Replication of the primary dog kidney-53 dengue 2 virus vaccine candidate in Aedes aegypti is modulated by a mutation in the 5' untranslated region and amino acid substitutions in nonstructural proteins 1 and 3.

Previous studies have demonstrated reduced replication of the cell culture-adapted Dengue-2 virus (DENV-2) vaccine candidate, primary dog kidney (PDK)-53, compared with the parental DENV-2 strain, 16681, in C6/36 cells. Various DENV-2 mutants incorporating PDK-53 substitutions singly and in combination into the 16681 genetic backbone were used to identify the genetic basis for impaired replication of the vaccine candidate in vitro in Aedes aegypti cell culture (Aag2 cells) as well as the reduced in vivo infectivity and transmissibility within Ae. aegypti infected by intrathoracic inoculation. 5' untranslated region (UTR-c57t) and nonstructural protein 1 (NS1-G53D) mutations were required to completely attenuate in vitro replication. In contrast, incorporation of the PDK-53-specific NS3-250V mutation into the 16681 virus resulted in reduced replication in mosquitoes but had no effect on in vitro replication. Further, reversion of the PDK-53 NS3-250 site to that of the wild-type 16681 virus (NS3-V250E) failed to increase either in vitro or in vivo replication. Intrathoracic inoculation of Ae. aegypti with mutants containing the PDK-53 NS1 substitution exhibited in vivo replication indistinguishable from the parental PDK-53 virus, implicating this mutation as the dominant determinant for impaired mosquito replication of the PDK-53 candidate; however, further attenuation of in vivo replication was magnified in mutants including the additional 5'UTR-c57t mutation.

Identification of Culex pipiens complex mosquitoes in a hybrid zone of West Nile virus transmission in Fresno County, California.

Culex pipiens sensu lato mosquitoes were collected from 24 gravid traps (mid-June to mid-October, 2005) in Fresno County, CA. Captured gravid females were allowed to oviposit before sibling species identification by Ace.2 PCR and detection of West Nile virus (WNV) RNA by RT-PCR were performed on the mother and her offspring. Of the 442 Cx. pipiens s.l. female mosquitoes collected, 88 were positive for WNV viral RNA (peaked in August) with no significant differences among complex members or habitat. Vertical transmission was detected in 4 out of 20 families originating from WNV-positive mothers, however, in only a small number of offspring from each family. Out of 101 families that had PCR-based maternal and offspring identifications, the offspring from 15 families produced inexplicable amplicon patterns, suggesting ambiguities in the PCR assay identifications. Male genitalia (DV/D ratio) and Ace.2 PCR identifications revealed numerous discrepancies in our ability to accurately determine the identity of Cx. pipiens complex members in the hybrid zone of Fresno County.

Effects of time after infection, mosquito genotype, and infectious viral dose on the dynamics of Culex tarsalis vector competence for western equine encephalomyelitis virus.

The vector competence of Culex tarsalis Coquillett for the BFS 1703 strain of western equine encephalomyelitis virus (WEEV) changed significantly as a function of time after infection, mosquito genotype, and infectious virus dose. After ingesting a high virus dose (5 log10 plaque-forming units [PFU]/0.1 ml), female of the susceptible high virus producer (HVP) strain rapidly amplified the virus, developed a disseminated infection, and efficiently transmitted WEEV by 4 days postinfection (dpi). The quantity of virus expectorated peaked at 4 dpi (mean 3.4 log10 PFU), and the percentage of females transmitting per os peaked at 7 dpi (80%); both measures of transmission subsequently decreased to low levels throughout the remainder of infected life. HVP females imbibing a low virus dose (3 log10 PFU/0.1 ml) were infected less frequently and took longer to amplify virus to levels recorded for the high virus dose group and did not transmit virus efficiently, thereby indicating midgut infection and escape barriers were dose and time dependent. These data emphasized the importance of elevated avian viremias in Cx. tarsalis vector competence. Females from the WEEV-resistant (WR) strain and two wild-type strains from Kern and Riverside counties were significantly less susceptible to infection at both high and low doses than was the HVP strain. Overall, females with a high virus titer more frequently had a disseminated infection, but there did not seem to be a distinct threshold demarcating this relationship. In marked contrast, all infected females transmitting virus had body titers >4.3 log10 PFU, and most had titers >4.8 log10 PFU. These data indicated that not all females with a disseminated infection transmitted virus because of the presence of one or more salivary gland barriers.

Phylogenetic analysis of North American West Nile virus isolates, 2001-2004: evidence for the emergence of a dominant genotype.

The distribution of West Nile virus has expanded in the past 6 years to include the 48 contiguous United States and seven Canadian provinces, as well as Mexico, the Caribbean islands, and Colombia. The suggestion of the emergence of a dominant genetic variant has led to an intensive analysis of isolates made across North America. We have sequenced the pre-membrane and envelope genes of 74 isolates and the complete genomes of 25 isolates in order to determine if a dominant genotype has arisen and to better understand how the virus has evolved as its distribution has expanded. Phylogenetic analyses revealed the continued presence of genetic variants that group in a temporally and geographically dependent manner and provide evidence that a dominant variant has emerged across much of North America. The implications of these findings are discussed as they relate to transmission and spread of the virus in the Western Hemisphere.

Blinded laboratory comparison of the in situ enzyme immunoassay, the VecTest wicking assay, and a reverse transcription-polymerase chain reaction assay to detect mosquitoes infected with West Nile and St. Louis encephalitis viruses.

A blinded laboratory evaluation compared the accuracy, sensitivity, and specificity of an in situ enzyme immunoassay (EIA), VecTest wicking assay, and reverse transcription-polymerase chain reaction (RT-PCR) to detect and distinguish West Nile (WN) and St. Louis encephalitis (SLE) viruses in pools of 50 mosquitoes. Adult female Culex tarsalis Coquillett were inoculated with either WN or SLE viruses, held for 0-11 d at 28 degrees C, killed by freezing, and then were added to 49 or 48 uninfected mosquitoes to make up 14 pools positive for WN virus, 14 positive for SLE virus, 14 positive for both WN and SLE viruses, and 14 negative for virus. Pools were number coded and tested blindly. Virus was not detected in known negative pools. VecTest and RT-PCR assays were comparably sensitive and accurate, detecting virus in pools containing females held for 3 d postinoculation; only RT-PCR detected SLE virus in pools on days 0-1. The VecTest and RT-PCR produced a single false-positive result for WN and SLE, respectively. RT-PCR detected RNA in samples positive by the VecTest, indicating that the detergent in the wicking buffer did not prevent RT-PCR from confirming VecTest results. Detector antibodies used in the in situ EIA cross-reacted between SLE and WN viruses, reducing accuracy. Both the VecTest and RT-PCR provided rapid and specific results, but they detected only those viruses known to be present. Plaque assay on Vero cells was comparably sensitive and had the added benefit of detecting newly emerging viruses, but this method required virus culture followed by identification, thereby delaying reporting.

Encephalitis virus persistence in California birds: experimental infections in mourning doves (Zenaidura macroura).

After-hatching and hatching year, mourning doves were infected by inoculation with either western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses; some birds in each group also were treated with the immunosuppressant cyclophosphamide before and during infection. Cyclophosphamide treatment significantly increased the WEE viremia but did not alterthe antibody response. In contrast, cyclophosphamide-treated and -untreated doves did not develop a detectable SLE viremia but became antibody positive. Antibody peaked at 10 wk after inoculation for both viruses and remained detectable in most birds throughout the 26-wk study. When treated with cyclophosphamide the following spring, birds did not relapse and develop a detectable viremia. Previously infected birds were protected when challenged with conspecific virus (i.e., none produced a detectable viremia), but there was no anamnestic antibody response to reinfection. In agreement with our failure to detect relapses, all birds were negative for viral RNA when sera, spleen, lung, and kidney tissues were tested by reverse transcriptase-polymerase chain reaction after necropsy. Our results indicated that adult mourning doves were an incompetent host for SLE virus and probably do not serve as a suitable overwintering or dispersal host for either WEE and SLE viruses.

Effects of immunosuppression on encephalitis virus infection in the house finch, Carpodacus mexicanus.

Immunosuppression of house finches was attempted by blood feeding Culex tarsalis Coquillett mosquitoes or by injecting birds with the corticosteroid dexamethasone or the immunosuppressant drug cyclophosphamide before and after inoculation with western equine encephalomyelitis or St. Louis encephalitis viruses. Mosquito bites (8-37 females blood feeding on each bird over a 3-d period) did not enhance the viremia response or increase the frequency of chronic infection. In contrast, dexamethasone and cyclophosphamide enhanced the amplitude and duration of the viremia response, but had no consistent effect on the antibody responses as measured by enzyme immunoassay or plaque reduction neutralization assay. Elevated viremias were followed by increases in the frequency of chronic infections with St. Louis encephalitis, but not western equine encephalomyelitis. Immunosuppression may provide a useful tool to study the chronic infection process of flaviviruses in vertebrates.