Rearing of Culex spp. and Aedes spp. Mosquitoes.

Mosquito-transmitted pathogens cause major public health problems and contribute substantially to the global burden of disease. Aedes mosquitoes transmit dengue, Zika, yellow fever, and Chikungunya viruses; Culex mosquitoes transmit West Nile, Japanese encephalitis, and Saint Louis encephalitis viruses, among others. Experiments utilizing laboratory-reared colonized mosquitoes can address many issues such as vector biology, vector competence, vector-pathogen interaction, and vector control. The establishment of healthy and standardized mosquito colonies requires generation and implementation of protocols, attention to detail, and an understanding of the factors that affect mosquito fitness, such as temperature and humidity, nutrient quality and availability, population density, blood feeding and mating behavior, and egg-laying requirements. Here, we present a standard protocol for the rearing of Culex spp. and Aedes spp. mosquitoes and maintenance of the mosquito colony.

Rabies direct fluorescent antibody test does not inactivate rabies or eastern equine encephalitis viruses.

An examination using the routine rabies direct fluorescent antibody test was performed on rabies or Eastern equine encephalitis positive mammalian brain tissue to assess inactivation of the virus. Neither virus was inactivated with acetone fixation nor the routine test, thus laboratory employees should treat all samples as rabies and when appropriate Eastern equine encephalitis positive throughout the whole procedure.

Detection Protocols for West Nile Virus in Mosquitoes, Birds, and Nonhuman Mammals.

West Nile virus is the most widespread mosquito-borne virus in the world, and the most common cause of encephalitis in the USA. Surveillance for this medially important mosquito-borne pathogen is an important part of public health practice. Here we present protocols for testing environmental samples such as mosquitoes, nonvertebrate mammals, and birds for this virus, including RT-PCR, virus isolation in cell culture, and antigenic assays, as well as serologic assays for antibody detection.

Detection of West Nile virus.

West Nile virus (WNV; Flavivirus, Flaviviridae) is a spherical enveloped virion containing single-stranded, positive-sense RNA, approximately 11 kb in length. The virus is the most widely distributed flavivirus in the world. Genetic analysis reveals two major lineages of virus, I and II, and several possible newly recognized lineages. Lineage I strains are most commonly associated with outbreaks of neurologic disease, although lineage II virus has led to large epidemics of fever, as in South Africa in 1974. Infection with WNV leads to a wide range of diseases from mildly febrile to severely neurologic, but asymptomatic -infections occur most frequently. Approximately one in 140 infected individuals develop neurologic -disease. The virus is maintained in an enzootic cycle, where it is transmitted between ornithophilic mosquitoes of the Culex genus and predominantly passeriform birds. Equines and humans are considered incidental hosts since they do not mount high enough viremia for mosquitoes to become infected -following feeding. Laboratory diagnosis of WNV infection is predominantly serological, although -caution is advised because of the high degree of cross-reactivity among flaviviruses. Field specimens, especially mosquitoes and dead birds, collected as part of surveillance programs, are tested for the presence of viral nucleic acid, viral antigen, or infectious virus. Rapid test protocols have been developed in response to the expansion of WNV in the United States. Since WNV is classified as a Biosafety Level-3 (BSL-3) agent by CDC, it is recommended that once this virus is identified in a diagnostic specimen, all infectious virus should be handled in a BSL-3 laboratory in Class II biosafety cabinets by laboratory staff who are trained to work at this level of containment. Assay protocols are described and the necessary equipment and supplies listed.

Crow deaths caused by West Nile virus during winter.

In New York, an epizootic of American crow (Corvus brachyrhynchos) deaths from West Nile virus (WNV) infection occurred during winter 2004-2005, a cold season when mosquitoes are not active. Detection of WNV in feces collected at the roost suggests lateral transmission through contact or fecal contamination.

Characterization of a small plaque variant of West Nile virus isolated in New York in 2000.

A small-plaque variant (SP) of West Nile virus (WNV) was isolated in Vero cell culture from kidney tissue of an American crow collected in New York in 2000. The in vitro growth of the SP and parental (WT) strains was characterized in mammalian (Vero), avian (DF-1 and PDE), and mosquito (C6/36) cells. The SP variant replicated less efficiently than did the WT in Vero cells. In avian cells, SP growth was severely restricted at high temperatures, suggesting that the variant is temperature sensitive. In mosquito cells, growth of SP and WT was similar, but in vivo in Culex pipiens (L.) there were substantial differences. Relative to WT, SP exhibited reduced replication following intrathoracic inoculation and lower infection, dissemination, and transmission rates following oral infection. Analysis of the full length sequence of the SP variant identified sequence differences which led to only two amino acid substitutions relative to WT, prM P54S and NS2A V61A.

Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture.

West Nile Virus (WNV) is a mosquito-borne flavivirus that was introduced into the U.S. in the New York City area in 1999. Despite its successful establishment and rapid spread in a naive environment, WNV has undergone limited evolution since its introduction. This evolutionary stability has been attributed to compromises made to permit alternating cycles of viral replication in vertebrate hosts and arthropod vectors. Outbreaks of a close relative of WNV, St. Louis encephalitis virus (SLEV), occur in the U.S. periodically and are also characterized by limited genetic change overtime. We measured both phenotypic and genotypic changes in WNV and SLEV serially passaged in mosquito cell culture in order to clarify the role of an individual host cell type in flavivirus adaptation and evolution. Genetic changes in passaged WNV and SLEV were minimal but led to increased relative fitness and replicative ability of the virus in the homologous cell line C6/36 mosquito cells. Similar increases were not measured in the heterologous cell line DF-1 avian cells. These phenotypic changes are consistent with the concept of cell-specific adaptation in flaviviruses.