RNAi Targeting of West Nile Virus in Mosquito Midguts Promotes Virus Diversification

West Nile virus (WNV) exists in nature as a genetically diverse population of competing genomes. This high genetic diversity and concomitant adaptive plasticity has facilitated the rapid adaptation of WNV to North American transmission cycles and contributed to its explosive spread throughout the New World. WNV is maintained in nature in a transmission cycle between mosquitoes and birds, with intrahost genetic diversity highest in mosquitoes. The mechanistic basis for this increase in genetic diversity in mosquitoes is poorly understood. To determine whether the high mutational diversity of WNV in mosquitoes is driven by RNA interference (RNAi), we characterized the RNAi response to WNV in the midguts of orally exposed Culex pipiens quinquefasciatus using high-throughput, massively parallel sequencing and estimated viral genetic diversity. Our data demonstrate that WNV infection in orally exposed vector mosquitoes induces the RNAi pathway and that regions of the WNV genome that are more intensely targeted by RNAi are more likely to contain point mutations compared to weakly targeted regions. These results suggest that, under natural conditions, positive selection of WNV within mosquitoes is stronger in regions highly targeted by the host RNAi response. Further, they provide a mechanistic basis for the relative importance of mosquitoes in driving WNV diversification.     

Migrating Birds as Dispersal Vehicles for West Nile Virus

Whereas migrating birds have been implicated in the spread of West Nile virus (WNV), there is no direct evidence of birds actively migrating while infectious. The role of birds in WNV dispersal is difficult to assess in the field. However, this role can be evaluated experimentally because birds in migratory disposition display increased locomotor activity or restlessness under captive conditions. We tested the following hypotheses: (1) migrating passerine birds continue to exhibit migratory activity while infectious with WNV and (2) the migratory state of the individual affects the magnitude of viremia. We examined the migratory activity of two neoarctic-neotropical passerine migrants, Swainson’s thrush (Catharus ustulatus) and gray catbird (Dumetella carolinensis), during acute WNV infection. All gray catbirds and six of nine Swainson’s thrushes exhibited migratory activity while infectious. Moreover, migratory status did not appear to influence viremia titers, as might be expected if individuals were immunosuppressed during migration. Therefore, we demonstrate that migrating passerine birds are potential dispersal vehicles for WNV.     

Comparing Competitive Fitness of West Nile Virus Strains in Avian and Mosquito Hosts

Enzootic transmission of West Nile virus (WNV; Flaviviridae, Flavivirus) involves various species of birds and ornithophilic mosquitoes. Single nucleotide substitutions in the WNV genome may impact viral fitness necessary for WNV adaptation and evolution as previously shown for the WN02 genotype. In an effort to study phenotypic change, we developed an in vivo fitness competition model in two biologically relevant hosts for WNV. The House Finch (HOFI; Haemorhous mexicanus) and Culex tarsalis mosquitoes represent moderately susceptible hosts for WNV, are highly abundant in Western North America and frequently are infected with WNV in nature. Herein, we inoculated HOFIs and Cx. tarsalis competitively (dually) and singly with infectious-clone derived viruses of the founding California isolate COAV997-2003 (COAV997-IC), the founding North American isolate NY99 (NY99-IC), and a 2004 field isolate from California (CA-04), and compared the replicative capacities (fitness) of these viruses to a genetically marked virus of COAV997 (COAV997-5nt) by measuring RNA copy numbers. COAV997 and COAV997-5nt exhibited neutral fitness in HOFIs and Cx. tarsalis, and the temperature-sensitive phenotype of COAV997 did not affect replication in HOFIs as none of the infected birds became febrile. The NY99 and CA-04 isolates demonstrated elevated fitness in HOFIs compared to COAV997-5nt, whereas all viruses replicated to similar titers and RNA copies in Cx. tarsalis, and the only fitness differences were related to infection rates. Our data demonstrated that competitive replication allows for the sensitive comparison of fitness differences among two genetically closely related viruses using relevant hosts of WNV while eliminating host-to-host differences. In conclusion, our approach may be helpful in understanding the extent of phenotypic change in fitness associated with genetic changes in WNV.     

Ecological Niche Modelling of Potential West Nile Virus Vector Mosquito Species and Their Geographical Association with Equine Epizootics in Italy

In Italy, West Nile virus (WNV) equine outbreaks have occurred annually since 2008. Characterizing WNV vector habitat requirements allows for the identification of areas at risk of viral amplification and transmission. Maxent-based ecological niche models were developed using literature records of 13 potential WNV Italian vector mosquito species to predict their habitat suitability range and to investigate possible geographical associations with WNV equine outbreak occurrence in Italy from 2008 to 2010. The contribution of different environmental variables to the niche models was also assessed. Suitable habitats for Culex pipiens, Aedes albopictus, and Anopheles maculipennis were widely distributed; Culex modestus, Ochlerotatus geniculatus, Ochlerotatus caspius, Coquillettidia richiardii, Aedes vexans, and Anopheles plumbeus were concentrated in north-central Italy; Aedes cinereus, Culex theileri, Ochlerotatus dorsalis, and Culiseta longiareolata were restricted to coastal/southern areas. Elevation, temperature, and precipitation variables showed the highest predictive power. Host population and landscape variables provided minor contributions. WNV equine outbreaks had a significantly higher probability to occur in habitats suitable for Cx. modestus and Cx. pipiens, providing circumstantial evidence that the potential distribution of these two species coincides geographically with the observed distribution of the disease in equines.     

West Nile Virus Surveillance in 2013 via Mosquito Screening in Northern Italy and the Influence of Weather on Virus Circulation

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.     

The Effect of West Nile Virus Perceptions and Knowledge on Protective Behavior and Mosquito Breeding in Residential Yards in Upstate New York

A knowledge, attitudes, and practices (KAP) questionnaire combined with entomological surveys of residential mosquito-breeding sites were conducted in two Upstate New York neighborhoods. We tested the hypothesis that “correct” West Nile virus (WNV) knowledge and perceptions correspond with the use of practices that prevent mosquitoes from breeding and biting. Our results demonstrate that perceptions of WNV relate to the number of positive containers in yards and the use of mosquito preventive measures. In contrast, WNV knowledge was not related. Culex pipiens and Cx. restuans were common species found breeding in containers. Aedes japonicus was the most abundant species in 77% of positive containers (buckets, flower pots, and birdbaths). This new, invasive mosquito together with the Culex species identified in this study represent significant potential as vectors of WNV and other arboviruses affecting human and animal health. We conclude that more training and education programs should focus on WNV control strategies and recognizing mosquito breeding in residential yards. This is the first study to directly investigate the relationship between KAP and breeding of WNV vectors in residential yards.     

Effect of Wolbachia on Replication of West Nile Virus in a Mosquito Cell Line and Adult Mosquitoes

Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.     

Immunization of Mice with Recombinant Mosquito Salivary Protein D7 Enhances Mortality from Subsequent West Nile Virus Infection via Mosquito Bite

Background

Mosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of proteins in saliva responsible for immunomodulation and counteracting their effects on host immune response is a potential strategy to protect against arboviral disease. We selected a member of the D7 protein family, which are among the most abundant and immunogenic in mosquito saliva, as a vaccine candidate with the aim of neutralizing effects on the mammalian immune response normally elicited by mosquito saliva components during arbovirus transmission.

Methodology/Principal Findings

We identified D7 salivary proteins of Culex tarsalis, a West Nile virus (WNV) vector in North America, and expressed 36 kDa recombinant D7 (rD7) protein for use as a vaccine. Vaccinated mice exhibited enhanced interferon-γ and decreased interleukin-10 expression after uninfected mosquito bite; however, we found unexpectedly that rD7 vaccination resulted in enhanced pathogenesis from mosquito-transmitted WNV infection. Passive transfer of vaccinated mice sera to naïve mice also resulted in increased mortality rates from subsequent mosquito-transmitted WNV infection, implicating the humoral immune response to the vaccine in enhancement of viral pathogenesis. Vaccinated mice showed decreases in interferon-γ and increases in splenocytes producing the regulatory cytokine IL-10 after WNV infection by mosquito bite.

Conclusions/Significance

Vector saliva vaccines have successfully protected against other blood-feeding arthropod-transmitted diseases. Nevertheless, the rD7 salivary protein vaccine was not a good candidate for protection against WNV disease since immunized mice infected via an infected mosquito bite exhibited enhanced mortality. Selection of salivary protein vaccines on the bases of abundance and immunogenicity does not predict efficacy.     

西尼罗病毒研究进展

西尼罗病毒 (West Nile virus,WNV) 首先在1937年乌干达的西尼罗地区的Omogo镇的一位发热病人血液中分离到,从而得名[1].西尼罗病毒属于黄病毒科黄病毒属.     

Mosquito saliva causes enhancement of West Nile virus infection in mice

West Nile virus (WNV) is transmitted to vertebrate hosts primarily by infected Culex mosquitoes. Transmission of arboviruses by the bite of infected mosquitoes can potentiate infection in hosts compared to viral infection by needle inoculation. Here we examined the effect of mosquito transmission on WNV infection and systematically investigated multiple factors that differ between mosquito infection and needle inoculation of WNV. We found that mice infected with WNV through the bite of a single infected Culex tarsalis mosquito exhibited 5- to 10-fold-higher viremia and tissue titers at 24 and 48 h postinoculation and faster neuroinvasion than mice given a median mosquito-inoculated dose of WNV (10(5) PFU) by needle. Mosquito-induced enhancement was not due to differences in inoculation location, because additional intravenous inoculation of WNV did not enhance viremia or tissue titers. Inoculation of WNV into a location where uninfected mosquitoes had fed resulted in enhanced viremia and tissue titers in mice similar to those in mice infected by a single infected mosquito bite, suggesting that differences in where virus is deposited in the skin and in the virus particle itself were not responsible for the enhanced early infection in mosquito-infected mice. In addition, inoculation of mice with WNV mixed with salivary gland extract (SGE) led to higher viremia, demonstrating that mosquito saliva is the major cause of mosquito-induced enhancement. Enhanced viremia was not observed when SGE was inoculated at a distal site, suggesting that SGE enhances WNV replication by exerting a local effect. Furthermore, enhancement of WNV infection still occurred in mice with antibodies against mosquito saliva. In conclusion, saliva from C. tarsalis is responsible for enhancement of early WNV infection in vertebrate hosts.     

Seasonality and Outbreaks in West Nile Virus Infection

In this paper we analyze the impact of seasonal variations on the dynamics of West Nile Virus infection. We are interested in the generation of new epidemic peaks starting from an endemic state. In many cases, the oscillations generated by seasonality in the dynamics of the infection are too small to be observable. The interplay of this seasonality with the epidemic oscillations can generate new outbreaks starting from the endemic state through a mechanism of parametric resonance. Using experimental data we present specific cases where this phenomenon is numerically observed.     

Probability of Disease Extinction or Outbreak in a Stochastic Epidemic Model for West Nile Virus Dynamics in Birds

Acta Biotheoretica - Thresholds for disease extinction provide essential information for the prevention and control of diseases. In this paper, a stochastic epidemic model, a continuous-time Markov...     

西尼罗病毒易感性研究现状

西尼罗病毒可引起鼠、人类及其他动物严重脑炎,近年来再度流行。基因研究分析表明近年来西尼罗病毒突变的速度逐渐加快,突变主要发生在基因编码区,这些突变可导致病毒蛋白E区、NS1区、NS2区及NS5区氨基酸序列的改变。西尼罗病毒感染主要发生于老龄及免疫缺陷动物或人群。西尼罗病毒的易感染性除与突变有重要关系外,与CCR5、OAS及CXCL10等受体及因子也有一定的关系。     

Modelling the dynamics of West Nile Virus

In this work we formulate and analyze a mathematical model for the transmission of West Nile Virus (WNV) infection between vector (mosquito) and avian population. We find the Basic Reproductive Number in terms of measurable epidemiological and demographic parameters. is the threshold condition that determines the dynamics of WNV infection: if the disease fades out, and for the disease remains endemic. Using experimental and field data we estimate for several species of birds. Numerical simulations of the temporal course of the infected bird proportion show damped oscillations approaching the endemic value.