Isolation of a new flavivirus related to cell fusing agent virus (CFAV) from field-collected flood-water Aedes mosquitoes sampled from a dambo in central Kenya.

Cell fusing agent virus (CFAV) is an RNA insect virus that was isolated from a line of Aedes aegypti mosquito cells and has been assigned to the family Flaviviridae, genus Flavivirus. We report here the first isolation of a CFA-like virus from field-collected mosquitoes. Mosquito larvae and pupae were sampled from flooded dambos in Central Province, Kenya during the short rain season of 1999. Specimens were reared to adults, identified and pooled by species and were tested for the presence of virus. Two virus isolates were obtained from two pools of Aedes macintoshi mosquitoes. The virus isolates replicated only in invertebrate cells in culture and not in vertebrate cells or in mice. The virus isolates did not antigenically cross-react with known arboviruses but were identified to family by reverse-transcriptase polymerase chain reaction (RT-PCR) performed using primers specific to alphaviruses, bunyaviruses and flaviviruses; only the flavivirus-specific primers produced a DNA fragment of the expected size. Nucleic acid sequencing of this fragment showed the two isolates to be nearly identical. Comparison of sequences to the GenBank database using BLAST identified the virus as most closely related to CFAV. Results from cross-neutralization tests suggested that, although the BLAST search indicated homology to CFAV, the virus isolated represented a new insect flavivirus. Detailed characterization of this new virus, described in Crabtree et al. [7], further supports this finding. We propose this new flavivirus be designated Kamiti River virus (KRV). This is the first isolation of a CFA-like virus from field-collected mosquitoes and indicates the presence of this group of viruses in nature.

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.

The growing threat of arbovirus transmission and outbreaks in Kenya: a review.

OBJECTIVE: To review the trend in arbovirus outbreaks and activity in Kenya in the last ten years. DATA SOURCE: Published reports of past outbreak investigations and more recent data available at the Arbovirology and Viral haemorrhagic fevers reference centre, Centre for Virus Research, Nairobi. STUDY SELECTION: Past and recent outbreaks and active transmission reports of arboviruses of medical importance in Kenya including Yellow fever (YF), Rift Valley Fever (RVF), Dengue and Crimean Congo haemorrhagic fever. SYNTHESIS: Each of the viruses was reviewed providing critical information on classification, incidence, outbreak, and activity in Kenya, mode of transmission, recognition of cases, management and control. CONCLUSION: There is increased frequency of outbreaks and detection of arbovirus activity in humans and vectors in the last ten years including re-emergence of YF virus as a public health concern in Kenya. The importance of recognition of cases and diagnosis (especially in malaria endemic areas) is critical to management and control. Effective countrywide surveillance backed by diagnostic centres is highly recommended.

A reassortant bunyavirus isolated from acute hemorrhagic fever cases in Kenya and Somalia.

In late 1997 and early 1998, a large outbreak of hemorrhagic fever occurred in East Africa. Clinical samples were collected in Kenya and southern Somalia, and 27 of 115 (23%) hemorrhagic fever patients tested showed evidence of acute infection with Rift Valley fever (RVF) virus as determined by IgM detection, virus isolation, detection of virus RNA by reverse transcription-polymerase chain reaction (RT-PCR), or immunohistochemistry. However, two patients (one from Kenya and the other from Somalia) whose illness met the hemorrhagic fever case definition yielded virus isolates that were not RVF. Electron microscopy suggested these two virus isolates were members of the family Bunyaviridae. RT-PCR primers were designed to detect bunyavirus RNA in these samples. Regions of the S and L segments of the two isolates were successfully amplified, and their nucleotide sequences exhibited nearly complete identity with Bunyamwera virus, a mosquito-borne virus not previously associated with severe human disease. Unexpectedly, the virus M segment appeared to be reassorted, as the sequences detected exhibited 32-33% nucleotide and 28% amino acid differences relative to the corresponding M segment sequence of Bunyamwera virus. The association of this reassortant bunyavirus, proposed name Garissa virus, with severe disease is supported by the detection of the virus RNA in acute-phase sera taken from 12 additional hemorrhagic fever cases in the region.

Molecular and biological changes associated with HeLa cell attenuation of wild-type yellow fever virus.

Six passages of the mosquito-borne flavivirus yellow fever (YF) wild-type strain Asibi in HeLa cells attenuated the virus for monkeys and newborn mice and resulted in loss of mosquito competence. Attenuation after the passage in HeLa cells was not unique to YF virus strain Asibi as demonstrated by the HeLa passage attenuation of wild-type YF virus strain French viscerotropic virus and YF vaccine virus 17D-204 for newborn mice. In contrast, wild-type strain Dakar 1279 and the French neurotropic vaccine virus remained virulent for newborn mice after six passages in HeLa cells. Thus not all strains of YF virus can be attenuated by passage in HeLa cells. Attenuation of YF virus strains Asibi and French viscerotropic virus was accompanied by alterations in the antigenic and biological properties of the viruses, including changes to envelope protein epitopes. Attenuation for newborn mice was coincidental with the acquisition by the HeLa-passaged viruses of the vaccine-specific envelope protein epitope recognized by monoclonal antibody H5. This suggests that this conformational change may play a role in the attenuation process. Wild-type Dakar 1279, which remained virulent for newborn mice after passage in HeLa cells, retained its wild-type antigenic character. The genome of Asibi HeLa p6 virus differed from wild-type Asibi virus by 29 nucleotides that encoded 10 amino acid substitutions: 5 in the envelope protein, 1 in NS2A, 3 in NS4B, and 1 in NS5. The substitution at NS4B-95 is seen in three different attenuation processes of wild-type YF virus, leading us to speculate that it is involved in the attenuation of virulence of wild-type strain Asibi.

Yellow fever in Kenya: the need for a country-wide surveillance programme.

Since the emergence of yellow fever (YF) as a public health threat in Kenya in 1992-1993, low level transmission of the virus to humans has continued to occur. A programme of YF surveillance has been instrumental in the monitoring of YF activity and has clearly demonstrated an expansion of the zone of virus activity into regions that were not affected in the 1992-1993 epidemic. This is of major concern for the approximately 29 million Kenyans who are unvaccinated and therefore at risk of infection. A revision of the surveillance programme is underway to create a more efficient system of recognition of suspect YF cases, laboratory diagnosis and reporting to the appropriate authorities for action. In addition, a research programme to study YF ecology in Kenya will benefit the surveillance programme, enabling it to target potential 'hotspots' of YF activity. As it may not be possible, for financial reasons, to incorporate YF vaccination into the Kenya Expanded Programme of immunization in the immediate future, the need for continued surveillance to monitor the emergence of YF in Kenya is vital.

Mapping amino acids of the measles virus hemagglutinin responsible for receptor (CD46) downregulation.

We compared the amino acid sequences of groups of receptor (CD46) downregulating and nondownregulating measles virus (MV) hemagglutinins (Hs) and identified seven group-specific differences as candidates for the mediation of the observed differential effects. Using site-directed mutagenesis, we mutated the chosen amino acids of the H of MV-strain WTF (WTF-H), a nondownregulating H, and Introduced the corresponding amino acids of Edmonston-H (Edm-H), a downregulating H. We identified four amino acids, 211G, 243R, 451V, and 481Y, which influenced the downregulative function when introduced into WTF-H. The double mutation 451V and 481Y in WTF-H led to a degree of CD46 downregulation comparable to that of Edm-H. Conversely, introducing amino acids 451E and 481N into Edm-H resulted in a loss of the downregulative function. These results indicate that these amino acids play a decisive role in the H-CD46 interaction.

Receptor (CD46) modulation and complement-mediated lysis of uninfected cells after contact with measles virus-infected cells.

Recently, it has been observed that the infection of human target cells with certain measles virus (MV) strains leads to the downregulation of the major MV receptor CD46. Here we report that CD46 downregulation can be rapidly induced in uninfected cells after surface contact with MV particles or MV-infected cells. Receptor modulation is detectable after 30 min of cocultivation of uninfected cells with MV-infected cells and is complete after 2 to 4 h, a time after which newly synthesized MV hemagglutinin (MV-H) cannot be detected in freshly infected target cells. This contact-mediated receptor modulation is also induced by recombinant MV-H expressed by vaccinia virus and is inhibitable with antibodies against CD46 and MV-H. By titrating the effect with MV Edmonston strain-infected cells, a significant contact-mediated CD46 modulation was detectable up to a ratio of 1 infected to 64 uninfected cells. As a result of CD46 downregulation, an increased susceptibility of uninfected cells for complement-mediated lysis was observed. This phenomenon, however, is MV strain dependent, as observed for the downregulation of CD46 after MV infection. These data suggest that in acute measles or following measles vaccination, uninfected cells might also be destroyed by complement after contacting an MV-infected cell.

Differential downregulation of CD46 by measles virus strains.

Recently, we found that several lymphotropic wild-type isolates of measles virus (MV) did not lead to the downregulation of CD46 following infection. We hypothesized that either the site of virus isolation, e.g., throat swab versus peripheral blood mononuclear cells, or the cell type used for the isolation may exert selective pressure on a mixed population of viruses, resulting in isolates with the differential properties observed. This hypothesis has been tested by simultaneously isolating MV from a throat swab and peripheral blood mononuclear cells from a single patient by cultivation on B95 and Vero cells. We report that neither the source of MV nor the cell type used for isolation directly influenced the capacity for CD46 modulation of these MV isolates.

Measles virus replication in neural cells.

Measles virus gene expression is attenuated in neural cells by mechanisms that affect both viral transcription and translation. Host enzymes that hypermutate viral genes, and those induced by cytokines, may act cooperatively to slow viral replication and to favor persistent measles virus infections in the human central nervous system.

Moesin, and not the murine functional homologue (Crry/p65) of human membrane cofactor protein (CD46), is involved in the entry of measles virus (strain Edmonston) into susceptible murine cell lines.

Membrane cofactor protein (CD46) has been firmly established as the major high affinity receptor for measles virus (MV). In addition, another protein, moesin, has been shown to be linked with the susceptibility of human cells to MV infection. Murine cells are largely resistant to MV infection, although a number of cell types can be productively infected. As murine cells do not express CD46 an additional mechanism for the uptake of MV is likely. Murine cells possess a functional homologue of CD46 (Crry/p65) in addition to murine moesin, which has nucleotide and amino acid homology to human moesin. We report that anti-moesin monoclonal antibodies 119 and 38/87 reduce the number of infectious centres attributed to MV in murine cell lines NS20Y and L929, whereas polyclonal antisera specific for Crry/p65 and CD46 had no effect on MV infection of these cells. We suggest that moesin may be important in the non-CD46-mediated uptake of MV strain Edmonston by susceptible murine cell lines.

Pathogenetic aspects of measles virus infections.

In this review we briefly summarise virological and immunological aspects of acute and persistent measles virus (MV) infections as found in man and further investigated in animal models and tissue culture. Several mechanisms contributing to MV persistence are discussed. Recent findings, especially the analysis of CD46 and moesin as receptor acting molecules for MV, correlate well with the tropism of MV and open new fields for the investigation of MV associated immunosuppression and central nervous system (CNS) complications.

Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains.

Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon.

Physical association of moesin and CD46 as a receptor complex for measles virus.

Recently, two cellular membrane proteins, the membrane cofactor protein CD46 and the membrane-organizing external spike protein, moesin, have been identified to be functionally associated with measles virus (MV) infectivity of cells. We investigated the functional consequences of binding of monoclonal antibodies to both molecules individually and combined on MV attachment, fusion, and plaque formation and the putative direct physical interaction of moesin and CD46. We found that antibodies to moesin or CD46 separately inhibited MV-cell interactions to a high percentage in the plaque test, by approximately 85 and 75%, respectively. The inhibition by combinations of antibodies was additive at low concentrations and complete at high concentrations. This indicates that similar sites of interaction were blocked by steric hindrance. Furthermore, antimoesin antibodies blocked the infection of CD46-negative mouse cell lines with MV. Chemical cross-linking of cell surface proteins indicated the close proximity of CD46 and moesin in the membrane of human cells, and coimmunoprecipitation of moesin with CD46 suggested their physical interaction. Immunohistochemically by electron microscopy, CD46 and moesin were found to be localized at sites of the cellular membrane where MV particles adsorbed. These data support a model of direct interaction of CD46 and moesin in the cellular membrane and suggest that this complex is functionally involved in the uptake of MV into cells.

Measles virus-induced down-regulation of CD46 is associated with enhanced sensitivity to complement-mediated lysis of infected cells.

CD46, the major component of the measles virus (MV) receptor complex and a member of the regulators of complement activity (RCA) gene cluster, is down-regulated in MV-infected cells. We investigated whether the reduction of surface CD46 correlates with enhanced sensitivity of lymphoid and monocytic cells to lysis by activated complement. On human U937 cells, acutely or persistently infected with MV-Edmonston (ED) vaccine strain, infection-dependent down-regulation of CD46 confers sensitivity to activated complement, regardless of the pathway of activation and the specificity of the activating antibodies. Interestingly, down-regulation of CD46 alone is sufficient to confer susceptibility of cells to complement lysis despite the continued surface expression of other RCA proteins such as CD35 and CD55. In primary cultures, both peripheral blood lymphocytes and macrophages are efficiently lysed in the presence of complement activated via the alternative pathway after MV infection. In contrast to the MV-ED infection, infection of cells with the lymphotropic MV wild-type strain WTF does not down-regulate CD46. Cells infected with MV-WTF do not exhibit enhanced susceptibility to complement lysis. These data suggest that MV strains similar to WTF that do not down-regulate CD46 may have an enhanced potential for replication and dissemination within the human host, whereas complement-mediated elimination of cells infected with CD46-down-regulating strains of MV, such as ED, may limit the spread of MV infection, and could thus represent an attenuating factor for MV.

Moesin: a cell membrane protein linked with susceptibility to measles virus infection.

Measles virus is a highly contagious virus causing acute and persistent diseases in man, the receptor of which is still not well characterized. We have isolated a monoclonal antibody (mAb), designated mAb 119, which specifically inhibits measles virus infection of susceptible cell lines in a dose-dependent manner. This antibody precipitates a protein with an apparent molecular mass of 75 kDa from 125I surface-labeled cells and its epitope is present on human peripheral blood mononuclear cells, human cell lines, and the African green monkey cell line Vero. Affinity chromatography of detergent-solubilized cell membrane proteins over a Sepharose column with covalently bound mAb 119 led to the partial purification of the 75-kDa protein. Preincubation of measles virus with this affinity-purified protein inhibited measles virus infection dose dependently. Amino acid microsequencing of this protein revealed its identity with the human membrane-organizing extension spike protein moesin, a protein intra- and extracellularly associated with the plasma membrane of cells. Subsequently, an antibody raised against purified moesin (mAb 38/87) was also found to specifically inhibit measles virus infection of susceptible cells and confirmed our data obtained with mAb 119. Our data suggest that moesin is acting as a receptor for measles virus.

Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.

Yellow fever 17DD vaccine virus is temperature sensitive when grown in mosquito C6-36 cells.

Twenty-three yellow fever (YF) vaccine viruses and three wild-type YF viruses were propagated independently in human adenocarcinoma (SW13) cells and mosquito Aedes albopictus C6-36 cells. The three YF 17DD vaccine viruses were found to be slightly temperature sensitive (ts) at 39.5 degrees C versus 37.0 degrees C (efficiency of plaquing 0.04 to 0.1) following propagation in C6-36 but not in SW13 cells. A plaque-purified preparation of the 17DD vaccine manufactured in Brazil was ts when grown in C6-36 cells and remained ts when passaged back into the SW13 cell line.

Primer extension dideoxy chain termination nucleotide sequencing of partially purified RNA virus genomes: a technique for investigating low titre viruses with extensive genome secondary structure.

A modified version of the primer extension dideoxy chain termination nucleotide sequencing technique (Sanger et al., 1977) is described. This method has advantages over existing molecular cloning and primer extension techniques in that it allows the genome of RNA viruses to be directly sequenced from partially purified RNA preparations. Thus, viruses growing at unacceptably low titres in tissue culture can now be partially purified from infected mouse brain and sequenced. The technique also incorporates steps for the denaturation of secondary structure which has previously provided difficulties for primer extension sequencing.