- Volume 75, Issue 12, 1994

It is not fully understood how antigenic drift of the haemagglutinin of type A influenza virus in man occurs in the presence of the expected polyclonal antibody response to the five antigenic sites, A to E. Here we show that 12 % (11/92) of sera from mice which had mounted a secondary immune response to inactivated influenza virus were able to select escape mutants. No escape mutant was selected with serum from nonimmunized mice (0/65). Selection required only a single passage, and escape mutants were identified by their reaction with monoclonal antibodies (MAbs); all but one had altered reactivity at site A. Most of the site A escape mutants (7/10) were conventional in character and did not react in haemagglutination-inhibition (HI) or neutralization assays with the identifying MAb. The HA genes of three of these were part sequenced and had a predicted single amino acid substitution (Gly-144 → Glu) in site A. The other escape mutants (3/10) had a small (2-fold) reduction in HI and neutralization to the site A MAb, but no amino acid substitution in site A. The final mutant was a conventional site B escape mutant. To model antisera which selected escape mutants, we constructed ‘pseudo-immune sera’ using mixtures of two neutralizing MAbs in which the first MAb was held at a constant high concentration (1000 HIU/ml). Escape mutants could be selected to the first MAb when the titre of the second MAb was reduced to a low but still inhibiting concentration (1 to 3 HIU/ml). Mixtures of three MAbs also selected escape mutants with similar facility provided that the second and third MAbs were reduced to a similar low concentration. Thus it is possible that the ability of an antiserum to select escape mutants is due to the neutralizing antibody response being biased to an epitope/cross-reacting epitopes within a single antigenic site. However, when escape mutants were reacted in HI assay with their selecting antiserum, the maximum difference from the titre with wt virus was 75 %. The findings of this study may be relevant to the understanding of antigenic drift in type A human influenza virus, and to immune-driven antigenic variation in other virus infections.

Influenza C virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 6. Unspliced mRNA from this RNA segment, which has not been previously identified, can potentially encode a polypeptide that contains an additional 132 amino acids on the carboxy terminus of the M1 protein. Here the nucleotide sequences of RNA segment 6 of four influenza C strains, isolated in Japan between 1964 and 1988, were compared with the previously determined sequence of C/Ann Arbor/1/50. The results indicated that the deduced amino acid sequence of the carboxy-terminal 132 amino acid domain is conserved fairly well although it is more divergent than the M1 protein sequence. Examination of RNA segment 6-specific mRNAs also showed that unspliced mRNA is present, although in small quantities (~ 13 % of spliced mRNA), in influenza C virus-infected cells. To search for a polypeptide encoded by the unspliced mRNA, the extra carboxy-terminal domain was expressed in Escherichia coli as the glutathione 5-transferase fusion protein, and rabbit immune serum was raised against the purified fusion protein. Immunoprecipitation experiments with this antiserum revealed that a previously unrecognized protein of apparent M r ~ 18000, designated CM2, is synthesized in influenza C virus-infected cells.

Recent field isolates of measles virus (MV) obtained by using B95-8 cells have been reported not to agglutinate African green monkey red blood cells (AGM-RBC). Vero cell-adapted, plaque-forming strains derived from three field isolates at the third passage in Vero cell cultures (T8Ve-3, T11Ve-3 and N13Ve-3) also exhibited markedly decreased binding activity, as determined by infectivity-absorption and haemadsorption tests. On the other hand, binding activity of the respective strains at the twentieth passage (T8Ve-20, T11Ve-20 and N13Ve-20) increased to practically the same level as that of the Edmonston strain, a standard strain of MV passaged long-term. A membrane immunofluorescence test revealed that the decreased binding activity to AGM-RBC of T8Ve-3, T11Ve-3 and N13Ve-3 was not due to decreased expression of the haemagglutinin (H) protein on the cell surface. The deduced amino acid sequence of the H protein synthesized in T11Ve-3-infected cells was identical to that in T11Ve-20-infected cells, although a single amino acid alteration was observed when T8Ve-3 was compared with T8Ve-20. Similarly, approximately half of the N13Ve-20-infected cells synthesized an H protein identical to that produced in N13Ve-3-infected cells, and nevertheless, exhibited markedly increased haemadsorption. The present results suggest that a viral protein(s) other than the H protein contributed to the binding activity of MV to AGM-RBC.

We established HeLa cell lines that constitutively expressed the fusion (F) and/or haemagglutinin-neuraminidase (HN) glycoproteins of human parainfluenza virus type 4A (PIV-4A) and used them to analyse the roles of these glycoproteins in virus-induced cell fusion. No syncytium formation occurred, even in HeLa cells expressing both the F and HN proteins (HeLa-4aF+HN cells). Also no syncytium was found in a mixed culture of cells expressing the F protein (HeLa-4aF) and the HN protein (HeLa-4aHN). Syncytia were observed in HeLa-4aF cells transfected with the HN gene, but no syncytium formation was found in HeLa-4aHN cells transfected with the F gene. Co-cultivation of HeLa-4aF + HN cells with HeLa-4aF cells generated large polykaryocytes, whereas co-cultivation with HeLa-4aHN cells induced no cell fusion. Infection of HeLa-4aF cells with PIV-4A generated large syncytia and degenerated nuclei, whereas little or no polykaryocytes were found in HeLa-4aHN cells infected with PIV-4A. From the above findings, the following conclusions were drawn: (i) the expression of both the F and HN proteins in the same cell is necessary for cell fusion; (ii) the expression of the F protein alone enhances susceptibility to cell fusion; (iii) the constitutive expression of the HN protein promotes resistance to paramyxovirus-induced cell fusion.

Following infection of BALB/c fibroblastic (BF) cells with simian virus 5 (SV5) only low levels of infectious virus were produced and the majority of cells survived the infection. However at 1 day post-infection (p i ), near normal levels of all the virus proteins were synthesized and the virus genome was replicated. RNA analysis of the infected cells revealed that the levels of viral genomic RNA remained high over 5 days of infection, but that viral mRNA levels were significantly reduced by 3 days p.i. There was no evidence for the accumulation of defective genomes over this period. The reduction in mRNA levels was reflected by a concomitant decrease in the rate of ongoing viral protein synthesis. Despite the apparent decrease in viral transcription, comparative measurements of the relative levels of the different virus proteins at various times p.i. revealed that the levels of the P and NP proteins were similar at 1 and 5 days p.i. but the levels of Y, M and F declined. Immunofluorescence analysis supported this data showing that at later times p.i., although there were some cells which were positive for all the viral proteins, a high proportion of cells were strongly positive for NP and P but negative for M, F and HN proteins. In these cells, NP and P were often located in discrete cytoplasmic foci. A series of cell lines were established from BF cells that had been infected at high multiplicity. Immunofluorescence studies showed that only a minority of cells in these cell lines were infected. This suggests that upon cell division, in a proportion of cells, virus replication was not taking place; otherwise it would be expected that all the daughter cells would remain infected. However, upon co-cultivation of these cells with Vero cells (cells that are fully permissive for SV5 replication), non-defective virus could be recovered. Virus cytoplasmic inclusion bodies could still be detected in a small proportion of BF cells that had been infected at high m.o.i. and passaged 10 times over a 12 week period, and again low levels of infectious virus could be recovered from these cells. It is proposed that in these persistently infected cells, the majority of virus genomes reside in an inactive form in cytoplasmic inclusion bodies but from which virus may occasionally be reactivated. Studies on cell clones obtained from a Vero cell line persistently infected with SY5 for over 100 passages (in which there are large numbers of defective genomes) also showed that not all daughter cells remained infected upon cell division. At later times p.i. in BF cells, when the majority of cells were positive for only NP and P, the cells became more resistant to cell-mediated immune lysis. The significance of these results in terms of the biology and immunology of paramyxovirus infections is discussed.

TsW16B is a temperature-sensitive mutant of vesicular stomatitis virus. Others have shown that it is temperature-sensitive for replication in vivo and for transcription in vitro and that these phenotypes are probably due to mutation of the N (nucleocapsid) gene. Five independent revertants were isolated from tsW16B based on their ability to grow at 39 °C. The thermosensitivity of in vitro transcription by these revertants was similar to that of the wild-type virus [Wt (HR) ] from which tsW16B was derived. Fractionation-reconstitution studies of two revertants indicated that the reversion was in the N or P (phosphoprotein) gene. The N and P genes of ts(HR), tsW16B, and these two revertants were sequenced. There were no differences between the P genes. Comparison of the predicted N protein sequences of ts (HR), tsW16B and the two revertants indicated that the growth and in vitro transcription phenotypes of ttW16B were due to a change of amino acid residue 238 from threonine to isoleucine. The amino acid at position 238 in the other three revertants also showed an exact reversion to threonine. Amino acid residue 238 lies in a domain of the N protein which is highly conserved among vesiculoviruses.

Respiratory syncytial viruses (RSVs) cause serious respiratory tract disease in infants and children worldwide and similar disease in calves. Strains have been isolated from other ruminant animals such as sheep and goats, but these viruses have not been characterized at the molecular level. In this study, we report the cloning and sequencing of four structural genes coding for the phosphoprotein, nucleocapsid (N) protein, matrix (M) protein and 22K protein of an ovine RSV strain. Comparisons of these sequences with those of bovine and human RSV show that the M and N proteins are the most conserved between ruminant RSV strains and the N protein is the most conserved protein between human and ruminant RSV strains. The attachment G glycoprotein and the small hydrophobic protein are the most divergent proteins among human and ruminant RSV subgroups.

The measles virus (MV) haemagglutinin (HA) is a class 2 glycoprotein by means of which the virus particle attaches to the host cell receptor. We have previously expressed this glycoprotein as a vaccinia recombinant virus and have shown that the HA glycoprotein synthesized is indistinguishable from that coded by MV. In the present study, we report that in RK13 cells a soluble form (sHA) of the HA is secreted into the medium. We show by SDS-PAGE and sucrose density gradient centrifugation that the sHA is a dimer and is smaller than the cell-associated form. Using a variety of inhibitors the production of sHA was shown to be a late event, probably occurring at the membrane; only fully glycosylated molecules were found in sHA. Finally, we demonstrate that sHA retains its antigenicity with conformation-dependent MAbs and its receptor recognition function. We conclude that sHA is a valuable tool for use in studies of the structure and function of the MV HA glycoprotein.

We have cloned and sequenced the entire fusion (F) protein gene of the RBOK vaccine strain of rinderpest virus and the coding regions for the F genes of two mild field isolates of the virus from Africa. Analysis of the nucleotide and the predicted amino acid sequences showed that the vaccine virus was more than 99% identical in the protein coding region to the virulent Kabete O strain from which it was derived, whereas the field isolates differed by 10 to 12% from each other and from the vaccine strain. No changes were found in the F protein which could explain attenuation of the vaccine; however, each of the mild field isolates had amino acid changes in important functional areas which may be related to their attenuated phenotype.

We reported previously that the antigenicity of the haemagglutinin-esterase (HE) glycoprotein of the human influenza C virus strain C/Nara/1/85 was indistinguishable from that of strain C/Nara/82. However, the ribonuclease T1-oligonucleotide map of total virion RNA of C/Nara/1/85 differed remarkably from the map of C/Nara/82, resembling instead the map of C/Nara/ 2/85, which has an HE antigenicity dissimilar to C/Nara/82 and C/Nara/1/85. This observation raised the possibility that C/Nara/1/85 might have arisen by reassortment from two viruses closely related to C/Nara/ 82 and C/Nara/2/85, respectively. Here, we compared the total nucleotide sequence of the HE gene and partial sequences of the other genes of C/Nara/1/85 with those of C/Nara/82 and C/Nara/2/85. The results suggest that C/Nara/1/85 has inherited HE and NP genes from a C/Nara/82-related virus and the PB2, PB1, PA, M and NS genes from a C/Nara/2/85-related virus.

The genomic sequences of many hepatitis C virus (HCV) isolates have been reported and a variety of virus genotypes have been classified based on homology in the conserved regions. We have previously identified five distinct genotypes (1a, 1b, 2a, 2b and 3b) in Japanese patients with chronic HCV infection by comparing the sequences of the NS5 region. The complete nucleotide sequence for five genotypes (1a, 1b, 1c, 2a and 2b) have already been reported and we report here the complete nucleotide sequence of genotype 3b. The isolate (HCV Tr) was 9439 nucleotides long, excluding the poly(U) tract at its 3′ end, and encodes a single long open reading frame of 3023 amino acids. Total nucleotide sequence homologies were 68·4 to 68·7%, 68·3 to 69·0%, 67·2%, 65·8 % and 65·6 % compared with type 1a, 1b, 1c, 2a and 2b genomes, respectively. The amino acid sequences of these five genotypes were highly homologous in the core, NS3 and NS5B regions, but the E2/NS1 region, which contains hypervariable regions 1 and 2, and the NS5A region were poorly conserved. Although it was possible to detect antibody against the relatively homologous core and NS3 regions by ELISA, the presence of divergent protein structures must be taken into account in the development of a vaccine.

The gene encoding the outer capsid protein, VP2, of African horsesickness virus serotype 3 (AHSV-3) has been sequenced in its entirety from cDNA clones of the segment 2 RNA, and compared with the previously published VP2 gene sequence of AHSV-4. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50·5 % identity to the amino acid sequence of AHSV-4 VP2. Two areas of high variability (approximately 35 % identity) were identified. The N-proximal variable region (amino acids 128 to 309) exhibited significant hydro-philicity, suggesting a possible role in the determination of the serotype-specific immune response. VP2 of AHSV-3 has furthermore been expressed in a baculo- virus expression system. The expressed protein was shown to react specifically with an anti-AHSV-3 serum in Western blots. Antibodies raised in rabbits and guinea-pigs against the recombinant VP2 neutralized the virus in a plaque reduction assay, confirming the identity of VP2 as the major neutralization-specific antigen of AHSV.

The nucleotide sequences of the genomic RNA segments 5 (gene 5) encoding the non-structural protein NSP1 of rotavirus strains M37 and ST3, isolated from neonates with asymptomatic infection, were determined. The sequences were similar overall (95 % identity) as were the deduced amino acid sequences of NSP1 (93 %). However, the M37 and ST3 NSP1 proteins shared only 82% and 81% identity, respectively, with the corresponding protein from another strain isolated from a neonate with asymptomatic infection (1321). Differences (of between 15% and 31 %) were found in comparison with NSP1 sequences of rotaviruses isolated from older children with symptomatic infection (Wa, DS1 and IGV-80–3). Using an M37 gene 5-derived probe, Northern hybridization analysis of total genomic RNA extracted from viruses isolated from older children (Wa, RV4, RV5 and P) and neonates (M37, ST3, RV3 and 1076), representatives of the most common human G and P types, further indicated that while the gene 5 alleles of strains M37, ST3 and RV3 had a high degree of identity, no significant identity between 1076 and M37 was observed. In addition, cross-hybridization between the M37 probe and RNA of strains from older children (Wa, RV4 and P) was evident. Thus, neonatal human rotavirus strains do not carry a common NSP1 gene.

By using a focus reduction assay in CrFK feline fibroblast cells, virus neutralizing antibodies (VNA) to feline immunodeficiency virus (FIV) were demonstrated in cats that had been naturally or experimentally infected with FIV. The antigenic relatedness of four strains of FIV, divergent in nucleotide sequence within the env gene, was investigated by neutralization following adaptation of each virus for growth in CrFK cells. Two of the viruses were from The Netherlands (FIV/AM-4 and AM-6), one was from the U.K. (FIV/GL-8) and one was from the U.S.A. (FIV/PET). Reaction of the viruses in the neutralization assay with cat antibodies to homologous or heterologous strains indicated that while there was a degree of cross-reactivity between all four, there were consistent differences suggesting the existence of FIV neutralization subtypes. In particular, FIV/PET and FIV/AM-6 were closely related but FIV/PET and FIV/GL-8 were clearly distinct. VNA from naturally infected cats in the field showed a pattern of reactivity against FIV/PET and FIV/GL-8 that confirmed the antigenic diversity of FIV.

Feline immunodeficiency virus (FIV) provokes a disease in cats characterized by histopathological lesions similar to those observed in AIDS patients. In order to determine whether endothelial cells from brain microvessels are involved in the central nervous system disease to the same extent as macrophages and microglia, cells were isolated from healthy cat brains, cultured and infected in vitro with the FIV Villefranche IFFA 1/88 strain. The isolated cells displayed typical endothelial cell ultrastructural features and were characterized further by von Willebrand factor-labelling and the binding of specific lectins such as Ulex europaeus lectin on their membrane. They were also able to take up acetylated low density lipoproteins. Two weeks after infection, significant amounts of FIV p24 antigen were detected by indirect immunofluorescence in syncytia and single cells. Concomitantly, the same antigen could be detected in the culture medium of the infected cells by an ELISA technique. Numerous viral particles as well as different steps in the process of viral budding were observed under transmission electron microscopy. The synthesis of FIV p24 antigens still occurred in cells in which replication was blocked in the G2 phase with taxol. Our results suggest the possibility of a productive infection of brain microvascular endothelial cells by FIV in vivo, which could lead to important perturbations in the functions of the blood-brain barrier.

Proviral DNA was obtained from ex vivo peripheral blood mononuclear cells of 75 human T cell leukaemia/ lymphoma virus type I (HTLV-I)-infected individuals who were either asymptomatic or had adult T cell leukaemia or tropical spastic paraparesis/HTLV-I-asso- ciated myelopathy. Amplified long terminal repeats (LTRs) were analysed for restriction fragment length polymorphisms (RFLPs). The results, together with previously published LTR data (a total of 180 specimens analysed), showed the presence of 12 different RFLP profiles with four major molecular subtypes. Furthermore, a fragment of 413 bp (nucleotides 22 to 434) of the U3/R region was sequenced for 12 new HTLV-I specimens originating from Central and West Africa (8 cases), Iran (1 case), Caribbean (2 cases) and Reunion Island (1 case). Phylogenetic analysis using three different techniques (maximum parsimony, neighbourjoining and UPGMA) comparing these 12 strains (including four new African HTLV-I variants) with the 30 published partial HTLV-I LTR sequences (nt 120 to 434) showed the existence of clusters of molecular variants in discrete geographical areas. The topology of the phylogenetic trees is thought to reflect HTLV-I evolution and the migrations of virally infected populations in the recent or distant past. Furthermore, there was a nearly perfect concordance between the clustering based on the LTR sequence homologies and the LTR RFLP subtypes suggesting that this rapid and simple technique is well suited to the investigation of HTLV-I molecular epidemiology. These results allow a new phylogenetic classification of HTLV-I genotypes into five major molecular subtypes: Cosmopolitan (C) subtype widespread all over the world, Japanese (J) subtype, West African (WA) subtype, Central African (CA) subtype and Melanesian (M) subtype.

The nucleotide sequence of a 2·2 kb genome region of the baculovirus Cryptophlebia leucotreta granulosis virus (C1GV) encompassing the granulin gene and a second open reading frame, designated ORF909, was determined. The putative granulin ORF comprises 744 nucleotides encoding a polypeptide with a predicted M r of 29·3K. The 5′ leader of the granulin gene contains a canonical late baculovirus promoter (ATAAG) and differs only in two nucleotide positions from the sequence of Cydia pomonella granulosis virus (CpGV). Sequence comparison with the granulin genes of different granulosis viruses indicated a very close relationship between C1GV and CpGV of about 96% amino acid identity. ORF909 is 909 nucleotides in length and encodes a potential protein of M r 36·2K. It contains two zinc finger-like motifs, similar to ME53, which was previously described for Autographa cali- fomica multiple nucleocapsid nuclear polyhedrosis virus. ORF909 protein is significantly shorter than ME53 protein, lacking more than 105 amino acids of the amino terminus of ME53. Both genes contain an early and a late promoter motif suggesting a similar temporal regulation. Homologous sequences to ORF909 are also present upstream of the granulin genes of other granulosis viruses (GVs) indicating that this ORF is common in GVs.

A modified hepatitis B virus (HBV) surface antigen, the SA-28 protein, was constructed and expressed by recombinant vaccinia virus in mammalian cells. This protein was composed of a PreSl region-derived peptide (amino acids 21 to 47) that contained the hepatocyte receptor-binding site, joined to the C terminus of the major S protein at amino acid position 223. This modified surface antigen could be efficiently assembled into particles with a density of 1·23 g/ml and could be secreted from several mammalian cell lines. The results of immunoprecipitation revealed that the SA-28 protein was recognized by both the anti-S protein antibody and the anti-PreSl antibody. A strong antibody response, against both the S protein and PreSl epitopes, was induced in BALB/c mice immunized by the SA-28 particles, indicating good immunogenicity. These results suggested that the HBV surface antigen consisting of the SA-28 protein could be a promising candidate as a new HBV vaccine with higher efficacy.

We have previously demonstrated that efficient replication of mutant herpes simplex virus which fails to synthesize the polypeptide ICP34.5 is cell type and cell state dependent. ICP34.5 negative viruses do not grow in stationary state mouse embryo fibroblast 3T6 cells whereas the growth kinetics in BHK cells are indistinguishable from those of wild-type. We now demonstrate that this defect is not due to an inability of mutant virus to adsorb to 3T6 cells but rather to an inability to spread from the initially infected cells. Electron microscopic studies with wild-type HSV in both BHK and 3T6 cells revealed virus particles equally distributed between nucleus and cytoplasm, and additionally in the extracellular matrix. In BHK cells infected with the ICP34.5 negative mutant 1716, virus is likewise distributed between nucleus and cytoplasm but in 50 % of the infected cells there is marked delamination and swelling of the nuclear membrane. In addition there is evidence of a significant number of particles trapped between the nuclear lamellae. When 1716 is used to infect 3T6 cells, over 90% of the virus particles are confined to the nuclei and the number of infected cells remains constant between 24 and 48 h with no increase in the proportion of extracellular virus. Failure to express ICP34.5 appears therefore to result in a defect in virus maturation and egress from the nuclei of infected cells. Egress of HSV from the nuclei to the extracellular space is thought to occur via two pathways. We postulate that lack of expression of ICP34.5 results in one of these pathways being blocked. In BHK cells this leads to overloading of the alternative pathway with a buildup of particles in the nuclear lamellae and associated endoplasmic reticulum. In stationary state 3T6 cells, it appears that there is no functional alternative pathway. We conclude that ICP34.5 exerts an effect on HSV maturation by controlling the passage of virus through infected cells.