Development and characterization of West Nile virus replicon expressing secreted Gaussia Luciferase

We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secreted into the culture medium from cells transfected with Gluc replicon RNA, and the medium can be assayed directly for luciferase activity. Using a known Flavivirus inhibitor (NITD008), we demonstrated that the Gluc-WNV replicon could be used for antiviral screening. The Gluc-WNV-Rep will be useful for research in antiviral drug development programs, as well as for studying viral replication and pathogenesis of WNV.     

西尼罗病毒研究进展

西尼罗病毒（West Nile virus,WNV）属黄病毒科,为正单链RNA病毒。它在人类中的感染导致以发热为主要症状的传染性疾病,主要由蚊虫叮咬传播。自20世纪50年代首例报告西尼罗病毒自然感染所致脑炎后的几十年内,西尼罗病毒脑炎在欧洲及中亚地区散在、小规模流行。西尼罗病毒脑炎于1999年在美国的爆发及随后几年在北美的流行引起了极大的关注。这次爆发流行中新出现的种种迹象,如其中间宿主——野生鸟类的大量死亡,人类感染者中中枢神经系统受损比例的增高等,提示近期的遗传变异已使西尼罗病毒感染的病理学与流行病学发生了较显著的变化。另外,随着感染的流行,蚊虫叮咬以外的传播途径,如输血、器官移植、母婴传播等日益受到人们重视。同时,人们对阻止疫情所急需的疫苗的研制也在进行之中。本文就近几年来对西尼罗病毒的感染、免疫与流行病学方面的研究进展进行了综述。     

Environmental risk factors associated with West Nile virus clinical disease in Florida horses

The objective of this study was to examine the extrinsic risk factors of West Nile virus (WNV) clinical disease in Florida horses as established from confirmed and negative horses tested within the state from 2001 to 2003. An Arboviral Case Information Form (ACF) was submitted by a referring veterinarian at the time of testing to the Florida Department of Agriculture and Consumer Services on every horse suspected of a viral encephalitis in Florida. A follow‐up survey that focused on arbovirus prevention and farm ecology was created and mailed to the owner of each tested horse. Data from the follow‐up survey indicated peak WNV prevalence in the late summer months in Florida. Quarter horses were the most commonly affected breed. The WNV vaccine was highly protective and natural water on the property also had a protective association. Factors that increased the risk of WNV to horses were the use of fans and a stable construction of solid wood or cement. Some risk indicators were dead birds on the property and other ill animals on the property. Data from this retrospective study have helped identify factors associated with WNV transmission in equines in Florida. Horses that have not been vaccinated and show clinical signs of arboviral infection from June to November should be tested for WNV. Horses that have been vaccinated and show clinical signs should be tested when the vaccination was administered within 1 month or greater than 6 months prior to the onset of clinical symptoms associated with WN infection.     

新疆伊犁地区马脑组织西尼罗病毒感染的调查

目的了解新疆伊犁地区草原放养马群中西尼罗病毒(W estN ile virus,WNV)中枢感染的流行状况。方法采用一步法实时荧光定量逆转录聚合酶链反应(Real Tim e RT-PCR)对采自新疆伊犁地区草原放养、未接种WNV疫苗的189例马脑组织进行WNV包膜蛋白(E)基因片段检测。结果被检马脑组织标本中未发现WNV E基因片段。结论目前尚没有证据表明我国新疆伊犁地区的放养马中存在WNV脑炎的感染,提示该地区出现WNV脑炎流行的可能性小。     

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.     

Persistence of West Nile virus

West Nile virus (WNV) is a widespread global pathogen that results in significant morbidity and mortality. Data from animal models provide evidence of persistent renal and neurological infection from WNV; however, the possibility of persistent infection in humans and long-term neurological and renal outcomes related to viral persistence remain largely unknown. In this paper, we provide a review of the literature related to persistent infection in parallel with the findings from cohorts of patients with a history of WNV infection. The next steps for enhancing our understanding of WNV as a persistent pathogen are discussed.     

Multi-species interactions in West Nile virus infection

In this paper, we analyse the interaction of different species of birds and mosquitoes on the dynamics of West Nile virus (WNV) infection. We study the different transmission efficiencies of the vectors and birds and the impact on the possible outbreaks. We show that the basic reproductive number is the weighted mean of the basic reproductive number of each species, weighted by the relative abundance of its population in the location. These results suggest a possible explanation of why there are no outbreaks of WNV in Mexico.     

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。     

西尼罗病毒的RT-PCR检测与鉴定

建立西尼罗病毒敏感、特异、快速的RT-PCR检测方法用于实验室诊断和流行病学监测。采用一步RT-PCR和套式PCR法对西尼罗病毒感染的乳鼠脑和细胞培养上清进行扩增,并对扩增产物进行序列测定。两种方法均可分别从两种组织中扩增出与预期大小相一致的片段,套式PCR法比一步RT-PCR法更为敏感,该扩增片段与西尼罗病毒埃及Eg101株相应序列的同源性为99％。     

Molecular evidence for trypanosomatids in Culex mosquitoes collected during a West Nile virus survey

Adult mosquitoes were previously collected and tested for West Nile virus during an intense WNV outbreak in 2003-2004 along the Cache la Poudre River in Colorado, USA. A subset of these mosquitoes was also tested for infection with trypanosomatids using nested PCR to amplify 18S rRNA. Of the 69 pools of Culex pipiens that were screened for both pathogens, 4.3% were positive for WNV and 11.6% tested positive for trypanosomes; no pools were found to be co-infected with both pathogens. One hundred and forty-three pools of Culex tarsalis, considered to be the principal WNV vector in this area, were tested in the same manner. 7.7% were positive for WNV and 20.3% of these pools tested positive for trypanosomes. Five pools of C. tarsalis were found to be co-infected with both pathogens, which was approximately 2.2 times more frequent than would be expected if these pathogens are independent of each other. Sequencing and maximum parsimony analysis of 18S rRNA revealed that four of the isolates arise in or near clades of described avian trypanosomes, likely indicating that these are vectored pathogens between birds and mosquitoes. Unexpectedly, the majority (24/28, 86%) of our positive samples form their own separate clade within the order Trypanosomatida with 100% bootstrap support. We have identified a potential new clade of trypanosomatids that exist within important mosquito vectors and discuss the potential ecological connections between these trypanosomes, arboviruses and mosquitoes.     

Evidence of vertical transmission of West Nile virus in field‐collected mosquitoes

Male and nulliparous female mosquitoes were surveyed for evidence of vertical WNV infection in East Baton Rouge Parish, Louisiana. Adult male mosquitoes collected by trapping and aspiration, and adult male and nulliparous female mosquitoes reared from field‐collected larvae were tested. Adult male Culex spp., female Aedes albopictus (Skuse), and female Culex quinquifasciatus Say mosquitoes that were collected as larvae were test‐positive for WNV RNA. Infectious WNV was detected using virus isolation in field‐collected male Aedes triseriatus Say and Culex salinarius Coquillett; these data represent the first field evidence of vertical transmission of WNV in Ae. triseriatus and Cx. salinarius.     

Apoptosis in mosquito midgut epithelia associated with West Nile virus infection

The mosquito Culex pipiens pipiens is a documented vector of West Nile virus (WNV, Flaviviridae, Flavivirus). Our laboratory colony of C. p. pipiens, however, was repeatedly refractory to experimental transmission of WNV. Our goal was to identify if a cellular process was inhibiting virus infection of the midgut. We examined midguts of mosquitoes fed control and WNV-infected blood meals. Three days after feeding, epithelial cells from abdominal midguts of mosquitoes fed on WNV fluoresced under an FITC filter following Acridine Orange staining, indicating apoptosis in this region. Epithelial cells from experimental samples examined by TEM exhibited ultrastructural changes consistent with apoptosis, including shrinkage and detachment from neighbors, heterochromatin condensation, nuclear degranulation, and engulfment of apoptotic bodies by adjacent cells. Virions were present in cytoplasm and within cytoplasmic vacuoles of apoptotic cells. No apoptosis was detected by TEM in control samples. In parallel, we used Vero cell plaque assays to quantify infection after 7 and 10 day extrinsic incubation periods and found that none of the mosquitoes (0/55 and 0/10) which imbibed infective blood were infected. We propose that programmed cell death limits the number of WNV-infected epithelial cells and inhibits disseminated viral infections from the mosquito midgut.     

Climate change impacts on West Nile virus transmission in a global context

West Nile virus (WNV), the most widely distributed virus of the encephalitic flaviviruses, is a vector-borne pathogen of global importance. The transmission cycle exists in rural and urban areas where the virus infects birds, humans, horses and other mammals. Multiple factors impact the transmission and distribution of WNV, related to the dynamics and interactions between pathogen, vector, vertebrate hosts and environment. Hence, among other drivers, weather conditions have direct and indirect influences on vector competence (the ability to acquire, maintain and transmit the virus), on the vector population dynamic and on the virus replication rate within the mosquito, which are mostly weather dependent. The importance of climatic factors (temperature, precipitation, relative humidity and winds) as drivers in WNV epidemiology is increasing under conditions of climate change. Indeed, recent changes in climatic conditions, particularly increased ambient temperature and fluctuations in rainfall amounts, contributed to the maintenance (endemization process) of WNV in various locations in southern Europe, western Asia, the eastern Mediterranean, the Canadian Prairies, parts of the USA and Australia. As predictions show that the current trends are expected to continue, for better preparedness, any assessment of future transmission of WNV should take into consideration the impacts of climate change.     

实验感染的三带喙库蚊和来亨鸡体内西尼罗病毒的分离与鉴定

采用C6/36细胞培养分离活病毒、间接免疫荧光染色检测病毒抗原、RT-PCR扩增病毒基因片段和PCR产物测序等方法,对实验感染的三带喙库蚊Culex tritaeniorhynchus和来亨鸡血液样本中的西尼罗病毒进行分离和鉴定。结果表明,接种实验感染蚊虫研磨液和来亨鸡血液样本的C6/36细胞出现细胞融合、空泡形成的病变效应; 用西尼罗病毒抗血清进行间接免疫荧光染色,感染病毒的细胞呈现黄绿色荧光,为阳性反应; 采用3对不同引物的RT- PCR体系扩增分别出现预期的408 bp、498 bp和559 bp的基因片段,序列测定证实扩增序列与实验所用毒株相应的基因序列基本相同。从而证实实验感染三带喙库蚊和来亨鸡体血液内的西尼罗病毒与实验感染所用的西尼罗病毒Chin-01株一致。     

Peptide derivatives as inhibitors of NS2B-NS3 protease from Dengue,West Nile,and Zika flaviviruses

Currently, more than 70 flaviviruses were identified and reported in the literature, whose Dengue (DENV), Zika (ZIKV), and West Nile (WNV) viruses have been responsible for millions of cases of infections worldwide, mainly in developing countries. These viruses are transmitted by the bite of mosquitoes from genus Aedes, or Culex and, in some cases, Stegomyia. Despite numerous efforts to identify a selective, safe, and effective antiviral agent, there is no currently approved drug for the treatment of flaviviral infections. Then, current pharmacological therapy has the objective to treat the clinical symptoms. Various peptidomimetics and peptide-derivatives have been synthesized and evaluated against several biological targets from flaviviruses with different applications, such as diagnosis, E protein inhibitors, entry inhibitors, virucidal inhibitors, and also viral replication inhibitors. Flaviviral replication depends on the NS3^pro that is completely activated when it is complexed to its cofactor, NS2B; forming a viral enzymatic complex. The development of NS2B-NS3^pro inhibitors is considered a challenging work due to its active site is shallow and open-pocket. In this work, we report all advances involving peptidomimetics, peptide-derived, and peptide-hybrids found in the literature. In sense, we discuss the influence of different functional groups in the activity and selectivity. Moreover, the first inhibitors reported in the literature as covalent ligands, comprising two basic residues followed by an electrophilic moiety that binds to the catalytic serine (Ser¹³⁵–O^?) are also discussed in details, such as trifluoromethyl ketones, aldehydes, and boronic acids. Furthermore, it is presented the influence of introducing transition metals, providing metallopeptide inhibitors; and cyclization of linear peptides, generating cyclic and macrocyclic peptide inhibitors. Finally, we provide the most accurate state of the art found in the literature, which can be utilized to design new and effective antiviral agents.     

The backward bifurcation in compartmental models for West Nile virus

In all of the West Nile virus (WNV) compartmental models in the literature, the basic reproduction number serves as a crucial control threshold for the eradication of the virus. However, our study suggests that backward bifurcation is a common property shared by the available compartmental models with a logistic type of growth for the population of host birds. There exists a subthreshold condition for the outbreak of the virus due to the existence of backward bifurcation. In this paper, we first review and give a comparison study of the four available compartmental models for the virus, and focus on the analysis of the model proposed by Cruz-Pacheco et al. to explore the backward bifurcation in the model. Our comparison study suggests that the mosquito population dynamics itself cannot explain the occurrence of the backward bifurcation, it is the higher mortality rate of the avian host due to the infection that determines the existence of backward bifurcation.     

Reconciling West Nile virus with the autophagic pathway

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for recurrent outbreaks of meningitis and encephalitis. Several studies analyzing the interactions of this pathogen with the autophagic pathway have reported opposite results with evidence for and against the upregulation of autophagy in infected cells. In this regard, we have recently reported that minimal genetic changes (single amino acid substitutions) in nonstructural proteins of WNV can modify the ability of the virus to induce autophagic features such as LC3 modification and aggregation in infected cells. We think that these results could help explain some of the previously reported discrepancies. These findings could also aid in deciphering the interactions of this pathogen with the autophagic pathway at the molecular level aimed to develop feasible antiviral strategies to combat this pathogen, and other related flaviviruses.