Emergence of Zika Virus in Culex tritaeniorhynchus and Anopheles sinensis Mosquitoes in China

Zika virus(ZIKV) has been isolated from mosquitoes such as Aedes, Mansonia uniformis, and Culex perfuscus; However,the isolation of ZIKV from Anopheles sinensis and Culex tritaeniorhynchus has not yet been reported. In June and July2018, 22,985 mosquitoes and 57,500 midges were collected in Jiangxi Province in southeastern China. Among them, six strains of ZIKV were isolated from mosquitoes: four from An. sinensis and two from Cx. tritaeniorhynchus. Molecular genetic analysis showed that the ZIKV isolated from An. sinensis and Cx. tritaeniorhynchus belonged to genotype 2 in the Asian evolutionary branch of ZIKV. In addition, the ZIKV strains isolated from An. sinensis and Cx. tritaeniorhynchus had amino acid substitutions identical to ZIKV strains prevalent in South America since 2015. This study is the first to isolate ZIKV from mosquito specimens collected in the wild of Jiangxi Province, China; This is also the first time that ZIKV has been isolated from An. sinensis and Cx. tritaeniorhynchus. Given that An. sinensis and Cx. tritaeniorhynchus have a very wide geographical distribution in China and even in eastern and southern Asia, the isolation of several strains of ZIKV from these two mosquitoes poses new challenges for the prevention and control of ZIKV infection in the mainland of China and countries and regions with the same distribution of mosquitoes.     

Association of vectors and environmental conditions during the emergence of Peruvian horse sickness orbivirus and Yunnan orbivirus in northern Peru

Since 1983, cases of diseased donkeys and horses with symptoms similar to those produced by alphaviruses were identified in two departments in northern Peru; however serological testing ruled out the presence of those viruses and attempts to isolate an agent were also unproductive. In 1997, also in northern Peru, two new orbiviruses were discovered, each recognized as a causative agent of neurological diseases in livestock and domestic animals and, at the same time, mosquitoes were found to be infected with these viruses. Peruvian horse sickness virus (PHSV) was isolated from pools of culicid mosquitoes, Aedes serratus and Psorophora ferox, and Yunnan virus (YUOV) was isolated from Aedes scapularis in the subtropical jungle (upper jungle) located on the slope between the east side of the Andes and the Amazonian basin in the Department of San Martín. Both viruses later were recovered from mosquitoes collected above the slope between the west side of the Andes and the coast (Department of Piura) in humid subtropical areas associated with the Piura River basin. In this region, PHSV was isolated from Anopheles albimanus and YUOV was isolated from Ae. scapularis. We discuss the ecology of vector mosquitoes during the outbreaks in the areas where these mosquitoes were found.     

The use of direct sequencing of dengue virus cDNA from individual field-collected Aedes aegypti for surveillance and epidemiological studies

The relative efficiencies of four methods to extract viral RNA from individual dengue-2 virus (D-2V)-infected mosquitoes, Aedes aegypti (L.) (Diptera: Culicidae), were compared. The most efficient of these methods was then used to extract viral RNA for the preparation of cDNA from the abdomens of six engorged D-2V-infected mosquitoes and sera from three dengue fever (DF) patients collected in an isolated rural town in Colombia. Comparisons of viral envelope (E) gene sequences from each of these strongly suggested that the D-2V population which circulated in this study area was a homogeneous genotype which was unrelated to any of the D-2 viruses isolated from elsewhere in the world. When coupled with our rapid method to identify viruses in individual mosquitoes (Romero-Vivas et al. (1998) Medical and Veterinary Entomology, 12, 101-105), the methodology we describe should be useful for epidemiological and surveillance studies of dengue viruses and other arboviruses.     

Evaluation of the pathogenicity and infectivity of entomopathogenic hypocrealean fungi,isolated from wild mosquitoes in Japan and Burkina Faso,against female adult Anopheles stephensi mosquitoes

We investigated the potential of entomopathogenic hypocrealean fungi that naturally occur in or on adult mosquitoes for use as biocontrol agents of vector mosquitoes. The fungi were isolated from wild mosquitoes collected in Japan and Burkina Faso using two isolation methods (with and without surface sterilization). Detected fungal species included Beauveria bassiana sensu lato, Isaria spp., Paecilomyces spp., Lecanicillium spp., and Simplicillium spp. These isolates were used in bioassays against adult female Anopheles stephensi mosquitoes. The median survival time ranged from 5.8 to 14.9 d (control, 17.0 d). Reduced survival times were observed in the isolates from surface-sterilized mosquitoes from Japan, with the isolate B. bassiana s.l. 60-2 exhibiting the highest virulence. This study indicates that adult mosquitoes are naturally infected with various entomopathogenic hypocrealean fungi, and that some of these isolates have the potential for use as fungal pesticides to control vector mosquitoes.     

Virus isolations from mosquitoes in southern Ontario, 1976 and 1977

Following the 1975 epidemic of St. Louis encephalitis (SLE) in Ontario, programs were instituted to monitor virus activity in mosquito populations during 1976 and 1977. Mosquitoes were trapped with CDC light traps and CO2 cone traps, pooled by species, and tested for virus by intracerebral inoculation of suckling mice. In 1976, 51 175 mosquitoes were tested. SLE virus was isolated from two mixed pools of Culex pipiens--C. restuans mosquitoes. Five isolations of California serogroup viruses were made. Three of these were trivittatus virus, which has not been demonstrated previously in Canada, and the other two were snowshoe have virus. Other viruses isolated in 1976 were a virus antigenically identical to the virus of infectious bursal disease of chickens and 34 Flanders viruses. In 1977, 34 428 mosquitoes were tested. Flanders virus was isolated most frequently, from pools of mixed C. pipiens--C. restuans mosquitoes. The only other isolate was a Bunyamwera group virus, Cache Valley virus. This virus has not been reported previously in Ontario.     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.     

An orbivirus of mosquitoes which induces CO2 sensitivity in mosquitoes and is lethal for rabbits.

An orbivirus, JKT-7400, isolated from Culex mosquitoes in Indonesia, replicated to a high titer and induced cytopathic effects in Aedes albopictus cell cultures. The virus produced lethal sensitivity to carbon dioxide in Culex and Aedes mosquitoes as well as in Drosophila melanogaster fruit flies but was not the agent of the hereditary sensitivity to carbon dioxide previously described for Culex quinquefasciatus. When injected intravenously in high doses, JKT-7400 virus was lethal for rabbits, apparently without replicating to a significant extent. It was not pathogenic for adult mice inoculated intravenously or for adult or suckling mice inoculated intracerebrally and intraperitoneally. Unlike an orbivirus isolated from Culex mosquitoes in China, JKT-7400 did not interfere with the replication of Japanese encephalitis virus in mosquitoes.     

白纹伊蚊和埃及伊蚊对基孔肯雅病毒的易感性和传播性的研究

用４株基孔肯雅病毒经口感染白纹伊蚊和埃及伊蚊,进行了易感性和传播性的研究。结果表明,这两种蚊虫对基孔肯雅病毒易感。无论白纹伊蚊或埃及伊蚊,感染后第５－６天即可通过吸血将病毒传播给乳鼠,至第８－１３天,传播率可高达５５．５５％－１００％。感染蚊亦可经叮咬将病毒传播给小鸡。埃及伊蚊的易感性和传播率高于白纹伊蚊。实验还发现,不同来源毒株之间存在一定差异,如分离自云南白纹伊蚊的Ｍ８１株的感染率和传播率均高于其它毒株。这些结果表明,白纹伊蚊和埃及伊蚊在基孔肯雅病毒的保存和传播中起重要作用。     

云南省4种伊蚊的乙型脑炎病毒分离物的研究

在云南省西南边境９县市捕获伊蚊属雌性成蚊１６种１９３６７只,用细胞法和乳鼠法分离病毒。从１８５批６４９１只白纹伊蚊中分离到病毒２株,从５０批１６０５只剌扰伊蚊中分离到病毒２株,从２３批７７２只窄翅伊蚊中分离到病毒２株,从４批１０３只阿萨姆伊蚊中分离到病毒１株。其它１２种共１０３９６只伊蚊的病毒分离物为阴性。分离到的７株病毒经免疫荧光、酶免疫、血凝抑制和中和试验鉴定,均为乙型脑炎病毒（ＪＥｖｉｒｕｓ）。白纹伊蚊是野外竹林的优势蚊种。分析认为白纹伊蚊在当地乙型脑炎病毒保存和传播中起重要作用,刺扰伊蚊、窄翅伊蚊和阿萨姆伊蚊亦可参与该病毒的传播。     

Synthesis of erythro-5,6-diacetoxyhexadecanoic acid,a novel fatty acid from mosquito eggs

The synthesis of erythro-5,6-diacetoxyhexadecanoic acid, a major component of diglycerides isolated from the eggs of Culex tarsalis and related species and reported to influence oviposition of these mosquitoes, is described.     

Parasite-induced suppression of the immune response in Aedes aegypti by Brugia pahangi

The melanization response against intrathoracically inoculated Brugia pahangi and Dirofilaria immitis microfilariae (mff) isolated from vertebrate host blood was evaluated in both uninfected Aedes aegypti black-eyed Liverpool strain and in mosquitoes harboring a developing B. pahangi infection. The immune response against inoculated mff of either species was significantly reduced by 28-47% in infected as compared with uninfected mosquitoes. Attempts to passively transfer this suppression factor(s) by inoculating naive mosquitoes with 0.1-0.2 microliter of hemolymph from B. pahangi-infected mosquitoes produced equivocal results. The role this parasite-induced immune suppression might play in aiding parasite survival in compatible vectors is discussed.     

Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes

The mechanism of death in densovirus infected mosquitoes remains unexplored. This study investigated the cellular consequences of densovirus infection in Aedes aegypti mosquitoes after a second generation challenge with a densovirus isolated from adult Aedes albopictus mosquitoes in Thailand (AThDNV). Specimens were analyzed by TUNEL assay, fluorescent in situ hybridization (FISH) and a calorimic assay to detect activation of caspase 3-like activity. After challenge, moribund mosquitoes showed considerable evidence of TUNEL positive cells. The caspase 3-like activity assay showed that the presence of TUNEL positive cells was associated with increased levels of activated caspase 3-like activity in AThDNV infected mosquitoes.     

Cache Valley virus: isolations from mosquitoes in Saskatchewan, 1972-1974.

Eighteen isolations of Cache Valley virus (Bunyaviridae) were obtained from a total of 113,694 mosquitoes collected in Saskatchewan during the summers of 1972 to 1974. Most of the isolations were from mosquitoes collected during August. Culiseta inornata, the most abundant mosquito (38% of total collected), had the highest minimum vector-infection rate (0.83 isolations per 1000 mosquitoes). The virus was also isolated from Culex tarsalis and Aedes vexans. It is indicated in the isolations that the prairie grasslands of the province are enzootic for Cache Valley virus.     

Evidence of Culiseta mosquitoes as vectors for Plasmodium parasites in Alaska

Mosquito vectors play a crucial role in the distribution of avian Plasmodium parasites worldwide. At northern latitudes, where climate warming is most pronounced, there are questions about possible changes in the abundance and distribution of Plasmodium parasites, their vectors, and their impacts to avian hosts. To better understand the transmission of Plasmodium among local birds and to gather baseline data on potential vectors, we sampled a total of 3,909 mosquitoes from three locations in south‐central Alaska during the summer of 2016. We screened mosquitoes for the presence of Plasmodium parasites using molecular techniques and estimated Plasmodium infection rates per 1,000 mosquitoes using maximum likelihood methods. We found low estimated infection rates across all mosquitoes (1.28 per 1,000), with significantly higher rates in Culiseta mosquitoes (7.91 per 1,000) than in Aedes mosquitoes (0.57 per 1,000). We detected Plasmodium in a single head/thorax sample of Culiseta, indicating potential for transmission of these parasites by mosquitoes of this genus. Plasmodium parasite DNA isolated from mosquitoes showed a 100% identity match to the BT7 Plasmodium lineage that has been detected in numerous avian species worldwide. Additionally, microscopic analysis of blood smears collected from black‐capped chickadees (Poecile atricapillus) at the same locations revealed infection by parasites preliminarily identified as Plasmodium circumflexum. Results from our study provide the first information on Plasmodium infection rates in Alaskan mosquitoes and evidence that Culiseta species may play a role in the transmission and maintenance of Plasmodium parasites in this region.     

Genomic Characterisation of Three Mapputta Group Viruses,a Serogroup of Australian and Papua New Guinean Bunyaviruses Associated with Human Disease

The Mapputta serogroup tentatively contains the mosquito-associated viruses Mapputta, Maprik, Trubanaman and Gan Gan. Interestingly, this serogroup has previously been associated with an acute epidemic polyarthritis-like illness in humans; however, there has been no ensuing genetic characterisation. Here we report the complete genome sequences of Mapputta and Maprik viruses, and a new Mapputta group candidate, Buffalo Creek virus, previously isolated from mosquitoes and detected by serology in a hospitalised patient. Phylogenetic analyses indicate that the group is one of the earliest diverged groups within the genus Orthobunyavirus of the family Bunyaviridae. Analyses show that these three viruses are related to the recently sequenced Australian bunyaviruses from mosquitoes, Salt Ash and Murrumbidgee. A notable feature of the Mapputta group viruses is the absence of the NSs (non-structural) ORF commonly found on the S segment of other orthobunyaviruses. Viruses of the Mapputta group have been isolated from geographically diverse regions ranging from tropical Papua New Guinea to the semi-arid climate of south-eastern Australia. The relevance of this group to human health in the region merits further investigation.     

Human immune responses that reduce the transmission of Plasmodium falciparum in African populations

Malaria-infected individuals can develop antibodies which reduce the infectiousness of Plasmodium gametocytes to biting Anopheles mosquitoes. When ingested in a bloodmeal together with gametocytes, these antibodies reduce or prevent subsequent parasite maturation in the insect host. This transmission-blocking immunity is usually measured in human sera by testing its effect on the infectivity of gametocytes grown in vitro. Here we evaluate evidence of transmission-blocking immunity in eight studies conducted in three African countries. Plasmodium falciparum gametocytes isolated from each individual were fed to mosquitoes in both autologous plasma collected with the parasites, and permissive serum from non-exposed donors. Evidence of transmission reducing effects of autologous plasma was found in all countries. Experiments involving 116 Gambian children (aged 0.5-15 years) were combined to determine which factors were associated with transmission reducing immune responses. The chances of infecting at least one mosquito and the average proportion of infected mosquitoes were negatively associated with recent exposure to gametocytes and sampling late in the season. These results suggest that effective malaria transmission-reducing antibodies do not commonly circulate in African children, and that recent gametocyte carriage is required to initiate and/or boost such responses.     

Multiple Cytochrome P450 genes: their constitutive overexpression and permethrin induction in insecticide resistant mosquitoes, Culex quinquefasciatus

Four cytochrome P450 cDNAs, CYP6AA7, CYP9J40, CYP9J34, and CYP9M10, were isolated from mosquitoes, Culex quinquefasciatus. The P450 gene expression and induction by permethrin were compared for three different mosquito populations bearing different resistance phenotypes, ranging from susceptible (S-Lab), through intermediate (HAmCq(G0), the field parental population) to highly resistant (HAmCq(G8), the 8(th) generation of permethrin selected offspring of HAmCq(G0)). A strong correlation was found for P450 gene expression with the levels of resistance and following permethrin selection at the larval stage of mosquitoes, with the highest expression levels identified in HAmCq(G8), suggesting the importance of CYP6AA7, CYP9J40, CYP9J34, and CYP9M10 in the permethrin resistance of larva mosquitoes. Only CYP6AA7 showed a significant overexpression in HAmCq(G8) adult mosquitoes. Other P450 genes had similar expression levels among the mosquito populations tested, suggesting different P450 genes may be involved in the response to insecticide pressure in different developmental stages. The expression of CYP6AA7, CYP9J34, and CYP9M10 was further induced by permethrin in resistant mosquitoes. Taken together, these results indicate that multiple P450 genes are up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, thus increasing the overall expression levels of P450 genes.     

Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae)

Amblyospora species and other aquatic Microsporidia were isolated from mosquitoes, black flies, and copepods and the small subunit ribosomal RNA gene was sequenced. Comparative phylogenetic analysis showed a correspondence between the mosquito host genera and their Amblyspora parasite species. There is a clade of Amblyospora species that infect the Culex host group and a clade of Amblyospora that infect the Aedes/Ochlerotatus group of mosquitoes. Parathelohania species, which infect Anopheles mosquitoes, may be the sister group to the Amblyospora in the same way that the Anopheles mosquitoes are thought to be the sister group to the Culex and Aedes mosquitoes. In addition, by sequence analysis of small subunit rDNA from spores, we identified the alternate copepod host for four species of Amblyospora. Amblyospora species are specific for their primary (mosquito) host and each of these mosquito species serves as host for only one Amblyospora species. On the other hand, a single species of copepod can serve as an intermediate host to several Amblyospora species and some Amblyospora species may be found in more than one copepod host. Intrapredatorus barri, a species within a monotypic genus with Amblyospora-like characteristics, falls well within the Amblyospora clade. The genera Edhazardia and Culicospora, which do not have functional meiospores and do not require an intermediate host, but which do have a lanceolate spore type which is ultrastructurally very similar to the Amblyospora spore type found in the copepod, cluster among the Amblyospora species. In the future, the genus Amblyospora may be redefined to include species without obligate intermediate hosts. Hazardia, Berwaldia, Larssonia, Trichotuzetia, and Gurleya are members of a sister group to the Amblyospora clades infecting mosquitoes, and may be representatives of a large group of aquatic parasites.     

Phylogenetic relationships of Pleistophora-like microsporidia based on small subunit ribosomal DNA sequences and implications for the source of trachipleistophora hominis infections

The microsporidian Trachipleistophora hominis was isolated in vitro from the skeletal muscle of an AIDS patient. Since its discovery several more cases of myositis due to Trachipleistophora have been diagnosed but the source of infection is unknown. Morphologically, T. hominis most closely resembles Pleistophora and Vavraia, which undergo polysporous sporogony in sporophorous vesicles, but differs from these genera in the mode of formation of sporoblasts and in the morphology of the sporophorous vesicles. Alignment and analyses of the small subunit ribosomal DNA sequences of T. hominis and several other polysporoblastic genera indicated that its closest phylogenetic relationships were with species of the genera Pleistophora and Vavraia, in line with morphological predictions. The type species of the latter two genera are Pleistophora typicalis and Vavraia culicis; these are parasites of fish and mosquitoes, respectively. These results suggest two possible routes and sources of infection to AIDS patients, these being perorally by ingestion of inadequately cooked fish or crustaceans or percutaneously during a bloodmeal taken by a haematophagous insect. Support for an insect source has been provided by recent detection of a microsporidium from mosquitoes in human corneal tissue.     

Identification of super-infected Aedes triseriatus mosquitoes collected as eggs from the field and partial characterization of the infecting La Crosse viruses

Background

La Crosse virus (LACV) is a pathogenic arbovirus that is transovarially transmitted by Aedes triseriatus mosquitoes and overwinters in diapausing eggs. However, previous models predicted transovarial transmission (TOT) to be insufficient to maintain LACV in nature.

Results

To investigate this issue, we reared mosquitoes from field-collected eggs and assayed adults individually for LACV antigen, viral RNA by RT-PCR, and infectious virus. The mosquitoes had three distinct infection phenotypes: 1) super infected (SI+) mosquitoes contained infectious virus, large accumulations of viral antigen and RNA and comprised 17 of 17,825 (0.09%) of assayed mosquitoes, 2) infected mosquitoes (I+) contained no detectable infectious virus, lesser amounts of viral antigen and RNA, and comprised 3.7% of mosquitoes, and 3) non-infected mosquitoes (I-) contained no detectable viral antigen, RNA, or infectious virus and comprised 96.21% of mosquitoes. SI+ mosquitoes were recovered in consecutive years at one field site, suggesting that lineages of TOT stably-infected and geographically isolated Ae. triseriatus exist in nature. Analyses of LACV genomes showed that SI+ isolates are not monophyletic nor phylogenetically distinct and that synonymous substitution rates exceed replacement rates in all genes and isolates. Analysis of singleton versus shared mutations (Fu and Li's F*) revealed that the SI+ LACV M segment, with a large and significant excess of intermediate-frequency alleles, evolves through disruptive selection that maintains SI+ alleles at higher frequencies than the average mutation rate. A QTN in the LACV NSm gene was detected in SI+ mosquitoes, but not in I+ mosquitoes. Four amino acid changes were detected in the LACV NSm gene from SI+ but not I+ mosquitoes from one site, and may condition vector super infection. In contrast to NSm, the NSs sequences of LACV from SI+ and I+ mosquitoes were identical.

Conclusions

SI+ mosquitoes may represent stabilized infections of Ae. triseriatus mosquitoes, which could maintain LACV in nature. A gene-for-gene interaction involving the viral NSm gene and a vector innate immune response gene may condition stabilized infection.