- Volume 81, Issue 2, 2000

Recombinant Erns glycoprotein of bovine viral diarrhoea virus (BVDV) has been tagged with a marker epitope or linked to an immunoglobulin Fc tail and expressed in insect and mammalian cell lines. The product was shown to be functional, both having ribonuclease activity and binding to a variety of cells that were permissive and non-permissive for replication of BVDV. Addition of soluble Erns to the medium blocked replication of BVDV in permissive cells. Binding of epitope-tagged Erns to permissive calf testes (CTe) cells was abolished and virus infection was reduced when cells were treated with heparinases I or III. Erns failed to bind to mutant Chinese hamster ovary (CHO) cells that lacked glycosaminoglycans (pgsA-745 cells) or heparan sulphate (pgsD-677 cells) but bound to normal CHO cells. Erns also bound to heparin immobilized on agarose and could be eluted by heparin and by a high concentration of salt. Flow cytometric analysis of Erns binding to CTe cell cultures showed that glycosaminoglycans such as heparin, fucoidan and dermatan sulphate all inhibit binding but dextran sulphate, keratan sulphate, chondroitin sulphate and mannan fail to inhibit binding. The low molecular mass polysulphonated inhibitor suramin also inhibited binding to CTe cells but poly-l-lysine did not. Furthermore, suramin, the suramin analogue CPD14, fucoidan and pentosan polysulphate inhibited the infectivity of virus. It is proposed that binding of Erns to cells is through an interaction with glycosaminoglycans and that BVDV may bind to cells initially through this interaction.

Healthy carriers of hepatitis C virus (HCV) infection exhibit a specific antibody response against all HCV antigens, which could play a role in disease control. Generation of panels of human antibodies may permit a thorough characterization of this response and further identify particular antibodies with potential clinical value. To this effect, we have established a human phage-display antibody library from a patient exhibiting a high antibody response against HCV antigens and no clinical symptoms of disease. This library was screened against a recombinant core antigen [amino acids (aa) 1–119] produced in E. coli. Two recombinant Fab-carrying phages (rFabCs) were isolated and characterized. Both rFabC3 and rFabC14 recognize aa 1–48 on core antigen, but rFabC14 is competed out by a synthetic peptide, C2–20 (aa 1–20), at much lower concentrations than rFabC3. In order to identify more precisely the recognition sites of these antibodies, we produced soluble forms of the rFabs (sFabs), and used them to pan a random phage-display peptide library. A single peptide sequence, QLITKPL, was identified with sFabC3, while two equally represented sequences, HAFPHLH and SAPSSKN, were isolated using sFabC14. The QLITKPL sequence was partially localized between aa 8 and 14 of core protein, but no clear homology was found for the two sFabC14 peptides. However, we confirmed the specificity of these peptides by competition experiments with sFabC14.

Chikungunya (CHIK) virus is a member of the genus Alphavirus in the family Togaviridae. Serologically, it is most closely related to o’nyong-nyong (ONN) virus and is a member of the Semliki Forest antigenic complex. CHIK virus is believed to be enzootic throughout much of Africa and historical evidence indicates that it spread to other parts of the world from this origin. Strains from Africa and Asia are reported to differ biologically, indicating that distinct lineages may exist. To examine the relatedness of CHIK and ONN viruses using genetic data, we conducted phylogenetic studies on isolates obtained throughout Africa and Southeast Asia. Analyses revealed that ONN virus is indeed distinct from CHIK viruses, and these viruses probably diverged thousands of years ago. Two distinct CHIK virus lineages were delineated, one containing all isolates from western Africa and the second comprising all southern and East African strains, as well as isolates from Asia. Phylogenetic trees corroborated historical evidence that CHIK virus originated in Africa and subsequently was introduced into Asia. Within the eastern Africa and southern Africa/Asia lineage, Asian strains grouped together in a genotype distinct from the African groups. These different geographical genotypes exhibit differences in their transmission cycles: in Asia, the virus appears to be maintained in an urban cycle with Aedes aegypti mosquito vectors, while CHIK virus transmission in Africa involves a sylvatic cycle, primarily with Ae. furcifer and Ae. africanus mosquitoes.

The calicivirus rabbit haemorrhagic disease virus (RHDV) possesses a 3C-like protease which processes the RHDV polyprotein. In order to monitor the proteolytic activity of the RHDV 3C-like protease on its putative target sequences, i.e. the 10 EG dipeptide bonds distributed along the large polyprotein, a new approach was carried out. Preliminary experiments showed that the luciferase gene when fused in-frame with a long gene yielded a fusion protein almost devoid of luciferase activity. This reporter system was used to test which EG dipeptide bonds were cleaved by the RHDV protease when the coding sequences of the dipeptides and their flanking sequences were inserted at the junction between the protease and luciferase genes. The coding sequences of the fusion proteins were cloned downstream of the T7 promoter and the proteolytic activity was evaluated by measuring the luciferase activity in both in vitro and ‘in vivo’ systems. The EG dipeptide bonds at positions 718–719, 1108–1109 and 1767–1768 were confirmed as cleavage sites and a new cleavage site EG (143–144) was identified.

Transmissible gastroenteritis coronavirus (TGEV) agglutinates erythrocytes of several species by virtue of sialic acid binding activity of the surface protein S. We have isolated and characterized five haemagglutination-defective (HAD) mutants. In contrast to the parental virus, the mutants were unable to bind to porcine submandibulary mucin, a substrate rich in sialic acid. Each of the mutants was found to contain a single point mutation in the S protein (Cys155Phe, Met195Val, Arg196Ser, Asp208Asn or Leu209Pro), indicating that these amino acids are affecting the sialic acid binding site. In four of the HAD mutants a nearby antigenic site is affected in addition to the sialic acid binding site, as indicated by reactivity with monoclonal antibodies. The parental virus was found to have an increased resistance to the detergent octylglucoside compared to the HAD mutants. This effect depended on cellular sialoglycoconjugates bound to the virion. If the binding of sialylated macromolecules was prevented by neuraminidase treatment, the parental virus was as sensitive to octylglucoside as were the HAD mutants. We discuss the possibility that the sialic acid binding activity helps TGEV to resist detergent-like substances encountered during the gastrointestinal passage and thus facilitates the infection of the intestinal epithelium. An alternative function of the sialic acid binding activity – accessory binding to intestinal tissues – is also discussed.

Porcine reproductive and respiratory virus (PRRSV) primarily infects and destroys alveolar macrophages of the pig. The aim of the present study was to characterize the changes of leukocyte populations in the broncho-alveolar lavage fluid (BALF) of PRRSV-infected pigs. Piglets were inoculated intranasally with PRRSV strain LV ter Huurne. On various days post-infection the piglets were sacrificed and the lungs removed, washed semi-quantitatively and analysed by flow cytometry. The total number of recovered BALF cells increased approximately 10 times between day 10 and day 21 of infection and decreased thereafter. The number of small low-autofluorescent cells (SLAC), i.e. lymphocytic and monocytic cells, increased very strongly from day 2 until day 21 of infection; in contrast, the number of large highly autofluorescent cells (LHAC), i.e. mostly macrophages, remained constant until day 14 of infection, increased slightly on day 21 and then decreased. On day 21 of infection in specific-pathogen-free piglets approximately 60% of the SLAC consisted of CD2+CD8+CD4−γδTCR− cells, which were partly CD8+CD6+ and partly CD8+CD6−. These phenotypes correspond to that of cytotoxic T-cells and natural killer cells respectively. From these results we can conclude that during a PRRSV infection the total number of BALF cells increases mainly due to an influx of lymphocytic cells with a cytolytic phenotype.

The pathogenic properties of four primary human immunodeficiency virus type 2 (HIV-2) isolates and two primary HIV-2 biological clones were studied in an in vivo human-to-mouse chimeric model. The cell-associated viral load and the ability to reduce the severity of the induced graft-versus-host disease symptoms, the CD4/CD8 ratio and the level of repopulation of the mouse tissues by the graft, were determined. All HIV-2 strains, irrespective of their in vitro biological phenotype, replicated to high titres and significantly reduced graft-versus-host disease symptoms as well as the CD4/CD8 ratios. Reduction of graft repopulation caused by infection with the respective HIV-2 strains showed that the in vitro replication rate, syncytium-inducing capacity and ability to infect human macrophages did influence the in vivo pathogenic potential whereas broadening of coreceptor usage did not.

To identify new subtype G human immunodeficiency virus type 1 (HIV-1) strains and AG recombinant forms, we collected 28 serum samples from immigrants to the Netherlands from 12 countries throughout Africa. Based on the gag sequences 22 isolates were identified as subtype A or G. Phylogenetic analysis of discontinuous regions ofthe gag (726 nt), pol (1176 nt) and env (276 nt) genes revealed 13 AG recombinants with the mosaic structure A gag /G pol /A env , three with A gag /G pol /G env and one other with A gag /G pol /G env , in addition to ‘pure’ subtypes A gag /A pol /A env (n=1) and G gag /G pol /G env (n=4). To analyse the crossover points in more detail, a new RT–PCR was developed resulting in a large contiguous sequence of 2600 nt from the gag region to half the pol region. All the 13 A gag /G pol /A env recombinants appeared to belong to the circulating recombinant form (CRF) AG (IbNG). The three A gag /G pol /G env recombinants differed from the CRF AG (IbNG) subtype, suggesting the identification of a new CRF subtype. The recovery of AG recombinants from African countries a thousand miles apart indicates the active spread of new recombinants.

The antiviral effects of recombinant ovine interferon-τ (roIFN-τ) were studied in 26 lambs inoculated with ovine lentivirus (OvLV) or mock-infected. Six of the OvLV-infected lambs and three of the mock-infected lambs were treated with 106 antiviral units (AVU) per kg roIFN-τ daily for 30 days starting at day 0 post-inoculation (p.i.) and twice a week thereafter (early treatment). Six of the OvLV-infected lambs and three of the mock-infected lambs were treated with 106 AVU/kg roIFN-τ daily for 30 days starting at day 150 p.i. and twice a week thereafter (late treatment). Six OvLV-infected and two mock-infected lambs were treated either early or late with placebo. Cell-associated viraemia was quantified by an end-point dilution method. The weekly antibody response against OvLV proteins was studied by ELISA. All experimental animals were killed at 27 weeks p.i. and histological sections of lung were scored for the degree of lymphoid interstitial pneumonia (LIP). A 90% reduction in OvLV titres was detected at 4 weeks post-treatment in lambs that received early roIFN-τ treatment (P<0·01). Differences in virus titres were also found at weeks 2 and 6 (P<0·05). Scores for LIP degree were higher in infected lambs treated with placebo or late roIFN-τ than in the mock-infected lambs or in the infected lambs that received early roIFN-τ (P<0·05). LIP scores were not different between mock-infected lambs and infected lambs that received early roIFN-τ. These results indicate that roIFN-τ curtails OvLV replication in vivo and reduces the likelihood of development of lentivirus-induced LIP when infected lambs are treated during the initial phases of OvLV infection.

Two serotypes, I and II, have been identified for infectious bursal disease virus (IBDV), a member of the family Birnaviridae. Here, the generation by reverse genetics of IBDV chimeras in segment A of the bisegmented genome is reported. The 5- and 3′-noncoding regions (NCRs) of a serotype II strain were exchanged with the NCRs of a full-length cDNA clone of segment A of a serotype I strain. Isolated chimeric viruses were characterized in cell culture and susceptible chickens. The results show that IBDV chimeras in segment A were able to replicate in cell culture and that VP1 encoded by a serotype I segment B is functionally active with serotype I NCRs as well as with serotype II NCRs. Chimeric viruses infected susceptible chickens and caused mild depletion of bursal cells. Thus, the noncoding regions of segment A are not responsible for the different pathotypes of IBDV serotypes I and II.

This study aimed to assess the role of specific human papillomavirus type 16 (HPV-16) variants, in combination with p53 codon 72 polymorphism genotypes, in cervical carcinogenesis. An initial sequence analysis of HPV-16 long control, E6 and E7 regions of 53 well-defined cervical samples containing HPV-16 revealed that a T to G transition at nucleotide position 350 within the E6 open reading frame was the most common variation, the frequency of which seemed to decrease with increasing severity of the lesion. Therefore, a total of 246 cervical samples of residents of The Netherlands was specifically analysed for HPV-16 350G/T variants and/or p53 codon 72 genotypes. These comprised HPV-negative normal cervical scrapes (n=40), normal cervical scrapes containing HPV-16 (n=46), scrapes containing HPV-16 from women with abnormal cervical cytology participating in a non-intervention follow-up study without (n=38) and with (n=51) a histologically proven cervical intraepithelial neoplasia (CIN) III lesion at the end of the study, and cervical squamous cell carcinomas (n=71). Neither specific HPV-16 350G/T variants nor specific p53 genotypes were associated with a higher risk of developing CIN III or cervical cancer. However, HPV-16 350T variants were significantly over-represented in p53 Arg homozygous women with cervical cancer. This suggests that, in p53 Arg/Arg women, infection with HPV-16 350T variants confers a higher risk of cervical cancer.

Recently, α6 integrin has been proposed as the epithelial cell receptor for papillomavirus. This study investigated whether α6 integrin is the cellular receptor for bovine papillomavirus type 4 (BPV-4), which is strictly epitheliotropic and infects the mucous epithelium of the upper digestive tract. Primary bovine mucosal keratinocytes from the palate of a foetus (PalK) displayed high levels of α6 integrin; matched primary fibroblasts from the same biopsy (PalF) expressed almost no α6 integrin. However, BPV-4 bound both PalK and PalF to similar, saturable levels. Native BPV-4 virions infected PalK in vitro, as detected by RT–PCR of E7 RNA. Infection could be blocked by excess virus-like particles (VLPs) and by neutralizing antisera against L1–L2 and L1 VLPs or by denaturation of the virions, supporting the view that infection in vitro mimics the process in vivo. α6 integrin-negative human keratinocyte cell lines were derived from patients affected by junctional epidermolysis bullosa presenting genetic lesions in their hemidesmosomes. The level of α6 integrin expression was determined in these cell lines by in situ immunofluorescence and FACS. Despite the absence of α6 integrin expression by BO-SV cells, they were bound by BPV-4 to similar, saturable levels as normal keratinocytes, KH-SV. Furthermore, BO-SV and KH-SV cells were both infected by BPV-4 to apparently the same extent as PalK cells. These results are consistent with the conclusion that α6 integrin is not the obligatory receptor for a bovine mucosotropic papillomavirus.

Aleutian mink disease parvovirus (ADV), causes an immune disorder with a persistent infection of lymphoid organs in adult mink. We studied replication of ADV in gel-supported histocultures prepared from adult mink mesenteric lymph node (MLN). Evidence of virus replication in the histocultures was first observed by indirect immunofluorescence 72 h after incubation with virus. Cells resembling lymphocytes and macrophages contained both ADV capsid (VP2) and nonstructural (NS1 and NS2) proteins, and were present in a distribution suggestive of infected cells within germinal centres. ADV replicative form and encapsidated virion DNA were also detected in infected histocultures at time-points after 72 h. In addition, we were able to passage ADV-Utah to a new round of histocultures. These results suggested that the infected cells were actual target cells for ADV replication and that productive ADV-Utah replication, complete with the generation of virus, was occurring in the histocultures. The mink MLN histocultures provide a system to study the replication and molecular pathogenesis of ADV in target tissues.

Infections with viruses of the feline parvovirus subgroup such as feline panleukopenia virus (FPV), mink enteritis virus (MEV) and canine parvovirus (CPV-2) [together with its new antigenic types (CPV-2a, CPV-2b)] have been reported from several wild carnivore species. To examine the susceptibility of different species to the various parvoviruses and their antigenic types, samples from wild carnivores with acute parvovirus infections were collected. Viral DNA was amplified, and subsequently analysed, from faeces or formalin-fixed small intestines from an orphaned bat-eared fox (Otocyon megalotis), a free-ranging honey badger (Mellivora capensis), six captive cheetahs (Acinonyx jubatus), a captive Siberian tiger (Panthera tigris altaica) and a free-ranging African wild cat (Felis lybica). Parvovirus infection in bat-eared fox and honey badger was demonstrated for the first time. FPV-sequences were detected in tissues of the African wild cat and in faeces of one cheetah and the honey badger, whereas CPV-2b sequences were found in five cheetahs and the bat-eared fox. The Siberian tiger (from a German zoo) was infected with a CPV-type 2a virus. This distribution of feline parvovirus antigenic types in captive large cats suggests an interspecies transmission from domestic dogs. CPV-2 sequences were not detected in any of the specimens and no sequences with features intermediate between FPV and CPV were found in any of the animals examined.

Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCVE promoter and to a lesser extent the JCVL promoter. Mutations in the NF-1 sites in the JCVE promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.

The avian polyomavirus budgerigar fledgling disease virus (BFDV) encodes an unusual set of four agnoproteins in its late upstream region. Of the two pairs of these proteins, which overlap each other in two different reading frames, the pL1-promoted agnoprotein-1a (agno-1a) is the dominant species and is able to support virus propagation in the absence of the other three polypeptides. Viral BFDV agno-1a, and also agno-1a expressed via an influenza virus vector, consists of a complex series of electrophoretically separable subspecies that can be reduced by phosphatase action down to a primary unphosphorylated protein with an apparent molecular mass of 31 kDa. Through peptide mass spectrometry and site-directed mutagenesis, the positions of four serine and three threonine residues have been determined as phosphate-accepting groups, which are partially modified by the combined action of three different cellular kinases. Since extensively phosphorylated agno-1a is required for its intracellular function, control over VP protein expression, and unphosphorylated agno-1a is observed as an additional component in the BFDV virion, both extreme subspecies appear to be drawn from that complex mixture, which also includes the intermediate stages of phosphorylation.

Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3–15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38–48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.

Hepatitis B virus (HBV) was partitioned into type, subtype and isolate categories and the average evolutionary distances within and between categories was plotted at each of 54 points along the genome. The graphs showed alternating variable and conserved domains within and between HBV subtypes and revealed that some specimens assigned to different groups are more similar across several contiguous intervals than specimens belonging to the same group. Isolates were screened individually to determine their conformation to type and mosaic structure was identified in 14/65 specimens. Two entire clades (six specimens) of genotype B had a B/C sequence switch in the core gene region, whereas six genotype D specimens showed D/A switching in one or more regions of the genome. Genotype E was not separate from genotype D in the X and C subgenomic regions. The nature and distribution of polymorphic sites in mosaic regions was mapped at both the nucleotide and protein levels and the position of the variant fragments was related to mutational hot spots and linear epitopes of HBV. Mosaic structure was demonstrated statistically in 11 isolates using bootstrap resampling and recombination, rather than random change, appeared to be the mechanism responsible. The sequence between and including the two DR regions was represented in all putative recombinants. The distribution of genetic distances over subgenomic regions showed that substitution rates are not constant among the lineages of HBV in the preS regions. Genotype F is the most diverse group. Only genotypes A, C and F partition consistently into subtypes.