Recognition of heparan sulfate by clinical strains of dengue virus serotype 1 using recombinant subviral particles.

Dengue is the most important arthropod-borne viral disease in humans, with an estimated 3.6 billion people at risk for infection and more than 200 million infections per year. Identification of the cellular receptors for dengue virus (DV), the causative agent of dengue, is important toward understanding the pathogenesis of the disease. Heparan sulfate (HS) has been characterized as a DV receptor in multiple model systems, however the physiological relevance of these findings has been questioned by observations that flaviviruses, including DV, can undergo cell culture adaptation changes resulting in increased binding to HS. It thus remains unclear whether HS is utilized by clinical, non-cell culture-adapted strains of DV. To address this question, herein we describe a set of methodologies using recombinant subviral particles (RSPs) to determine the utilization of HS by clinical strains of DV serotype 1 (DV1). RSPs of clinically isolated strains with low cell culture passage histories were used to study HS interaction. Biochemically characterized RSPs showed dose-dependent binding to immobilized heparin, which could be competed by heparin and HS but not structurally related glycosaminoglycans chondroitin sulfate A and hyaluronic acid. The relevance of heparin and HS biochemical interactions was demonstrated by competition of RSP and DV binding to cells with soluble heparin and HS. Our results demonstrate that clinical strains of DV1 can specifically interact with heparin and HS. Together, these data support the possibility that HS on cell surfaces is utilized in the DV-human infection process.

Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins.

Classical swine fever virus (CSFV) harbors three envelope glycoproteins (E(rns), E1 and E2). Previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic CSFV mutants. Here we analyzed the effects of lack of glycosylation of baculovirus-expressed E(rns), E1, and E2 proteins on immunogenicity. Interestingly, E(rns), E1, and E2 proteins lacking proper post-translational modifications, most noticeable lack of glycosylation, failed to induce a detectable virus neutralizing antibody (NA) response and protection against CSFV. Similarly, no NA or protection was observed in pigs immunized with E1 glycoprotein. Analysis of E(rns) and E2 proteins with single site glycosylation mutations revealed that detectable antibody responses, but not protection against lethal CSFV challenge is affected by removal of specific glycosylation sites. In addition, it was observed that single administration of purified E(rns) glycoprotein induced an effective protection against CSFV infection.

Identification of an NTPase motif in classical swine fever virus NS4B protein.

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive-sense single-stranded RNA virus within the Pestivirus genus of the Flaviviridae family. Here, we have identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV non-structural (NS) protein NS4B. Expressed NS4B protein hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NS4B NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. Similar mutations introduced into the genetic backbone of a full-length cDNA copy of CSFV strain Brescia rendered no infectious viruses or viruses with impaired replication capabilities, suggesting that this NTPase activity is critical for the CSFV cycle. Recovered mutant viruses retained a virulent phenotype, as parental strain Brescia, in infected swine. These results have important implications for developing novel antiviral strategies against CSFV infection.

Diversified reassortant H9N2 avian influenza viruses in chicken flocks in northern and eastern China.

According to our previous study of the M genes of H9N2 avian influenza viruses (AIV) in infected chickens, A/Quail/Hong Kong/G1/97 (G1 97)-like M genes newly emerged in northern and eastern China in addition to the existing A/chicken/Hong Kong/Y280/97 (Y280)-like lineage M genes. To systematically track the genesis and evolution of H9N2 viruses in this region, whole genome sequences of seventeen H9N2 isolates were obtained and their phylogenetic properties were determined. Phylogenetic analysis revealed several newly emerged lineages of gene segments in addition to the Y280-like and A/chicken/Shanghai/F/98(F 98)-like lineages, which are prevailing in northern and eastern China according to previous reports. Reassortments among these gene segments generated five novel genotypes of H9N2 viruses that have not been reported before in China. The emerging genotypes of H9N2 viruses in this region indicate that H9N2 virus genes undergo active evolution, particularly their internal genes, which raises concern for their likely contribution to gene reassortment and production of AIVs with new properties. Our study provides valuable insight into the prevalence of H9N2 viruses in northern and eastern China and demonstrates the need of long-term monitoring of the evolution of H9N2 AIV.