- Volume 71, Issue 11, 1990

This review illustrates how molecular and structural analyses have contributed to a greater understanding of the rich biological diversity seen in the picornaviruses. In many cases the second phase of studies, based on increasingly powerful genetic engineering, biochemical and immunological techniques is now well under way and beginning to reveal the depth of the vast store of information which still lies encoded cryptically in the sequences and structures which have been determined. The advances made already suggest that the next few years will be even more fruitful than those since 1981 when the first picornavirus sequence was published.

Strains of poliovirus type 3 isolated in Finland in 1984 and 1985 (P3/Fin/84) are known to differ considerably from the type 3 vaccine strains in both nucleotide sequence and antigenic properties. In the search for the origin of the outbreak we first tested 80 type 3 strains that had been isolated elsewhere in the world during the years 1953 to 1986. An oligonucleotide probe complementary to a highly variable 17 nucleotide interval in the 5′ non-coding region of the genomic RNA of P3/Fin/84 reacted with five strains. Also it was revealed that two of the latter five strains were related to the P3/Fin/84 strains in two separate genomic regions compared after partial RNA sequencing. One of them was isolated in Switzerland in 1980 and the other in Turkey in 1981. The Swiss strain was from a patient who had recently returned from a journey to various Mediterranean countries. Consequently, 16 other strains isolated in the late 1970s and early 1980s in Europe or in the Mediterranean countries were studied in detail by partial genomic sequencing and with neutralizing monoclonal antibodies. Two separate regions of the genome were compared by sequencing and corresponding dendrograms were constructed. The Switzerland and Turkey strains were found to be the strains most closely related to the viruses of the 1984 Finland epidemic. These results indicate that type 3 poliovirus strains related to P3/Fin/84 had been circulating in Mediterranean countries since the late 1970s.

Chimeric polioviruses have been prepared in which part of the antigenic site 1-encoding sequence of the Sabin strain of type 1 poliovirus has been replaced by sequences based on those found in the homologous region of the Sabin type 3 strain. The chimeras were analysed for their reaction with polyclonal and monoclonal antibodies raised against type 1 and type 3 viruses, and with polyclonal antipeptide sera, as well as for their immunogenicity in animals. The effectiveness with which the type 3 site was presented antigenically varied in ways which were partially predictable, based on the behaviour of type 3 mutants with monoclonal antibodies. However, other factors were implicated which may include conformational effects and other components of the site in addition to those altered in the chimeras. The ability of the chimeras to induce antibodies reacting with type 3 polioviruses paralleled their antigenic reactivity, and evidence is presented for the induction of strain-specific antibodies.

The 3C protease of poliovirus is distinguished from that of all other picornaviruses in that it only cleaves at Gln-Gly amino acid pairs within the viral polyprotein. To determine whether this strict cleavage specificity is an intrinsic property of the poliovirus 3C protease, amino acid substitutions were introduced at one of the Gln-Gly cleavage sites. Oligonucleotide-directed site- specific mutagenesis of an infectious poliovirus type 1 (Mahoney strain) cDNA was used to change the Gln-Gly site at the 3C/3D junction of the polyprotein into Gln-Val, Gin-Ala, Gln-Ser or Gin-Pro. The effects of these substitutions were studied in vivo after transfection of primate cells by the mutated cDNAs. The Gln-Gly to Gin-Pro substitution was lethal for virus growth, and the corresponding altered 3CD polypeptide expressed in insect cells using a recombinant baculovirus vector did not appear to undergo autocleavage. The Gln-Gly to Gln-Val change was also lethal, although production of virus was occasionally observed as a result of reverse mutations. Mutants with Gin-Ala and Gln-Ser sequences were viable, indicating that these dipeptides can be cleaved by the poliovirus protease in vivo. However, processing at the 3C/3D junction occurred relatively inefficiently in the case of the Gln-Ser virus. Furthermore, the Gln-Gly to Gin-Ala substitution seemed to result in an additional cleavage event within the N-terminal part of polypeptide 3D.

After intracerebral inoculation of Borna disease virus (BDV), Lewis rats develop a persistent infection of the central nervous system which is pathohistologically represented by perivascular encephalitic lesions predo-minantly in the grey matter. In previous studies it has been shown that a cell-mediated immune response causes Borna disease (BD). In order to define further the immune cell responsible for this immunopathologi-cal disease, a BDV-specific T cell line, NM1, was established and cultured in vitro. Phenotypically this T cell line was characterized by cytofluorometry as CD4-positive (CD4+). Proliferation assays with syngeneic and allogeneic antigen-presenting cells, and blocking experiments with monoclonal antibodies, revealed major histocompatibility complex class II antigens to be restriction elements. After passive transfer of this virus-specific CD4+ T cell into immunosuppressed BDV-infected recipients, full-blown disease could be induced. Immunohistological examination of the cells involved in perivascular inflammatory infiltrates in BDV-infected rats and in recipients of the NM1 T cell line revealed a dominance of macrophages and CD4+ T cells. The presence of these cells in encephalitic lesions strongly suggests a delayed type of hypersensitivity reaction as the pathogenetic mechanism of BD.

The sites of multiplication in the mouse central nervous system (CNS) of the virulent L10 strain of Semliki Forest virus (SFV) and the L10-SFV-derived demyelinating M9 mutant were determined using both BALB/c and SJL mouse strains. In situ hybridization (ISH), using a cRNA probe to an SFV non-structural sequence, and immunogold-silver staining (IGSS), using polyclonal anti-SFV rabbit IgG, were the techniques utilized. For L10-SFV, viral RNA and antigen were detected in neurons and glial cells of both mouse strains. For BALB/c mice infected with M9-SFV, both neuronal and glial cell infection was less extensive than that obtained with L10. ISH or IGSS were generally not sensitive enough to detect viral RNA and antigen, respectively, in M9-SFV-infected SJL mice. M9-SFV multiplied to a similar titre in primary cultures of glial cells derived from either BALB/c or SJL mice. Following infection with M9-SFV, small plaques of demyelination in the CNS and occasional small aggregates of mononuclear leukocytes in the leptomen- inges persisted for up to 12 months in SJL mice but not BALB/c mice. This was not associated with detectable persistence of infectious virus, viral antigen or viral RNA in the CNS.

We have previously shown that the antiviral state of explanted mouse peritoneal macrophages (PM) decays during in vitro culture and that this decay is much more rapid in Lps d PM than it is in Lps n PM. Moreover, Lps n PM can transfer the antiviral state to other cells, whereas Lps d PM cannot. In vitro treatment of Lps n PM with different agents [i.e., bacterial lipopolysaccharide (LPS), interferon (IFN)-γ, tumour necrosis factor (TNF)-α, macrophage colony-stimulating factor (M-CSF) and antibody to Mac-1 antigen] induced an antiviral state to vesicular stomatitis virus (VSV) which was inhibited by antibodies to IFN-β. Treatment of Lps n PM with LPS or IFN-γ resulted in greater accumulation of IFN-β mRNA, whereas no change in the barely detectable levels of IFN-α mRNA was observed. Marked accumulation of IFN-β mRNA was also observed in PM after TNF-α treatment. M-CSF and IFN-γ (but not LPS) also induced an IFN-mediated antiviral state in Lps d PM. Low levels of spontaneous transcription of IFN-β mRNA were detected in nuclei from Lps d PM. Treatment of Lps d PM with IFN-γ for 3 h resulted in the accumulation of IFN-β mRNA without any concomitant increase in the transcription of the IFN-β gene, as determined by run-on transcription assays with isolated nuclei. The addition of as little as 1 international unit/ml of IFN-γ to PM resulted in a 100-fold inhibition of VSV yield. As antibodies to IFN-α/β inhibited only a portion of the IFN-γ-induced antiviral state, such an antiviral state might reflect the synergism between IFN-γ and endogenous IFN-β. In fact, the addition of low doses of both IFN-γ and IFN-β to either Lps n or Lps d PM resulted in synergistic antiviral effects. In vivo treatment of Lps d mice with granulocyte-macrophage (GM)-CSF, M-CSF, IFN-γ or Newcastle disease virus rendered peritoneal cells capable of transferring an antiviral state. These results indicate that (i) various stimuli can induce IFN-β production by PM, (ii) Lps d PM spontaneously transcribe low levels of IFN-β mRNA, even though they cannot transfer an antiviral state, (iii) different stimuli, but not LPS, induce a normal IFN response in Lps d PM, (iv) IFN-γ increases the accumulation of IFN-β mRNA in Lps d PM by post-transcriptional mechanisms and (v) IFN-γ may act synergistically with endogenous IFN-β in inducing a potent antiviral state to VSV in PM.

Hybridomas producing monoclonal antibodies (MAbs) to rabbit haemorrhagic disease virus (RHDV) were prepared. Using Western blot (WB) analysis, the MAbs obtained were divided into two groups, one reacting with the major structural proteins of M r 61K and 38K, and the other giving negative reactions. Both groups of MAbs, however, reacted specifically with RHDV in ELISA and by immunoperoxidase (IP) and immunofluorescence (IF) tests with infected cells. As demonstrated by WB using RHDV-specific MAbs and a MAb to feline calicivirus (FCV) strain F9, the major structural (capsid) proteins of RHDV and FCV have very similar sizes (M r 61K and 38K compared to 62K to 64K and 40K respectively). No cross-reactions of MAbs with proteins of the other virus were observed in WB analysis, ELISA, IP tests or IF. The high specificity and sensitivity of RHDV-specific MAbs make them suitable for the routine IP and IF diagnosis of RHDV in liver cells of rabbits dying after natural or experimental infections.

The genome organization of porcine respiratory coronavirus (PRCV), a newly recognized agent which has a close antigenic relationship to the enteropathogenic transmissible gastroenteritis virus (TGEV), was studied. Genomic RNA from cell-cultured PRCV (French isolate RM4) was used to produce cDNA clones covering the genomic 3′ end to the start of the spike (S) glycoprotein gene (7519 nucleotides). Six open reading frames (ORFs) were identified that allowed the translation of three coronavirus structural proteins and three putative non-structural (NS) polypeptides, homologous to TGEV ORFs designated NS3-1, NS4 and NS7. Pairwise alignment of PRCV nucleotide and amino acid sequences with sequence data available for three TGEV strains revealed a 96% overall homology. However, the genome of PRCV exhibited two important distinctive features. The first was that the S gene lacked 672 nucleotides in the 5′ region and encoded a truncated form of the S polypeptide, and secondly, the first NS ORF downstream of the S gene was predicted to be non-functional as a consequence of a double deletion. The significance of genomic deletions with respect to tissue tropism and evolution of coronaviruses is discussed.

Antibody-binding sites were mapped on all overlapping nonapeptides of the major core protein p24 of human immunodeficiency virus type 1 (HIV-1) using murine monoclonal antibodies (MAbs) and sheep and rabbit polyclonal antibodies raised against HIV-1/H9 (strain IIIB) viral lysate and antibodies obtained from humans infected with HIV-1. The binding sites were mapped to various distinct regions of this protein. After superimposition of the antibody-binding sites on a proposed model of p24 of HIV-1, these sites appeared to be located on the surface of the protein on loops, turns and coils of p24 but, unexpectedly, not on the major part of the predicted ‘puff. Little reaction was found with the inaccessible anti-parallel β-barrel. These results are the first experimental evidence for the validity of the structure proposed for p24 of HIV-1.

Cross-reactive neutralization epitopes on VP4 of human rotavirus (HRV) were analysed by the use of VP4-specific neutralizing monoclonal antibodies (N-MAbs) and MAb-resistant mutants. Seven anti-VP4 N-MAbs obtained in this study by using HRV serotypes 1 and 3 as immunizing antigens showed a variety of cross-reactivity patterns to 20 HRV strains with different serotype specificity in neutralization tests and a broader cross-reactivity to them was found for four N-MAbs in an enzyme-linked immunosorbent assay. On the basis of the reactivity patterns against rotaviruses in neutralization tests, these seven N-MAbs were classified into four groups. Cross-neutralization tests using a total of 12 pairs of MAbs and resistant mutants, including five pairs which had been prepared previously, showed that VP4 of HRV (strain KU) contained two independent antigenic regions. One, region C1, was recognized by a single MAb (YO-2C2) and the other was made up of two antigenic regions (C2 and C3) which overlapped operationally. Identification of amino acid substitution sites on VP4 of representative mutants of HRV strain KU indicated that amino acid positions 385 or 392 and 428 or 433 were critical for the C2 and C3 regions, respectively. These results suggested that regions C2 and C3 exist as conformational antigenic sites.

Bovine rotavirus (BRV) VI005 is serologically distinct from rotavirus serotypes 1, 2, 3, 4, 5, 6, 8 and 9. BRV VI005 showed cross-reactions with BRV B223, the American prototype of serotype 10 rotavirus, and with BRV E4049, a British serotype 10 isolate. BRV V1005 was, however, not neutralized by four monoclonal antibodies directed against VP7 of BRV B223. Twoway cross-reactions were observed between BRV VI005 and a reassortant rotavirus containing the VP4 from BRV UK. In addition the major tryptic cleavage product of VP4, VP5*, from BRV V1005 is indistinguishable by peptide mapping and its isoelectric point from the homologous protein of BRV UK, but is clearly different from VP5* of BRV NCDV. The peptide map of VP7 from BRV VI005 differed from that obtained for VP7 of BRV UK.

More than 100 isolates were plaque-purified to examine the genetic variations in four wild stocks of Spodoptera litura. Nuclear polyhedrosis virus (NPV) collected in Japan. These isolates were characterized by their in vitro host range in three established insect cell lines, growth characteristics, polyhedral protein, DNA restriction endonuclease pattern and DNA hybridization. The isolates were separated into four distinct groups: (I) isolates corresponding to Autographa californica NPV, (II and IV) two different groups of isolates of S. littoralis NPV which had been previously characterized and (III) isolates with no correspondence to any reported virus group. Of the S. litura NPV wild stocks, two were mixtures of more than two different groups of NPVs. We have discussed the advantage of having a mixture of different NPV groups in the same wild virus stocks.

We studied the genetic expression of gp85 of avian leukaemia virus (ALV) subgroup A in a baculovirus/insect cell system. 5′-terminal sequences of the gag gene were added to precede the ALV gp85 sequence and a stop codon was introduced at the boundary of gp85 and gp37. The resulting construct was then cloned into the baculovirus transfer vector pAcYM1, which contains the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Cells of the insect Spodoptera frugiperda (Sf9) were cotransfected with the resulting recombinant transfer vector pAc85 and infectious AcNPV/E2 DNA. After cotransfection, recombinant baculovirus that lacked the polyhedrin gene and expressed gp85 was selected from the supernatant and used to infect Sf9 cells. The expression of the gp85 gene peaked 3 days after infection, but expression products were not released into the culture medium even though the signal peptide had been cleaved. Owing to incomplete N-glycosylation in the insect cells the largest gp85 product had an M r of only 65000. In immunofluorescence tests and immunoblots the recombinant gp85 products reacted with polyclonal and monoclonal antibodies directed against ALV gp85 of subgroup A. Chickens inoculated with crude lysates of Sf9 cells infected with gp85-expressing recombinant baculovirus developed antibodies directed against ALV gp85. These antibodies were not capable of neutralizing ALV.

The major core protein, VP7, of bluetongue virus serotype 10 (BTV-10) has been purified from insect cells infected with a genetically manipulated recombinant baculovirus. The high level expression of VP7 (in excess of 100 mg per litre of culture) and its presence in the soluble fraction of infected cells following lysis by detergent has allowed the purification of the protein virtually to homogeneity (95%) by a simple two-step procedure of ammonium sulphate fractionation and ion-exchange chromatography. The purified antigen is highly immunogenic and has been shown in an ELISA to be reactive with antisera of 24 BTV serotypes (1 to 24) as well as with an antiserum raised to African horsesickness virus type 4 (AHSV-4), a representative of another serogroup of orbiviruses. In confirmation of these data a monospecific antiserum raised with the expressed product has been shown by Western blot analyses to react with other BTV serotypes as well as with two serotypes of epizootic haemorrhagic disease virus (EHDV-1 and EHDV-2), a closely related orbivirus. The data indicated that VP7 is a highly conserved protein amongst BTV serotypes and at least partly conserved amongst three serogroups of orbiviruses.

Infectious hypodermal and haematopoietic necrosis (IHHN) is one of the most important viral diseases of cultured penaeid shrimps and is potentially a limiting factor in the development of farming projects for some species of these shrimps. Although the IHHN agent was recognized early as being viral in origin, attempts to characterize it were inconclusive because of difficulties in obtaining sufficient amounts of purified virions to permit its characterization. Recent improvements of purification procedures have allowed the physic chemical characterization of this virus. Purified IHHNV is a non-enveloped icosahedral particle averaging 22 nm in diameter, exhibiting a mean buoyant density of 1·40 g/ml in CsCl. The genome is a single molecule of ssDNA with an estimated size of 4·1 kb by molecule length measurement in transmission electron microscopy. As determined by SDS-PAGE, the particle contains four polypeptides with M rs of 74K, 47K, 39K and 37·5K, respectively. From its characteristics, this virus could be a member of the Parvoviridae family.

We have expressed a number of polypeptides derived from the capsid proteins of the human parvovirus B19 in Escherichia coli. These include native VP1 (84K) and VP2 (58K) proteins and also fusions to β- galactosidase containing differing amounts of the amino terminus of the VP 1/2 polypeptide. Although each of these was expressed at high levels and the majority were produced as full-length proteins, only one was soluble. This soluble polypeptide, pi32, is a β- galactosidase fusion protein that includes 145 amino acids from B19 which are entirely derived from the region unique to VP1. Despite containing such a small portion of VP1, which itself constitutes only 4% of total capsid protein, p132 reacted with all our known anti-B19 IgM-positive human serum samples. We conclude that this region contains epitopes which must be prominently exposed on the intact virus. We have demonstrated the use of this recombinant antigen in a simple diagnostic assay for B19-specific antibodies which can be used for initial screening of human serum samples. In a survey of 103 serum specimens, our ELISA positively identified all samples (19/19) which were positive by IgM antibody capture radioimmunoassay. The recombinant p132 antigen is efficiently produced and readily purified from E. coli, and its use as a diagnostic antigen should increase the availability of routine clinical testing for human parvovirus infection.

Murine rhonoclonal antibodies (MAbs) were made to the 52000 (gp52) and the 93000 to 130000 Mr (gp93–130) glycoproteins from a human cytomegalovirus (HCMV) glycoprotein complex designated gC-I or the gB homologue. MAbs recognizing either gp52 or gp93–130 could immunoprecipitate unreduced gC-I complexes from non-ionic detergent extracts of HCMV. Western blotting was performed with immunoaffmity- purified gC-I complexes which were reduced prior to analysis. MAbs made against gp52 recognized gp52 and a 158000 Mr glycoprotein (gpl58). MAbs which recognized gp93–130 in a Western blot also reacted with gpl58, which is a gC-I precursor glycoprotein. The origin of gp93–130 was demonstrated by the reactivity of our gp93–130 MAbs with a recombinant protein containing the N-terminal portion of the gB gene. These data are consistent with the hypothesis that gp52 and gp93–130 are generated from the same high Mr precursor by proteolysis. MAbs recognizing either gp52 or gp93–130 neutralized Towne strain HCMV, but MAbs recognizing gp52 required complement to neutralize whereas MAbs recognizing gp93–130 did not. It was also determined that gp93–130 and gpl58 have detectable amounts of 0-linked glycans but gp52 does not, showing a difference in the glycosyla- tion of these glycoproteins. Analysis of gC-I disulphide bonds showed that two types were present, one which was very susceptible to reduction and a second which was less susceptible. These complexes could consist of very susceptible inter-complex disulphide bonds and less susceptible intra-complex disulphide bonds.

Varicella-zoster virus (VZV) gene 62 encodes a protein with a predicted Mr of 140000 (VZV 140K) that shares considerable amino acid homology with the immediate early (IE) regulatory protein Vmwl75 of herpes simplex virus type 1 (HSV-1) and is believed to be its functional equivalent. We have tested this hypothesis by insertion of VZV gene 62 (expressed from the HSV- 1 IE3 promoter) into both IE3 gene loci in the short region repeats of the HSV-1 genome. The parent virus used for this manipulation was D30EBA, which is a variant of HSV-1 from which the majority of the Vmwl75 coding sequences have been deleted. Like other HSV-1 viruses lacking Vmwl75 function, D30EBA is able to grow only in cell lines which express Vmwl75 constitutively. The resulting recombinant virus, HSV-140, is able to propagate (but unable to form obvious plaques) on normal cell lines. The properties of HSV-140 were studied by monitoring the time course of polypeptide expression and DNA replication during normal infection. We found that at high multiplicity HSV-140 synthesized apparently normal amounts of many viral polypeptides but that the expression of certain late genes was reduced; this slight defect may be related to less efficient DNA replication by HSV-140. At low multiplicity HSV-140 expressed viral proteins inefficiently. Surprisingly, VZV 140K was produced in large amounts at later times of a normal infection, indicating that the polypeptide fails to autoregulate the IE3 promoter. The results strongly suggest that VZV 140K is able to perform most of the functions of Vmwl75 during growth of HSV-1, but that differences in detail lead to less efficient virus growth.

Three murine major histocompatibility complex (MHC) class II-restricted T cell determinants were identified in the major capsid protein L1 of human papillomavirus (HPV) type 16. Peptides derived from HPV-16 L1, which contain putative T cell epitopes located by a predictive algorithm, were synthesized and tested for lymphoproliferative activity by direct immunization, followed by in vitro assay of responses to peptides or recombinant HPV-16 L1. The MHC restriction of the stimulatory peptides was determined using blocking monoclonal antibodies against class II molecules. The responses, which were specific for the priming peptides alone, cross-reacted with recombinant L1 but not with analogous peptides derived from other HPV types.