Effect of incubation at overwintering temperatures on the replication of West Nile Virus in New York Culex pipiens (Diptera: Culicidae)

We examined the effect of simulated overwintering temperatures on West Nile (WN) virus replication in Culex pipiens L. derived from mosquitoes collected during the autumn 1999 WN epizootic in New York. The WN virus was a strain isolated from a dead crow also collected during this outbreak. Virus was recovered from most mosquitoes held exclusively at 26 degres C. In contrast, none of the mosquitoes held exclusively at the lower temperatures had detectable infections. When mosquitoes were transferred to 26 degrees C after being held at 10 degrees C for 21-42 d, infection and dissemination rates increased with increased incubation at 26 degrees C. Future studies involving the attempted isolation of WN virus from overwintering mosquitoes may benefit from holding the mosquitoes at 26 degrees C before testing for infectious virus.     

Isolation of arboviruses from mosquitoes (Diptera: Culicidae) collected from the Gulf Plains region of northwest Queensland,Australia

As part of investigations into Japanese encephalitis (JE) virus and related flaviviruses in northern Australia, 153,529 mosquitoes were collected and processed for virus isolation from the Gulf Plains region of northwest Queensland. Collections from within 30 km of each of the townships of Croydon, Normanton and Karumba yielded 3,087 (2.0%), 66,009 (43.0%), and 84,433 (55.0%) mosquitoes, respectively, from which 16 viruses were isolated. Four isolates of Murray Valley encephalitis (MVE), two of Kunjin (KUN), three of Ross River (RR), and one of Sindbis (SIN) viruses were obtained from Culex sitiens subgroup mosquitoes. Molecular identification of the mosquito species composition of these virus positive pools revealed that most isolates were from pools containing mainly Culex annulirostris Skuse and low numbers of Culex palpalis (Taylor). Only three pools, one each of MVE, KUN, and RR, were from mosquitoes identified exclusively as Cx. annulirostris. Other viruses isolated include one Edge Hill virus from Ochlerotatus normanensis (Taylor), an isolate of SIN from Anopheles meraukensis Venhuis, two isolates of RR from Anopheles amictus Edwards, and single isolates of RR from Anopheles bancroftii Giles andAedes lineatopennis (Ludlow). The isolate of RR from Ae. lineatopennis was the first reported from this species. The public health implications of these isolations in the Gulf Plains region are discussed briefly.     

Use of enzyme immunoassay and nucleic acid hybridization for detecting Sindbis virus in infected mosquitoes

Aedes aegypti mosquitoes were inoculated intrathoracically with prototype Sindbis virus, held at 26.7 degrees C for from 0-95 h and placed at -70 degrees C. Individual mosquitoes were tested for virus by plaque assay in Vero cells, for viral RNA by nucleic acid hybridization using a cloned cDNA probe, and for viral protein by enzyme-linked immunosorbent assay. Virus was detected by plaque assay as early as 8 h after infection. Sindbis virus RNA was detected by nucleic acid hybridization 18 h after infection and by enzyme-linked immunosorbent assay 10 h after infection. The results of these comparisons suggest that both nucleic acid hybridization and enzyme-linked immunosorbent assay are applicable to direct detection of Sindbis virus in mosquitoes containing virus at levels usually found during arbovirus epidemics.     

Specific determination of flaviviruses by molecular hybridization with synthetic deoxyoligonucleotide probes

Molecular probes were designed for the purpose of specific determination of flavioviruses transmitted by the ticks of tick-borne encephalitis (TBE) and Omsk hemorrhagic fever (OHV) as well as by mosquitoes of Japanese encephalitis (JE), North Nile (NN), Murrey Valley encephalitis (MVE), Saint-Lois encephalitis (SLE), dengue 1-4 and of yellow fever (YF). The probes are synthetic deoxyoligonucleotides with the 18-20 long basis and complementary for the RNA fragments defined by computer analysis. The thus obtained probes, which specifically hybridize themselves with the sets of the TBE virus or of the OHV virus and do not hybridize themselves with other TBE viruses' sets. Group-specific probes for YE and dengue viruses as well as virus-specific probes, which are able to detect each of the above viruses without any cross effects, were suggested for indexing and identifying the flaviviruses transmitted by mosquitoes.     

Stability of St. Louis encephalitis viral antigen detected by enzyme immunoassay in infected mosquitoes.

The use of enzyme immunoassay to detect St. Louis encephalitis (SLE) viral antigen in vector mosquitoes enhances the effectiveness of surveillance because infected mosquitoes can be identified more rapidly than with conventional virus isolation systems and because it is a simple and accessible procedure. Infectivity among mosquitoes experimentally infected with SLE virus was lost within 24 h after the mosquitoes were stored at 27 degrees C and 80% relative humidity; however, viral antigen remained stable under these conditions and could be detected by enzyme immunoassay 2 weeks later. Desiccation further extended the period during which antigen could be detected to 6 weeks. Absorbances were higher in infected mosquitoes stored at 27 degrees C than in mosquitoes frozen continuously. Absorbances in infected mosquitoes also increased after repeated freezing and thawing and sonication. Both phenomena may be related to the release of antigen from decaying or disrupted cells. The relative stability of SLE viral antigen at ambient temperatures lends flexibility to schemes which use direct antigen detection to identify vectors. Surveillance systems can be designed without regard to collecting living mosquitoes, and a cold chain in unnecessary to preserve specimens, thus reducing the cost of surveillance and expanding the geographic areas to which it is accessible.     

RNA interference, arthropod-borne viruses, and mosquitoes

RNA interference (RNAi) probably functions as an antiviral mechanism in most eukaryotic organisms. Variations in the activity of this antiviral pathway in mosquitoes could explain, in part, why some mosquitoes are competent vectors of medically important, arthropod-borne viruses (arboviruses) and others are not. There are three lines of evidence that show the RNAi pathway exists in Aedes species that transmit arboviruses. The first is that recombinant Sindbis viruses expressing a RNA fragment from a genetically unrelated dengue-2 virus (DENV-2) interfere with DENV-2 replication in Aedes aegypti mosquitoes by a mechanism similar to virus-induced gene silencing described in plants. The second is that transfection of C6/36 (Aedes albopictus) cells with either double-stranded RNA or synthetic small interfering RNAs derived from an arbovirus genome interferes with replication of the homologous virus. The third is that a hairpin DENV-2-specific RNA transcribed from a plasmid can generate virus-resistant C6/36 cells. We hypothesize that genetically modified mosquitoes can be generated that transcribe a flavivirus-specific dsRNA, triggering the RNAi response soon after ingestion of a blood meal. This could induce the RNAi pathway in the midgut prior to establishment of virus infection and profoundly change vector competence. Towards this goal, we are developing transgenic A. aegypti lines that are refractory to DENV by exploiting the RNAi pathway.     

Effect of triethylamine on the recovery of selected South American alphaviruses,flaviviruses, and bunyaviruses from mosquito (Diptera: Culicidae) pools

We evaluated the effect of triethylamine (TEA) on the recovery of infectious virus from pools of mosquitoes for two South American alphaviruses (eastern equine encephalomyelitis and Venezuelan equine encephalomyelitis subtypes IIIC and ID), one flavivirus (Ilheus) and two bunyaviruses (Mirim [Guama group] and Itaqui [group C]). Mosquitoes were inoculated intrathoracically with virus, held for 7-10 d at 26 degrees C, and handled under one of four regimens before testing for the presence of virus by plaque assay. Mosquitoes were killed by freezing at - 70 degrees C for 3 min and tested immediately for the presence of virus; killed by freezing at -70 degrees C for 3 min and then held at room temperature for 1 h before testing for the presence of virus; anesthetized with TEA and assayed immediately for the presence of virus; or anesthetized with TEA and then held at room temperature for 1 h before being assayed for the presence of virus. For each of the viruses tested, viral titers in mosquitoes anesthetized with TEA were similar to those in mosquitoes killed by freezing at-70 degrees C. Likewise, there was no significant difference in viral titers in mosquitoes anesthetized with TEA and held at room temperature for 1 h or in mosquitoes frozen at -70 degrees C and held at room temperature for 1 h before being processed for virus by isolation. Triethylamine is advantageous for the handling of mosquitoes in a field environment. The elimination of the need for a cold chain, without compromising virus recovery, increases the feasibility of conducting research projects requiring the isolation of live virus from mosquitoes in remote tropical environments.     

Reduced infection in mosquitoes exposed to blood meals containing previously frozen flaviviruses

The increased difficulty and expense of using live animals for delivering infectious blood meals in arthropod-borne virus vector competence experiments has resulted in an increase in the use of artificial feeding systems. Compared to live hosts, artificial systems require higher viral titers to attain mosquito infection, thereby limiting the utility of such systems with low or moderate titer virus stocks. Based on the report that freshly propagated virus is more infectious than previously frozen virus, we determined whether such a preparation would enhance the ability to use artificial feeding systems. Culex quinquefasciatus and Aedes aegypti mosquitoes were offered blood in artificial membrane feeders containing freshly collected or previously frozen St. Louis encephalitis and dengue serotype-2 viruses (family Flaviviridae), respectively. Infection rates and estimates of vector competence were significantly lower (P<0.05) for mosquitoes feeding on blood meals containing frozen-thawed compared to freshly collected virus. We indicate that the use of freshly propagated virus in artificial feeding systems can be an effective blood delivery method for low-titer viruses and viruses that are otherwise inefficient at infecting vectors in such systems. Fresh viruses used in artificial feeding systems may be a viable alternative to the heavily regulated and expensive use of live animals.     

Brugia malayi microfilariae (Nematoda: Filaridae) enhance the infectivity of Venezuelan equine encephalitis virus to Aedes mosquitoes (Diptera: Culicidae)

We examined the potentially conflicting effects that microfilarial (MF) enhancement of viral infectivity and MF-induced mortality in mosquitoes have on the vectorial capacity of Aedes aegypti (L.), Aedes triseriatus (Say), and Aedes taeniorhynchus (Wiedemann) for Venezuelan equine encephalitis virus (VEE) when mosquitoes feed on gerbils co-infected with Brugia malayi (Buckley). Groups of mosquitoes were fed on gerbils that were either dually infected (VEE plus B. malayi MF) or singly infected (VEE only). Mosquito mortality was recorded daily, and 5-8 d later, surviving mosquitoes were assayed for disseminated viral infection. The contrasting effects of MF enhancement and MF-induced mortality differed among mosquito species and were determined by the nature and consequences of MF penetration through the mosquito midgut, but not to differences in mosquito susceptibilities to parenterally introduced virus. In Ae. aegypti, MF-induced mortality was high and tended to eliminate any significant effect of MF enhancement. In Ae. triseriatus, MF-induced mortality was low, and feeding on dually infected hosts resulted in 9 times as many mosquitoes with disseminated viral infections as did feeding on singly-infected hosts. In Ae. taeniorhynchus, MF-induced mortality was extremely high, yet under our experimental conditions, feeding on a dually infected hosts resulted in nearly 30 times as many disseminated infections as did feeding on singly infected hosts. The final outcome on vectorial capacity depended on the specific combination of MF, virus, and mosquito species involved. Therefore, future efforts toward understanding MF enhancement should be directed toward mosquito-virus-parasite species combinations that occur together in nature.     

A simplified new method for the identification of arboviruses: virus detection using enzyme immunoassay on cultured cells

A simple new method is reported for the identification of arbovirus isolates and the titration of arboviruses. Horseradish peroxidase conjugated Staphylococcus aureus protein A was used for the indirect staining of virus antigens grown in an Aedes albopictus cell line, C6/36 cells. Thirty-five isolates from Culex tritaeniorhynchus mosquitoes were examined by this method and 20 of these were identified as Japanese encephalitis virus (JEV). These results were confirmed by immunofluorescent assay and plaque neutralization test. Comparative titration of JEV, Murray Valley encephalitis (MVE) and Kunjin viruses showed that this method was as sensitive as the chick embryo plaque assay. Specific enzymatic reactions on these virus-infected cells began to appear before day 3 and reached the end-point on day 5 post-infection of C6/36 cells, whereas the cytopathic effect (CPE) appeared about 2 days later than the positive enzymatic reactions. Fixation with 10% formalin for 0.5-8 h did not damage the positive reaction in infected cells and did not increase the background colour of uninfected cells.     

Effect of temperature on the infectivity of the iridovirus and densonucleosis virus of blood-sucking mosquitoes

Mosquito densonucleosis virus and iridovirus were used to study the influence of temperature on their infectivity. Biological assays of mosquito densonucleosis virus were carried out in larvae of I-II instar of Aedes aegypti mosquitoes, those of mosquito iridovirus in larvae of Galleria mellonella. The rate of inactivation was found to be directly dependent on the dose of the heat treatment. Treatment at 60 degrees C led to complete loss of iridovirus infectivity; the virus activity declined considerably after treatment at 50 degrees and 40 degrees C for 60 min. Mosquito densonucleosis virus infectivity was eliminated completely at 65 degrees C. The decline of infectivity of both viruses was intermittent.     

Construction of a replicon and an infectious cDNA clone of the Sofjin strain of the Far-Eastern subtype of tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV) causes severe encephalitis in humans. The Sofjin-HO strain is the prototype strain of the TBEV Far-Eastern subtype and is highly pathogenic in a mouse model. In this study, we constructed replicons and infectious cDNA clones of the Sofjin-HO strain. The replication of the replicon RNA was confirmed, and infectious viruses were recovered from the infectious cDNA clone. The recombinant viruses showed similar virulence characteristics to those of the parental virus. While characterizing the replicon and infectious cDNA, several amino acid differences derived from cell culture adaptations were analysed. The amino acids differences at E position 496 and NS4A position 58 were found to affect viral replication. The Gly- or Ala-to-Glu substitution at E position 122 was shown to increase neuroinvasiveness in mice. These replicons and infectious cDNA clones are useful in revealing the viral molecular determinants involved in the replication and pathogenicity of TBEV.     

The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein.

To study the role of the precursor to the membrane protein (prM) in flavivirus maturation, we inhibited the proteolytic processing of the Murray Valley encephalitis (MVE) virus prM to membrane protein in infected cells by adding the acidotropic agent ammonium chloride late in the virus replication cycle. Viruses purified from supernatants of ammonium chloride-treated cells contained prM protein and were unable to fuse C6/36 mosquito cells from without. When ammonium chloride was removed from the cells, both the processing of prM and the fusion activity of the purified viruses were partially restored. By using monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of MVE virus, we found that at least three epitopes were less accessible to their corresponding antibodies in the prM-containing MVE virus particles. Amino-terminal sequencing of proteolytic fragments of the E protein which were reactive with sequence-specific peptide antisera or MAb enabled us to estimate the site of the E protein interacting with the prM to be within amino acids 200 to 327. Since prM-containing viruses were up to 400-fold more resistant to a low pH environment, we conclude that the E-prM interaction might be necessary to protect the E protein from irreversible conformational changes caused by maturation into the acidic vesicles of the exocytic pathway.     

Failure of dengue-2 virus antibody to interfere with the isolation of dengue-2 virus from Aedes aegypti (Diptera: Culicidae)

When isolating dengue virus (DEN) from mosquitoes collected in endemic areas, pools may contain both anti-dengue antibodies from freshly engorged females and virus from DEN infected females. To determine if these antibodies may interfere with virus isolation, we simulated the isolation procedure using Aedes aegypti (L.) that we infected with the 16,681 strain of dengue type 2 virus by intrathoracic inoculation. At 7 d postinfection, we allowed females to engorge on immunized or normal mouse blood. Virus in a mixture of anti-dengue-2 antibodies and dengue-2 virus became inactive after incubation at 37 degrees C for 1 h, but remained infective without incubation. Therefore, at ambient conditions antibodies would not interfere with virus isolation from field-collected Ae. aegypti from endemic areas. In addition, DEN antibodies enhanced virus replication when inoculated into Ae. aegypti, but not C6/36 cells. The mechanism for this in vitro antibody enhancement of infection remains unclear.     

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.     

Defined epitope blocking with Murray Valley encephalitis virus and monoclonal antibodies: laboratory and field studies.

In an attempt to develop a specific serological test for Murray Valley encephalitis (MVE) virus antibodies, a panel of MVE monoclonal antibodies was utilised in defined-epitope blocking ELISA tests. In sera of mice immunised singly and in combinations of MVE, Alfuy (ALF), and Kunjin (KUN) viruses, blocking patterns usually distinguished MVE infections from those of the other flaviviruses. When blocking tests with selected MAbs were applied to 468 flavivirus antibody positive sera collected from human subjects throughout New South Wales, sera with blocking patterns consistent with previous MVE infection were found in 18 subjects. All were long-term residents of areas previously frequented by MVE, and all were of an age to have been exposed to the virus in past epidemics. No such sera were found in subjects living in coastal areas of NSW where MVE has never been reported.     

Adaptation of an Aedes aegypti mosquito cell line to growth at 15 degrees C and its response to infection by Sindbis virus

Aedes aegypti mosquito cells, usually cultured at 28 to 30 degrees C, were adapted to grow at 15 degrees C. They were designated A. aegypti (c) cells, and had an estimated doubling time of 10 days. Sindbis virus (SV) replicated in these cells to peak titres of over 1.0 x 10(9) p.f.u./ml 8 to 10 days after inoculation. These, or about 10-fold lower titres, continued to be produced over a 130 day test period without causing visible cell damage. Continuous virus proliferation and the yield of uniformly large plaque forming progeny viruses are the two most important features which differentiate infection with this virus in A. aegypti (c) cells from that of A. aegypti cells grown at 28 degrees C (Peleg & Stollar, 1974). Absence of homologous interference vis-à-vis cell-virus coexistence suggests that homologous interference is not a prerequisite for maintaining cell-virus coexistence. Preinoculation of A. aegypti (c) cultures with a small plaque forming Sindbis virus (SV-S) leads, under certain conditions, to the establishment of homologous interference.