- Volume 75, Issue 9, 1994

A restriction map of the genomic DNA of Lactobacillus casei phage PL-1 was constructed using the restriction endonucleases BamHI EcoRI, HindIII, KpnI, NruI and XhoI. The PL-1 genome was 42·2 kb in size and had complementary cohesive ends forming a ring-like monomer. The cohesive ends, analysed with exonuclease III and SI nuclease, were 3 -terminated single strands protruding from both ends. The nucleotide sequence of the cohesive ends, determined by the dideoxynucleotide method, comprised four A + T and 10 G + C pairs: 5′ GAGGCCGACCGTTC 3′ /3′ CTCCGGCTGGCA- AG 5′. Thus, the cohesive ends of PL-1 DNA were higher in G + C content than those of other known bacteriophage DNAs.

Two filamentous phages of Xanthomonas campestris pv. vesicatoria and Xanthomonas oryzae pv. oryzae were isolated and designated ϕXv and ϕXo, respectively. They were similar to other filamentous phages of Xanthomonas in (i) shape, (ii) restrictive host specificity, (iii) high stability, (iv) an ssDNA genome, (v) a dsDNA as the replicative form (RF), (vi) propagation without lysis of host cells and (vii) ability to integrate into the host chromosome. These phages showed sequence homology to filamentous phage ϕLf of X. c. pv. campestris. ϕXv was inactivated by antisera against ϕXv, ϕXo and ϕLf, whereas ϕ Xo and ϕLf were inactivated only by their respective antisera and the anti-ϕXv serum. Both the single-stranded phage DNAs and the RF DNAs of ϕXv, ϕXo and ϕLf were able to transfect X. c. pv. vesicatoria, X. o. pv. oryzae and X. c. pv. campestris. Physical maps of ϕXv and ϕXo were constructed for the RF DNAs. Genome sizes were estimated, based on mapping data, to be 6·8 kb for ϕXv and 7·6 kb for ϕXo, larger than that of the ;ϕLf genome (6·0 kb). The difference in genome sizes appeared to result from insertions of large DNA fragments. These fragments and the regions mediating integration were localized in the physical maps.

To determine whether the aetiological agent of bovine spongiform encephalopathy (BSE) is pathogenic for mink, standard dark mink were inoculated with coded homogenates of bovine brain from the U.K. Two homogenates were from cows affected with BSE. The third was from a cow that came from a farm with no history of having had BSE or having been fed ruminant- derived, rendered by-products, the proposed vehicle for introduction of the BSE agent. Each homogenate was inoculated intracerebrally into separate groups of mink and a pool of the three was fed to a fourth group. Signs of neurological disease appeared in mink an average of 12 months after intracerebral inoculation and 15 months after feeding. Decreased appetite, lethargy and mild to moderate pelvic limb ataxia were the predominant clinical signs, quite unlike the classic clinical picture of transmissible mink encephalopathy (TME). Microscopic changes in brain sections of most affected mink were those of a scrapie-like spongiform encephalopathy. Vacuolar change in grey matter neuropil was accompanied by prominent astrocytosis. Varying greatly in severity from one mink to another, the degenerative changes occurred in the cerebral cortex, dorsolateral gyri of the frontal lobe, corpus striatum, diencephalon and brainstem. Although resembling TME, the encephalopathy was distinguishable from it by less extensive changes in the cerebral cortex, by more severe changes in the caudal brainstem and by sparing of the hippocampus. The results of this study extend the experimental host range of the BSE agent and demonstrate for the first time the experimental oral infection of mink with a transmissible spongiform encephalopathy agent from a naturally infected ruminant species.

Diagnosis of Crimean-Congo haemorrhagic fever (CCHF) virus infections is hampered by the problems of handling this human pathogen, which requires the highest levels of biological containment. Recombinant antigens were examined for their potential as nonhazardous diagnostic reagents. The nucleocapsid (N) gene of the Greek AP92 isolate of CCHF virus was sequenced from cloned PCR products and the open reading frame was identified by homology to the N protein of a Chinese isolate of CCHF virus. The N protein was expressed to high levels in a baculovirus expression system. Three N protein-derived peptides were expressed in Escherichia coli as fusions with glutathione S-transferase and the antigenicities of these proteins and the baculovirus-expressed protein were tested by ELISA. When tested with laboratory animal sera representing all seven serogroups of nairoviruses, the only reactive sera were those raised to CCHF virus (Greek, Nigerian and Chinese isolates) and, more weakly, Hazara virus. When tested with a panel of known positive and negative human sera, the baculovirus-expressed N protein, and the peptide derived from the central region of the N protein, proved to be the best for identifying CCHF virus-specific IgG.

Human CD46, a member of the family of regulators of complement activation, has been shown recently to act as a measles virus (MV) receptor, interacting with the virus envelope glycoprotein haemagglutinin (HA). Owing to alternative RNA splicing, several CD46 isoforms are co-expressed in all tissues except erythrocytes. The optional exons encode extracellular serine-, threonine- and proline-rich regions of CD46 (designated STP-A, -B and -C) which are located proximal to the plasma membrane, and alternative cytoplasmic tails (CYT1 or CYT2). The ability of the BC-CYT2, B-CYT2 and BC-CYT1 CD46 isoforms, expressed in rodent Chinese hamster ovary (CHO) cells, to mediate MV infection was tested. Every isoform was recognized by a monoclonal antibody (MAb), MCI20.6, which recognizes the MV-binding site on CD46. CHO cells expressing any of these CD46 isoforms were able to bind MV, the level of binding correlating with the CD46 expression level. Likewise, MV infection induced the cell-cell fusion of all CD46-expressing CHO cells but not of the parental CHO cells. Accordingly, MV replication was observed after infection of CHO cells expressing each CD46 isoform but not after infection of parental CHO cells. Finally, cell surface expression of every isoform was decreased after infection by MV. Altogether these data showed that the specific STP regions of CD46 played no major role in HA-mediated MV binding to CD46, virus infection and virus-induced down-regulation of CD46. Moreover, the CYT1 and CYT2 cytoplasmic tails of CD46 are either functionally similar although having distinct amino acid sequences or are dispensable for interaction with HA of MV.

The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome, (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties, (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface, (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not oligomerize and were retained in the endoplasmic reticulum. In conclusion, cysteine residues 287, 300, 381, 394, 494, 579 and 583 appear to play a particularly critical role in the antigenic structure and processing of the H molecules and they probably participate in the inter- or intramolecular disulphide bonding.

Pandemic strains of influenza A virus arise by genetic reassortment between avian and human viruses. Pigs have been suggested to generate such reassortants as intermediate hosts. In order for pigs to serve as ‘mixing vessels’ in genetic reassortment events, they must be susceptible to both human and avian influenza viruses. The ability of avian influenza viruses to replicate in pigs, however, has not been examined comprehensively. In this study, we assessed the growth potential of 42 strains of influenza virus in pigs. Of these, 38 were avian strains, including 27 with non-human-type haemagglutinins (HA; H4 to H13). At least one strain of each HA subtype replicated in the respiratory tract of pigs for 5 to 7 days to a level equivalent to that of swine and human viruses. These results indicate that avian influenza viruses with or without non-human-type HAs can be transmitted to pigs, thus raising the possibility of introduction of their genes into humans. Sera from pigs infected with avian viruses showed high titres of antibodies in ELISA and neutralization tests, but did not inhibit haemagglutination of homologous viruses, cautioning against the use of haemagglutination-inhibition tests to identify pigs infected with avian influenza viruses. Co-infection of pigs with a swine virus and with an avian virus unable to replicate in this animal generated reassortant viruses, whose polymerase and HA genes were entirely of avian origin, that could be passaged in pigs. This finding indicates that even avian viruses that do not replicate in pigs can contribute genes in the generation of reassortants.

A model of long term viral persistence has been established by selecting a spontaneous mutant strain of influenza C/Ann Arbor/1/50 virus in a permanent carrier culture of MDCK cells. Infectivity and cell tropism are mainly determined by the multifunctional viral membrane glycoprotein (HEF). HEF analysis was aimed at identifying a putative correlation between sequence and function, i.e. receptor binding, enzymatic activity, antigenicity and rate of infection. The current experimental picture is summarized by the following findings: (i) C/Ann Arbor/1/50 persistent virus carries a modified receptor-binding sequence, (ii) receptor-binding activity is altered, as indicated by a higher efficiency in recognizing low amounts of the receptor determinant N-acetyl-9-O-acetylneuraminic acid, (iii) direct attachment to cell surfaces differs from that of wild-type virus, as measured by slower kinetics of viral elution, (iv) receptor-destroying enzymatic activity is diminished, (v) characteristic features of virion surface morphology are altered or unstable, (vi) persistent-type HEF epitopes are distinguishable by monoclonal antibodies from wild- type and (vii) viral infectivity is intensified for cells bearing a low number of receptors. The sum of these changes highlights a structurally and functionally modified HEF glycoprotein that allows long term viral persistence. In order to clarify which of the described points are required for the persistent viral phenotype, a working concept is presented.

Brefeldin A (BFA) is a macrolide antibiotic that blocks membrane traffic through the vesicular system and affects the glycosylation of viral glycoproteins. Treatment of HeLa cells infected with Semliki Forest virus (SFV) with BFA enhances the synthesis of late viral proteins. Proteolytic cleavage of p107 is partially blocked and viral glycoproteins accumulate in BFA-treated cells. This enhanced synthesis of late SFV proteins is due, at least in part, to an increase in the formation of the subgenomic SFV 26S mRNA. Since BFA blocks the replication of poliovirus genomes without affecting the cleavage of the translation initiation factor p220, protein synthesis was analysed in doubly infected cells. HeLa cells infected with SFV and poliovirus at the same multiplicity predominantly synthesize poliovirus proteins. But if these cells are treated with BFA they synthesize significant amounts of SFV capsid protein C for several hours, despite the fact that p220 has been degraded.

The VP4, VP7, NS53 and VP6 genes of two porcine rotavirus variants which differ in their in vitro growth properties and pathogenicity have been cloned and sequenced. The VP4 genes show only 67·2% nucleic acid and 70·6% amino acid identity. The VP4 gene of one variant (4S) is closely related to that of the bovine UK rotavirus strain, whereas the VP4 gene of the other variant (4F) is only distantly related to known VP4 genes and is likely to represent a new P serotype. In contrast the NS53 (VP5), VP6 and VP7 genes of the 4F and 4S variants show greater than 99 % nucleotide and amino acid identity, indicating that the two viruses are genetically related by a reassortment event. The implications for the role of VP4 in the determination of in vitro growth characteristics and pathogenicity are discussed.

Processing and assembly of bovine leukaemia virus-like particles were studied in African green monkey kidney cells using recombinant vaccinia viruses (rWs) expressing regions of the bovine leukaemia virus genome. Unprocessed gag precursor protein (Pr44) was detected in immunoblot analysis of lysed cells and particles sedimented from culture supernatants after infection with a rW carrying the gag and truncated protease (pro) gene. Processing of Pr44 was observed after infection of cells with a rW carrying the gag and pro gene or a rW expressing the gag, pro and polymerase (pol) gene. Reverse transcriptase activity was detected only in association with particles produced by gag-,pro- andpol- expressing recombinants. Thin section electron microscopic analysis of infected cells and pelleted particles revealed that Pr44 and processed gag proteins assembled at the cell membrane. Pr44 was released into the cell culture media as immature virus-like particles, whereas processed gag proteins from rWs expressing gag and pro or gag, pro and pol formed mature particles.

Peripheral blood mononuclear cells (PBMCs) from bovine leukaemia virus (BLV) -seronegative cattle and from BLV-seropositive cows either with normal haematological values or persistent lymphocytosis were tested for their proliferative response to BLV antigens. Cells from only BLV-infected cattle with normal lymphocyte counts were stimulated to a detectable level by the fetal lamb kidney cell supernatant containing BLV antigens. Proliferation assays performed with the purified major core protein p24 indicated that this protein has to be processed through a chloroquine-sensitive compartment before being recognized by CD4+ T lymphocytes. Forty-one 15-mer overlapping peptides spanning the entire p24 sequence were synthesized and analysed for their stimulating potential. It appeared that two regions included T cell epitopes recognized by PBMCs from three of five animals tested. These regions were represented by amino acids 31 to 55 (PGSQVWIQTL-RLAILQADPTPADLE) and 141 to 165 (AESYVE-FVNRLQISLADNLPDGVPK). The possible implication of this cell-mediated immune response in BLV pathogenesis and vaccine development is discussed.

Human T cell leukaemia virus type 1 (HTLV-1) protease (PR14) was expressed in bacteria and purified by gel filtration. A continuous spectrophotometric assay was used to measure the kinetic parameters of substrate hydrolysis by PR14. Several peptide substrates containing HTLV-1 sequences known to be cleaved by PR14 were used. Cleavage analysis showed that the affinity with which PR14 binds these substrates is higher than that previously reported for HTLV-1 Gag peptides. Also, the affinities of peptides containing the sites involved in autocleavage of protease from its precursor are higher than for the peptides containing sites required for structural protein maturation. This suggests that the autocatalysis of protease from its own precursor has priority over other cleavage reactions and supports similar observations of an ordered hierarchy of processing events by retroviral proteases. As the N- and C-terminal regions of retroviral aspartic proteases are known to contribute to stability of the dimer by forming antiparallel β-strands, short peptides corresponding to these terminal sequences of HTLV-1 protease were tested for their ability to inhibit cleavage of substrates by PR14. Inhibition was seen with a C-terminal peptide corresponding exactly to the C-terminal 11 amino acids of the processed PR14, whereas a peptide containing a sequence situated further from the C terminus was less effective. An inhibitor of the protease of human immunodeficiency virus type 1, Ro 31-8959, was found to be a poor inhibitor of PR14.

Our previous studies have shown that human immunodeficiency virus type 1 (HIV-1), with mutations in accessory genes such as vif vpr or vpu, can generate persistent infection of MT-4 cells, whereas infection by wild-type or nef mutant HIV-1 causes extensive cell death. The possibility of generating a naturally attenuated form of HIV-1 with reduced cytopathogenicity in MT-4 cells was examined by in vitro serial passage of the wild-type and a nef mutant form of HIV-1, each derived from the infectious molecular clone pNL432. The ability to cause persistent infection was observed after four passages of wild-type HIV-1 with the frequency of persistence becoming progressively higher with serial passage. In contrast, persistent infection was not observed even after 50 passages of the nef mutant virus. Sequence analysis of the accessory gene loci in genomes recovered from the persistent infections caused by passaged virus revealed mutations in vif and vpr, but not in vpu. The processing of the Env precursor to mature forms was not modified in any of the passages of either wild-type or nef mutant HIV-1. However, when compared with acute infections caused by similarly passaged virus of both wild-type and ne/mutant HIV-1, persistent infections by passaged wild-type HIV-1 showed a significant decrease in the cell surface expression and function of Env. Cell surface CD4 was only partially down-regulated on cells acutely infected with the passaged viruses, whereas on cells persistently infected with passaged wild-type HIV-1 it was completely down- regulated. These results suggest that, during serial passage of HIV-1, mutations accumulate at least in the accessory genes vif and vpr in parallel with a lesser interaction between cell surface Env and CD4 molecules, and lead to the generation of less cytopathogenic viruses capable of persistent infection. Our results also suggest an important role for the nef gene product in the generation of HIV-1 strains that are less cytopathogenic.

Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) long terminal repeat (LTR) revealed a novel cis-acting positive and a negative regulatory element in the U3 region, located upstream of the enhancer-promoter region. These elements acted in a cell type-specific manner, being most active in human lymphocytic CEM cells, more active in Jurkat cells than in human monocytic U937 cells and least active in epithelioid HeLa cells. The down-modulatory effect of the negative regulatory element was abolished by HIV-2 Tat, suggesting the involvement of upstream DNA elements in optimal Tat-mediated trans-activation. The sequence elements that respond to T cell activation signals were also located in the upstream U3 region. Notably, the magnitude of the effect of the upstream regulatory elements depended on the basal activity of the LTR, which was also cell type-dependent. This emphasizes the importance of the cell-specific transcriptional factors and other effectors in regulating HIV gene expression. These observations may be relevant to the cell type-specific restriction of virus replication in vivo.

The three avian polyomaviruses budgerigar fledgling disease virus types 1 to 3 (BFDV−1 to −3) contain genomes of identical size, 4981 bp. With differences of up to only 15 bp between the three genomes, these viruses show distinct tropism for cultured cells of various avian species: infection of chicken embryo (CE) cells with BFDV−1 and −2 results in virus propagation, whereas BFDV-3 is not replicated; all three viruses replicate, with different efficiencies, in infected Muscovy duck cells. Transfection of CE cells with BFDV-3 DNA results in a single productive cycle. As shown by construction of hybrid virus genomes and site-directed mutagenesis, a single amino acid difference (glycine instead of valine or alanine) within the common region of the minor structural proteins VP2/VP3 is responsible for this type of abortive infection of CE cells. Further experiments indicate a defect in one of the early steps during infection, at or prior to uncoating.

Baculovirus-expressed human papillomavirus type 11 (HPV-11) major capsid protein (LI) virus-like particles (VLPs) were produced in insect cells and purified on CsCl density gradients. The VLPs retained conformational neutralizing epitopes that were detected by a series of HPV-11-neutralizing monoclonal antibodies. Electron microscopy determined that the HPV-11 LI

VLPs were variable in size with a surface topography similar to that of infectious HPV-11. The VLPs were very antigenic, and induced high titres of neutralizing antibodies in rabbits and mice when used as an immunogen without commercial preparations of adjuvant. These VLP reagents may be effective vaccines for protection against HPV infections.

Forty-two women attending a colposcopy clinic for evaluation of abnormal cervical cytology and 13 normal controls were studied for the presence of lymphocyte proliferation (LP) cell-mediated immune (CMI) responses and serological reactivity to E7 peptides of human papillomavirus type 16 (HPV-16). HPV was typed by Southern blot hybridization of exfoliated cervicovaginal cell DNA. Positive LP responses (stimulation index ⩾ 5·0) to one or more E7 peptides were observed in 28·6% (12 of 42) of patients and 23·1 % (three of 13) of controls. Of patients infected with HPV-16,-31 or -33, 63·6% (seven of 11) showed a positive LP response compared with 14-3 % (two of 14) of women infected with other HPV types (P = 0·02), 17·6 % (three of 17) negative for HPV (P = 0·02) and 23·1 % (three of 13) of controls (HPV status unknown) (P = 0·05). C-terminal peptide 109 (amino acids 72 to 97) elicited positive LP responses in 45-4% (five of 11) of patients infected with HPV -16, -31 or -33 compared with 71 % (one of 14) patients infected with other HPVs (P = 0·04), 5·9 % (one of 17) of women negative for HPV (P = 0·02) and 7·7% (one of 13) of controls (P = 0·05). HPV-16 group-specific LP responses of borderline significance were also observed against E7 peptides 103,105 and 108 (17−37, 37−54 and 62−80) (P = 0·07). ELISA reactivity (IgG) to E7 peptide 109 (72−97) was present in 7·7% (one of 13) of controls, 35·3 % (six of 17) of HPV-negative patients, 42·9 % (six of 14) of patients infected with other HPVs, and only 9·1 % (one of 11) of patients infected with HPV-16, -31 or -33. CMI responses to C-terminal HPV-16 E7 peptide 109 (72−97) were thus significantly related to ongoing cervical infection with HPV-16 and closely related types, whereas serological reactivity to E7 peptides was not HPV type-specific.

The wasp Campoletis sonorensis injects a segmented, double-stranded DNA polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. After parasitization, CsPDV protects the wasp egg and larva by selectively disabling the host's cellular immune response. Other host physiological systems including growth and development are affected to the apparent benefit of the parasite. To begin the characterization of the biochemical effects and mode of action of CsPDV on host growth, the titre of a developmentally regulated insect storage protein, arylphorin, was studied. Parasitized or virus-infected insects had substantially less circulating arylphorin than control insects. Fat bodies from parasitized larvae also synthesized less arylphorin in vitro. However, Northern blots of total RNA from parasitized and non-parasitized, control insects showed that the arylphorin transcript level was unaffected by parasitization suggesting a biochemical block at the translational level. In vitro translation followed by immunoprecipitation of arylphorin indicated that the mRNA was present and translatable at equal levels in both parasitized and control insects. Injection of purified virus elicited the response observed in naturally parasitized larvae, demonstrating that the effect on arylphorin synthesis is mediated, either directly or indirectly, by polydnavirus gene product(s).