- Volume 69, Issue 4, 1988

Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for the nucleocapsid protein gene of Hantaan virus. Two different cDNA clones representing the small genome segment of Hantaan virus were inserted into the transfer vector pAcYM1, and recombinants were generated by replacement of a portion of the baculovirus polyhedrin gene with the foreign, Hantaan virus gene. Recombinants containing both the first and second ATG initiation codons of the Hantaan virus gene produced nucleocapsid protein, while those containing only the second codon did not. The expressed nucleocapsid protein was evaluated as a potential diagnostic antigen with a variety of hantavirus-immune sera. The high levels of expression obtained, specific serological reactivity with immune sera and the low level of biological containment required for production of this protein all suggest a significant advantage over authentic viral antigen for diagnosis of hantavirus infection.

A new picorna-like virus, provisionally named aphid lethal paralysis virus (ALPV), was isolated from the aphid Rhopalosiphum padi. The virus particles are isometric with a diameter of 26 nm, a sedimentation coefficient of 164S and a density in caesium chloride of 1.34 g/ml. Virions contain a 9.7 kb polyadenylated, ssRNA and four polypeptides: three major polypeptides of M r 34400, 32000 and 31200 and one minor polypeptide of M r 40800. ALPV is serologically unrelated to R. padi virus of aphids but is serologically related to cricket paralysis virus. The properties of this virus indicate that it should be classified in the family Picornaviridae.

Bovine leukaemia virus (BLV) induces syncytia in productively infected ovine and bovine monolayer cells. Expression of the env gene directly determines the syncytium-forming activity, since the expression of a cloned env gene directed by the simian virus 40 (SV40) early promoter efficiently induced syncytia in transfected ovine embryonic (OE) cells. However a BLV long terminal repeat (LTR)-directed expression plasmid (pLTRenv) failed to induce syncytia in transfected OE cells, suggesting insufficient promoter activity of the LTR sequences. To assess the role of the XBL genes, which are located in the 3′ distal region of the genome, in viral gene expression we constructed SV40 early promoter-directed expression plasmids. These contained open reading frames (ORFs) in the XBL region, and were examined for syncytium-inducing activity by cotransfection with pLTRenv. The results suggest that both XBL-I (the longest ORF: x-lor) and XBL-II (a shorter overlapping ORF: x-sor) are trans-acting genes which cooperatively activate LTR-directed viral gene expression.

Core proteins VP7 and VP3 have been localized in bluetongue virus (BTV) and BTV-infected cells by immunoelectron microscopy. Gold-labelled monoclonal antibodies to VP7 gave intense labelling with purified BTV core particles and weaker labelling with both directly visualized viral particles, which require no purification, and purified virus particles. It is believed that VP7 is, in a small number of viruses, accessible from the outer surface. The intensity of labelling by anti-VP7 antibodies was markedly increased by treatment of the virus with methanol. Intracellularly, VP7 antibodies also reacted with virus-like particles which appeared to be leaving virus inclusion bodies (VIB), the presumed site of virus synthesis and assembly. These antibodies also reacted with virus-like particles which were bound to the cytoskeleton and did not appear to be virus cores because they also reacted with gold-labelled antibody to the outer coat protein VP2. VP3 was not detected immunologically in either virus or core particles nor in cytoskeleton-associated virus-like particles suggesting an inner core location. VP7 and to a lesser extent VP3 were localized within the matrix of VIB. Virus tubules, a major structure found in infected cells and known to contain the non-structural protein NS1, were found to react with antibodies to both VP3 and VP7.

The wild-type ZH501 strain of Rift Valley fever (RVF) virus and two small-plaque strains (T1 and T46) derived from it were characterized by plaque size, pathogenicity for hamsters and ability to replicate in Vero cells. Additionally, a mutagenized, attenuated, large-plaque, vaccine-candidate strain of RVF virus (ZH548-M12) was also studied. Infections with either the ZH501 or T46 strain were uniformly fatal to hamsters. In contrast, nearly all hamsters infected with either the T1 or ZH548-M12 strains survived and were immune to challenge with 105 LD50 of the ZH501 strain. Both of these attenuated strains failed to replicate in Vero cells maintained at 41 °C, whereas the more virulent strains (ZH501 and T46) replicated at this temperature. The low virulence and ability to induce protection against lethal RVF virus challenge that is associated with the T1 and ZH548-M12 strains make them potential vaccine candidates.

The size and antigenic relationships among structural proteins (VPs) of canine parvovirus (CPV), feline parvovirus (FPV), porcine parvovirus (PPV), minute virus of mice (MVM) and bovine parvovirus (BPV) were determined by SDS-PAGE of radiolabelled, purified virus and immunoprecipitated viral proteins. Mature virions of CPV, FPV, PPV and MVM were composed of three VPs designated VP1, VP2 and VP3. The corresponding proteins of each virus were similar in molecular weight [79000 to 82500 (VP1), 65000 to 66000 (VP2), 62000 to 63500 (VP3)]. Additional similarities among VPs were indicated by antigenic relationships which included precipitation of VPs of CPV, FPV and PPV by both homologous antisera and antisera raised to each of the other two viruses and by precipitation of VPs of MVM by cat anti-FPV sera. A non-structural protein identified in lysates of cells infected with FPV and CPV was precipitated by cat anti-FPV and dog anti-CPV sera only. Mature virions of BPV were composed of four VPs [74500 (VP1), 67000 (VP2), 60000 (VP3), 57500 (VP4)] which were antigenically unrelated to those of the other parvoviruses tested. However, the possibility that swine are sometimes infected with a virus which is antigenically related to BPV was suggested by the finding that sera from conventionally raised swine, irrespective of their serological status for PPV, precipitated VPs of BPV, whereas neither pre-exposure sera nor anti-PPV sera from gnotobiotic pigs did so.

Of the biological activities of influenza C virus, haemagglutination, receptor inactivation and fusion, only the latter has been conclusively correlated with its surface glycoprotein (gp). We have purified the gp by octylglucoside treatment of influenza C virions followed by centrifugation into a sucrose gradient. Evidence was obtained that gp also represents the receptor-destroying enzyme of influenza C virus, which has been characterized as a neuraminate 9-O-acetylesterase: (i) it inactivated the receptors for influenza C virus on chicken erythrocytes; (ii) it had acetylesterase activity as indicated by the release of acetate from bovine submandibulary mucin; (iii) monoclonal antibodies directed against gp inhibited the acetylesterase activity of influenza C virus. Although purified gp was unable to agglutinate chicken red blood cells, it blocked haemagglutination by viruses. This finding as well as the haemagglutination inhibition activity of monoclonal anti-gp antibodies indicate that gp is also responsible for the haemagglutinating activity of influenza C virus. Thus, as the influenza C glycoprotein is the only myxovirus glycoprotein with three different activities, we propose the designation HEF in order to describe its function as a haemagglutinin (H), an esterase (E) and a fusion factor (F).

The antibody response to herpes simplex virus (HSV) is complex and involves antibody to at least 33 virus-induced polyeptides. Serum IgG contains four isotypes in mice and it is known that the isotypes differ in their biological functions and that individual antigenic proteins may preferentially elicit restricted isotype responses. We therefore examined the anti-polypeptide isotypes induced in immune mouse serum. By ELISA, we found that the total serum virus-specific antibody activity was 51% IgG1, 39% IgG2a, 11% IgG2b and 1% IgG3 in immune ICR strain mice and 51%, 45%, 4% and 0.4% respectively in strain BALB/c mouse immune serum. These proportions are significantly different from those reported for other virus infections. Sepharose-Protein A affinity-purified isotypes were also studied and showed IgG1 > IgG2a ⩾ IgG2b ≫ IgG3 activity per µg of isotype, indicating that competition between isotypes present in high concentrations did not significantly alter the results. Immunoblotting studies of the purified isotypes showed that the major immunogenic HSV-1 proteins (VP155, gC, gB, pgB, gD and nucleocapsid proteins 42K and 44K) induced all isotypes. However, the isotype responses were not uniform among the glycoproteins and some other proteins. In addition neutralization assays of the purified isotypes indicated that IgG2a and IgG2b had significantly greater neutralizing capacity than IgG1, suggesting that less of the IgG1 was directed against neutralizing virion epitopes. These data are discussed with respect to the biological implications in host defence.

The US7 open reading frame of herpes simplex virus type 1 (HSV-1), previously identified by nucleotide sequencing, has been expressed in a recombinant vaccinia virus (US7-VAC). Antiserum raised against HSV-1 reacted with a 66K glycoprotein in US7-VAC-infected cells and this polypeptide was present on both nuclear and cell surface membranes. In the presence of tunicamycin the protein was reduced in size to 58K showing it contained N-linked sugar residues. Antisera from animals vaccinated with US7-VAC recognized 66K and 58K polypeptides in HSV-1-infected cells and neutralized HSV-1 infectivity in vitro in the presence of complement.

Glycoproteins D1 (gD1) and D2 (gD2) of herpes simplex virus type 1 and type 2, respectively, were purified from infected HEp-2 cells labelled with [3H]glucosamine for 14 h followed by a 3 h chase using HD1 monoclonal antibody linked to Sepharose. O-linked oligosaccharides were found to be present in both glycoproteins. The identification of N-acetyl [3H]galactosaminitol as the major labelled component in the oligosaccharides generated by mild alkaline borohydride treatment demonstrated that these chains have N-acetylgalactosamine at the reducing end. These oligosaccharides consist of mono- and disialylated species with a predominance of the latter species in gD1. Size analysis and radioactive amino sugar composition strongly suggest a structure in which the galactosyl-N-acetylgalactosamine core is substituted with one or two sialic acid residues. In terms of [3H]glucosamine-derived radioactivity, O-linked oligosaccharides are less represented than N-linked oligosaccharides. The O-linked oligosaccharide number determination showed that gD1 and gD2 carry two and three chains, respectively.

Earlier studies have suggested that successful parasitism by certain braconid parasitoids may depend on the presence in host insect larvae of both polydnavirus and venom. We have shown that venom from the braconid parasitoid, Cotesia melanoscela, was required for in vivo persistence of polydnavirus DNA in host larvae. In parallel studies using an in vitro system, we observed that in the presence of venom nucleocapsids were released into the cytoplasm and subsequently uncoated at nuclear pores; in the absence of venom, this sequence of events was not observed.

A virus associated with turkey rhinotracheitis was purified and its morphology and structural polypeptides were compared with those of the bovine, human and murine members of the genus Pneumovirus. The isolate possessed surface projections 13 to 14 nm in length and a helical nucleocapsid 14 nm in diameter with a pitch of 7 nm. Approximately seven presumed viral polypeptides were observed. Their apparent molecular weights were 200 × 103 (200K), 84K, 54K, 42K, 37K, 31K and 14K; two of these, the 84K and 54K polypeptides, were glycosylated. The virus was shown to possess many features that were similar to established pneumoviruses and can therefore be regarded as a possible member of this genus.

Turkey rhinotracheitis (TRT) virus is a paramyxovirus associated with recent outbreaks of acute respiratory disease in turkeys. On morphological criteria it resembles the pneumoviruses more than the other members of the paramyxovirus family. In this communication we report the identification of five virus-induced polypeptides in TRT virus-infected BS-C-1 cells. The M r of these polypeptides were estimated as 38K, 35K, 30K, 19K and 15K from their electrophoretic mobility in SDS-polyacrylamide 6 to 15% gradient gels. The virus specificity of four (the 38K, 35K, 30K and 19K M r polypeptides) of these five was confirmed by their predominance as products of in vitro translation of mRNA from TRT virus-infected BS-C-1 cells. An additional virus-specific polypeptide with an estimated M r of 22K was revealed by in vitro translation and may be either a non-structural polypeptide or a minor structural protein. All six polypeptides were immunoprecipitated by a murine antiserum. Three other polypeptides (129K, 57K and 45K) were also immunoprecipitated. The 57K, 45K and 15K M r polypeptides were glycosylated, and the 57K glycopolypeptide may be a disulphide-bonded dimer of the 45K and 15K glycopolypeptides. Another major glycopolypeptide of 83K M r was observed but its virus-specificity could not be confirmed. Taken together our results tentatively identify eight or nine TRT virus-specified polypeptides and are consistent with the classification of TRT virus as a new member of the genus Pneumovirus.

The elevation of culture temperatures from 35 °C to 39°C led to the cessation of the synthesis of the fusion (F) protein of measles virus. This effect was caused by inhibition of the translation of the corresponding mRNA rather than by a decrease in the synthesis or stability of the mRNA or by increased degradation of the F protein at elevated temperatures. The haemagglutinin (H) and F mRNAs were distributed differently in gradients on which polysomes were fractionated. The H mRNA was present almost exclusively in the largest polysomes whereas the F mRNA was more evently distributed over large and small polysomes. The distribution was not affected by a temperature shift. The inhibition of F protein synthesis thus appeared to be related to a cessation of elongation of the nascent polypeptide chain rather than to a defect in initiation of the translation of the F mRNA at 39 °C.

Thirteen monoclonal antibodies (MAbs) were prepared against human parainfluenza virus type 2 (PIV2). These MAbs reacted with the haemagglutinin-neuraminidase glycoprotein with an M r of 84K. The MAbs defined one antigenic site which could be divided into five epitopes. A correlation between haemagglutination inhibition (HI) and neutralization activity could be seen although one MAb, which recognized a distinct epitope, showed neutralization and no HI activity to PIV2. The reactivity of the MAbs was tested against Sendai virus, parainfluenza virus type 3, simian virus 5 (SV5), mumps virus, Newcastle disease virus, measles virus and canine distemper virus. Only one MAb showed any cross-reaction with a low HI titre to SV5.

Seven neutralizing monoclonal antibodies (MAbs) to the rotavirus simian agent 11 were produced. Although displaying variable degrees of haemagglutination-inhibiting activity, they were shown by radioimmunoprecipitation and Western blot analyses to react with the major outer capsid glycoprotein (VP7). In competition binding assays, MAbs defined two distinct VP7 epitopes, which appeared to be close to each other or partially overlapping. In addition, MAbs of the two epitope groups enhanced binding of a broadly reactive, non-neutralizing, MAb specific for rotavirus group antigen.

The complete nucleotide sequence of the RNA segment 10 of Australian BTV serotype 1 has been deduced from a combination of sequencing cDNA inserts cloned into pBR322 and synthetic deoxynucleotide priming on purified double-stranded RNA molecules. The gene segment was 822 nucleotides in length and capable of coding for two proteins on either 229 or 216 amino acids, having net charges of either +4.5 or +5.5 respectively at neutral pH. Comparison with the RNA segment 10 from BTV serotype 10 revealed a high degree of amino acid sequence conservation as well as regions of nucleic acid sequence conservation.

Infection of Vero cells with Tacaribe virus stocks containing a high ratio of standard (plaque-forming) viruses to defective interfering particles (DIP) induced inhibition of the host cell Ca2+ ATPase (Ca2+ pump) and the ouabain-sensitive Na+/K+ ATPase (Na+/K+ pump). The Mg2+ ATPase which is not involved in cation transport was not affected. The presence of DIP in the inocula protected the cells from alteration of the transport-associated ATPases induced by standard viruses.

We describe a method of sample preparation to detect scrapie-associated fibril (SAF) proteins in small amounts of scrapie-infected mouse tissues by Western blot analysis using an antiserum to a synthetic peptide that corresponds to the N-terminal region of hamster prion protein. SAF proteins were efficiently detected in brain tissue by this procedure. The proteins were also detected in preparations from spleen and lymph node. SAF proteins were detected in brain samples at 24 weeks after intraperitoneal infection. Using spleen samples, the proteins were detected from mice in the preclinical stage (from 4 weeks after infection), clinical symptoms of scrapie were observed in some mice from 22 weeks after infection.

The grey tremor mouse is an autosomal recessive mutant characterized by a phenotype of unusual pigmentation, neurological abnormalities and early death. These mice have a spongiform encephalopathy similar to scrapie and Creutzfeldt-Jakob disease. Although the disease is clearly heritable, the grey tremor mouse spongiform pathology has also been transmitted by inoculation of genetically normal mice with diseased brain homogenates. The possibility that a scrapie-like agent is involved has been proposed. We examined brain homogenates from grey tremor mice, scrapie-affected mice and normal mice for the presence of the mouse scrapie agent protein (MoSp33–37) and its normal cellular homologue. All untreated homogenates contained one or both isoforms of this protein as detected on immunoblots. Grey tremor mouse brain homogenates, when protease-treated, showed no evidence of MoSp33–37. A purification method for MoSp33–37 concentrated it in samples from scrapie-affected mice, but this protein was not detected in grey tremor or normal mice. These results suggest that it is unlikely that the scrapie agent is involved in grey tremor disease.

A head-to-tail dimer of cloned DNA from a Swedish isolate of wheat dwarf virus (WDV) was integrated between the T-DNA border sequences of a broad host range binary vector and transferred into cells of wheat seedlings using an Agrobacterium-mediated delivery system. Two-thirds of the inoculated plants developed a systemic infection. Extracts of infected leaves were shown to contain the virus double-stranded (supercoiled, open circular and linear) and single-stranded DNA forms of unit genome length and the virus capsid polypeptide. The results demonstrate the infectivity of a previously sequenced clone of WDV DNA.

Phaseolus vulgaris ‘Top Crop’ plants inoculated with a mixture of cloned tandem (head to tail) dimers of each component of bean golden mosaic virus (BGMV) developed yellow mosaic symptoms identical to those induced by inoculation with BGMV particles or BGMV ssDNA. Progeny virus particles isolated from plants infected with such cloned DNAs resembled BGMV particles by electron microscopy, reacted with anti-BGMV serum and contained circular unit-length ssDNA. Southern blot analysis of DNA extracted from plants infected with cloned DNA dimers detected virus-specific ssDNA and dsDNA species similar to those found in cells infected following inoculation with BGMV particles.