Joel M. Dalrymple

Antigenic Relationships between Flaviviruses as Determined by Cross-neutralization Tests with Polyclonal Antisera

The recently established virus family Flaviviridae contains at least 68 recognized members. Sixty-six of these viruses were tested by cross-neutralization in cell cultures. Flaviviruses were separated into eight complexes [tick-borne encephalitis (12 viruses), Rio Bravo (six), Japanese encephalitis (10), Tyuleniy (three), Ntaya (five), Uganda S (four), dengue (four) and Modoc (five)] containing 49 viruses; 17 other viruses were not sufficiently related to warrant inclusion in any of these complexes.

Coding strategy of the S genome segment of Hantaan virus

Hantaan virus is the type species of the recently recognized Hantavirus genus of Bunyaviridae. The small (S) RNA segment of the negative-sense, tripartite genome was molecularly cloned and the nucleotide sequence was determined. The RNA sequence derived from the cDNA copy was found to contain 1696 nucleotides. A single open reading frame of sufficient size to encode the virus nucleocapsid protein was detected in the cDNA corresponding to viral complementary-sense RNA. RNA transcripts of the cDNA were synthesized with SP6 polymerase and were used to program cell-free reticulocyte lysate translation systems. Viral complementary-sense transcripts served as efficient messages in translation systems and generated Hantaan nucleocapsid protein. No translation products were detected when lysates were programmed with viral-sense transcripts. This coding assignment of the nucleocapsid protein to the viral complementary-sense RNA of the S genome segment is consistent with those of other members of this family. Unlike other Bunyaviridae, which encode both a nucleocapsid protein and a nonstructural (NSs) protein of similar sizes, a NSs protein has not been identified for Hantaan virus. Furthermore, other than the nucleocapsid protein gene sequence, the only potential open reading frame in Hantaan S RNA encoded a short, 48-amino acid polypeptide which initiated two codons beyond the termination of the nucleocapsid protein in the same reading frame. These data demonstrate that the coding strategy of the Hantaan virus S RNA is different than those reported for other viruses in this family.

Characterization of Hantaan virus envelope glycoprotein antigenic determinants defined by monoclonal antibodies.

A panel of 24 monoclonal antibodies (MAbs) to the G1 or G2 envelope glycoproteins of Hantaan virus were used to determine the surface topography and functional properties of antigenic sites. Nine distinct, partially overlapping antigenic sites, two on G1 and seven on G2, were demonstrated by competitive binding assays. Analyses of the antigenic sites by haemagglutination (HA) inhibition and plaque-reduction neutralization tests showed that all of the sites, except one on G1, were related to viral HA. Only one of the G1 antigenic sites and two of the G2 sites were involved in virus neutralization. These results suggest that certain epitopes related to HA were not critical for virus neutralization. The nine antigenic sites could be further divided into 13 based upon the serological cross-reactivity of MAbs with viruses representative of each of the four known antigenic groups within the Hantavirus genus of Bunyaviridae, i.e. Hantaan, Seoul, Puumala and Prospect Hill viruses.

Baculovirus expression of the M genome segment of Rift Valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins.

Autographa californica nuclear polyhedrosis viral recombinants containing coding information for the Rift Valley fever virus (RVFV) envelope glycoproteins (G1 and G2) and varying amounts of preglycoprotein coding sequences were prepared by using transfer vectors pAc373 or pAcYM1. Expression products were processed to yield proteins indistinguishable from authentic G1 and G2 by gel electrophoresis. The immunogenic properties of the expressed proteins were assessed by immunizing mice and challenging with RVFV. A single inoculation with lysates of cells infected with recombinants expressing both G1 and G2 induced neutralizing antibody responses in mice and protected them from an otherwise lethal challenge with RVFV. Lysates of cells infected with a recombinant expressing only G2 also induced a protective response after two immunizations. Survivors displayed elevated antibody titers to G1 and G2 and also developed antibodies to the RVFV nucleocapsid protein, the latter allowing discrimination from vaccinated mice and indicating that animals had survived infection. Nonimmune mice were protected from lethal RVFV infection by passive transfer of sera from animals immunized with recombinant antigens, indicating that a humoral immune response is sufficient to protect against RVFV.

Preparation of candidate vaccinia-vectored vaccines for haemorrhagic fever with renal syndrome

Two vaccinia-vectored candidate vaccines for haemorrhagic fever with renal syndrome were prepared by inserting cDNA, representing the medium (M) genome segment, or the M and the small (S) genome segments of Hantaan virus into the thymidine kinase gene of the Connaught vaccine strain of vaccinia virus. In the single recombinant, the M segment was placed under control of the vaccinia virus 7.5 kDa promoter. In the double recombinant, the M and S segments were placed under control of the vaccinia virus 7.5 kDa and 11 kDa promoters, respectively. An immunoplaque assay technique was developed to select recombinants without the need for expression of irrelevant genes or use of potential mutagens. Proteins indistinguishable from authentic viral envelope glycoproteins and nucleocapsid protein were observed by immunoprecipitation with antibodies to Hantaan virus. The recombinant expressing both the M and the S segments was selected for further development and testing as a human vaccine.

Comparison of hantavirus isolates using a genus-reactive primer pair polymerase chain reaction

RNA of more than 40 hantavirus isolates, originating from rodents and humans of widely separated geographical areas, was copied to cDNA using reverse transcriptase and amplified by polymerase chain reaction (PCR). A genus-reactive oligonucleotide primer pair, flanking a 365 bp region of the G2 glycoprotein gene, was chosen for genus-reactive PCR. DNA products were digested with 20 restriction endonucleases and cleavage patterns were analysed. For strains of known sequence, the restriction patterns observed were consistent with those predicted from sequence data, demonstrating that the amplified products originated from target virus RNA. Further analyses suggested that all amplified viruses could be easily typed into one of five restriction patterns using only five enzymes. The categories identified by restriction analysis of PCR-amplified cDNA corresponded with serogroups established by plaque-reduction neutralization tests. This method may greatly simplify the identification of new hantavirus isolates.

Prophylactic ribavirin treatment of dengue type 1 infection in rhesus monkeys.

The prophylactic efficacy of the broad-spectrum antiviral nucleoside analog ribavirin against flavivirus infection in non-human primates was investigated in a blinded, placebo-controlled study of rhesus monkeys infected with dengue virus. Both placebo- and ribavirin-treated monkeys developed viremia, as measured by direct plaque assay on Aedes albopictus C6/36 cells. Peak viremia occurred between days 3 and 9 after infection. No significant differences in time of onset, duration, or level of viremia were observed between placebo- and ribavirin-treated monkeys. Ribavirin induced predictable and reversible anemia and thrombocytosis. Serum ribavirin reached maximum levels of 30 microM by day 4, which approximates the in vitro minimum inhibitory concentration for dengue virus. Ribavirin appeared ineffective as a prophylactic drug for dengue type 1 viral infection, as evaluated by the magnitude of viremia in this monkey model.

Hantaan Virus Replication: Effects of Monensin, Tunicamycin and Endoglycosidases on the Structural Glycoproteins

The monovalent ionophore monensin, which interferes with cellular transport pathways, and the antibiotic tunicamycin, which prevents glycosylation of newly synthesized proteins, were used to examine Hantaan virus particle formation and polypeptide synthesis. Viral replication in the presence of either drug resulted in reduced antigen production as well as reduced yields of both intracellular and extracellular infectious virus. Analysis of viral polypeptides synthesized in the presence of the drugs suggested differential effects of monensin and tunicamycin on Hantaan virus. Although reduced levels of the three major structural proteins were detected with increasing concentrations of monensin, the electrophoretic migrations of the polypeptides synthesized were unaltered. In contrast, after tunicamycin treatment, G1 was no longer detectable and G2 displayed both a quantitative reduction and an apparent molecular weight reduction of approximately 3000. Both G1 and G2 were sensitive to endoglycosidases H and F with resultant electrophoretic mobility shifts corresponding to molecular weights of approximately 7000 for G1 and 3000 for G2. Oligosaccharides appeared to be mostly, but not entirely, of the high-mannose type.

Conservation of Antigenic Properties and Sequences Encoding the Envelope Proteins of Prototype Hantaan Virus and Two Virus Isolates from Korean Haemorrhagic Fever Patients

Viruses isolated from the blood of two Korean haemorrhagic fever patients were propagated in cell culture and compared to prototype Hantaan virus which was isolated from Apodemus mice. The antigenic properties of the human isolates were found to be closely related to Hantaan virus by plaque reduction neutralization, haemagglutination inhibition and fluorescent antibody staining with both polyclonal and monoclonal antibodies. The medium genome segment of each human isolate was sequenced and compared to that of Hantaan virus. Nucleotides comprising the Hantaan virus G1 and G2 envelope protein-coding regions differed from those of the other viruses by only 5.4% and 5.7%. The human isolates differed from one another by 1.6%. The nucleotide differences resulted in predicted amino acid variations of 1.3% to 2.3% among the three viruses, with the majority occurring as conservative substitutions in G1.

BIOCHEMICAL CHARACTERIZATION OF HANTAAN VIRUS

Publisher Summary Hantaan virus is the prototype of a group of viruses implicated as the etiologic agents of clinically similar diseases, which have collectively been termed as hemorrhagic fever with renal syndrome (HFRS). Three well recognized diseases in the syndrome include Korean hemorrhagic fever, nephropathia epidemica, and epidemic hemorrhagic fever, various forms of which are endemic throughout Korea, Scandinavia, Europe, and China. The physical characteristics of Hantaan virions as well as a tripartite, single stranded RNA genome and virion associated polymerase , were supportive of the previous morphological observations that Hantaan virus was similar to the Bunyaviridae. This chapter describes an experiment that compares the RNAs of Hantaan virus to those of other serologically related viruses to confirm that Hantaan virus is the representative of a group of viruses that are similar to but unique from other Bunyaviridae. In the experiment, three common RNA denaturants, glyoxal, methyl-mercury, and acid urea, in conjunction with agarose gel electrophoresis, were used to compare the RNAs of Hantaan virus strain 76–118 and two of the serologically related rodent viruses.