- Volume 73, Issue 6, 1992

During the 1988/1989 influenza season, five antigenic reassortant influenza A (H1N2) viruses not previously isolated from man were isolated in Hebei province, People’s Republic of China. All isolates contained haemagglutinins (HAs) and neuraminidases (NAs) which were antigenically similar to those of the recent Russian (H1N1) and Hong Kong influenza A (H3N2) viruses, respectively. The results of antigenic and nucleotide sequence analyses revealed that the genes encoding the polymerase, nucleoprotein, NA, matrix and non-structural proteins of the reassortant A/Hebei/24/89 (H1N2) virus were derived from the H3N2 parent virus, whereas its HA gene was from the H1N1 parent virus. The nucleotide sequences of the HA (encoding the HA1 subunit) and NA genes of the reassortant viruses were also determined. Phylogenetic trees constructed from these data by the neighbour-joining method revealed that the HA gene of the reassortant virus was closely related to those of recent human H1N1 viruses, whereas the NA gene was related to a recent human Hong Kong (H3N2) virus lineage.

A T cell epitope of the influenza virus NS1 molecule was identified and shown to be a determinant used in class II major histocompatibility complex-restricted T cell responses to infectious virus. An I-Ed-restricted BALB/c mouse T hybridoma clone recognizing influenza virus A/Puerto Rico/8/34 (PR8; subtype H1N1) but not A/Udorn/72 (subtype H3N2) secreted lymphokines in response to purified recombinant NS1 or fusion proteins containing amino acids 1 to 81 or 1 to 42 of NS1. As expected for recognition of a non-virion protein, the clone failed to respond to u.v.-inactivated virus. The antigenic determinant was localized by synthetic peptides to amino acids 13 to 32 of NS1, explaining the lack of recognition of A/Udorn/72 virus which has an alanine to valine substitution at position 23 within the determinant. A single intranasal dose of infectious PR8 virus was found to elicit T cells that responded to peptide NS1 13–32, suggesting that this determinant is a significant target of T cells in normal infections. To stimulate helper T cell responses similar to those achieved with infectious virus, influenza virus vaccines may therefore have to include NS1 in addition to virion components.

Ultraviolet light-inactivated, non-infectious influenza virus is pyrogenic; virion components are probably responsible for this pyrogenicity. To try to identify the pyrogenic component, influenza virions were disrupted with either bromelain or sodium deoxycholate (DOC). Treatment of infectious virions with bromelain, under conditions that removed the surface glycoproteins (spikes), destroyed their pyrogenicity. The supernatant, containing non-aggregated and modified glycoproteins, was also non-pyrogenic. Disruption of virions with DOC considerably reduced pyrogenicity; however, some was retained by the sub-viral cores. Viral nucleoprotein and matrix protein, purified from the supernatant, were non-pyrogenic. Aggregated stellate clusters of surface glycoproteins separated on sucrose gradients were pyrogenic in half of numerous tests performed with different batches of material. Treatment of virus with ether resulted in complete loss of pyrogenicity. Liposomes made from extracted viral lipid were non-pyrogenic. In contrast, virosomes made from the viral lipid and the aggregated stellate clusters of surface glycoproteins were pyrogenic. Hence, optimum pyrogenicity depends upon the integrity of the virus particle, but haemagglutinin and/or neuraminidase appear essential, and lipid may be involved.

Negative-stranded virion RNA and oligonucleotide primers complementary to fusion (F) protein gene sequences were used to generate cDNA clones, revealing that the gene 5′-proximal to the F protein corresponded to the M2 (22K) gene, as in respiratory syncytial (RS) virus. The transcription start signal, GGGACAAGU, was identical to that of the F and matrix (M) proteins of turkey rhinotracheitis virus (TRTV). There were two sequences with the potential to function as transcription termination/poly(A) signals, located at nucleotides 751 to 762 and 777 to 787; 15 clones derived from mRNA indicated that the first of these sequences formed the major signal. Part of the next downstream (5′) gene was sequenced; unlike mammalian pneumoviruses the TRTV M2 gene did not overlap the beginning of the 5′-proximal gene. Northern blotting indicated that infected Vero cells contained less M2 mRNA than F mRNA and that about half of the M2 mRNA was present as a F-M2 dicistronic mRNA. The M2 gene contained two overlapping open reading frames (ORFs 1 and 2), as with RS virus. ORF 1 comprised 558 nucleotides with the coding potential for a 186 amino acid polypeptide, M r 20959, eight or nine residues shorter than for human RS virus strains. The overall amino acid identity was 40%, the N-terminal one-third of the proteins sharing 62% of residues, the remainder 29%. A hydropathy plot of the TRTV M2 protein had close similarity to that of the M2 of RS virus. The protein was predicted to have a basic character with no N-terminal signal sequence or other major highly hydrophobic sequences. In vitro translation of a transcript comprising both ORFs 1 and 2 produced a single product of apparent M r 23000, corresponding to the M2 product of ORF 1. Site-directed mutagenesis confirmed that this product was derived from ORF 1 and that frameshifting was not involved. The second ORF was expressed only from a transcript which lacked the AUG codons of ORF 1 and, although occupying a similar position to that in the RS virus M2 gene, had virtually no amino acid identity in its 73 residue length and was approximately 25% shorter than the corresponding RS virus ORF 2. The hydropathy plot of the potential products of the second ORFs of TRTV and RS virus showed little resemblance. Taken together these results suggest that ORF 2 is unlikely to be expressed in vivo. Our accumulated data show that TRTV has the partial gene order 3′ M-F-M2 5′, whereas the corresponding RS virus genes are arranged 3′ M-SH-G-F-M2 5′.

A pairwise comparison of the nucleic acid sequence of 168 bases from 152 wild-type or unique cell culture-adapted strains of hepatitis A virus (HAV) revealed that HAV strains can be differentiated genetically into seven unique genotypes (I to VII). In general, the nucleotide sequence of viruses in different genotypes differs at 15 to 25% of positions within this segment of the genome. Viruses from four of the genotypes (I, II, III and VII) were recovered from cases of hepatitis A in humans, whereas viruses from the other three genotypes (IV, V and VI) were isolated only from simian species developing a hepatitis A-like illness during captivity. Among non-epidemiologically related human HAV strains, 81 were characterized as genotype I, and 19 as genotype III. Within each of these major genotypes, there were two distinct groups (sub-genotypes), which differed in sequence at approximately 7.5% of base positions. Each genotype and sub-genotype has a characteristic amino acid sequence in this region of the polyprotein, with the most divergent genotypes differing at 10 of 56 residues. Strains recovered from some geographical regions belonged to a common (endemic) genotype, whereas strains from other regions belonged to several, probably imported, genotypes. Thus, HAV strains recovered in North America were for the most part closely related at the nucleotide sequence level, whereas in other regions, such as Japan and Western Europe, HAV strains were derived from multiple genotypes or sub-genotypes. These data indicate that patterns of endemic transmission can be differentiated from situations in which infections are imported due to travel.

The role of natural killer (NK) cells in the early immune response to a pancreatropic isolate of coxsackievirus B4 (CVB4) was investigated in a murine model of pancreatitis. Endogenous (background) NK cell activity in fresh spleen effector cells from eight mouse strains was compared with virus-augmented NK cell activity 4 days post-infection (p.i.). A significant virus-induced increase (P < 0.003) in NK cell activity was seen in seven of eight infected mouse strains, when virus titres in the pancreas were beginning to fall. Lesions in the exocrine pancreas were least extensive in the three strains with the highest endogenous NK cell activity. In C3H/HeJ mice that had been depleted of NK cells prior to infection with a low virus concentration, resistance to infection of the pancreas was completely abolished; myocarditis was also observed in one of these animals. Thus, NK cells may limit virus replication in the pancreas and play a role in resistance in C3H/HeJ mice. Virus-specific neutralizing antibody was not detected in the serum until 5 to 6 days p.i. in most strains and did not appear to influence pancreatic virus titres. It may be significant that CVB4 infection did not induce the expression of major histocompatibility complex (MHC) class I molecules on target acinar cells. With certain tumour cells, an inverse relationship between MHC class I expression and susceptibility to NK cell-mediated lysis is well documented.

The course of infection of a pancreas-adapted isolate of coxsackievirus B4 was followed over a 10 month period in a murine model. Following intraperitoneal inoculation a typical acute infection was seen in nine of 10 inbred mouse strains. Virus rapidly infected the exocrine pancreas, titres peaking 3 to 4 days post-infection (p.i.). Lesions were almost exclusively confined to pancreatic acinar cells and varied in severity among the inbred strains. Virus shed into the blood-stream was not cell-associated. Evidence of persistent infection was found in nine mouse strains and infective virus was recovered from the pancreas of seven strains for up to 10 months p.i. Approximately 28% of pancreases examined beyond the acute phase showed focal inflammation and 22% showed focal necrosis (cell death). Virus was occasionally recovered from other organs (heart, liver and spleen), but lesions were rarely seen. Virus-specific antigen was localized to small groups of pancreatic acinar cells using an indirect immunogold silver staining technique. These observations suggested that the virus persists in pancreatic tissues because it seems unlikely that virus disseminated from distant sites would cause such localized infection. In three of these strains, the course of infection may have been influenced by superinfection with mouse hepatitis virus.

The physical state of the human papillomavirus (HPV) genome is usually different in malignant lesions of the skin, in which it is generally found in episomal form, and genital mucosa, in which it is frequently integrated with disruption of the E2 gene. Using chimeric or natural HPV promoters in the presence of the bovine papillomavirus type 1 E2 gene product, we observed transcription activation or repression, depending on the distance of E2-binding motifs from the start site. We found a clear difference in the positions of E2-binding motifs in cutaneous and genital HPVs that may partly explain the selective pressure for genome integration of genital HPV types in malignant lesions.

We have constructed a recombinant Oka varicella vaccine virus expressing hepatitis B virus (HBV) surface antigen (HBsAg). HBsAg was synthesized as 26K and 30K proteins in infected cells and secreted into the culture supernatant as 30K and 35K proteins. Inhibitors and glycosidase treatments, and pulse-chase labelling experiments, revealed the glycosylation process of HBsAg. The latter was synthesized as a non-glycosylated 26K protein and subjected to N-linked glycosylation to form a 30K protein with high mannose glycans. Three species of dimers composed of 26K and 30K subunits were then formed with disulphide bonds. Both subunits of the dimers were further subjected to O-linked glycosylation and conversion from high mannose glycans to complex glycans followed by sialylation. Three species of dimers composed of 30K and 35K subunits were secreted into the culture supernatant as HBsAg particles. HBsAg was synthesized, glycosylated with both N- and O-linked glycans, sialylated, and then secreted into the culture supernatant within 1 h. These modifications of HBsAg by glycans might stabilize its structure and enhance its immunogenicity as a live HBV vaccine.

Epstein—Barr virus (EBV) is a major human pathogen for which the development of an effective vaccine remains an important goal. Rabbits were immunized with one of a set of 10 fusion proteins representing protein fragments from the EBV receptor-binding ligand and candidate subunit vaccine gp340. Sera from recipients of fragments from the amino-terminal half of the polypeptide chain bound gp340 in Western blot assays and ELISA but were not virus-neutralizing. The fine epitope specificity of these sera, and of EBV-neutralizing rabbit sera raised against whole EBV and gp340-containing immune-stimulating complexes, were assessed in a peptide ELISA. All but two of these sera bound peptides located between positions 236 and 327 in the 907 amino acids of the gp340 polypeptide chain. Among these it was possible to identify regions containing candidate virus-neutralizing B cell epitopes. The use of a gp340 fusion protein affinity column to isolate antibodies from EBV-neutralizing rabbit sera specific for this region suggests the presence of both continuous and discontinuous B cell epitopes with potential roles in EBV neutralization.

The amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) has protein kinase (PK) activity and properties similar to those of growth factor receptor kinases which can be activated to transforming potential. DNA sequences that encode the PK domain cause neoplastic transformation of immortalized cells. The studies described in this report used a spontaneous mutant (ts5-152) temperature-sensitive for the synthesis of ICP10 and the previously described ICP10 expression vectors to study the role of ICP10 expression in HSV-2 growth and neoplastic potential. The titres of the ts5-152 mutant are 1000-fold lower at 39 °C compared to 34 °C after 12 h post-infection. The efficiency of plaquing is 0.003. The growth defect at 39 °C correlates with decreased ICP10 synthesis. Sequence analysis of the PK domain of the ts5-152 ICP10 gene identified a pair of frameshift mutations resulting in a 19 amino acid residue substitution at positions 275 to 293 and a downstream single base pair mutation causing a substitution at position 309. Cloning of the mutant ICP10 gene from ts5-152 into a wild-type HSV-2 isolate resulted in a recombinant (859/152) with growth properties and rates of ICP10 synthesis at 39 °C similar to those of ts5-152. Cells transformed with u.v.-inactivated ts5-152, or the recombinant 859/152, have significantly decreased cloning efficiency in agarose at 39 °C, but only during the first 250 post-transfer population doublings. Anchorage-independent growth was observed in cells transfected with expression vectors pJW17 or pJW32 that express ICP10 or its PK domain, respectively. Cells transfected with the frameshift mutant pJW21 or the ICP10 carboxy-terminal vector pJW31 did not form clones in agarose.

The adenovirus (Ad) hexon contains both group- and type-specific antigenic determinants. To identify the latter, peptides were synthesized corresponding to residues 281 to 292 from loop 1 and 441 to 455 from loop 2 of the Ad2 hexon. These sequences display type-specific variation and have been shown by X-ray crystallography to be present on the surface of the virion. Antisera raised against the peptides bound both peptide and the native hexon in ELISA, and blocked virus infectivity, as determined by immunofluorescence or neutralization assays. The loop 1 peptide was shown to inhibit binding of the corresponding antiserum to the native hexon in ELISA and to abolish its neutralizing activity. Neither the loop 1- nor loop 2-specific antiserum neutralized the infectivity of Ad4 or Ad40. Neutralization did not appear to result from aggregation of virus particles and thus their inability to attach to the cell, because virions treated with immune serum were internalized to the same extent as those treated with preimmune serum. Examination of the immune response elicited by Ad2 infection revealed that antibodies directed against the L1 and L2 epitopes were also present in human serum. Thus, the variable regions exposed on the surface of the Ad2 hexon represent type-specific neutralizing antigenic determinants.

Two new mutants of Autographa californica multiple nuclear polyhedrosis virus affected in the morphogenesis of their polyhedra, designated M276 and M934, were investigated. Marker transfer experiments demonstrated that the observed phenotype was due exclusively to alterations in the polyhedrin gene. M276 contained a 229 base insertion near the carboxyl terminus coding region which resulted in synthesis of a truncated protein; M934 had a point mutation substituting phenylalanine for leucine at amino acid 183. Both mutations occurred in highly conserved regions of the protein and prevented the occlusion of virus particles, but did not affect targeting for the intranuclear ring zone. M276 was distinct in that it had prominent cytosolic condensations of polyhedrin, although these were probably due to decreased protein solubility. M934 polyhedrin condensations associated prematurely with calyx material such that it became incorporated into the condensation rather than at the surface. Results confirm that occlusion size and shape are features inherent to the polyhedrin protein, and suggest that polyhedrin conformation may help regulate the occlusion process.

The temporal expression of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin and p10 genes in Spodoptera frugiperda cells was studied using virus recombinants in which either gene was replaced by the juvenile hormone esterase (JHE) gene of Heliothis virescens. The JHE served as a highly specific and sensitive reporter for gene expression. Activation of the p10 gene followed a pattern different to that of polyhedrin. The p10 gene was activated a few hours earlier than the polyhedrin gene, but its expression reached a lower maximum level. Northern blot analysis complemented and confirmed the results obtained from the JHE assays. Co-infection of sense recombinants and those containing an anti-sense copy of the JHE gene in place of the polyhedrin or p10 gene resulted in reduced levels of JHE gene expression. These experiments independently supported the hypothesis that the p10 gene promoter is more active at earlier times post-infection than that of the polyhedrin gene. The results also highlight the potential of the antisense strategy as an experimental approach for the study of baculovirus gene regulation and possibly insect metabolism.

Restriction endonuclease analysis was performed on the genomic DNA of granulosis viruses isolated from noctuid species of six genera: Xestia c-nigrum, Autographa gamma, Hydraecia amurensis, Celaena leucostigma, Aletia pallens and Pseudaletia separata. All of the isolates gave very similar restriction endonuclease profiles with only minor variations. An isolate obtained from X. c-nigrum was chosen as the reference genotype, and a genomic library was constructed for this isolate using plasmid vectors. The genome was mapped using EcoRI, BamHI and BglII, and Southern hybridization; the size of the genome was estimated to be 179 kbp. Hybridization of labelled clones to fragments of other isolates revealed that genotypic variation among isolates resulted from changes in restriction sites, and from deletion or insertion of DNA. Comparative restriction mapping revealed that all of the isolates were variants of one virus, even though they originated from different host species.

A 6.4 kb region from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome was sequenced and found to contain open reading frames (ORFs) homologous to the polyhedron envelope (PE) protein coding sequence, and the C-terminal half of ORF 1, which is a gene located upstream of the PE protein gene in other baculoviruses. The proteins predicted from the LdMNPV genes encoding the PE protein, and ORF 1 demonstrated 27 and 34% amino acid sequence identity, respectively, with the corresponding genes in the Autographa californica multicapsid nuclear polyhedrosis virus.

A 1.85 kb region, containing an open reading frame (ORF) homologous to the baculovirus p39-capsid gene, was sequenced from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome. Analysis of the p39-capsid gene demonstrated that it was 39% and 47% identical in amino acid sequence with the homologous genes in the Autographa californica and Orgyia pseudotsugata MNPVs, respectively. Two late promoter elements located upstream of the p39 gene in the LdMNPV genome are conserved with two other baculoviruses, whereas an ORF located downstream is not conserved.

A bacterial lacZ gene was inserted into an isolate of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV). The transfer vector was constructed by site-directed mutagenesis of the translation start site of the LdMNPV polyhedrin gene, within the BglII E fragment of the viral genome. A multiple cloning sequence was inserted at this start site and used for the insertion of the lacZ gene into the transfer plasmid. Liposome transfection was used to cotransfect L. dispar tissue culture cells with viral DNA and the transfer plasmid. Recombinant LdMNPV isolates were purified by isolation of plaques producing β-galactosidase but not polyhedra. Restriction enzyme fragment profiles were used to determine the site of the lacZ gene insertion, and DNA sequencing of the 5′ and 3′ ends of the lacZ gene insert and the adjoining polyhedrin promoter and coding regions was performed to identify its precise location. Expression of the lacZ gene was examined by studying virus-induced protein using [35S]methionine pulse-labelling, SDS-PAGE fractionation and autoradiography. Expression of β-galactosidase was examined in tissue culture cells using colorimetric assays. The maximum rate of β-galactosidase production was approximately 50 international units (IU)/106 tissue culture cells/day between 3 and 4 days post-infection (p.i), and the peak total expression was 158 IU/106 cells 5 days p.i. β-Galactosidase activity was first detected 48 h p.i. in haemolymph samples from fourth instar L. dispar larvae injected with 106 p.f.u. of virus. The peak β-galactosidase activity in larval haemolymph samples was 1931 IU/ml of haemolymph at 11 days p.i., just prior to death.

A recombinant baculovirus was constructed containing a copy of the hepatitis B virus (HBV) genome which was inserted to produce an in-frame fusion of the precore (pre-C) coding region with the first 11 amino acids of the polyhedrin gene. The recombinant baculovirus expressed the 25K pre-C protein and two novel proteins, of approximately 93K and 72K. Both the 93K and 72K proteins are recognized by an anti-polymerase monoclonal antibody. Northern blot analysis of the mRNA produced during infection of Spodoptera frugiperda cells by the HBV recombinant baculovirus detected only one HBV mRNA species, suggesting that the three HBV-specific proteins expressed are translated from the same mRNA. No larger fusion proteins cross-reacting with either anti-core or polymerase antibodies were detected. These findings suggest that the two proteins encoded within the HBV polymerase gene are not produced via a core-polymerase fusion intermediate but by internal binding of ribosomes. These results are the first clear demonstration of efficient expression of two bona fide unprocessed polymerase proteins in a 1:1 ratio from an unspliced pre-C mRNA-like transcript. With the successful expression of the polymerase gene in insect cells it is now possible to produce large amounts of these proteins, allowing a more detailed structural and functional analysis of these proteins.