serogroup (Bunyaviridae:Bunyavirus). Aedes squamiger, a coastal salt marsh mosquito, was an efficient vector of a California encephalitis (CE)-like virus isolated from its habitat (89% of the pledget-fed females became infected and 61% transmitted virus). Aedes dorsalis, a coastal mosquito, and Ae. melanimon, an inland valley mosquito, were competent vectors of prototype CE virus (98% and 100% of the pledget-fed females became infected and 56% and 30%, respectively, transmitted virus). Aedes squamiger and Ae. dorsalis transmitted both viruses vertically to one or more of 20 of their progeny. Culiseta inornata was susceptible to infection with both viruses, but 5% or less transmitted virus perorally. Alpine mosquitoes, Ae. cataphylla, Ae. increpitus, and Ae. tahoensis, became infected with both CE and CE-like viruses, but 3% or less transmitted virus. All species of mosquitoes were more efficient vectors of both viruses following intrathoracic inoculation than following pledget feeding, suggesting the presence of mesenteronal barriers.     

Mosquito surveys carried out on Green Island, Orchid Island, and Penghu Island, Taiwan, in 2003

Field surveys of mosquitoes were carried out on Green, Orchid, and Penghu Islands in 2003 to ascertain the status of mosquito vectors. Eighteen species of mosquitoes were collected, including three species of Anopheles, four species of Aedes, eight species of Culex, two species of Armigeres, and one species of Malaya. Seventeen previously recorded species were not collected in this study but 11 species collected had not previously been recorded. Ten newly recorded species, An. maculatus, An. takasagoensis, Ae. alcasidi, Ae. lineatopennis, Ae. vexans vexans, Ar. omissus, Cx. vishnui, Cx. halifaxii, Cx. hayashii, and Cx. neomimulus, were collected on Green Island and one previously unrecorded species, Ar. subalbatus, was collected on Orchid Island. Potential vectors An. maculatus and An. sinensis, malaria vectors in Korea and Mainland China, Ae. albopictus, a vector of dengue in Taiwan and West Nile virus in the USA, Cx. vishnui and Cx. tritaeniorhynchus, Japanese encephalitis vectors in Taiwan, Ae. vexans vexans, an eastern equine encephalitis vector in the USA, and Cx. quinquefasciatus, a vector of filariasis in Taiwan and West Nile virus in the USA, were among the mosquito species collected.