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At least 19 recordsLinked to original sources
PubMed2004

Use of recombinant E protein domain III-based enzyme-linked immunosorbent assays for differentiation of tick-borne encephalitis serocomplex flaviviruses from mosquito-borne flaviviruses.

The serological diagnosis of infection by flaviviruses is complicated by the presence of flavivirus cross-reactive antibodies that produce false-positive results for flavivirus infections, especially in regions where more than one virus is endemic. Current diagnostic reagents for tick-borne flavivirus infection have been found to cross-react with yellow fever- or dengue virus-positive sera. This study utilized recombinant flavivirus E protein domain 3 (rE-D3) as a diagnostic reagent to differentiate between infection by mosquito- and tick-borne flaviviruses. This study found that the use of rE-D3 in an enzyme-linked immunosorbent assay (ELISA)-based format allowed the differentiation between serum specific for either mosquito- or tick-borne flaviviruses, but not among the members of the tick-borne encephalitis (TBE) serocomplex of flaviviruses. Sera derived against several TBE serocomplex rE-D3 were found to cross-react with heterologous rE-D3 within the TBE serocomplex, but not with those from mosquito-borne flaviviruses, in both Western blots and ELISAs. Mouse hyperimmune sera generated against TBE serocomplex viruses were also found to react specifically with TBE serocomplex rE-D3, but not with rE-D3 from mosquito-borne viruses and vice versa. When a similar test using virus-derived antigen was performed, a loss of both specificity and sensitivity was observed. These results indicate that flavivirus rE-D3 would be a useful reagent for the detection of infection by TBE serocomplex flaviviruses, several of which are potential biothreat agents, but would not provide the ability to differentiate among infections by separate members of the serocomplex.

Amino Acid Sequence↗
PubMed2001

Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences.

Arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. Because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by PCR would be a useful first-line diagnostic method. The six published Flavivirus genus primer pairs localized in the NS1, NS3, NS5, and 3' NC regions were evaluated in terms of specificity and sensitivity with flaviviruses (including the main viruses pathogenic for humans) at a titer of 10(5) 50% tissue culture infectious doses (TCID(50)s) ml(-1) with a common identification step by agarose gel electrophoresis. Only one NS5 primer pair allowed the detection of all tested flaviviruses with the sensitivity limit of 10(5) TCID(50)s ml(-1). Using a heminested PCR with new primers designed in the same region after an alignment of 30 different flaviviruses, the sensitivity of reverse transcription-PCR was improved and allowed the detection of about 200 infectious doses ml(-1) with all of the tick- and mosquito-borne flaviviruses tested. It was confirmed that the sequenced amplified products in the NS5 region allowed predictability of flavivirus species by dendrogram, including the New York 99 West Nile strain. This technique was successfully performed with a cerebrospinal fluid sample from a patient hospitalized with West Nile virus encephalitis.

Animals↗
PubMed1998

Identification of Brazilian flaviviruses by a simplified reverse transcription-polymerase chain reaction method using Flavivirus universal primers.

We report a simplified reverse transcription-polymerase chain reaction (RT-PCR) method for identification of Brazilian flaviviruses based on the patterns of electrophoretic separation of the amplicons. The RT-PCR was done on the culture fluids of Aedes albopictus C6/36 cells infected with Brazilian flaviviruses, without previous extraction of viral RNA, using Flavivirus universal primers that anneal to highly conserved sequences within the nonstructural protein 5 and 3'- non translated region of the virus genome. Genomes of 13 Brazilian Flavivirus isolates were amplified. It was not possible to amplify the genome of Bussuquara virus. Analysis of the RT-PCR products gave reproducible results and three distinct amplicon patterns were observed. Cacipacoré (800-850 basepairs [bp]) and yellow fever viruses (600 bp) yielded a single amplicon; dengue virus types 1 and 2 (650 and 550 bp), dengue virus type 4 (550 and 450 bp), Iguape (650-600 bp and 750-700 bp), St. Louis encephalitis (700 and 650-600 bp), and Rocio viruses (600 and 500-550 bp) yielded two amplicons; and Ilheus virus yielded five amplicons, two larger than 1,000 bp, one 650-700 bp, one 550-600 bp, and one 450-500 bp. The analysis of amplicon DNA sequences of six viruses showed homology with the 3'- nontranslated region of Flavivirus genome. The use of the Flavivirus universal primers in this simple RT-PCR technique is suitable as a screening test for the genus Flavivirus, with the exception of Bussuquara virus, in Brazilian isolates in tissue culture fluid.

Aedes↗
PubMed1993

Genetic studies of flavivirus resistance in inbred strains derived from wild mice: evidence for a new resistance allele at the flavivirus resistance locus (Flv).

Studies of genetic resistance to flavivirus infection in laboratory mice have led to the development of a single model in which resistance is conferred by an autosomal dominant gene designated Flvr. Because of evidence suggesting that wild mice carry virus resistance genes which are not present in laboratory mice, we compared flavivirus resistance in the inbred strains CASA/Rk, CAST/Ei, and MOLD/Rk, which are derived directly from wild mice, and the congenic strains C3H/RV (Flvr/Flvr) and C3H/HeJ (Flvs/Flvs). Resistance to the Murray Valley encephalitis virus strain OR2 and the 17D vaccine strain of yellow fever virus was assessed by determining the lethality of intracerebral infection and by measuring virus replication in the brain. The resistance of the CASA/Rk and CAST/Ei strains resembled the resistance of C3H/RV mice, whereas the resistance of the MOLD/Rk strain was intermediate between those of C3H/RV and C3H/HeJ mice. Genetic analyses showed that resistance in both the CASA/Rk and MOLD/Rk strains is conferred by single autosomal dominant alleles at the Flv locus. Our data indicate that flavivirus resistance in the CASA/Rk strain is due to a gene which is similar or identical to Flvr, whereas resistance in the MOLD/Rk strain is due to a previously undescribed gene which we designate Flvmr to indicate minor resistance to flavivirus infection. Since genetic resistance to flaviviruses is rare in laboratory mice, the CASA/Rk and MOLD/Rk strains will be valuable for further investigation of this phenomenon.

Alleles↗
PubMed1998

Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4.

Langat virus (LGT) strain TP21 is the most attenuated of the tick-borne flaviviruses for humans. Even though LGT has low-level neurovirulence for humans, it, and its more attenuated egg-passage derivative, strain E5, exhibit significant neurovirulence and neuroinvasiveness in normal mice, albeit less than that associated with tick-borne encephalitis virus (TBEV), the most virulent of the tick-borne flaviviruses. We sought to reduce or ablate these viral phenotypes of TP21 and E5 by using a strategy that had been used successfully in the past to reduce neurovirulence and abolish neuroinvasiveness of TBEV, namely substitution of structural protein genes of the tick-borne flavivirus for the corresponding genes of dengue type 4 virus (DEN4). In pursuit of these objectives different combinations of LGT genes were substituted into the DEN4 genome but only chimeras containing LGT structural proteins premembrane (preM) and envelope glycoprotein (E) were viable. The infectious LGT(preM-E)/DEN4 chimeras were restricted in replication in simian cell cultures but grew to moderately high titer in mosquito cell culture. Also, the chimeras were at least 5,000 times less neurovirulent than their parental LGT virus in suckling mice. Significantly, the chimeras lacked detectable evidence of neuroinvasiveness after i.p. inoculation of Swiss mice or the more permissive SCID mice with 10(5) or 10(7) plaque-forming units (PFU), respectively. Nonetheless, i.p. inoculation of Swiss mice with 10 or 10(3) PFU of either chimeric virus induced LGT neutralizing antibodies and resistance to fatal encephalitis caused by i.p. challenge with LGT TP21. The implications of these observations for development of a live attenuated TBEV vaccine are discussed.

Amino Acid Sequence↗
PubMed1999

Mosquito feeding modulates Th1 and Th2 cytokines in flavivirus susceptible mice: an effect mimicked by injection of sialokinins, but not demonstrated in flavivirus resistant mice.

Culex pipiens and Aedes aegypti mosquitoes were fed on C3H/HeJ mice and systemic cytokine production was quantified from stimulated lymphocytes harvested four to ten days after feeding. Mosquito feeding on C3H/HeJ mice significantly down regulated IFN gamma production seven to ten days post feeding by Cx. pipiens and seven days after Ae aegypti feeding. Th2 cytokines, IL-4 and IL-10, were significantly up regulated 4-7 days after Cx. pipiens and Ae. aegypti feeding. The immunosuppressive effect of Cx. pipiens feeding on systemic cytokine production was not evident in congenic flavivirus resistant (C3H/RV) mice, as systemic IFN gamma and IL-2 were significantly up regulated at days 7 and 10, correlating with a significant decrease in IL-4 10 days after feeding by Cx. pipiens mosquitoes. Inoculation of 5-1000 ng of sialokinin-I into C3H/HeJ mice mimicked the effect of Ae. aegypti feeding by down regulating Th1 cytokines and significantly up regulating Th2 cytokines four days post inoculation. Injections of sialokinin-II resulted in only moderate effects on IFN gamma and IL-4 production seven and ten days after injection. Thus natural feeding by two arbovirus vectors had a profound T cell modulatory effect in vivo in virus susceptible animals which was not demonstrated in the flavivirus resistant host. Moreover, sialokinin-I and sialokinin-II mimicked the effect of mosquito feeding by modulating the host T cell response. These results may lend new insight into specific aspects of the role of the mosquito vector in potentiating virus transmission in the mammalian host.

Aedes↗
PubMed2001

Salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa B activation.

Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-kappaB), and salicylates have been shown to inhibit NF-kappaB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-kappaB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IkappaBalpha-DeltaN, a dominant-negative mutant that antagonizes NF-kappaB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-kappaB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IkappaBalpha-DeltaN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-kappaB pathway.

Animals↗
PubMed1996

Evaluation of monoclonal antibodies for generic detection of flaviviruses by ELISA.

Three monoclonal antibodies (Mabs) specific for the envelope (E) protein of flaviviruses were evaluated for use in an antigen capture ELISA. Three combinations of Mabs and a combination of polyclonal antibodies (Pabs) were evaluated in antigen capture ELISAs for their ability to detect 18 flaviviruses. The Mab ELISAs detected 50% of flavivirus antigens with a sensitivity between 1 and 9 x 10(4)/ng viral protein/ml, however, none of the ELISAs evaluated proved to be useful for generic detection of flaviviruses, being unable to detect tick-borne flaviviruses and some mosquito-borne flaviviruses. The inability of the ELISAs to detect tick-borne flaviviruses is thought to be due to the conformation of surface epitopes, which the Mabs were unable to recognise. This was again observed using recombinant TBE virus prM/E protein as antigen in direct and antigen capture ELISAs. The Mabs reacted with the prM/E protein when it was denatured by binding directly onto the solid phase, but the antibodies were unable to detect the native protein in antigen capture ELISAs. The antigen capture ELISAs evaluated in this study were considered to be unsuitable for the generic detection of flaviviruses, but may provide a sensitive diagnostic assay for specific flavivirus infection.

Animals↗
PubMed2003

Chimeric flaviviruses: novel vaccines against dengue fever, tick-borne encephalitis, and Japanese encephalitis.

Many arthropod-borne flaviviruses are important human pathogens responsible for diverse illnesses, including YF, JE, TBE, and dengue. Live, attenuated vaccines have afforded the most effective and economical means of prevention and control, as illustrated by YF 17D and JE SA14-14-2 vaccines. Recent advances in recombinant DNA technology have made it possible to explore a novel approach for developing live attenuated flavivirus vaccines against other flaviviruses. Full-length cDNA clones allow construction of infectious virus bearing attenuating mutations or deletions incorporated in the viral genome. It is also possible to create chimeric flaviviruses in which the structural protein genes for the target antigens of a flavivirus are replaced by the corresponding genes of another flavivirus. By combining these molecular techniques, the DNA sequences of DEN4 strain 814669, DEN2 PDK-53 candidate vaccine and YF 17D vaccine have been used as the genetic backbone to construct chimeric flaviviruses with the required attenuation phenotype and expression of the target antigens. Encouraging results from preclinical and clinical studies have shown that several chimeric flavivirus vaccines have the safety profile and satisfactory immunogenicity and protective efficacy to warrant further evaluation in humans. The chimeric flavivirus strategy has led to the rapid development of novel live-attenuated vaccines against dengue, TBE, JE, and West Nile viruses.

Amino Acid Sequence↗
PubMed2002

Complete genome sequence of Montana Myotis leukoencephalitis virus, phylogenetic analysis and comparative study of the 3' untranslated region of flaviviruses with no known vector.

Montana Myotis leukoencephalitis virus (MMLV), a virus isolated from bats, causes an encephalitis in small rodents reminiscent of flavivirus encephalitis in humans. The complete MMLV genome is 10690 nucleotides long and encodes a putative polyprotein of 3374 amino acids. The virus contains the same conserved motifs in genes that are believed to be interesting antiviral targets (NTPase/helicase, serine protease and RNA-dependent RNA polymerase) as flaviviruses of clinical importance. Phylogenetic analysis of the entire coding region has confirmed the classification of MMLV in the clade of the flaviviruses with no known vector (NKV) and within this clade to the Rio Bravo branch (both viruses have the bat as their vertebrate host). We have provided for the first time a comparative analysis of the RNA folding of the 3' UTR of the NKV flaviviruses (Modoc, Rio Bravo and Apoi viruses, in addition to MMLV). Structural elements in the 3' UTR that are preserved among other flaviviruses have been revealed, as well as elements that distinguish the NKV from the mosquito- and tick-borne flaviviruses. In particular, the pentanucleotide sequence 5' CACAG 3', which is conserved in all mosquito- and tick-borne flaviviruses, is replaced by the sequence 5' C(C/U)(C/U)AG 3' in the loop of the 3' long stable hairpin structure of all four NKV flaviviruses. The availability of this latter sequence motif allows us to designate a virus as either an NKV or a vector-borne flavivirus.

3' Untranslated Regions↗
PubMed2004

Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes.

The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231.

Amino Acid Sequence↗
PubMed1998

Immune mediated and inherited defences against flaviviruses.

BACKGROUND: Flavivirus infection elicits an abundant immune response in the host which is directed against a number of the viral proteins. Resistance to flavivirus-induced disease can also be controlled via a non-immune mechanism involving the product of a naturally occurring murine gene, Flv. OBJECTIVES: To review studies that have reported the mapping of epitopes on flavivirus proteins that elicit T- or B-cell immune responses in mice or humans and to discuss a possible mechanism for flavivirus-specific genetic resistance. STUDY DESIGN: Purified viral proteins and synthetic peptides were used to map B-cell epitopes. Purified proteins, vaccinia-expressed viral protein fragments and synthetic peptides were used to map T-cell epitopes. Congenic-resistant, C3H/RV and congenic susceptible, C3H/He mice and cell cultures were used to study the mechanism of genetic resistance to flavivirus infection. RESULTS: T- and B-cell epitopes have been mapped to the E, NS1 and NS3 proteins of several flaviviruses. Immune responses to the C, PreM, NS2a, NS4a, and NS5 proteins have also been documented. Data suggest that the Flv gene product acts intracellularly to suppress the synthesis of viral genomic RNA. CONCLUSIONS: Although flavivirus infection elicits an abundant immune response, this response is not always rapid enough to protect the host from developing encephalitis. During secondary infections both the humoral and cellular flavivirus-specific responses can confer protection. Dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) appear to be caused by an overly vigorous immune response. In genetically resistant animals reduced production of virus results in a slower spread of the infection, which in turn allows time for the immune response to develop and to clear the infection before disease symptoms appear.

Animals↗
PubMed2002

Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA.

Flavivirus infections are a significant public health problem, since several members of the Flaviviridae family are highly pathogenic to humans. Accurate diagnosis and differentiation of the infecting virus is important, especially in areas where many flaviviruses are circulating. In this study we evaluated a newly developed commercially available immunofluorescence assay (IFA) (INDX, Baltimore, MD, USA) for the detection of IgM and IgG antibodies against dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus. IFA was compared with standard diagnostic enzyme immunoassays (EIAs) specific for the detection of IgM and IgG antibodies against these viruses. Forty-seven serum samples from patients with a defined flavivirus infection were tested. As controls, serum samples from individuals with antibodies against tick-borne encephalitis virus and hepatitis C virus as well as healthy individuals were included. The results obtained from this study indicate that IFA showed a significantly better discrimination for flavivirus specific IgM antibodies than did the standard IgM specific EIAs (the overall cross-reactivity varied between 4 and 10% by IFA and 30-44% by EIA for the respective viruses). In contrast, the detection of flavivirus specific IgG antibodies showed high cross-reactions in both IFA and EIAs (overall cross-reactivity 16-71 and 62-84%, respectively). This study clearly stated the complexity of flavivirus diagnosis, showing that one cannot rely on one assay or search for one virus only. The flavivirus IFA is a useful tool for the identification of flavivirus infections during the acute stage of disease. In particular, IFA can be an important diagnostic tool for testing samples from travellers who have been accidentally exposed to these viruses.

Antibodies, Viral↗
PubMed1992

The complete nucleotide sequence of cell fusing agent (CFA): homology between the nonstructural proteins encoded by CFA and the nonstructural proteins encoded by arthropod-borne flaviviruses.

Cell fusing agent (CFA) is an RNA virus originally isolated from a line of Aedes aegypti mosquito cells. Although our characterization of the virus many years ago showed that it resembled the flaviviruses, there was no detectable serological cross-reaction with members of the genus flavivirus. Furthermore, unlike the well-studied members of the genus flavivirus, CFA did not replicate in any of several vertebrate cell lines tested. We have now determined the nucleotide sequence of the CFA genome. Comparison of the predicted amino acid sequence of the CFA polyprotein with viral protein sequences in Genbank, has made it apparent that CFA should now be assigned to the family Flaviviridae, genus flavivirus. The homology between CFA proteins and those of other flaviviruses was highest for NS5 (45%) and NS3 (34%). Little homology was found for the structural proteins. Thus, CFA is only distantly related to the other flaviviruses for which there is sequence information; nevertheless, with respect to their hydrophobicity plots, the CFA polyprotein and the polyproteins of other flaviviruses are remarkably similar. We suggest that CFA is an insect virus, which was present in the embryos from which the Ae. aegypti cell line was established. Thus, CFA seems to be the first member of the family Flaviviridae, genus flavivirus, to be identified as an insect virus.

Amino Acid Sequence↗
PubMed1999

Development and characterization of new flavivirus-resistant mouse strains bearing Flv(r)-like and Flv(mr) alleles from wild or wild-derived mice.

A single genetic locus, flavivirus resistance (Flv), controls virus titres and severity of flavivirus infection in mouse brain. It has been mapped to mouse chromosome 5 and shown to include different allelic forms. While the majority of laboratory mouse strains are susceptible to flaviviruses and carry the Flv(s) allele, wild mice and laboratory mouse strains recently derived from them are resistant and carry flavivirus-resistance alleles including Flv(r)-like and Flv(mr) alleles. Although there is a mouse model of flavivirus resistance conferred by the Flv(r) allele, other resistance alleles have not been adequately studied due to a lack of appropriate animal models. In this paper we describe the development of new flavivirus-resistant mouse strains, C3H.M.domesticus-Flv(r) and C3H.MOLD-Flv(mr), which carry the novel resistance alleles Flv(r)-like and Flv(mr) on the genetic background of flavivirus susceptible C3H/HeJ mice. The new strains were created by 10 to 11 generations of backcrossing followed by brother-sister matings resulting in a generation of homozygous founder stocks. Genome analysis of the newly developed mouse strains has revealed chromosomal regions of approximately 9 and 11 cM, respectively, encompassing Flv on chromosome 5, which are derived from resistant donor mice. These segments are much smaller than the segment of approximately 31 cM described in the congenic resistant mouse strain C3H.PRI-Flv(r) (also known as C3H/RV). The new congenic mouse strains, which were created to carry the Flv(r)-like and Flv(mr) alleles on the standardized genetic background of susceptible mice, represent new animal models of flavivirus resistance conferred by these novel resistance alleles.

Alleles↗
PubMed2005

Differential induction of antiviral effects against West Nile virus in primary mouse macrophages derived from flavivirus-susceptible and congenic resistant mice by alpha/beta interferon and poly(I-C).

A cell model of primary macrophages isolated from the peritoneal cavity of flavivirus-susceptible and congenic resistant mice has been used to study the extent and kinetics of antiviral effects against West Nile virus upon priming with alpha/beta interferon (IFN-alpha/beta) or poly(I-C) (pIC). The pattern of flavivirus resistance expressed after priming of cells in this model was in good agreement with the pattern of flavivirus resistance described in the brains of the corresponding mouse strains. While priming with either IFN-alpha/beta or pIC completely blocked flavivirus replication in macrophages from resistant mice, it only transiently reduced flavivirus replication in macrophages from susceptible mice. It was only the combined pretreatment with IFN-alpha/beta and pIC that elicited strong antiviral responses that completely prevented flavivirus replication in macrophages from susceptible mice. Primary macrophages isolated from the blood of healthy human donors expressed a similar need for double-stranded RNA (dsRNA) cofactor in developing efficient antiviral responses against West Nile virus. These findings reveal that the inefficient IFN-alpha/beta-induced antiviral effects against flaviviruses in cells from susceptible hosts could be successfully complemented by an external dsRNA factor leading to the complete eradication of the virus. This treatment appears to compensate for the lack of an inborn resistance mechanism in cells from the susceptible host. Furthermore, it may also provide useful clues for the prevention and treatment of flavivirus infections.

Animals↗
PubMed2003

Genetic and phenotypic characterization of the newly described insect flavivirus, Kamiti River virus.

We have described in the accompanying paper by Sang, et al., ([57], Arch Virol 2003, in press) the isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in Central Province, Kenya. Among known flaviviruses, KRV was shown to be most similar to, but genetically and phenotypically distinct from, Cell fusing agent virus (CFAV). KRV was provisionally identified as an insect-only flavivirus that fails to replicate in vertebrate cells or in mice. We report here the further characterization of KRV. Growth in cell culture was compared to that of CFAV; although growth kinetics were similar, KRV did not cause the cell fusion that is characteristic of CFAV infection. The KRV genome was found to be 11,375 nucleotides in length, containing a single open reading frame encoding 10 viral proteins. Likely polyprotein cleavage sites were identified, which were most similar to those of CFAV and were comparable to those of other flaviviruses. Sequence identity with other flaviviruses was low; maximum identity was with CFAV. Possible terminal secondary structures for the 5' and 3' non-coding regions (NCR) were similar to those predicted for other flaviviruses. Whereas CFAV was isolated from insect cells in the laboratory, the isolation of KRV demonstrates the presence of an insect-only flavivirus in nature and raises questions regarding potential interactions between this virus and other mosquito-borne viruses in competent vector populations. Additionally, this virus will be an important tool in future studies to determine markers associated with flavivirus host specificity.

Aedes↗