Molecular determinants of virulence of West Nile virus in North America.

West Nile virus (WNV) is a mosquito-borne flavivirus that until very recently had not been found in the Americas. In 1999, there was an outbreak of West Nile encephalitis in New York and surrounding areas, involving 62 human cases, including 7 fatalities. The virus has subsequently become established in the United States of America (U.S.) with 4156 human cases, including 284 deaths, in 2002. The WNV strains found in the U.S. are members of "lineage I", a genetic grouping that includes viruses from Europe, Asia and Africa. Molecular epidemiologic studies indicate that two genetic variants of WNV emerged in 2002. The major genetic variant is found in most parts of the U.S., while the minor genetic variant has been identified only on the southeast coast of Texas. Investigation of WNV in mouse and hamster models demonstrated that strains from the U.S. are highly neurovirulent and neuroinvasive in these laboratory rodents. Other strains, such as Ethiopia 76a from lineage I, are not neuroinvasive and represent important viruses which can be used to elucidate the molecular basis of virulence and attenuation of WNV. To identify putative molecular determinants of virulence and attenuation, we have undertaken comparative nucleotide sequencing of Ethiopia 76a and strains from the U.S. The results show that the two viruses differ by 5 amino acids in the envelope (E) protein, including loss of the glycosylation site. Comparison of our panel of 27 WNV strains suggests that E protein glycosylation is a major determinant of the mouse neuroinvasive phenotype.

Nucleotide sequencing and serological evidence that the recently recognized deer tick virus is a genotype of Powassan virus.

Deer tick virus (DTV) is a recently recognized North American virus isolated from Ixodes dammini ticks. Nucleotide sequencing of fragments of structural and non-structural protein genes suggested that this virus was most closely related to the tick-borne flavivirus Powassan (POW), which causes potentially fatal encephalitis in humans. To determine whether DTV represents a new and distinct member of the Flavivirus genus of the family Flaviviridae, we sequenced the structural protein genes and 5' and 3' non-coding regions of this virus. In addition, we compared the reactivity of DTV and POW in hemagglutination inhibition tests with a panel of polyclonal and monoclonal antisera, and performed cross-neutralization experiments using anti-DTV antisera. Nucleotide sequencing revealed a high degree of homology between DTV and POW at both nucleotide (>80% homology) and amino acid (>90% homology) levels, and the two viruses were indistinguishable in serological assays and mouse neuroinvasiveness. On the basis of these results, we suggest that DTV should be classified as a genotype of POW virus.

Jatobal virus is a reassortant containing the small RNA of Oropouche virus.

Jatobal (JAT) virus was isolated in 1985 from a carnivore (Nasua nasua) in Tucuruí, Pará state, Brazil and was classified as a distinct member of the Simbu serogroup of the Bunyavirus genus, family Bunyaviridae on the basis of neutralization tests. On the basis of nucleotide sequencing, we have found that the small (S) RNA of JAT virus is very similar (>95% identity) to that of Oropouche (ORO) virus, in particular, the Peruvian genotype of ORO virus. In comparison, limited nucleotide sequencing of the G2 protein gene, encoded by the middle (M) RNA, of JAT and ORO viruses, revealed relatively little identity (<66%) between these two viruses. Neutralization tests confirmed the lack of cross-reactivity between the viruses. These results suggest that JAT virus is a reassortant containing the S RNA of ORO virus. JAT virus was attenuated in hamsters compared to ORO virus suggesting that the S RNA of ORO virus is not directly involved in hamster virulence.

Nucleotide sequence and deduced amino acid sequence of the medium RNA segment of Oropouche, a Simbu serogroup virus: comparison with the middle RNA of Bunyamwera and California serogroup viruses.

The Bunyavirus genus of the family Bunyaviridae contains 18 serogroups. To date nucleotide sequence data has been obtained for three serogroups, Bunyamwera, California and Simbu, based on analysis of the small (S) RNA segment. In comparison, there is only nucleotide sequence data for the large and medium (M) RNA segments for members of the Bunyamwera and California serogroups. In this paper we report the nucleotide sequence of the M RNA of Oropouche (ORO) virus, a member of the Simbu serogroup. The M RNA was 4396 nucleotides in length with G1, G2 and NSm proteins similar in size to those reported for members of the Bunyamwera and California serogroups. However, there was limited nucleotide (50-52%) and amino acid (30-32%) homology between ORO virus M RNA and those of published members of the other two serogroups. The Bunyamwera and California serogroups are more closely related to each other than the Simbu serogroup virus Oropouche. These data were consistent with that previously reported for the S RNA (Saeed et al., 2000. J. Gen. Virol. 81, 743-748). It has been noted previously that three of four potential N-linked glycosylation sites of the Bunayamwera and California serogroups are conserved in G1 and G2 proteins. In contrast, ORO virus was found to have only three potential N-linked glycosylation sites of which only one, in G1, was conserved with members of the other two serogroups. Comparison of M RNA sequences of different strains of ORO virus revealed genetic variation consistent with that reported previously for the S RNA.

Genetic variation in the 3' non-coding region of dengue viruses.

The 3' non-coding region (3'NCR) of strains of dengue 1 (DEN 1), DEN 2, DEN 3, and DEN 4 viruses, isolated in different geographical regions, was sequenced and compared to published sequences of the four dengue viruses. A total of 50 DEN 2 strains was compared: 7 West African strains, 3 Indonesian mosquito strains, 1 Indonesian macaque isolate, and 39 human isolates from Southeast Asia, the South Pacific, and the Caribbean and Americas. Nucleotide sequence alignment revealed few deletions and no repeat sequences in the 3' NCR of DEN 2 viruses and showed that much of the 3' NCR was well conserved. The strains could be divided into two groups, sylvatic and human/mosquito/macaque, based on nucleotide sequence homology. A hypervariable region was identified immediately following the NS5 stop codon, which involved a 2-10 nucleotide deletion in human, mosquito, and macaque isolates compared with the sylvatic strains. The DEN 2 3'NCR was also compared with 3'NCR sequences from strains of DEN 1, DEN 3, and DEN 4 viruses. DEN 1 was found to have four copies of an eight nucleotide imperfect repeat following the NS5 stop codon, while DEN 4 virus had a deletion of 75 nucleotides in the 3'NCR. We propose that the variation in nucleotide sequence in the 3'NCR may have evolved as a function of DEN virus transmission and replication in different mosquito and non-human primate/human host cycles. The results from this study are consistent with the hypothesis that DEN viruses arose from sylvatic progenitors and evolved into human epidemic strains. However, the data do not support the hypothesis that variation in the 3'NCR correlates with DEN virus pathogenesis.

Epitopes on the dengue 1 virus envelope protein recognized by neutralizing IgM monoclonal antibodies.

Three of 41 IgM monoclonal antibodies derived from dengue 1 virus immunized mice neutralized dengue 1 infection in vitro. All three neutralizing monoclonal antibodies reacted with spatially related epitopes on the E protein of dengue 1 which were also recognized by antibodies in sera from dengue patients. Two neutralization-resistant populations of dengue 1 virus, D1-M10 and D1-M17, were selected by sequential passage of virus in C6/36 cells in the presence of neutralizing IgM monoclonal antibodies M10 and M17, respectively. Single nucleotide changes occurred in the E protein gene of each of these virus populations resulting in single amino acid substitutions at E279 (Phe-Ser) in D1-M10 and at E293 (Thr-Ile) in D1-M17. Both neutralization-resistant populations of virus were more sensitive to elevated temperature than was the wild-type dengue 1 virus and the infectivity and haemagglutinating ability of the neutralization-resistant populations decreased more slowly than that of wild-type virus when exposed to pH in the range 5.8 to 7.0. These are the first epitopes involved in neutralization to have been identified in dengue 1 virus and the first outside domain III of the E protein on any dengue virus.

Analysis of a recombinant dengue-2 virus-dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus system.

In a step towards a tetravalent dengue virus subunit vaccine which is economical to produce, highly immunogenic and stable, a hybrid dengue virus envelope (E) protein molecule has been constructed. It consists of 36 amino acids from the membrane protein, the N-terminal 288 amino acids of the dengue-2 virus E protein plus amino acids 289-424 of the dengue-3 virus E protein. It has been engineered for secretory expression by fusion to a mellitin secretory signal sequence and truncation of the hydrophobic transmembrane segment. Using the baculovirus expression system and serum-free conditions, more than 95% of recombinant dengue-2 virus-dengue-3 virus hybrid E protein (rD2D3E) was secreted into the cell culture supernatant in a stable form with multiple features indicative of preserved conformation. The hybrid molecule reacted with a panel of dengue virus- and flavivirus-specific MAbs which recognize linear or conformational epitopes on dengue virions. Human dengue virus-specific antisera also reacted with the protein. The hybrid rD2D3E protein was able to inhibit the in vitro binding of dengue-2 and dengue-3 viruses to human myelomonocytic cells, suggesting that the receptor-binding epitope(s) was preserved. Adjuvant-free immunization with the hybrid protein induced an antibody response to both dengue-2 and dengue-3 virus in outbred mice, comparable in strength to that of individual rD2E and rD3E proteins. Notably, these antibody responses were primarily of the IgG2a and IgG2b isotype. A strong dengue virus cross-reactive T cell response was also induced in the mice, suggesting that dengue virus hybrid E proteins could form the basis of an efficacious multivalent dengue virus vaccine.