我国西尼罗病毒和Tahyna病毒的发现与流行

已发现100余种蚊传虫媒病毒在世界各地流行,其引发的人兽共患病是全世界关注的公共卫生问题。长期以来我国仅发现乙型脑炎和登革热两种蚊传虫媒病毒病,但近年来新发现西尼罗病毒和Tahyna病毒及其感染疾病流行。从我国新疆维吾尔自治区采集的蚊虫标本中分离到西尼罗病毒,大量血清学研究证明当地不仅存在西尼罗病毒感染所致疾病,还发生过西尼罗病毒感染引发的病毒性脑炎流行。目前已从新疆维吾尔自治区、青海省和内蒙古自治区采集的蚊虫标本中分离到Tahyna病毒,并发现其在自然界动物中的循环和导致的人类感染流行。西尼罗病毒和Tahyna病毒及其相关感染性疾病的发现为我国虫媒病毒及虫媒病毒病的预防与控制提出了新的挑战。     

Climate-based models for West Nile <Emphasis Type="Italic">Culex</Emphasis> mosquito vectors in the Northeastern US

Climate-based models simulating Culex mosquito population abundance in the Northeastern US were developed. Two West Nile vector species, Culex pipiens and Culex restuans, were included in model simulations. The model was optimized by a parameter-space search within biological bounds. Mosquito population dynamics were driven by major environmental factors including temperature, rainfall, evaporation rate and photoperiod. The results show a strong correlation between the timing of early population increases (as early warning of West Nile virus risk) and decreases in late summer. Simulated abundance was highly correlated with actual mosquito capture in New Jersey light traps and validated with field data. This climate-based model simulates the population dynamics of both the adult and immature mosquito life stage of Culex arbovirus vectors in the Northeastern US. It is expected to have direct and practical application for mosquito control and West Nile prevention programs.     

A continental risk assessment of West Nile virus under climate change

Since first introduced to North America in 1999, West Nile virus (WNV) has spread rapidly across the continent, threatening wildlife populations and posing serious health risks to humans. While WNV incidence has been linked to environmental factors, particularly temperature and rainfall, little is known about how future climate change may affect the spread of the disease. Using available data on WNV infections in vectors and hosts collected from 2003–2011 and using a suite of 10 species distribution models, weighted according to their predictive performance, we modeled the incidence of WNV under current climate conditions at a continental scale. Models were found to accurately predict spatial patterns of WNV that were then used to examine how future climate may affect the spread of the disease. Predictions were accurate for cases of human WNV infection in the following year (2012), with areas reporting infections having significantly higher probability of presence as predicted by our models. Projected geographic distributions of WNV in North America under future climate for 2050 and 2080 show an expansion of suitable climate for the disease, driven by warmer temperatures and lower annual precipitation that will result in the exposure of new and naïve host populations to the virus with potentially serious consequences. Our risk assessment identifies current and future hotspots of West Nile virus where mitigation efforts should be focused and presents an important new approach for monitoring vector‐borne disease under climate change.     

West Nile fever/encephalitis as one of the arboviral infections

West Nile virus maintains natural infection cycle between birds and mosquitoes. It has been known that about 200 species of birds are infected with West Nile virus and the virus is isolated from more than 40 species of mosquitoes. This suggests that West Nile virus has an ability to be transmitted by many species of mosquitoes and infect many kinds of animals. Approximately 20% of infected humans develop symptoms. West Nile fever, an acute febrile illness, is the main disease, and meningitis and encephalitis (meningoencephalitis) occasionally occur. Cases with flaccid paralysis or polyneuritis have been recently reported. Thus, West Nile virus causes multiple types of symptoms in humans. The endemic area has expanded in North America and Siberia. West Nile virus may enter Japan in the near future; therefore, we should keep paying attention to the endemic and epidemic situations in the world.     

Worldwide invasion of vector mosquitoes: present European distribution and challenges for Spain

An Asiatic mosquito species, Aedes albopictus, began to spread worldwide in the 1970s thanks to marine transport of tires and other goods, leading to colonization of many areas of the world. This species is a vector of major human diseases such as Dengue, Yellow Fever and the West Nile virus. In Europe, it was established in Albania and Italy and has been detected in other countries such as France; no records exist for Spain as yet. Colonization by Aedes albopictus is a major public health concern considering that the West Nile virus and several other viruses are known to circulate sporadically in the Mediterranean. Additionally, the parent species Aedes aegypti was the vector causing severe outbreaks of Dengue and Yellow Fever two centuries ago. Although Ae. aegypti was also introduced, it was eradicated from Spain. Both mosquitoes shared habitat types, diseases transmitted and many bionomic data. This article contains a review of the present Ae. albopictusdistribution range worldwide and discusses the likelihood of an establishment in Spain in view of climatological and geographical data. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of factors for rapid development of Culex quinquefasciatus in belowground stormwater treatment devices

Water samples from 11 belowground stormwater treatment Best Management Practices (BMPs) were evaluated for their capacity to support rapid development of the West Nile virus (WNV) mosquito vector, Culex quinquefasciatus. The observed minimum development time from egg to pupa ranged from six to over 30 days. Concentrations of potential food resources (total suspended solids and the particulate organic matter in water samples) were significantly correlated to development times. In addition, the rate of immature mosquito development was both site‐dependent and variable in time, suggesting that factors favorable to rapid development were strongly influenced by watershed characteristics and seasonal changes in temperature. Measured temperatures in belowground BMPs suggest that these structures may remain amenable to WNV virus activity longer each year than sites aboveground.     

Survival of West Nile virus‐challenged Southern house mosquitoes,Culex pipiens quinquefasciatus,in relation to environmental temperatures

We investigated the effect of West Nile virus (WNV) infection on survival in two colonies of Culex pipiens quinquefasciatus Say (Diptera: Culicidae) originating from Vero Beach and Gainesville, FL. Mosquitoes were fed West Nile virus‐infected blood and checked daily for survival. Exposure to WNV decreased survival among Cx. p. quinquefasciatus from Gainesville relative to unexposed individuals at 31° C. In contrast, exposure to WNV enhanced survival among Cx. p. quinquefasciatus from Vero Beach relative to unexposed individuals at 27° C. These results may suggest that exposure to WNV and associated infection could increase or decrease components of fitness, dependent on environmental temperature and intraspecific variation in Cx. p. quinquefasciatus. The relationship between lifespan (time of death in days) and WNV titer differed in the colonies at 31° C and 27° C, suggesting that intraspecific species variation in response to temperature impacts interactions with WNV. While further work is needed to determine if similar effects occur under field conditions, this suggests intraspecific variation in vector competence for WNV and adult survival of Cx. p. quinquefasciatus, both aspects of vectorial capacity.     

Culex pipiens and Stegomyia albopicta (= Aedes albopictus) populations as vectors for lineage 1 and 2 West Nile virus in Europe

The emerging disease West Nile fever is caused by West Nile virus (WNV), one of the most widespread arboviruses. This study represents the first test of the vectorial competence of European Culex pipiens Linnaeus 1758 and Stegomyia albopicta (= Aedes albopictus) (both: Diptera: Culicidae) populations for lineage 1 and 2 WNV isolated in Europe. Culex pipiens and S. albopicta populations were susceptible to WNV infection, had disseminated infection, and were capable of transmitting both WNV lineages. This is the first WNV competence assay to maintain mosquito specimens under environmental conditions mimicking the field (day/night) conditions associated with the period of maximum expected WNV activity. The importance of environmental conditions is discussed and the issue of how previous experiments conducted in fixed high temperatures may have overestimated WNV vector competence results with respect to natural environmental conditions is analysed. The information presented should be useful to policymakers and public health authorities for establishing effective WNV surveillance and vector control programmes. This would improve preparedness to prevent future outbreaks.     

Abundance of West Nile virus mosquito vectors in relation to climate and landscape variables

It is currently unclear if the potential for West Nile virus transmission by mosquito vectors in the eastern United States is related to landscape or climate factors or both. We compared abundance of vector species between urban and suburban neighborhoods of Henrico County, VA, in relation to the following factors: temperature, precipitation, canopy cover, building footprint, and proximity to drainage infrastructure. Mosquitoes were collected throughout the 2005, 2006, and 2007 seasons and tested for West Nile virus (WNV) in pools of 10–50. Test results of mosquito pools were compared to average site abundance from 37 sites in Henrico County, VA; abundance was then examined in relation to ecological variables. Urban infrastructure was positively correlated with the abundance of Culex pipiens L./Cx. restuans, and our findings implicate combined sewer overflow systems as large contributors to Culex vector populations. No measure of urbanization examined in our study was correlated with Aedes albopictus abundance. Our study showed that certain landscape variables identified using Geographic Information Systems are valuable for predicting primary WNV vector abundance in Virginia, and that temperature along with low precipitation are strong predictors of population growth. Our results support other regional studies that found WNV proliferates under drought conditions.     

Microsatellite loci from the northern house mosquito (Culex pipiens), a principal vector of West Nile virus in North America

Microsatellites were isolated and characterized in the northern house mosquito, Culex pipiens, a widespread pest species and important vector of diseases such as West Nile virus. An enrichment protocol yielded 150 positive clones. We designed primers to amplify 17 unique (GT)_n microsatellites, eight of which amplified cleanly and were polymorphic. A survey of 29 individuals showed that these loci are highly variable with the number of alleles ranging from seven to 19 and expected heterozygosity ranging from 0.66 to 0.93. These markers will be useful for studies of population structure and intraspecific variation in epidemiological characteristics of Cx. pipiens.     

Infection of mouse neurones by West Nile virus is modulated by the interferon-inducible 2'-5' oligoadenylate synthetase 1b protein

Over the past 7 years, West Nile zoonosis has been an emerging concern for public health in Europe, Middle East and more recently in North America. West Nile virus causes epidemic outbreaks in humans and infected patients may exhibit severe neurological symptoms. Because susceptibility and sensitivity to West Nile virus infections may depend on host genetic factors, a mouse model has been established to investigate the genetic determinism of host susceptibility to West Nile virus. A nonsense mutation in gene encoding the 1b isoform of the 2'-5'oligoadenylate synthetase (OAS1b) was constantly associated with the susceptibility of mouse strains to experimental West Nile virus infection. Oligoadenylate synthetase are interferon-inducible proteins playing a role in the endogeneous antiviral pathway. It was of interest to establish whether interferon-alpha and OAS 1B were sufficient to mediate resistance to West Nile virus infection. In the present study, we showed that interferon-alpha had the ability to modulate West Nile virus infection in mouse. In vitro, interferon-alpha protected mouse neuroblastoma cells against West Nile virus infection if cells have been pretreated with the cytokine for several hours. As a consequence of the presence of a stop codon, the Oas1b gene of the susceptible mice encodes a truncated and presumably inactive form, while resistant mice have a normal copy of the gene. Stable mouse neuroblastoma cell clones overexpressing mutant or wild-type OAS 1B were established. Replication of West Nile virus was less efficient in cells that produce the normal copy of OAS 1B as compared to those expressing the truncated form. Our data illustrate the notion that interferon-alpha and Oas genes may be critical for West Nile virus pathogenesis.     

Polymorphic microsatellite loci from the West Nile virus vector Culex tarsalis

Since its introduction in 1999, West Nile virus (WNV) has spread across North America. Culex tarsalis is a highly efficient WNV vector species. Many traits such as virus susceptibility, autogeny and host preference vary geographically and temporally in C. tarsalis. Culex tarsalis genomic libraries were developed and were highly enriched for microsatellite inserts (42–96%). We identified 12 loci that were polymorphic in wild C. tarsalis populations. These microsatellites are the first DNA‐based genetic markers developed for C. tarsalis and will be useful for investigating population structure and constructing genetic maps in this mosquito.     

Wide recognition of Culex pipiens and lack of detection of Culex torrentium through biomolecular differentiation of mosquitoes in the Emilia‐Romagna region,Northern Italy

The Culex pipiens complex includes species with reported differences in vector competence for arthropod‐borne viruses, many of which are of significant importance to human health such as the West Nile virus and the Sindbis virus. This group of mosquitoes is difficult to distinguish morphologically; particularly as adult females. In Europe, the two species of the complex, Culex pipiens Linnaeus 1758 and Culex torrentium Martini 1925, are often found sympatrically. With the aim to characterize the presence and spread of both species in the Emilia‐Romagna region, Northern Italy, mosquitoes of the complex – collected during the West Nile virus surveillance plans – were tested by multiplex real‐time PCR for the detection of the two species Cx. pipiens and Cx. torrentium. A total of 24 165 mosquitoes, collected between 2012 and 2014 from 105 sites, and sorted in 204 pools, were tested. All tested pools were found to be composed of Cx. pipiens, whereas Cx. torrentium was not detected. These results indicate a likely absence of Cx. torrentium mosquitoes within the surveyed territory, whereas Cx. pipiens is widely distributed in the area mentioned. This is in line with previous reports, which describe a pre‐alpine distribution of Cx. torrentium in Italy.     

Experimental infection of Eurasian collared‐dove (Streptopelia decaocto) with West Nile virus

The Eurasian collared‐dove (Streptopelia decaocto) has recently experienced a population explosion in North America. It is frequently infected with West Nile virus (WNV). To test the hypothesis that the Eurasian collared‐dove is competent to transmit WNV, we experimentally infected two cohorts of doves with two different strains of WNV, CO08, and NY99, respectively. Both virus strains induced a low‐level viremia, capable of infecting a small fraction of vector mosquitoes. We suggest that the Eurasian collared‐dove plays a relatively insignificant role as an amplifying host for WNV, but it may be important where it is locally abundant.     

Routes of inoculation and the immune response to a resolving genital flavivirus infection in a novel murine model

The prolonged, abnormal immune response patterns produced by many sexually transmitted viruses have been intensively studied. Because normal antiviral immune responses in the vagina are less well-defined, we developed a resolving murine model using vaginal inoculation with the flavivirus, West Nile virus. Infection resulted in 12% mortality, with sterile protective immunity to vaginal or systemic re-challenge. B-cell numbers increased in the vaginal mucosa from day 1-7 after primary infection, while similar increases in B220(+), CD4(+) and CD8(+) lymphocytes in the draining lymph node were delayed by 48 h. By day 4 postinfection, a MHC-II(+) dendritic cell population became depleted from the stroma and formed aggregates below the basement membrane at points of demonstrable epithelial infection. In contrast, primary systemic or intradermal inoculation resulted in 80-90% mortality, but also conferred protective sterile immunity to vaginal West Nile virus re-challenge. Intravaginal and intradermal immunization elicited comparable, accelerated accumulation of larger B-cell numbers in the mucosa and draining lymph node upon intravaginal re-challenge than systemic immunization. However, accumulation of CD4(+) T cells in both sites in the intradermally immunized group was significantly greater than in intravaginally or systemically immunized mice. Accelerated accumulation of dendritic cells occurred at periodic sub-basement membrane sites in the absence of detectable virus 1 day after vaginal re-challenge, irrespective of the route of immunization. These data illustrate the diversity of possible effective immune responses to West Nile virus in the vaginal mucosa. They show primary vaginal inoculation produces effective immunity to flavivirus infection with lower mortality than other routes and suggest a local role for vaginal mucosal dendritic cells in both primary and secondary responses.     

Evaluation of vector competence for West Nile virus in Italian Stegomyia albopicta (=Aedes albopictus) mosquitoes

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle that involves wild birds as reservoir hosts. The virus is responsible for outbreaks of viral encephalitis in humans and horses. In Europe, Culex pipiens (Diptera: Culicidae) is considered to be the main vector of WNV, but other species such as Stegomyia albopicta (=Aedes albopictus) (Diptera: Culicidae) may also act as competent vectors of this virus. Since 2008 human cases of WNV disease have been reported in northeast Italy. In 2011, new areas of southern Italy became involved and a first outbreak of WNV lineage 1 occurred on the island of Sardinia. On the assumption that a potential involvement of St. albopicta in WNV transmission cannot be excluded, and in order to evaluate the competence of this species for the virus, an experimental infection of an St. albopicta laboratory colony, established from mosquitoes collected in Sardinia, was carried out. The results were compared with those obtained in a colony of the main vector Cx. pipiens. The study showed St. albopicta collected on Sardinia to be susceptible to WNV infection, which suggests this Italian mosquito species is able to act as a possible secondary vector, particularly in urban areas where the species reaches high levels of seasonal abundance.     

Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus

West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.     

Ecological correlates of risk and incidence of West Nile virus in the United States

West Nile virus, which was recently introduced to North America, is a mosquito-borne pathogen that infects a wide range of vertebrate hosts, including humans. Several species of birds appear to be the primary reservoir hosts, whereas other bird species, as well as other vertebrate species, can be infected but are less competent reservoirs. One hypothesis regarding the transmission dynamics of West Nile virus suggests that high bird diversity reduces West Nile virus transmission because mosquito blood-meals are distributed across a wide range of bird species, many of which have low reservoir competence. One mechanism by which this hypothesis can operate is that high-diversity bird communities might have lower community-competence, defined as the sum of the product of each species’ abundance and its reservoir competence index value. Additional hypotheses posit that West Nile virus transmission will be reduced when either: (1) abundance of mosquito vectors is low; or (2) human population density is low. We assessed these hypotheses at two spatial scales: a regional scale near Saint Louis, MO, and a national scale (continental USA). We found that prevalence of West Nile virus infection in mosquito vectors and in humans increased with decreasing bird diversity and with increasing reservoir competence of the bird community. Our results suggest that conservation of avian diversity might help ameliorate the current West Nile virus epidemic in the USA     

NMR Analysis of the Dynamic Exchange of the NS2B Cofactor between Open and Closed Conformations of the West Nile Virus NS2B-NS3 Protease

Background

The two-component NS2B-NS3 proteases of West Nile and dengue viruses are essential for viral replication and established targets for drug development. In all crystal structures of the proteases to date, the NS2B cofactor is located far from the substrate binding site (open conformation) in the absence of inhibitor and lining the substrate binding site (closed conformation) in the presence of an inhibitor.

Methods

In this work, nuclear magnetic resonance (NMR) spectroscopy of isotope and spin-labeled samples of the West Nile virus protease was used to investigate the occurrence of equilibria between open and closed conformations in solution.

Findings

In solution, the closed form of the West Nile virus protease is the predominant conformation irrespective of the presence or absence of inhibitors. Nonetheless, dissociation of the C-terminal part of the NS2B cofactor from the NS3 protease (open conformation) occurs in both the presence and the absence of inhibitors. Low-molecular-weight inhibitors can shift the conformational exchange equilibria so that over 90% of the West Nile virus protease molecules assume the closed conformation. The West Nile virus protease differs from the dengue virus protease, where the open conformation is the predominant form in the absence of inhibitors.

Conclusion

Partial dissociation of NS2B from NS3 has implications for the way in which the NS3 protease can be positioned with respect to the host cell membrane when NS2B is membrane associated via N- and C-terminal segments present in the polyprotein. In the case of the West Nile virus protease, discovery of low-molecular-weight inhibitors that act by breaking the association of the NS2B cofactor with the NS3 protease is impeded by the natural affinity of the cofactor to the NS3 protease. The same strategy can be more successful in the case of the dengue virus NS2B-NS3 protease.     

基因免疫制备西尼罗病毒NS5蛋白特异性单抗

研究了NS5蛋白在西尼罗病毒的特异性检测方面的应用及NS5在黄病毒复制中的作用机理。采用RT．PCR方法扩增了西尼罗病毒株的NS5基因片段,将其克隆至真核表达载体pVAX1,构建真核表达质粒。以重组质粒免疫BALB／c小鼠后取脾脏进行杂交瘤细胞融合,建立能稳定分泌西尼罗NS5单克隆抗体的杂交瘤细胞株。构建了真核表达质粒pVAX1-WNV—NS5,免疫动物后获得了28289等4株稳定分泌特异性抗体的杂交瘤细胞株,均为IgM型。真核表达质粒免疫后成功地诱导了针对NS5蛋白的体液免疫应答,单抗特异性分析显示4株单抗与其他黄病毒存在一定交叉反应。