- Volume 68, Issue 3, 1987

Monoclonal antibodies were produced against two distinct Thai dengue-2 (DEN-2) virus strains isolated in 1980 from dengue haemorrhagic fever patients. Nine of 36 hybridomas produced monoclonal IgG antibodies which reacted in radioimmune precipitation assays with the NS1 non-structural protein (42000 mol. wt.) from DEN-2- infected C6/36 (Aedes albopictus) cells. The virus specificity of NS 1 -reactive monoclonal antibodies was determined by indirect immunofluorescence assays using LLC-MK2 cells infected with either the Thai 1980 DEN-2 isolates, prototype DEN viruses (four serotypes), Japanese encephalitis (JE), Murray Valley encephalitis, West Nile, Wesselbron or Tembusu viruses. Eight of the monoclonal antibody preparations were DEN-2-serotype specific. One preparation defined a special serological relationship between DEN-2 and JE viruses. Four preparations had detectable complement fixation titres using Thai DEN-2 virus antigen. Six spatially unique epitopes were identified using competitive binding assays.

Immunization of mice with the dengue 2 virus (DEN 2)-specified non-structural protein NS1 provided significant protection against intracerebral challenge with the virus in the absence of detectable neutralizing or other anti-virion antibody. NS1, purified from lysates of infected Vero cells by immunoaffinity chromatography, expressed an antigenic site(s) common to each of the four DEN serotypes, and hyperimmunization of rabbits with NS1 stimulated production of complement-fixing (CF) antibody with broad DEN serotype specificity. However, cross-protection was not observed: mice immunized with DEN 2 NS1 developed little or no heterologous CF antibody and were not protected against challenge with neurovirulent DEN 1. Induction of a protective immune response by NS1 suggests that it be considered for incorporation into possible synthetic or recombinant DNA DEN vaccines.

Strains of tick-borne encephalitis (TBE) virus isolated from ticks in natural foci in Austria were compared to strains isolated from the same foci 14 years previously. Comparative peptide mapping of the envelope (E) glycoproteins as well as analysis of the antigenic structure of the E proteins by the use of 14 monoclonal antibodies defining different epitopes did not provide evidence for antigenic variation. The same also holds true for isolates from a probably newly established natural focus in Western Austria. These results confirm previous data by showing that under natural ecological conditions TBE virus is quite stable and does not undergo major antigenic changes.

Inoculation of suckling mice with coxsackievirus B4 (CB4) results in the death of a majority of the animals. In this study we selected antigenic variants of CB4 in the presence of neutralizing monoclonal antibodies and tested them to see whether they were attenuated. Antigenic variants selected with a single antibody showed little or no attenuation by producing a high mortality (60 to 100 %). A double variant selected with two antibodies showed considerable attenuation by causing only 25% mortality. A triple variant selected with three antibodies was almost completely attenuated (killed only 5 % of the animals). Polypeptides from these variants were tested for their ability to interact with the monoclonal antibodies used for their selection. These studies showed that resistance of variant virus to neutralization in general was due to the inability of the antibody to bind to the virus. However, one of the antibodies could bind but not neutralize the virus, perhaps due to an alteration in the epitope. It is concluded that selection of CB4 variants using more than one neutralizing monoclonal antibody can lead to attenuation of the virus.

According to the localization of hepatitis B virus (HBV) core antigen (HBcAg), detected by the avidin-biotin complex method, infected hepatocytes were classified into three types, i.e. those having nuclear (type I), nuclear and cytoplasmic (type II) or only cytoplasmic (type III) antigen. HBcAg-positive hepatocytes of all specimens (three) from non-specific reactive hepatitis and of most (five of seven) from chronic persistent hepatitis (CPH) patients were only type I; the other two CPH samples and all (seven) chronic active hepatitis samples were composed of a mixture of types I, II and III. Linear correlations between the frequency of type I, as well as that of all types (I, II and III) of the HBcAg-positive hepatocytes, and the amount of HBV DNA in serum were found. The relative HBV production of HBcAg-positive hepatocytes (serum HBV DNA amount/frequency of HBcAg-positive cells) was 0·11 in type I and 0·07 in all hepatocytes including types I, II and III. HBV core particles and complete HBV particles were found in type I hepatocytes. On the other hand, these particles were not found in a predominantly type III liver specimen. These results suggest that type I hepatocytes are more involved in the propagation of HBV than types II and III.

The effect of gangliosides extracted from human group O Rh+ erythrocytes on haemagglutination by BK virus was investigated. Experiments were performed on both ganglioside mixtures and isolated fractions separated by column chromatography and characterized by thin-layer chromatography. These results were compared with those obtained with standard preparations of gangliosides, and the inhibiting activity was shown to be confined mainly to gangliosides with a R Flower than GM1. It was also observed that the insertion of gangliosides in liposomes increased the haemagglutination-inhibiting activity and that ganglioside coating restored the ability of glycosidase- treated human red blood cells to agglutinate.

We have compared the sequences of the putative polypeptides of the human pathogenic B19 parvovirus with protein sequences in the National Bethesda Research Foundation Library, and have discovered a significant homology between a B19 parvovirus non-structural (NS) protein and the T antigens of polyomaviruses and simian virus 40 (SV40) and the putative E1 proteins of papillomaviruses. The region of highest homology with the papovavirus proteins corresponds to the region that is most highly conserved in the NS1 proteins of several other parvoviruses. Studies with the T antigen of both polyomaviruses and SV40 have implicated this region as having an ATPase activity and nucleotide-binding function.

The genomic relationship between porcine parvovirus (PPV) and several other autonomous parvoviruses was examined by restriction site and hybridization analysis. Restriction site maps of the PPV genome were prepared by digesting the doublestranded replicative form of the viral DNA with each of eight restriction enzymes. Subsequent comparison of such maps with those previously reported for PPV, canine parvovirus (CPV), feline panleukopenia virus (FPV), minute virus of mice (M VM), H-1 virus (H-1) and bovine parvovirus (BPV) revealed that while the maps of CPV, FPV, MVM and H-1 had a number of features in common, those of PPV and BPV were substantially different. For hybridization analysis radioactive probes prepared by nick translation of PPV, CPV and BPV genomes were tested under conditions of both low and high stringency for homologous hybridization and for heterologous hybridization with each of the other two viruses and with FPV. The results of these tests indicated homology among the genomes of PPV, CPV and FPV, but little or no homology between the genome of BPV and those of any of the other viruses tested. Additional tests with restriction fragments of PPV and a CPV probe indicated that heterologous hybridization was confined primarily to a segment of the genome between 1·85 and 2·7 kb from the 3′ end. Based on transcriptional maps previously determined for several of the rodent parvoviruses, this interval is likely to include part of the coding sequences for both non-structural and structural proteins and may be the genetic basis for the replicative as well as the antigenic similarities between PPV and both CPV and FPV.

The polyhedrin gene region of the Autographa californica nuclear polyhedrosis virus (AcMNPV) morphology mutant M29 has been characterized by genetic and physical techniques. Recombination analysis of mutants M29 and AcM5polyl demonstrated that wild-type polyhedrin recombinants could be obtained and that the DNA restriction patterns of the recombinant viruses were identical to wild-type AcMNPV DNA. Marker rescue experiments using the wild-type AcMNPV EcoRI I fragment indicated that the morphology mutation responsible for the M29 phenotype was located in the 0·0 to 5·9% region of the genome. Direct DNA sequencing of the BamHI F fragment from M29 showed a single point mutation at position 253 of the polyhedrin gene. This mutation caused a substitution of phenylalanine for leucine at amino acid 84 of the M29 polyhedrin protein. These results indicated the necessity of amino acid conservation in the polyhedrin amino acid sequence for proper folding and assembly of the polypeptide into occlusion bodies.

The stability of the restriction endonuclease profile of herpes simplex virus type 1 strain SC 16 in mice was studied. Virus isolated from skin during acute infection was compared with that from latently infected ganglia and with that from recrudescent lesions induced by trauma. In another experiment virus serially passaged only in skin was compared with virus that had also replicated in the nervous system. The loss or gain of specific restriction sites was not observed but in some cases the mobility of certain fragments decreased.

It has been recently shown that VP-16–213, a semi-synthetic derivative of podophyllotoxin, inhibits the function of mammalian DNA topoisomerase II. In the present study, we examined the effects of VP-16–213 on the replication of herpes simplex virus type 2 (HSV-2). The compound did not inhibit the synthesis of early viral polypeptides at concentrations at which viral DNA synthesis was strongly suppressed, but induced double-strand breaks in newly synthesized HSV DNA. Electron microscopic examination of treated cells revealed the presence of a number of capsids with empty or partial cores. The level of topoisomerase II activity remained unaltered after infection, and all attempts to isolate VP-16–213-resistant mutants of HSV-2 have failed in spite of extensive efforts. It is suggested therefore that the mode of action of VP-16–213 may be inhibition of viral DNA synthesis by impairing the function of host cell topoisomerase II.

Negative staining electron microscopy was used to examine culture fluids from the H9/HTLV-III cell line after concentration by centrifugation. Characteristic retroviruslike particles bearing distinctive envelope projections were seen. The virion envelope was frequently extended in the form of a bleb or a tail. These particles were morphologically virtually indistinguishable from similar preparations of Friend murine leukaemia virus. H9/HTLV-III culture fluids contained, in addition, numerous comet-shaped particles with a dense head and flared tail. These particles were clumped by the addition of anti-HTLV-III-positive serum suggesting that they may represent intermediate forms of the virus.

A subline of 293 cells able to grow in suspension culture has been developed by passage of 293 cells through nude mice. This new line, designated 293N3S, grows with a doubling time of approximately 30 h, continues to express adenovirus 5 early region 1 (E1) antigens, and remains permissive for adenovirus 5 host range mutants defective in E1 functions.

The susceptibility of adenovirus to the inhibitory effect of human interferons in vitro was investigated. We tested recombinant human interferons-α 2, -β 1, and -γ against adenovirus serotypes 1 and 5 (group C), 3 and 7a (group B), and a wild strain isolated from an acutely ill child who later died. Pretreatment of WISH cells with interferon-γ for 24 h induced a dose-dependent inhibition of multiplication of all adenovirus strains tested, the TCID50 varying from 25 to 90 IU/ml. Human interferon-α 2 was unable to decrease adenovirus multiplication, while interferon-β 1, at 2000 IU/ml slightly lowered the yield of adenovirus. Similar results were obtained in HEp-2 and FS-4 cells, while A- 549 and peripheral blood mononuclear cells were insensitive to interferon-y. The difference between the effects of interferon-y and interferon-γ and β> on adenovirus multiplication in vitro suggests that its mechanism of antiviral action is different.

A recombinant interferon (IFN) hybrid has been found to have a broad host-range of activity in an antiviral assay (plaque reduction of vesicular stomatitis virus) and also high efficacy as an antiviral agent in at least 12 different animal cell species. The IFN hybrid consists of amino acids 1 to 60 from HuIFN-αB and amino acids 61 to 166 from HuIFN-αD. The profile of cross-species activity of the IFN-αB/D hybrid has been compared with that of HuIFN-αF, and of the parents HuIFN-αB and -αD. When both IFN-αB and -αD were active in a cell species, the hybrid IFN had comparable or better activity than the more active parental IFN. The hybrid shared a broad cross-species activity with IFN-αD. However, the IFN-αB/D hybrid was 10-fold more active on human cells, 30-fold more active on rabbit cells, and 50-fold more active on mouse cells than IFN-αD.

The linkage between the genome-linked protein VPg and the RNAs of cowpea mosaic virus has been analysed. The bond between VPg and the RNA chains was found to be resistant to mild acidic hydrolysis but sensitive to alkaline treatment. Hydrolysis of isolated VPg-phosphate with 5·6 m-HCl at 110 °C yielded phosphoserine as the sole phosphorylated amino acid. The data obtained show that VPg is linked to the 5′-terminal uridine of the genomic RNAs by the β-OH group of the serine located at the amino-terminal end of the protein.

Filamentous particles of rice grassy stunt virus, a member of the rice stripe virus group, were found to be associated with an RN A-dependent RNA polymerase activity. The general properties of this RNA polymerase were similar to those of that associated with particles of rice stripe virus. A minor polypeptide (mol. wt. 230000), which may be the polymerase, was detected in virus preparations.

Particles of festuca leaf streak virus (FLSV) contain three major proteins. One of these [mol. wt. 49000 (49K)] is the main constituent of the nucleocapsid, whereas the other two (mol. wt. 58K and 20K) were released from the nucleocapsid when particles were treated with non-ionic detergent. The 58K protein is glycosylated. The 58K, 49K and 20K proteins correspond to the G, N and M proteins of rhabdoviruses, respectively. Four minor proteins associated with the virus particles have mol. wt. of 189K, 117K, 101K and 41K. The 189K and 101K proteins are associated with the nucleocapsid, whereas the 117K protein was found in the soluble fraction after detergent treatment. Nucleic acid isolated from virus particles is probably RNA with an estimated mol. wt. of 4·3 × 106. The buoyant density of virus particles in sucrose was estimated to be 1·194 g/ml and the s 20 w, be 704S. The present results, together with previous information, make FLSV a definitive member of subgroup A of the plant rhabdovirus group of the family Rhabdoviridae.