Venezuelan equine encephalomyelitis (VEE) viruses are taxonomically classified within the genus Alphavirus of the family Togaviridae (formerly the Group A arboviruses). The VEE complex of viruses includes six antigenic subtypes (I–VI) divided by antigenic variants. Within subtype I there are five antigenic variants (variants AB–F). Originally, subtypes I-A and I-B were considered to be distinct variants, but they are now considered to be identical (I-AB). Antigenic variants I-AB and I-C are associated with epizootic/epidemic activity in equids and humans. The other three variants of subtype I (I-D, I-E, and I-F) and the other five subtypes of VEE (II–VI) circulate in natural enzootic cycles.

Venezuelan equine encephalitis.

Heparan sulfate (HS) proteoglycans (PGs) are ubiquitously expressed on cell surfaces and in the extracellular matrix of most animal tissues, having essential functions in development and homeostasis, as well as playing various roles in disease processes. The functions of HSPGs are mainly dependent on interactions between the HS-side chains with a variety of proteins including cytokines, growth factors, and their receptors. In a given HS polysaccharide, negatively charged sulfate and carboxylate groups are arranged in various types of domains, generated through strictly regulated biosynthetic reactions and with enormous potential for structural variability. The mode of HS-protein interactions is assessed through binding experiments using saccharides of defined composition in vitro, signaling assays in cell models where HS structures are manipulated, and targeted disruption of genes for biosynthetic enzymes in animals (mouse, zebrafish, Drosophila, and Caenorhabditis elegans) followed by phenotype analysis. Whereas some protein ligands appear to require strictly defined HS structure, others bind to variable saccharide domains without apparent dependence on distinct saccharide sequence. These findings raise intriguing questions concerning the functional significance of regulation in HS biosynthesis and the potential for development of therapeutics targeting HS-protein interactions.

Heparan Sulfate: Biosynthesis, Structure, and Function.

Sixty-two new Japanese encephalitis virus (JEV) isolates were obtained from mosquitoes, biting midges, human cerebrospinal fluid and human blood samples in China during 2002-2005. The E and prM genes were sequenced and phylogenetic analyses were performed with 38 JEV other isolates from China and 36 JEV strains from other countries. Phylogenetic trees based on the E and prM gene sequences were similar. The results indicate that: (i) recent JEV isolates from China are divided into two genotypes, genotype 1 and genotype 3; (ii) recent JEV isolates from China are grouped into the same clusters within genotypes 1 and 3; and (iii) genotype 1 JEV strains have been isolated in China since 1979, whilst genotype 3 JEV strains were isolated before the 1970s. The results suggest that genotype 1 JEV was introduced to China around 1979 and that JEV strains belonging to genotypes 1 and 3 circulate in China.

Molecular epidemiological analysis of Japanese encephalitis virus in China

Understanding the alphaviruses: recent research on important emerging pathogens and progress towards their control.

Japanese encephalitis (JE) is a seasonal epidemic disease with a 50-year recorded history in China. Its characteristics can be summarized as follows: (i) it is a seasonal epidemic disease; approximately 90% of cases are recorded in July, August, and September each year. The peak of JE onset is 1 month earlier in South China than in the north of the country; (ii) the disease is highly sporadic. It is rare for more than two cases to appear simultaneously in one family; (iii) most affected children are under 15 years old; (iv) the disease is widely distributed in all areas of the nation except Qinghai Province, Xinjiang Uygur Autonomous, and Tibet. Due to widespread application of the JE vaccine, the number of JE cases has decreased significantly nationwide, from 174,932 cases of morbidity in 1971 to 5,097 cases in 2005.

Japanese encephalitis in mainland china.

Cell culture-adapted strains of Sindbis virus (SINV) initially attach to cells by the ability to interact with heparan sulfate (HS) through selective mutation for positively charged amino acid (aa) scattered in E2 glycoprotein (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72: 7357-7366, 1998). Here we have further confirmed that interaction of E2 protein with HS is crucial for cellular infection of SINV based on the reverse genetic system of XJ-160 virus, a Sindbis-like virus (SINLV). Both SINV YN87448 and SINLV XJ-160 displayed similar infectivity on BHK-21, Vero, or C6/36 cells, but XJ-160 failed to infect mouse embryonic fibroblast (MEF) cells. The molecular mechanisms underlying the selective infectivity of XJ-160 were approached by substituting the E1, E2, or both genes of XJ-160 with that of YN87448, and the chimeric virus was denominated as XJ-160/E1, XJ-160/E2, or XJ-160/E1E2, respectively. In contrast to the parental XJ-160, all chimeric viruses became infectious to wild-type MEF cells (MEF-wt). While MEF-Ext(-/-) cells, producing shortened HS chains, were resistant not only to XJ-160, but also to YN87448 as well as the chimeric viruses, indicating that the inability of XJ-160 to infect MEF-wt cells likely due to its incompetent discrimination of cellular HS. Treatment with heparin or HS-degrading enzyme resulted in a substantial decrease in plaque formation by YN87448, XJ-160/E2, and XJ-160/E1E2, but had marginal effect on XJ-160 and XJ-160/E1, suggesting that E2 glycoprotein from YN87448 plays a more important role than does E1 in mediating cellular HS-related cell infection. In addition, the peptide containing 145-150 aa from E2 gene of YN87448 specifically bound to heparin, while the corresponding peptide from the E2 gene of XJ-160 essentially showed no binding to heparin. As a new dataset, these results clearly confirm an essential role of E2 glycoprotein, especially the domain of 145-150 aa, in SINV cellular infection through the interaction with HS.

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Interaction of E2 Glycoprotein with Heparan Sulfate Is Crucial for Cellular Infection of Sindbis Virus

An infectious clone (pBR-XJ160) was constructed using the full-length cDNA of the Sindbis-like XJ-160 virus. Two nucleotide mutations, causing amino acid changes at residue 169 from Lys to Arg and at residue 173 from Thr to Ile in the nonstructural protein (nsP) 1 coding region, strongly influenced the infectivity of in vitro-synthesized RNA. We used site-directed mutagenesis to obtain clones encoding a change to Arg at residue 169 of nsP1 (pBR-169), a change to Ile at residue 173 (pBR-173), or both changes (pBR-6973). Infectivity of RNA from pBR-169 was abolished, but viral forms BR-173 and BR-6973 were obtained from pBR-173 and pBR-6973, respectively. Further, BR-173 exhibited higher propagation than BR-XJ160 in cell culture and higher neurovirulence in a suckling mouse model. BR-6973 possessed an intermediate phenotype. BR-173 and BR-6973 showed increased sensitivity to 3-deazaadenosine (3-DZA), which inhibits S-adenosylhomocysteine hydrolase. Thus, mutagenesis at residue 169 in the nsP1 region of XJ-160 is lethal, but mutation at residue 173 from Thr to Ile enhances viral infectivity and neurovirulence and suppresses the lethal effect of the mutation at residue 169. These mutations might be associated with the RNA methyltransferase (MTase) activity of nsP1.

Substitutions of 169Lys and 173Thr in nonstructural protein 1 influence the infectivity and pathogenicity of XJ-160 virus

OBJECTIVE To analyze the genotype of newly isolated Japanese encephalitis viruses in Fujian province and the characteristics of amino acid sequence in the E gene. METHODS PrM and E segments of newly isolated Japanese encephalitis viruses were amplified by RT-PCR, the PCR products were cloned into T vector for sequencing. Phylogenetic analysis was carried out by PHYLIP program. RESULTS Newly isolated Japanese encephalitis viruses belonged to genotype III, the nucleotide and amino acid of E gene showed high homology to vaccine strain SA14-14-2, but had some difference in some domains. CONCLUSION Newly isolated viruses in Fujian province belonged to Japanese encephalitis virus genotype III. E protein of the isolates showed some differences as compared with vaccine strains.

Molecular characteristics of three new isolates of Japanese encephalitis virus in Fujian Province

The purpose of this study is to elucidate the molecular mechanism of one-way serological reaction between XJ-160 virus and SINV by recombinant viruses which exchanged the glycoprotein genes individually or simultaneously. Three recombinant viruses were obtained based on the whole-length infectious cDNA clone of XJ-160 virus. The infectivity and pathogenesis to BHK-21 cells and animals were studied and the gene which controlled this one-way serological reaction phenomenon was searched by MCPENT. The results showed that the E2 glycoprotein was the main factor which influenced the growth rate, plaque morphology and pathogenicity of BHK-21 cells and suckling mice. The results of MCPENT showed that the E2 glycoprotein of SINV played a major role in this one-way serological reaction phenomenon. Our study identified the SINE2 gene was the determined gene for one way serological reaction between XJ-160 virus and SINV, and this research laid the foundation for further analysis of the genomic structure and function of SINV.

Yi-liang Yang

Papers

Interaction of E2 Glycoprotein with Heparan Sulfate Is Crucial for Cellular Infection of Sindbis Virus

Substitutions of 169Lys and 173Thr in nonstructural protein 1 influence the infectivity and pathogenicity of XJ-160 virus

Molecular characteristics of three new isolates of Japanese encephalitis virus in Fujian Province

[Molecular basis of one-way serological reaction between SINV and XJ-160 virus].