Endurance, refuge, and reemergence of dengue virus type 2, Puerto Rico, 1986-2007.

To study the evolution of dengue virus (DENV) serotype 2 in Puerto Rico, we examined the genetic composition and diversity of 160 DENV-2 genomes obtained through 22 consecutive years of sampling. A clade replacement took place in 1994-1997 during a period of high incidence of autochthonous DENV-2 and frequent, short-lived reintroductions of foreign DENV-2. This unique clade replacement was complete just before DENV-3 emerged. By temporally and geographically defining DENV-2 lineages, we describe a refuge of this virus through 4 years of low genome diversity. Our analyses may explain the long-term endurance of DENV-2 despite great epidemiologic changes in disease incidence and serotype distribution.

West Nile virus from blood donors, vertebrates, and mosquitoes, Puerto Rico, 2007.

West Nile virus (WNV) was isolated from a human blood donor, a dead falcon, and mosquitoes in Puerto Rico in 2007. Phylogenetic analysis of the 4 isolates suggests a recent introduction of lineage I WNV that is closely related to WNV currently circulating in North America.

Manipulation of the yellow fever virus non-structural genes 2A and 4B and the 3'non-coding region to evaluate genetic determinants of viral dissemination from the Aedes aegypti midgut.

Although much is known about the ecology, epidemiology, and molecular biology of mosquito-borne viruses, the viral factors that allow transmission by mosquitoes to humans or animals remain unknown. Using infectious clones of disseminating (Asibi) and non-disseminating (17D) yellow fever viruses (YFV), we produced chimeric viruses to evaluate the role of different viral genes in dissemination. Previously, we showed that virus produced from an infectious clone containing the structural genes of 17D in Asibi disseminated from the mosquito midgut at a rate of 31%, indicating that some genetic determinants of dissemination must lie within the non-structural (NS) protein genes or 3' non-coding region (NCR). We chose to investigate the roles of NS2A, NS4B, and the 3'NCR in YFV dissemination. Substitution of the 17D NS2A or NS4B into Asibi significantly attenuated YFV dissemination, demonstrating that this is a multigenic property. There was no difference in dissemination after substitution of the 17D 3'NCR.

Growth characteristics of ChimeriVax-Den vaccine viruses in Aedes aegypti and Aedes albopictus from Thailand.

Four chimeric yellow fever (YF) 17D-dengue (DEN) candidate vaccine viruses (ChimeriVax-DEN; Acambis, Cambridge, MA) were characterized in Aedes aegypti and Ae. albopictus mosquitoes collected from Thailand. The four vaccine viruses contained the relevant prM and E genes of wild-type dengue viruses (DENV; serotypes 1-4) substituted for the equivalent genes in the YF vaccine virus (17D) backbone. Each chimera conferred protection against the homologous DENV serotype; a tetravalent mix of all four chimeras stimulates an immune response against all serotypes. Field-collected mosquitoes from Thailand were fed on blood containing each of the viruses under study and held 21 days after infection. Infection and dissemination rates were based on antigen detection in the body or head tissues, respectively. All four wild-type DENV serotypes infected and disseminated, but the candidate vaccine viruses were highly attenuated in mosquitoes with respect to infection and especially with respect to dissemination. Considering the low level viremias anticipated in humans vaccinated with these viruses, it is predicted that the risks of infection and transmission by mosquitoes in nature is minimal.

Determinants of vector specificity of o'nyong nyong and chikungunya viruses in Anopheles and Aedes mosquitoes.

The alphaviruses o'nyong nyong virus (ONNV) and chikungunya virus (CHIKV) provide a unique system to study the viral genes involved in vector specificity. ONNV infects both anopheline and culicine mosquitoes, whereas CHIKV infects only culicine mosquitoes. In this study, chimeric viruses were constructed that contained genes from both ONNV and CHIKV. These chimeras and previously described full-length infectious clones of ONNV and CHIKV were evaluated in Anopheles gambiae and Aedes aegypti mosquitoes. Virus derived from the infectious clones of ONNV and CHIKV retained the vector specificity of the parental viruses. All six of the chimeras were found to infect Ae. aegypti mosquitoes at high rates but only the chimera containing viral genes encoding all of the structural proteins of ONNV was able to infect An. gambiae mosquitoes. These data indicate that all of the viral structural proteins are necessary for ONNV to infect An. gambiae mosquitoes.

Development and characterization of a double subgenomic chikungunya virus infectious clone to express heterologous genes in Aedes aegypti mosquitoes.

Three full-length infectious cDNA clones based on the alphavirus chikungunya (CHIKV) were developed and characterized in vitro and in vivo. The full-length clone retained the viral phenotypes of CHIKV in both cell culture and in mosquitoes and should be a valuable tool for the study of virus interactions in an epidemiologically significant natural vector, Aedes aegypti. Two additional infectious clones were constructed that express green fluorescent protein (EGFP) in the midgut, salivary glands, and nervous tissue of Aedes aegypti mosquitoes following oral infection. The two constructs differed in the placement of the subgenomic promoter and the gene encoding EGFP. Viruses derived from the pCHIKic EGFP constructs (5' CHIKV EGFP and 3' CHIKV EGFP) expressed EGFP in 100% of the Ae. aegypti mosquitoes tested on days 7 and 14 post infection (p.i.). The 5' CHIKV EGFP disseminated to 90% of the salivary glands and nervous tissue by day 14 p.i. Dissemination rates of this new viral vector exceeds those of previous systems, thus expanding the repertoire and potential for gene expression studies on this important vector species.

Differential infectivities of o'nyong-nyong and chikungunya virus isolates in Anopheles gambiae and Aedes aegypti mosquitoes.

O'nyong-nyong virus (ONNV) and chikungunya virus (CHIKV) are closely related alphaviruses that cause human disease in Africa and Asia. Like most alphaviruses, CHIKV is vectored by culicine mosquitoes. ONNV is considered unusual as it primarily infects anopheline mosquitoes; however, there are relatively few experimental data to support this. In this study, three strains of ONNV and one strain of CHIKV were evaluated in Anopheles gambiae and Aedes aegypti mosquitoes and in four cell lines. As predicted, CHIKV was not infectious to An. gambiae, and we observed strain-variability for ONNV with respect to the ability of the virus to infect An. gambiae and Ae. aegypti. The species specificity in vivo was reflected by in vitro experiments using culicine and anopheline-derived cell lines.

Characterization of the antigen distribution and tissue tropisms of three phenotypically distinct yellow fever virus variants in orally infected Aedes aegypti mosquitoes.

Arbovirus dissemination from the midgut of a vector mosquito is a critical step in facilitating virus transmission to a susceptible host. We previously characterized the genetic determinants of yellow fever virus (YFV) dissemination from the Aedes aegypti mosquito midgut using 2 genetically and phenotypically distinct strains of YFV: the wild-type, disseminating YFV Asibi strain and the attenuated, midgut-restricted YFV 17D vaccine strain. We examined the process of viral dissemination in YFV-infected Ae. aegypti by characterizing the tissue tropisms of 3 YF viruses in Ae. aegypti: Asibi, 17D, and a chimeric virus (17D/Asibi M-E) containing the Asibi membrane (M) and envelope (E) structural protein genes and 17D nonstructural genes. Ae. aegypti were infected orally, and whole, sectioned mosquitoes were evaluated for antigen distribution at 3, 7, 10, 14, and 21 days postinfection by immunohistochemical staining. Virus antigen was consistently observed in the posterior and anterior midgut, cardial epithelium, salivary glands, fat body, and nervous tissues in Asibi- and 17D/Asibi M-E-infected Ae. aegypti following 10 or 14-day extrinsic incubation, respectively. Amplification of virus in the abdominal and thoracic fat body is hypothesized to facilitate YFV infection of the Ae. aegypti salivary glands. As expected, 17D infection was generally limited to the midgut following oral infection. However, there did not appear to be a direct correlation between distribution of infection in the midgut and dissemination to the secondary tissues.

Substitution of wild-type yellow fever Asibi sequences for 17D vaccine sequences in ChimeriVax-dengue 4 does not enhance infection of Aedes aegypti mosquitoes.

To address concerns that a flavivirus vaccine/wild-type recombinant virus might have a high mosquito infectivity phenotype, the yellow fever virus (YFV) 17D backbone of the ChimeriVax-dengue 4 virus was replaced with the corresponding gene sequences of the virulent YFV Asibi strain. Field-collected and laboratory-colonized Aedes aegypti mosquitoes were fed on blood containing each of the viruses under investigation and held for 14 days after infection. Infection and dissemination rates were based on antigen detection in titrated body or head triturates. Our data indicate that, even in the highly unlikely event of recombination or substantial backbone reversion, virulent sequences do not enhance the transmissibility of ChimeriVax viruses. In light of the low-level viremias that have been observed after vaccination in human volunteers coupled with low mosquito infectivity, it is predicted that the risk of mosquito infection and transmission of ChimeriVax vaccine recombinant/revertant viruses in nature is minimal.

Role of the yellow fever virus structural protein genes in viral dissemination from the Aedes aegypti mosquito midgut.

Live-attenuated virus vaccines are key components in controlling arboviral diseases, but they must not disseminate in or be transmitted by mosquito vectors. Although the cycles in which many mosquito-borne viruses are transmitted are well understood, the role of viral genetics in these processes has not been fully elucidated. Yellow fever virus (YFV) is an important arbovirus and the prototype member of the family Flaviviridae. Here, YFV was used in Aedes aegypti mosquitoes as a model to investigate the genetic basis of infection and dissemination in mosquitoes. Viruses derived from infectious clones and chimeric viruses with defined sequential manipulations were used to investigate the influence of specific sequences within the membrane and envelope structural protein genes on dissemination of virus from the mosquito midgut. Substitution of domain III of the envelope protein from a midgut-restricted YFV into a wild-type YFV resulted in a marked decrease in virus dissemination, suggesting an important role for domain III in this process. However, synergism between elements within the flavivirus structural and non-structural protein genes may be necessary for efficient virus escape from the mosquito midgut.

Characterization of an infectious clone of the wild-type yellow fever virus Asibi strain that is able to infect and disseminate in mosquitoes.

Infectious clone technology provides an opportunity to study the molecular basis of arthropod-virus interactions in detail. This study describes the development of an infectious clone of the prototype yellow fever virus Asibi strain (YFV-As) with the purpose of identifying sequences or domains that influence infection dynamics in the mosquito vector. The full-length cDNA of YFV-As virus was produced from RT-PCR products of parental viral RNA. These were cloned into a low-copy-number plasmid previously used to develop the YFV-17D infectious clone (pACNR/FLYF-17D). Virus recovered from the infectious clone exhibited biological characteristics similar to those of the parental YFV-As, including replication kinetics, reactivity to flavivirus cross-reactive and YFV-specific antibodies and infection and dissemination rates in Aedes aegypti, the principal mosquito vector of YFV. These data provide the basis for future studies with chimeric Asibi/17D viruses to identify the determinants of vaccine attenuation in the vector.