Dengue type-2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge.

The gene coding for the envelope (E) glycoprotein of dengue-2 virus was cloned into baculovirus (Autographa californica nuclear polyhedrosis virus). The recombinant virus contained the entire E protein gene, preceded by 38 nucleotides from the end of the prematrix glycoprotein gene and followed by the first 83 nucleotides of nonstructural protein 1. When expressed in Spodoptera frugiperda (Sf9) cells, approximately 1 mg of recombinant E antigen was made per 10(9) cells. This antigen reacted with polyclonal, anti-dengue type-2 antibody and a dengue type-2-specific, neutralizing monoclonal antibody. BALB/c mice immunized with the recombinant antigen produced only non-neutralizing antibody against dengue-2 virus, but were partially protected against morbidity and mortality after intracranial challenge with virulent dengue-2 virus.

Purification of native dengue-2 viral proteins and the ability of purified proteins to protect mice.

Both the envelope structural protein and the non-structural NS1 protein have been purified from the flavivirus dengue-2 by high-pressure liquid chromatography. These purified proteins maintain their reactivity with monoclonal antibodies. When tested in mice, the envelope protein elicited neutralizing antibodies and partially protected the animals against a lethal viral challenge. The mice responded to the non-structural protein by producing antibodies; however, these antibodies were not neutralizing and the mice were not protected.

Dengue-1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicits virus-neutralizing antibody in mice and protects them from virus challenge.

In order to test the feasibility of baculovirus (Autographa californica nuclear polyhedrosis virus, AcNPV) expression vectors for making immunogens against dengue-1 (DEN-1) virus, a portion of the envelope (E) glycoprotein gene of DEN-1 virus was cloned and expressed. The recombinant baculovirus contains 107 nucleotides from the 3' terminus of the DEN-1 matrix (M) gene, which encodes a hydrophobic signal peptide and extends through the first 1, 245 nucleotides of E, terminating 243 nucleotides before the 3' terminus of E. When the recombinant virus was grown in Spodoptera frugiperda cells, about 1 mg of E antigen was made per 10(9) cells. Recombinant E antigen reacted with E protein-specific monoclonal antibodies and stimulated production of DEN-1 virus neutralizing antibody in BALB/c mice. Mice immunized with recombinant E antigen or with heat-inactivated DEN-1 virus were protected significantly against lethal DEN-1 virus challenge. A dose/response effect was observed, with increasing amounts of recombinant antigen leading to increased survival. These results demonstrate the utility of baculovirus for producing immunogens against DEN-1 virus.

Protection of mice against lethal Japanese encephalitis with a recombinant baculovirus vaccine.

Genes coding for the E and NS1 glycoproteins of Japanese encephalitis virus (JEV) were cloned into baculovirus expression vectors. The recombinant baculoviruses obtained were used to infect Spodoptera frugiperda cells. The infected cells were used to immunize C57/B mice, which were then challenged with live JEV. Survival was increased from about 30% in unimmunized mice to 70% in E and polyprotein recipients (P less than 0.005), but was not increased in NS1 recipients despite the development of antibody against NS1 by these mice. Virus neutralizing antibody was demonstrated in 18/20 E glycoprotein recipients and 15/20 polyprotein recipients. The baculovirus expressed E glycoprotein stimulated antibody which was protective and neutralizing in this system.

Detection of dengue virus RNA using nucleic acid hybridization.

Conditions for using slot-blot nucleic acid hybridization to quantitatively detect dengue-2 virus using a radiolabelled cDNA probe, pVV17, were determined. As little as 11 plaque-forming units of virus were detected using a hybridization mixture without formamide and performing the test at 70 degrees C. While predominantly serotype-specific using stringent (65 degrees C) washing conditions, the probe detected all four dengue virus serotypes using astringent (28 degrees C) washing conditions. No significant qualitative differences were detected using Thai dengue-2 viruses isolated over a 10-year period. High titered, anti-flavivirus antibodies blocked virus detection by an antigen capture, enzyme-linked, immunosorbent assay or by intrathoracic inoculation of Toxorhyncites mosquitoes, but not by nucleic acid hybridization. The appearance of virus-specified RNA coincided with the detection of antigen in infected C6/36 (Aedes albopictus) cells by immunofluorescence, or in cell culture supernatants by the antigen capture method. The method has potential as a diagnostic tool for identifying dengue viruses in clinical and field specimens.

Hepatitis A virus in stool during clinical relapse.

Among 256 patients with acute hepatitis A, 17 (6.6%) had a relapse of the disease between 30 and 90 days after the primary episode. We studied 7 of these patients. Serologic testing showed mean alanine aminotransferase levels of 1668 IU/L during the acute stage, 107 IU/L during the early convalescence, and 1027 IU/L during the relapse. Tests for IgM antibody against hepatitis A virus were positive in the 7 patients at the onset of disease, with decreasing levels in 3 of the 4 patients tested during the evolution of the illness. Stools collected during the relapse phase showed hepatitis A virus by immune electron microscopy, radioimmunoassay, and molecular hybridization using a 32P-labeled cDNA-hepatitis A virus probe. Stools collected from 4 of these patients 6 to 12 months after the onset of disease were negative for the virus. The finding of hepatitis A virus in the stool of these patients during the relapse phase strongly implicates hepatitis A virus as the causative agent of the clinical relapse.

Partial sequence analysis of cloned dengue virus type 2 genome.

Dengue virus (DEN) is a member of flaviviruses and contains a single, (+)-strand RNA of approx. 11 kb. Complementary DNA copy of the RNA was synthesized using reverse transcriptase and oligo(dT) as primer. The double-stranded DNA copy was cloned at the PstI site of pUC13'-1 vector and was used to transform Escherichia coli JM83. Eleven transfomants were found to contain DEN insert as screened by colony hybridization. Three clones were chosen for further characterization by nucleotide (nt), sequence analysis. Two of these clones overlapped by 470 bp. Sequences of these three clones totalling about 4.6 kb were obtained. Translation of this DNA in all possible reading frames revealed the presence of long open reading frames spanning the entire length of the cDNA clones. The putative polypeptides derived from the nt sequence are 885 and 643 amino acids in length and show homology to the region of polyprotein coded by the yellow fever virus genome corresponding to the non-structural proteins [Rice et al., Science 229 (1985) 726-733]. The significant homology between these two viruses in the regions coding for the non-structural proteins NS3 and NS5 suggests an important role for these two proteins in the life cycle of these viruses.

Studies of the Epstein-Barr virus receptor found on Raji cells. I. Extraction of receptor and preparation of anti-receptor antibody.

Raji, a human lymphoblastoid cell line, expresses a membrane receptor (EBVR) specific for Epstein Barr virus (EBV). A component that binds EBV was extracted from this cell line by treatment of the cells for 3 hr on ice with Tris buffer containing 10% glycerol. The treatment reduced the capacity of the cells to bind virus, and after concentration the receptor extract (RE) inhibited both EBV binding and superinfection of fresh Raji cells. Similarly prepared extracts of EBVR- cells lacked such activity. An antibody was made to the extract (anti-RE), which after absorption with EBVR- cells, bound to the same percentages of EBVR+ lymphoblastoid cell lines, EBVR+ human/mouse somatic cell hybrids, and fresh peripheral B cells as the virus did. In reciprocal assays, preincubation of EBVR+ cells with anti-RE inhibited virus binding. Doubly stained patches were observed on membranes of EBVR+ cells that had been incubated simultaneously with virus and anti-RE and stained respectively with rhodaminated and fluoresceinated reagents. The major polypeptide immunoprecipitated by anti-RE from radiolabeled Raji cells had an approximate calculated m.w. of 150,000.

Studies of the Epstein Barr virus receptor found on Raji cells. II. A comparison of lymphocyte binding sites for Epstein Barr virus and C3d.

A comparison was made between the binding sites of two receptors that are believed to be closely associated on human B lymphocytes: complement receptor type two (CR2) that is specific for C3d fragments, and the receptor (EBVR) for Epstein Barr virus (EBV). Isolated fluid-phase CR2 bound to C3d on erythrocytes (EC3d) and inhibited both B cell-EC3d rosettes and the agglutination of EC3d by anti-C3d, it failed to inhibit either the binding or superinfection of B cells by EBV. By contrast, isolated fluid-phase EBVR inhibited EBV B cell binding activity and superinfection but had no CR2 activity. In addition, radiolabeled CR2 bound to EC3d and anti-CR2-Sepharose, whereas radiolabeled EBVR did not. Purified fluid-phase C3d fragments inhibited EC3d rosette formation with CR2+/EBVR+ cells but did not inhibit EBV binding. However, EBV binding to B cells did inhibit EC3d rosette formation. Clones of human/mouse somatic cell hybrids made from CR2+/EBVR+ human B lymphoblastoid cell and CR2-/EBVR- mouse myeloma cell parents expressed either EBVR or CR2 but only rarely expressed both EBVR and CR2. This suggested that the genes for EBVR and CR2 were located on two different human chromosomes. Thus it was concluded that CR2 is probably not the binding site for EBV.

Epstein-Barr virus polypeptides: identification of early proteins and their synthesis and glycosylation.

We have identified at least six early polypeptides induced by Epstein-Barr virus in cells or under conditions which are nonpermissive for Epstein-Barr virus DNA replication ranging in molecular weight from 140,000 to 26,000.

Epstein-Barr virus polypeptides: effect of inhibition of viral DNA replication on their synthesis.

After Epstein-Barr virus superinfection of the human lymphoblastoid cell line Raji, a Burkitt lymphoma-derived line that contains Epstein-Barr virus genomes in an episomal form, at least 40 polypeptides could be resolved by polyacrylamide gel electrophoresis. Eleven of the 40 polypeptides were immunoprecipitable by early antigen+/viral capsid antigen+ antiserum. The polypeptides could be divided into six classes, immediate-early, early, intermediate, late, very late, and persistent, depending upon the time of synthesis. Ten of the 40 polypeptides appeared to preexist before superinfection and persisted despite general cessation of host protein synthesis; none of the persistent proteins was immunoprecipitated by the Epstein-Barr virus antibody-containing serum. When viral DNA replication was blocked by a variety of inhibitors of DNA synthesis, a number of different patterns of polypeptide synthesis could be detected. The synthesis of six polypeptides was blocked by the most virus-specific inhibitors, acyclovir and phosphonoacetic acid. Additionally, in the presence of 1-beta-D-arabinofuranosylcytosine, 1-beta-D-arabinofuranosyladenine, and methotrexate, seven polypeptides showed oversynthesis.

Induction of Epstein-Barr virus-associated DNA polymerase by 12-O-tetradecanoylphorbol-13-acetate. Purification and characterization.

The diterpene ester promoter of mouse skin tumors, 12-O-tetradecanoylphorbol-13-acetate (TPA), efficiently induces Epstein-Barr virus (EBV)-associated DNA polymerase (DNA nucleotidyltransferase) activity in the EBV-producing lymphoblastoid cell line, P3HR-1. With the use of intervent dilution chromatography followed by sequential DEAE-cellulose and phosphocellulose column chromatography, the virus-associated enzyme has been isolated and purified 300-fold. The partially purified EBV DNA polymerase activity could be distinguished from cellular polymerases by its activation with salt and its degree of sensitivity to N-ethylmaleimide and phosphonoacetic acid. The enzyme showed maximum activity for copying activated calf thymus DNA in the presence of 100 mM ammonium sulfate. In the absence of salt, the enzyme utilized with high efficiency deoxyoligomer-homopolymer templates, but failed to copy poly(rA) . oligo(dT)10 and oligo(dT)10, showing that the enzyme had properties distinct from DNA polymerase gamma, reverse transcriptase, and terminal deoxynucleotidyltransferase. The partially purified enzyme is strongly inhibited by acyclovir triphosphate and thus has properties similar to herpes simplex virus DNA polymerase.

Polypeptide synthesis and phosphorylation in Epstein-Barr virus-infected cells.

Epstein-Barr virus superinfection of the human lymphoblastoid cell line Raji, a Burkitt lymphoma-derived line that contains Epstein-Barr virus genomes in an episomal form, results in the sequential synthesis of 29 detectable proteins, which range in molecular weight from approximately 155,000 to 21,000, and in the shutoff of the bulk of host protein synthesis within 6 to 9 h after infection. There are three classes of virus-induced proteins; these are an early class, consisting of eight proteins synthesized by 6 h postinfection, an intermediate class, containing two proteins synthesized 9 h postinfection, and a late class, consisting of five proteins synthesized 12 h postinfection. In addition, there is a fourth class of polypeptides, called persistent, that are found both before and after superinfection. The rates of synthesis of the proteins fall into three patterns; these are pattern A, in which the rate of synthesis decreases, pattern B, in which the rate of synthesis remains steady, and pattern C, in which the rate of synthesis increases after the initial appearance of the polypeptide. Both 9-(2-hydroxy-ethoxymethyl)guanine (acyclovir) and phosphonoacetic acid inhibit the appearance of one intermediate protein and at least three late proteins. Seven polypeptides are phosphorylated at different times after infection.