Sindbis Research Articles

Evolutionary genetics and vector adaptation of recombinant viruses of the western equine encephalitis antigenic complex provides new insights into alphavirus diversity and host switching

Western equine encephalitis virus (WEEV), Highlands J virus (HJV), and Fort Morgan virus (FMV) are the sole representatives of the WEE antigenic complex of the genus Alphavirus, family Togaviridae, that are endemic to North America. All three viruses have their ancestry in a recombination event involving eastern equine encephalitis virus (EEEV) and a Sindbis (SIN)-like virus that gave rise to a chimeric alphavirus that subsequently diversified into the present-day WEEV, HJV, and FMV. Here, we present a comparative analysis of the genetic, ecological, and evolutionary relationships among these recombinant-origin viruses, including the description of a nsP4 polymerase mutation in FMV that allows it to circumvent the host range barrier to Asian tiger mosquito cells, a vector species that is normally refractory to infection. Notably, we also provide evidence that the recombination event that gave rise to these three WEEV antigenic complex viruses may have occurred in North America.

Utility of the Sindbis replicon system as an Env-targeted HIV vaccine

Sindbis replicon-based vaccine vectors are designed to combine the immunostimulatory properties of replicating viruses with the superior safety profile of non-replicating systems. In this study we performed a detailed assessment of Sindbis (SIN) replicon vectors expressing HIV-1 envelope protein (Env) for the induction of cell-mediated and humoral immune responses in a small animal model. SIN-derived virus-like particles (VLP) elicited Env-specific antibody responses that were detectable after boosting with recombinant Env protein. This priming effect could be mediated by replicon activity alone but may be enhanced by Env attached to the surface of VLP, offering a potential advantage for this mode of replicon delivery for Env based vaccination strategies. In contrast, the Env-specific CTL responses that were elicited by SIN-VLP were entirely dependent on replicon activity. SIN-VLP priming induced more durable humoral responses than immunization with protein only. This is important from a vaccine perspective, given the intrinsic tendency of Env to induce short-lived antibody responses in the context of vaccination or infection. These results indicate that further efforts to enhance the magnitude and durability of the HIV-1 Env-specific immune responses generated by Sindbis vectors, either alone or as part of prime-boost regimens, are justified.

Phylogeographic Structure and Evolutionary History of Sindbis Virus

Sindbis (SIN) virus, Alphavirus, is a mosquito-borne and bird-associated virus with large geographic distribution in the Old World. We investigated the genetic diversity of 59 SIN strains after limited sequencing of their E2 glycoprotein genes. The SIN strains showed maximal diversity of 22.2% at the amino acid (aa) level, and formed five tentative genotypes. The SIN-I genotype included strains from Europe and Africa. Strains from Australia and East Asia formed SIN-II and SIN-III with about 12% and 15% aa divergence from SIN-I. The only isolate from New Zealand was distinct, and constitutes the SIN-V genotype. Isolates from Azerbaijan and China formed genotype SIN-IV with 15.6%-19.1% aa divergence from SIN-I to III and SIN-V. Phylogenetic analyses indicated that Aura virus was present before the recombinant alphavirus lineage arose. This is consistent with a South American origin of the SIN complex, and argue for a spread in North America before reaching Asia and Australia, followed by westward radiation into Africa and Europe. High levels of sequence identities were observed for geographic regions belonging to the same north-south axis, whereas the east-west genetic exchange appears to be limited. The observed phylogeographic structure was confirmed by distinct aa patterns within two-thirds of the structural protein-coding region of SIN virus strains from Saudi Arabia, Asia, and Australia. The present geography of the five SIN genotypes and subclusters within SIN-I correlate with major bird migration patterns.

Genomic sequencing of Highlands J virus: A comparison to western and eastern equine encephalitis viruses

Highlands J virus (HJV) is a member of the genus Alphavirus, family Togaviridae. HJV is the sole representative of the western equine encephalitis (WEE) serocomplex found in the eastern United States, and circulates in nature in an apparently identical transmission cycle as eastern equine encephalitis virus (EEEV). North American representatives of the WEE serocomplex [HJV, WEE virus (WEEV), and Fort Morgan virus (FMV)] are believed to be derived from a recombination event involving EEEV and a Sindbis (SIN)-like virus, such that the nonstructural polyprotein, the capsid, and the terminal end of the 3′ UTR are derived from EEEV, while the surface glycoproteins (E1 and E2) and small peptides (E3 and 6K) encoded in the subgenomic RNA are derived from the SIN-like virus. In this report, the complete nucleotide sequence of HJV is described, along with a comparative analysis of the HJV nonstructural polyprotein to WEEV and EEEV.

Serological evidence of antibodies to certain arboviruses in desert rodent sera in Egypt

Introduction : Small rodents inhabiting the wadis close to St Katherine in the mountains of the Sinai Peninsula, Egypt, were trapped and antibodies to Sindbis (SIN) , West Nile (WN) and Quaranfil (QRF) viruses in their sera were studied. Materials and Methods One mouse had Haemagglutination–inhibition (HI) antibodies against SIN antigen in his serum, 3 mice had HI antibodies against WN antigen in their sera. The titers is low to be of any significance. Conclusion: It seems that natural cycle of transmission of SIN and WN viruses involving rodents does not exist. Seven mice had Complement-fixation (CF) antibodies in their sera against Quaranfil antigen. Conclusion, the CF test results suggest the existence of another mammal vector cycle.

Serological evidence of antibodies to certain arboviruses in desert rodent sera in Egypt

Introduction : Small rodents inhabiting the wadis close to St Katherine in the mountains of the Sinai Peninsula, Egypt, were trapped and antibodies to Sindbis (SIN) , West Nile (WN) and Quaranfil (QRF) viruses in their sera were studied. Materials and Methods One mouse had Haemagglutination–inhibition (HI) antibodies against SIN antigen in his serum, 3 mice had HI antibodies against WN antigen in their sera. The titers is low to be of any significance. Conclusion: It seems that natural cycle of transmission of SIN and WN viruses involving rodents does not exist. Seven mice had Complement-fixation (CF) antibodies in their sera against Quaranfil antigen. Conclusion, the CF test results suggest the existence of another mammal vector cycle.

SEROPREVALENCE AND DISTRIBUTION OF FLAVIVIRIDAE, TOGAVIRIDAE, AND BUNYAVIRIDAE ARBOVIRAL INFECTIONS IN RURAL CAMEROONIAN ADULTS

Arboviruses from the families Flaviviridae, Togaviridae, and Bunyaviridae are suspected to cause widespread morbidity in sub-Saharan African populations, but little research been done to document the burden and distribution of these pathogens. We tested serum samples from 256 Cameroonian adults from nine rural villages for the presence of Dengue-2 (DEN-2), West Nile (WN), Yellow fever (YF), Chikungunya (CHIK), O'nyong-nyong (ONN), Sindbis (SIN), and Tahyna (TAH) infection using standard plaque-reduction neutralization tests (PRNT). Of these samples, 12.5% were DEN-2 positive, 6.6% were WN positive, 26.9% were YF positive, 46.5% were CHIK seropositive, 47.7% were ONN positive, 7.8% were SIN positive, and 36.3% were TAH positive. DEN-2, YF, and CHIK seroprevalence rates were lower among individuals living in dwellings with grass or thatched roofs versus corrugated tin and in villages isolated from urban centers. Seroprevalence rates of YF and CHIK increased with age. These results suggest that inter-epidemic arboviral infection is common in central African populations.

Formation of nsP3-Specific Protein Complexes during Sindbis Virus Replication

Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. Their replication proceeds in the cytoplasm of infected cells and does not directly depend on nuclei. Alphaviruses encode only four nonstructural proteins that are required for the replication of viral genome and transcription of the subgenomic RNA. However, the replicative enzyme complexes (RCs) appear to include cellular proteins and assemble on cellular organelles. We have developed a set of recombinant Sindbis (SIN) viruses with green fluorescent protein (GFP) insertions in one of the nonstructural proteins, nsP3, to further understand the RCs' genesis and structure. We studied the assembly of nsP3/GFP-containing protein complexes at different stages of infection and isolated a combination of cellular proteins that are associated with SIN nsP3. We demonstrated the following. (i) SIN nsP3 can tolerate the insertion of GFP into different fragments of the coding sequence; the designed recombinant viruses are viable, and their replication leads to the assembly of nsP3/GFP chimeric proteins into gradually developing, higher-order structures differently organized at early and late times postinfection. (ii) At late times postinfection, nsP3 is assembled into complexes of similar sizes, which appear to be bound to cytoskeleton filaments and can aggregate into larger structures. (iii) Protein complexes that are associated with nsP3/GFP contain a high concentration of cytoskeleton proteins, chaperones, elongation factor 1A, heterogeneous nuclear ribonucleoproteins, 14-3-3 proteins, and some of the ribosomal proteins. These proteins are proposed to be essential for SIN RC formation and/or functioning.

Replication and Clearance of Venezuelan Equine Encephalitis Virus from the Brains of Animals Vaccinated with Chimeric SIN/VEE Viruses

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.

Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and approximately 10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC+) that are active in plus-strand synthesis. Minus-strand synthesis and incorporation of [3H]uridine into replicative intermediates differed among PI replicons, depending on the location of the mutation in nsP2. Minus-strand synthesis by PI cells appeared normal; it was dependent on continuous P123 and P1234 polyprotein synthesis and ceased when protein synthesis was inhibited. The failure by the PI replicons to shut off minus-strand synthesis was not due to some defect in the PI cells but rather was due to the loss of some function in the mutated nsP2. This was demonstrated by showing that superinfection of PI cells with wt SFV triggered the shutdown of minus-strand synthesis, which we believe is a host response to infection with alphaviruses. Together, the results indicate alphavirus nsP2 functions to engage the host response to infection and activate a switch from the early-to-late phase. The loss of this function leads to continuous viral minus-strand synthesis and the production of unstable RC+.

Comparison of the transmission potential of two genetically distinct Sindbis viruses after oral infection of Aedes aegypti (Diptera: Culicidae).

Within mosquitoes, arboviruses encounter barriers to infection and dissemination that are critical determinants of vector competence. The molecular mechanisms responsible for these barriers have yet to be elucidated. The prototype Sindbis (SIN) strain, AR339, and viruses derived from this strain, such as TR339 virus, have limited infection and transmission potential in the medically important arthropod vector, Aedes aegypti (L.). However, the Malaysian SIN virus strain, MRE16, disseminates in nearly 100% of Ae. aegypti 14 d after oral infection. Here, we compare the spatial and temporal infection patterns of MRE16 and TR339 viruses in Ae. aegypti. The results indicate that a midgut escape barrier is primarily responsible for the significantly lower dissemination and transmission potentials observed after oral infection with TR339 virus. MRE16 and TR339 viruses now represent a well-characterized model system for the further study of virus determinants of vector infection, particularly determinants affecting the midgut escape barrier in Ae. aegypti.

DC-SIGN and L-SIGN can act as attachment receptors for alphaviruses and distinguish between mosquito cell- and mammalian cell-derived viruses.

C-type lectins such as DC-SIGN and L-SIGN, which bind mannose-enriched carbohydrate modifications of host and pathogen proteins, have been shown to bind glycoproteins of several viruses and facilitate either cis or trans infection. DC-SIGN and L-SIGN are expressed in several early targets of arbovirus infection, including dendritic cells (DCs) and cells of the reticuloendothelial system. In the present study, we show that DC-SIGN and L-SIGN can function as attachment receptors for Sindbis (SB) virus, an arbovirus of the Alphavirus genus. Human monocytic THP-1 cells stably transfected with DC-SIGN or L-SIGN were permissive for SB virus replication, while untransfected controls were essentially nonpermissive. The majority of control THP-1 cells were permissive when attachment and entry steps were eliminated through electroporation of virus transcripts. Infectivity for the DC-SIGN/L-SIGN-expressing cells was largely blocked by yeast mannan, EDTA, or a DC-SIGN/L-SIGN-specific monoclonal antibody. Infection of primary human DCs by SB virus was also dependent upon SIGN expression by similar criteria. Furthermore, production of virus particles in either C6/36 mosquito cells or CHO mammalian cells under conditions that limited complex carbohydrate content greatly increased SB virus binding to and infection of THP-1 cells expressing these lectins. C6/36-derived virus also was much more infectious for primary human DCs than CHO-derived virus. These results suggest that (i) lectin molecules such as DC-SIGN and L-SIGN may represent common attachment receptor molecules for arthropod-borne viruses, (ii) arbovirus particles produced in and delivered by arthropod vectors may preferentially target vertebrate host cells bearing these or similar lectin molecules, and (iii) a cell line has been identified that can productively replicate alphaviruses but is deficient in attachment receptors.

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.

Entomological investigations of an outbreak of Japanese encephalitis virus in the Torres Strait, Australia, in 1998.

Japanese encephalitis (JE) virus first appeared in Australia in 1995, when three clinical cases (two fatal) were diagnosed in residents on Badu Island in the Torres Strait, northern Queensland. More recently, two confirmed human JE cases were reported in the Torres Strait Islands and Cape York Peninsula, in northern Queensland in 1998. Shortly after JE virus activity was detected in humans and sentinel pigs on Badu Island in 1998, adult mosquitoes were collected using CO2 and octenol-baited CDC light traps; 43 isolates of JE virus were recovered. Although Culex sitiens group mosquitoes yielded the majority of JE isolates (42), one isolate was also obtained from Ochlerotatus vigilax (Skuse). Four isolates of Ross River virus and nine isolates of Sindbis (SIN) virus were also recovered from members of the Culex sitiens group collected on Badu Island in 1998. In addition, 3,240 mosquitoes were speciated and pooled after being anesthetized with triethylamine (TEA). There was no significant difference in the minimum infection rate of mosquitoes anesthetized with TEA compared with those sorted on refrigerated tables (2.8 and 1.6 per 1,000 mosquitoes, respectively). Nucleotide analysis of the premembrane region and an overlapping region of the fifth nonstructural protein and 3' untranslated regions of representative 1998 Badu Island isolates of JE virus reveled they were identical to each other. Between 99.1% and 100% identity was observed between 1995 and 1998 isolates of JE from Badu Island, as well as isolates of JE from mosquitoes collected in Papua New Guinea (PNG) in 1997 and 1998. This suggests that the New Guinea mainland is the likely source of incursions of JE virus in Australia.

Seroepidemiology of California and Bunyamwera serogroup (Bunyaviridae) virus infections in native populations of Alaska.

This study investigated the geographic distribution and prevalence of antibodies to California and Bunyamwera serogroup viruses in Native populations of Alaska, and demographic and ecologic risk factors associated with exposure. Sera (n = 1,635) from 18 communities were screened using an ELISA. All age groups were tested for antibodies to Jamestown Canyon (JC), Inkoo (INK), snowshoe hare (SSH), and Northway (NOR) viruses; persons > or = 45 years old (n = 90) from six communities were additionally tested for antibodies to Tahyna (TAH), Batai (BAT), Cache Valley (CV), and Sindbis (SIN) viruses. Thirty free-ranging mammals were tested by a plaque reduction neutralization test (PRNT) for antibodies to all eight viruses and to Getah (GET) virus. In Natives, overall antibody prevalence was 24.9% (JC = 17.6%, monotypic JC = 6.5%, INK = 11.1%, monotypic INK = 0.6%, SSH = 6.8%, monotypic SSH = 3.5%, and NOR = 6.2%). Five TAH, CV, and BAT virus exposures may be serologic cross-reactions, and no SIN virus antibodies were detected. Sindbis-like virus antibodies were found in 30% of the mammals. Most mammals had antibodies to NOR (83.3%) and California serogroup (70.0%) viruses; no GET virus exposures were found. Significant risk factors for human bunyavirus exposures were age group, ethnic-linguistic group, biotic province, climate zone, terrestrial vegetation, and presence of some ungulates and small mammals in communities. Sex was not a significant risk factor.

Geographic distribution and evolution of Sindbis virus in Australia.

The molecular epidemiology and evolution of Sindbis (SIN) virus in Australia was examined. Several SIN virus strains isolated from other countries were also included in the analysis. Two regions of the virus genome were sequenced including a 418 bp region of the E2 gene and a 484 bp region containing part of the junction region and the 5' end of the C gene. Analysis of the nucleotide and deduced amino acid sequence data from 40 SIN virus isolates clearly separated the Paleoarctic/Ethiopian and Oriental/Australian genetic types of SIN virus. Examination of the Australian strains showed a temporal rather than geographic relationship. This is consistent with the virus having migratory birds as the major vertebrate host, as it allows for movement of virus over vast areas of the continent over a relatively short period of time. The results suggest that the virus is being periodically redistributed over the continent from an enzootic focus of evolving SIN virus. However, SIN virus strains isolated from mosquitoes collected in the south-west of Australia appear to represent a new SIN virus lineage, which is distinct from the Paleoarctic/Ethiopian and Oriental/Australian lineages. Given the widespread geographic dispersal of the Paleoarctic/Ethiopian and Oriental/Australian lineages, it is surprising that the South-west genetic type is so restricted in its area of circulation. Nucleotide sequence data from the C gene of the prototype strain of the alphavirus Whataroa were also determined. This virus was found to be genetically distinct from the SIN virus isolates included in the present study; however, it is clearly SIN-like and appears to have evolved from a SIN-like ancestral virus.

Development of a Chimeric Sindbis Virus with Enhancedper OsInfection ofAedes aegypti

The TE/3′2J double subgenomic Sindbis (dsSIN) viruses have been used to stably express genes inAedes aegyptinerve and salivary gland tissues. However, because these viruses inefficiently infectAe. aegyptiwhen administered by theper osroute, TE/3′2J viruses must be intrathoracically inoculated into the mosquitoes to infect these tissues. A Malaysian Sindbis (SIN) virus isolate (MRE16) does efficiently infectAe. aegyptimidgut tissues after ingestion, and approximately 95% of these mosquitoes also develop disseminated infections within 14 days. We have sequenced the entire 26S RNA of MRE16 virus and have developed a chimeric SIN cDNA infectious clone, designated MRE1001, which contains sequence elements of TE/3′2J and MRE16 virus. MRE1001 virus efficiently infects midgut cells, and greater than 90% of infected mosquitoes develop disseminated infections after 14 days extrinsic incubation. The chimeric MRE1001 cDNA clone should allow identification of viral determinants of midgut infection and dissemination and lead to the development of new SIN virus expression systems.

Construction of hepatitis C-SIN virus recombinants with replicative dependency on hepatitis C virus serine protease activity

An in vivo assay system was developed for the serine protease of hepatitis C virus (HCV) using the sindbis (SIN) viral replication system in which HCV serine protease activity is essential for the replication of the HCV-SIN chimeric virus. Two chimeric viral cDNA clones were constructed by inserting the NS3/4A region and NS3/4A region with the putative helicase deleted, into the N-terminal region of SIN core protein. The constructs were named Tpro CT and Tpro T, respectively. BHK-21 cells transfected with the in vitro transcribed RNAs from Tpro CT and Tpro T showed specific cytopathic morphology and produced chimeric viruses, Vpro CT and Vpro T. In contrast, in vitro transcribed RNAs from Tpro CTI and Tpro TI, in which serine of catalytic triad of HCV protease was changed to alanine, were not infectious. When the chimeric viruses were passaged in BHK-21 cells at about 0.1 multiplicity of infection (MOI), Vpro T, but not Vpro CT, stably expressed HCV protease for up to five passages. Surprisingly, the cell culture media of BHK-21 cells infected with Vpro T, compared to wild-type sindbis virus, showed rapid pH changes by more than 0.8 pH degree at 72 h post-infection. HCV-SIN hybrid viruses could be used in screening the HCV protease-inhibitor in cell culture systems.

Alphavirus hybrid virion vaccines.

Many members of the alphavirus family are important human or veterinary pathogens. These viruses are extremely similar in molecular architecture yet differ in host range and in the pathological consequences of infection. Despite marked structural conservation, the various species are inefficient in eliciting immunological cross protection. Previously(l) we described a systematic approach for selection of epitope-cassettes of the E2 envelope of the alphavirus family that induce virus species specific protective immunity. This approach was tested and proved successful(2–5) with two viruses - Sindbis (SIN) and Semliki Forest (SF) - which are very remote phylogenetically. Vaccination was performed with recombinant peptide cassettes fused to a bacterial protein carrier (β-galactosidase) formulated with different adjuvants. In order to create a vaccine compatible with human use, we designed several live vector vaccines for presentation of the protective epitope cassette. Attenuated salmonella vaccine vector which can potentially allow presentation of many different cassettes and thus generation of a pan-alphavirus vaccine, proved to be inefficient. On the other hand the live attenuated viral vaccine based on a benign alphavirus such as SIN vector, appears to be promising. The principle of such vaccines is substitution of E2 epitopes of SIN virus vector with parallel protective epitopes from a different alphavirus. This system should have the advantage of presentation of specific virus epitopes in the context of the SIN alphavirus backbone which in turn could provide cross protective immunity on the basis of multiple common antigen determinants.KeywordsSemliki Forest VirusProtective EpitopeForeign EpitopeCross Protective ImmunityCellular Mediate ImmunityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The Expression of Chloramphenicol Acetyltransferase in Mosquitoes and Mosquito Cells Using a Packaged Sindbis Replicon System

Sindbis (SIN) replicon virus was used to express chloramphenicol acetyltransferase (CAT) in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes. RNA transcribed in vitro from a SIN replicon plasmid (pSINrep5/CAT) and from SIN virus helper constructs (pDH-EB or pDH(26S)5′SIN) was coelectroporated into BHK-21 cells to generate replicon viruses, designated rep5/CAT/EB and rep5/CAT/26S. C6/36 cells infected with rep5/CAT/EB or rep5/CAT/26S virus at a multiplicity of infection of 3, expressed 3.8 × 106 and 6.0 × 106 CAT trimers per cell, respectively, at 2 days postinfection (pi). Both viruses attained peak titers by Day 2 pi. Adult female A. triseriatus mosquitoes were intrathoracically inoculated with 7 × 104 IFU rep5/CAT/EB or 1 × 105 IFU rep5/CAT/26S virus. Virus titers remained at approximately 105 IEU/ml through Day 2 pi and decreased roughly 1 log by Day 10 pi. CAT enzyme activity was detected 2 days pi (rep5/CAT/EB, 1.49 × 10−4 units CAT/10 μg protein; rep5/CAT/26S, 2.03 × 10−5 units CAT/10 μg protein) and remained near these levels through Day 10 pi. CAT was detected in the head, salivary glands, midgut, and ovaries of inoculated mosquitoes by indirect immunofluorescence or CAT activity assays. These results suggest that packaged replicon viruses can be useful for expression of heterologous genes in mosquito cells and whole mosquitoes.