Experimental vertical transmission of West Nile virus by Culex pipiens (Diptera: Culicidae)

Despite the detection of West Nile (WN) virus in overwintering Culex pipiens L. in New York in February 2000, the mechanism by which this virus persists throughout the winter to initiate infections in vertebrate hosts and vectors the following spring remains unknown. After a blood meal, parous mosquitoes generally do not survive until spring and gonotrophic dissociation occurs in only a small percentage of the population. To investigate vertical transmission as a means of viral survival during interepizootics, we intrathoracically inoculated Cx. pipiens and Aedes albopictus (Skuse) with WN virus and subsequently tested their F1 progeny for the presence of virus. Among the Cx. pipiens, we recovered virus from two of 1,417 adult progeny that had been reared at 18 degrees C for a minimal filial infection rate (MFIR) of approximately 1.4/1,000 and four of 1,873 adult progeny reared at 26 degrees C (MFIR = 2.1/1,000). The mean titer of the positive pools was 10(5.6) plaque-forming units (PFU)/ml (=10(5.9) PFU/mosquito for positive mosquitoes) of virus. Overall, the MFIR was approximately 1.8/1,000 for Cx. pipiens. Although reports indicate that Ae. albopictus vertically transmit various viruses in the Japanese encephalitis virus complex, we did not detect WN virus in any of > 13,000 F1 progeny of WN virus-inoculated specimens. Female Cx. pipiens that are vertically infected during the late summer season and then survive the winter could serve as a source of WN virus to initiate an infection cycle the following spring.     

Drought-induced amplification and epidemic transmission of West Nile virus in southern Florida

We show that the spatial-temporal variability of human West Nile (WN) cases and the transmission of West Nile virus (WNV) to sentinel chickens are associated with the spatial-temporal variability of drought and wetting in southern Florida. Land surface wetness conditions at 52 sites in 31 counties in southern Florida for 2001-2003 were simulated and compared with the occurrence of human WN cases and the transmission of WNV to sentinel chickens within these counties. Both WNV transmission to sentinel chickens and the occurrence of human WN cases were associated with drought 2-6 mo prior and land surface wetting 0.5-1.5 mo prior. These dynamics are similar to the amplification and transmission patterns found in southern Florida for the closely related St. Louis encephalitis virus. Drought brings avian hosts and vector mosquitoes into close contact and facilitates the epizootic cycling and amplification of the arboviruses within these populations. Southern Florida has not recorded a severe, widespread drought since the introduction of WNV into the state in 2001. Our results indicate that widespread drought in the spring followed by wetting during summer greatly increase the probability of a WNV epidemic in southern Florida.     

Trafficking mechanism of West Nile (Sarafend) virus structural proteins

Previous studies have shown that West Nile (Sarafend) virus matured by budding at the plasma membrane, which differs from the usual intracellular maturation of other flaviviruses. The present study investigated the trafficking mechanism of the envelope (E) and capsid (C) proteins of West Nile (Sarafend) virus during the replication cycle. The use of time-based double-immunofluorescence labelling coupled with the Triton X-100 extraction procedure revealed that both the E and C proteins were transported from the perinuclear region towards the plasma membrane along the microtubules simultaneously. The strong association of these virus proteins with the microtubules was demonstrated further with Triton X-100 extraction procedure coupled with double immunogold-labelling. Extraction of infected cells with Triton X-100 in high salt also revealed that virus E proteins were associated with the microtubules via protein-protein interaction. The disruption of microtubules with vinblastine sulphate inhibited the trafficking of both the virus E and C proteins. Both virus structural proteins were observed to co-localise and retained within vinblastine sulphate-induced microtubulin paracrystals. Extracellular virus production was also reduced drastically by vinblastine sulphate at non-cytotoxic concentration. Subsequent studies revealed that the transportation of virus E protein was associated with the microtubules-based motor protein, kinesin.     

Mosquito (Diptera: Culicidae) bloodmeal sources during a period of West Nile virus transmission in Puerto Rico

Host bloodmeals of indigenous Caribbean mosquitoes have not been studied previously. We identified vertebrate DNA in 90 blood-engorged mosquitoes belonging to four genera (Aedes, Culex, Deinocerites, and Uranotaenia) and 12 species that were collected in Puerto Rico within a geographic and temporal focus of West Nile virus transmission in 2007. It was found that 62 (68.8%) bloodmeals were from reptiles, 18 (20.0%) from birds, and 10 (11.1%) from mammals. Only one bloodmeal of 18 derived from Culex (Culex) species was passerine, suggesting a preference for nonpasserine birds and other vertebrates (i.e., reptiles) among the candidate WNV vectors. We interpret the results with respect to vectorial capacity for West Nile virus, an emerging arbovirus throughout the Caribbean Basin.     

Actin filaments participate in West Nile (Sarafend) virus maturation process

West Nile (Sarafend) virus has previously been shown to egress by budding at the plasma membrane of infected cells, but relatively little is known about the mechanism involved in this mode of release. During the course of this study, it was discovered that actin filaments take part in the virus maturation process. Using dual-labeled immunofluorescence and immunoelectron microscopy at late infection (10 hr p.i.), co-localization of viral structural (envelope and capsid) proteins with actin filaments was confirmed. The virus structural proteins were also immunoprecipitated with anti-actin antibody, further demonstrating the strong association between the two components. Perturbation of actin filaments by cytochalasin B strongly inhibited the release of West Nile virus (approximately 10,000-fold inhibition) when compared with the untreated cells. Infectious virus particles were recovered after the removal of cytochalasin B. Further confirmation was obtained when nucleocapsid particles were found associated with disrupted actin filaments at the periphery of cytochalasin B-treated cells. Together, these results showed that actin filaments do indeed have a key role in the release of West Nile (Sarafend) virions.     

West Nile virus infection rates in Culex nigripalpus (Diptera: Culicidae) do not reflect transmission rates in Florida

We describe the first documented field transmission of West Nile (WN) virus by a North American mosquito. WN was first detected in northern Florida in 2001. An intensive mosquito trapping and surveillance program was conducted in this region for four nights to assess mosquito transmission of WN. Four mosquito traps, each with a single sentinel chicken, were placed at five different locations on each of four nights. A total of 11,948 mosquitoes was collected, and 14 mosquito pools were found to contain WN, giving a minimum infection rate between 1.08 and 7.54 per 1,000. Only one of the 80 sentinel chickens seroconverted to WN, demonstrating a single mosquito transmission event during the study and a mosquito transmission rate of between 0.8 and 1 per 1,000. Culex nigripalpus Theobald was responsible for WN transmission to the sentinel chicken, although both Cx. nigripalpus and Culex quinquefasciatus Say were found infected with WN. Mosquito transmission rates are reported in this study for the first time for a WN outbreak. This information is essential to determine risk of human and animal infection.     

Immunity to West Nile virus

Over the past five years, West Nile (WN) virus has emerged as an important public health concern in the United States. Recent studies from experimental models of WN virus infection have increased our understanding of its pathogenesis and immunity. These include the demonstration that the gene encoding 2'-5'oligoadenylate synthetase is responsible for murine susceptibility to WN virus, the elucidation of the contributions of B, CD8(+) and gamma T cells in the control of murine WN virus infection, and the use of active immunization with envelope protein and passive transfer of immunoglobulin for immunotherapy. These efforts will facilitate the development of effective vaccines and therapies to combat WN virus.     

Molecular evolution of West Nile virus

Phylogenetic analysis and estimation of the rate of evolution of West Nile virus (WNV) were conducted. Sixty-eight nucleotide sequences of WNV E protein were used for the analysis. The rate of nucleotide substitution accumulation was 2.5 × 10^?4 substitutions per site per year. Phylogenetic analysis and estimation of WNV evolution time using molecular-clock methodology demonstrated that the WNV genotypes 1, 2, and 4 with an estimated time of divergence from the common precursor of approximately 2360, 2800, and 5950 years, respectively, circulate on the territory of the European part of Russia. The ratio of frequencies of nonsynonymous substitutions (dN) to synonymous substitutions (dS) can vary within 0.022–0.275 for certain WNV strains grouped according to geographical and/or phylogenetic traits. The highest values of dN/dS ratio were found for modern WNV isolates in Russia and in North America, which appeared in new natural biocenoses of these regions in the last 14 years. dN/dS estimation for WNV species shows that indices of intraspecific dN/dS variability can be used for detecting the presence of accelerated evolution of new WNV isolates. All this confirms the hypothesis that favorable conditions exist for wide distribution and rapid evolution of different WNV genotypes (that arose 2000–6000 years ago) in modern natural and climatic conditions.     

West Nile virus in mosquitoes in Greece

Epidemics of West Nile virus (WNV) occurred for two consecutive years in Greece (in 2010 and 2011). A total of 16,116 adult Culex pipiens mosquitoes collected in two peripheries, Central Macedonia and Thessaly, were tested for WNV infection. WNV lineage 2 was detected in 6/296 mosquito pools, three in each year. The H₂₄₉P substitution in the NS3 protein, previously associated with increased pathogenicity and thermotolerance, was detected in all six WNV-positive mosquito pools. When 21 individual C. pipiens mosquitoes were tested for the locus CQ11 to distinguish between the two C. pipiens forms, pipiens and molestus, 71.4 % were identified as pipiens, 4.7 % as molestus, and 19 % as hybrid pipiens/molestus, giving the first evidence that both C. pipiens biotypes are present in Greece, with a significant proportion being hybrids. The exact role of the C. pipiens forms and hybrids in the WNV epidemiology, in combination or not with the H₂₄₉P substitution in the virus genome, remains to be elucidated.     

West Nile virus in the vertebrate world

Summary. West Nile virus (WNV), an arthropod-borne virus belonging to the family Flaviviridae, had been recognized in Africa, Asia and the south of Europe for many decades. Only recently, it has been associated with an increasing number of outbreaks of encephalitis in humans and equines as well as an increasing number of infections in vertebrates of a wide variety of species. In this article, the data available on the incidence of WNV in vertebrates are reviewed. Moreover, the role of vertebrates in the transmission of WNV, the control of WNV infections in veterinary medicine as well as future perspectives are discussed. A wide variety of vertebrates, including more than 150 bird species and at least 30 other vertebrate species, are susceptible to WNV infection. The outcome of infection depends on the species, the age of the animal, its immune status and the pathogenicity of the WNV isolate. WNV infection of various birds, especially passeriforms, but also of young chickens and domestic geese, results in high-titred viremia that allows arthropod-borne transmission. For other vertebrate species, only lemurs, lake frogs and hamsters develop suitable viremia levels to support arthropod-borne transmission. The role of vertebrates in direct, non-arthropod-borne transmission, such as via virus-contaminated organs, tissues or excretions is less well characterized. Even though direct transmission can occur among vertebrates of several species, data are lacking on the exact amounts of infectious virus needed. Finally, the increased importance of WNV infections has led to the development of killed, live-attenuated, DNA-recombinant and chimeric veterinary vaccines.     

Extrinsic incubation periods for horizontal and vertical transmission of West Nile virus by Culex pipiens pipiens (Diptera: Culicidae)

Culex pipiens pipiens L. (Diptera: Culicidae), infected per os from a membrane feeder, transmitted West Nile virus (family Flaviviridae, genus Flavivirus, WNV) at 26 degrees C horizontally during feeding to hamsters and suckling mice and vertically to F1 progeny during egg deposition. Horizontal transmission rates increased with extrinsic incubation, with 75-100% of the females transmitting on days 16 through 25 postinfection (pi). No females deposited eggs infected with WNV after the first bloodmeal on 3-8 d pi. Females vertically transmitted WNV during egg laying after their second, third, and fourth bloodmeals on days 13-33 pi. The vertical transmission rate was 4.7%. The vertical minimal infection rate was 0.52 infected F1 specimens/1,000 specimens tested from females feeding during their second and later bloodmeals on hamsters or suckling mice. The sequence of horizontal and vertical transmission is reported. A female may transmit WNV 1) horizontally to a host during feeding and subsequently vertically to her offspring during egg laying, 2) vertically to her offspring during oviposition without prior horizontal transmission to a host, and 3) horizontally to a host without vertically transmitting the virus. These two means of transmission by Cx. p. pipiens contribute to the relatively high minimum infection rates that are reached in late summer and to the survival of virus during winter and initiation of amplification in the spring in the northeastern United States.     

Overwintering of West Nile virus in Southern California

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded southern California during 2003, successfully overwintered, amplified to epidemic levels, and then dispersed to every county in the state. Although surveillance programs successfully tracked and measured these events, mechanisms that allowed the efficient overwintering and subsequent amplification of WNV have not been elucidated. Our current research provided evidence for three mechanisms whereby WNV may have persisted in southern California during the winters of 2003-2004 and 2004-2005: 1) continued enzootic transmission, 2) vertical transmission by Culex mosquitoes, and 3) chronic infection in birds. WNV was detected in 140 dead birds comprising 32 species, including 60 dead American crows, thereby verifying transmission during the November-March winter period. Dead American crows provide evidence of recent transmission because this species always succumbs rapidly after infection. However, WNV RNA was not detected concurrently in 43,043 reproductively active female mosquitoes comprising 11 species and tested in 1,258 pools or antibody in sera from 190 sentinel chickens maintained in 19 flocks. Although efficient vertical transmission by WNV was demonstrated experimentally for Culex tarsalis Coquillett infected per os, 369 females collected diapausing in Kern County and tested in 32 pools were negative for WNV. Vertical transmission was detected in Culex pipiens quinquefasciatus Say adults reared from field-collected immatures collected from Kern County and Los Angeles during the summer transmission period. Chronic infection was detected by finding WNV RNA in 34 of 82 birds that were inoculated with WNV experimentally, held for >6 wk after infection, and then necropsied. Frequent detection of WNV RNA in kidney tissue in experimentally infected birds >6 wk postinfection may explain, in part, the repeated detection of WNV RNA in dead birds recovered during winter, especially in species such as mourning doves that typically do not die after experimental infection. In summary, our study provides limited evidence to support multiple modes of WNV persistence i n southern California. Continued transmission andvertical transmission by Culex p. quinquefasciatus Say seem likely candidates for further study.