- Volume 80, Issue 8, 1999

A warm-water fish cell line developed from blotched snakehead caudal peduncle (BSN) was found to have persistent birnavirus infection. Purified virus particles were of icosahedral shape and had 57±1·6 nm diameter. The BSN virus was resistant to 5-iodo-2′-deoxyuridine and induced yellowish-green cytoplasmic inclusions when stained with acridine orange. The virus was resistant to chloroform, acid and alkaline pH and heat treatment at 56 °C for 2 h. Purified virions had a buoyant density of 1·33 g/ml in CsCl and contained two genomic segments with molecular masses of 2·56×106 and 2·00×106 Da and four structural polypeptides of 112 (polyprotein, PP), 91 (VP1), 44 (VP2) and 37 (VP3) kDa. Reciprocal β cross-neutralization tests incorporating four classical strains of infectious pancreatic necrosis virus (IPNV) (WB, Sp, Ab and TV-1) and the BSN virus established the complete serological distinctness of the virus from IPNV. Considering the uniqueness of the virus, the name blotched snakehead virus is proposed for this agent.

Two types of strains of serotype I of infectious bursal disease virus (IBDV) have been described, on the basis of their ability (IBDV-TC) or inability (IBDV-BU) to infect chicken embryonic cells in culture. However, both types infect B lymphocytes in the bursa of Fabricius of young chickens. To determine the molecular basis for tissue culture infectivity, virus recombinants with chimeric segments A were constructed from IBDV-TC and IBDV-BU by reverse genetics. The region responsible for the different phenotypes was located in VP2. Site-directed mutagenesis identified single amino acids that are responsible for the restriction in infectivity. However, the appropriate amino acid exchanges are strain-specific.

An ovine group B rotavirus (GBR) isolate, KB63, was isolated from faeces of a young goat with diarrhoea in Xinjiang, People’s Republic of China. Sequence determination and comparison of genes 6 and 11 with the corresponding sequences of GBR strains ADRV and IDIR showed that they were the cognate genes encoding NSP1 and NSP5, respectively. While the overall identities of nucleotide sequences between these two genes and the corresponding genes of strains ADRV and IDIR were in the range 52·6–57·2%, the identities of deduced amino acid sequences were only 34·9–46·3%. These results demonstrate that the substantial diversity of NSP1 observed among group A rotaviruses (GAR) also exists within GBRs and that a high degree of diversity also exists among NSP5 of GBRs, in contrast to GAR NSP5. The NSP1 gene of KB63 contains three ORFs, whereas the NSP1 genes of other GBR strains contain only two. ORFs 2 and 3 of the KB63 gene may be derived from a single ORF corresponding to ORF2 of other GBR strains by the usage of a stop codon created by an upstream single base deletion and single point mutations. In vitro expression studies showed that ORFs 1 and 2, but not 3, of gene 6 can be translated, suggesting that ORF2 may encode a C-terminally truncated, potentially functional product. It may play a role, together with the product of ORF1, in virus replication, as the virus can be passaged further in kids. Similarly, gene 11 can be translated in vitro. Like its counterpart in GARs, the protein encoded by gene 11 was shown to be phosphorylated in vitro.

Transcription of oncogenes E6 and E7 of human papillomavirus type 16 (HPV-16) from the P97 promoter is regulated by viral and cellular proteins. The transcription factor YY1 represses transcription through binding to cognate sequences in the long control region (LCR). In HPV-16 DNA from cervical carcinomas, mutations of YY1-binding sites have been identified that increase P97 activity 3–6-fold. A second, SP1-binding site has now been identified in the HPV-16 LCR (nt 7842–7847), which overlaps the YY1-binding site at positions 7840–7848. A point mutation within this YY1 site in viral DNA from a cervical cancer, previously shown to prevent YY1 binding, was shown to increase SP1 binding and P97 activity 4·7-fold. An engineered mutant eliminating SP1 binding showed only 1- to 1·6-fold increased P97 activity. It is concluded that competition between SP1 and YY1 for DNA binding plays a major role in YY1 repression mediated by the binding site at positions 7840–7848.

In this study, we characterized the 5′ regulatory region of the murine cyclin E gene and analysed activation of the gene by the E7 oncogene of human papillomavirus type 16 in transfection experiments. We found that the murine cyclin E promoter is composed of multiple regulatory elements, and we present evidence for at least two independent transcription units, designated P1 and P2. Overlapping binding sites for the cellular transcription factors Sp1 and E2F were identified in both promoters, and we found that E2F-mediated activation of transcription is inhibited by Sp1 in cotransfection experiments. The E2F/Sp1 binding sites contribute to transcriptional activation by E7, and the data suggest that the cyclin E gene is rendered E7-inducible through the combination of several cis-acting elements which display only weak intrinsic responsiveness to E7.

TT virus is a newly described agent infecting humans. Initially isolated from a patient (initials T.T.) with unexplained hepatitis, the virus has since been found in both normal and diseased individuals. In the present study, we utilized genomic-length sequences from distinct genotypes of TT virus to design PCR-based assays using conserved oligonucleotide primers from three independent regions of the virus genome. Each of the three assays was found to be superior to the PCR-based assays previously published. The most sensitive of the new assays was utilized to demonstrate the prevalence of TT virus to be at least 34·1% in volunteer blood donors, 39·6% in commercial blood donors, 59·6% in non-A–GB hepatitis cases, 81·7% in injectable drug users and 95·9% in haemophiliacs. In an attempt to identify a possible source of human infection, we found TT virus sequences to be present in 19% of chickens, 20% of pigs, 25% of cows and 30% of sheep. Sequence determination and phylogenetic analyses demonstrated that isolates from farm animals were not genetically distinct from those found in humans. This study clearly demonstrates that previously reported PCR assays dramatically underestimate the true prevalence of TT virus within the human population. Due to the high rate of infection in both blood donors and those with non-A–GB hepatitis, these results question the causal role of TT virus in cases of unexplained hepatitis. Further, it is possible that domesticated farm animals serve as a source of human infection.

In a search for monoclonal antibodies (MAbs) that can bind hepatitis B virus surface antigen (HBsAg) with amino acid substitutions in the immune dominant ‘a’ region (escape mutants) we investigated the epitope recognition site of the human MAb 4-7B. Pepscan analysis and experiments with alanine substitution as well as substitutions known from nature pointed to residues 178–186 in the small S protein with the amino acid sequence PFVQWFVGL (key amino acids in bold) as the minimal epitope. Single amino acid substitutions at positions 122(R/K)(d/y), 134(Y/F), 145(G/R), 148(T/A) and 160(K/R)(w/r), representing ‘a’ region variants in recombinant HBsAg COS-I cells, did not influence binding of MAb 4-7B. Synthetic peptides (residues 175–189) including the 4-7B epitope sequence were able to evoke an anti-HBs response in rabbits. According to established polypeptide models, the 4-7B epitope region is located in the lipid layer of 20 nm HBsAg particles. The present results, however, suggest that residues 178–186 are exposed on the surface of the 20 nm particle. This may change our view of the structure of HBsAg.

This paper describes the use of one-step growth conditions to study the kinetics of duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Synchronized infection was achieved using partially purified DHBV virions at an m.o.i. of 640 DHBV DNA-containing virions per cell, and these conditions were shown to produce a single cycle of infection. In this model, input purified DHBV DNA was rapidly internalized by cells at ⩾0·5 h, and localized to the nucleus by 4 h, but both covalently closed circular (CCC) DNA and single-stranded DNA were not detected until 48 h post-inoculation (p.i.), suggesting that there was a ⩾40 h delay between DHBV localization to the nucleus and formation of CCC DNA. In contrast, CCC DNA can be first detected in hepatocytes at 6 h p.i. in in vivo infection of ducks with the same DHBV strain. In an analysis of the nuclear transport of the DHBV genome, release of nuclear viral DNA from a particulate form to a soluble nucleoplasmic form was only 50% complete by 48 h p.i. However, this process occurred simultaneously with genome uncoating since all soluble nucleoplasmic DHBV DNA was free of nucleocapsid material; this suggests that nucleocapsid disassembly and genome uncoating may occur at the nuclear membrane and not within the nucleus. Quantitative analysis demonstrated inefficiency in a number of steps including virus uptake and internalization, translocation of nucleocapsid across the nuclear membrane and antigen expression from intranuclear viral DNA.

Gene A40R from vaccinia virus (VV) strain Western Reserve has been characterized. The open reading frame (ORF) was predicted to encode a 159 amino acid, 18152 Da protein with amino acid similarity to C-type animal lectins and to the VV A34R protein, a component of extracellular enveloped virus (EEV). Northern blotting and S1 nuclease mapping showed that gene A40R is transcribed early during infection from a position 12 nucleotides upstream of the ORF, producing a transcript of approximately 600 nucleotides. Rabbit anti-sera were raised against bacterial fusion proteins containing parts of the A40R protein. These were used to identify an 18 kDa primary translation product and N- and O-glycosylated forms of 28, 35 and 38 kDa. The A40R proteins were detected early during infection, formed higher molecular mass complexes under non-reducing conditions and were present on the cell surface but absent from virions. The proteins partitioned with integral membrane proteins in Triton X-114. Canine pancreatic microsomal membranes protected in vitro-translated A40R from proteinase K digestion, suggesting the A40R protein has type II membrane topology. A mutant virus with the A40R gene disrupted after amino acid 50, so as to remove the entire lectin-like domain, and a revertant virus were constructed. Disruption of the A40R gene did not affect virus plaque size, in vitro growth rate and titre, EEV formation, or virus virulence in a murine intranasal model.

This study describes the outcome of herpes simplex virus type 1 (HSV-1) infection in an organotypic raft culture of spontaneously immortalized HaCat keratinocytes and human fibroblasts, as related to the virus load and epithelial stratification and differentiation. In this model, a confluent monolayer of HaCat keratinocytes was formed 60 h after seeding. Inoculation of HSV-1 before induction of differentiation by lifting of the culture to the air–liquid interface always resulted in a productive infection, but the virus yield was highest when the inoculation took place 72 h after seeding. Even at 0·1 p.f.u. per culture, the HaCat cultures became HSV positive. Infection of the full-thickness epithelium at 5 p.f.u. per culture resulted in a productive infection of the whole epithelium. The HaCat cells were about 10 times more sensitive to HSV-1 infection than the Vero cells in which the virus stocks were titrated. The raft cultures infected 30 min after lifting were negative by HSV-1 culture, and no HSV-1 antigen was detected by immunocytochemistry. PCR showed the presence of HSV-1 DNA and in situ hybridization showed reactivity with a latency-associated RNA probe, indicating the presence of a non-productive infection. Two different patterns of virus spread in epithelia were found: (i) lateral spread through the superficial layers of the epithelium and (ii) a demarcated infection throughout the whole thickness of the epithelium at the margins of the culture.

A rabbit polyclonal antiserum was raised against a recombinant 6×His–UL3 fusion protein expressed in Escherichia coli and used to examine the intracellular localization of the UL3 protein of herpes simplex virus type 2 (HSV-2). The antiserum reacted specifically with 31 and 34 kDa proteins in HSV-2 186-infected Vero cells and with 31 and 35 kDa proteins in UL3-expressing COS-7 cells. The UL3 protein localized both in the cytoplasm and in five to ten bright fluorescent granules in the nucleus close to the nuclear membrane at 4 h post-infection (p.i.). These structures became bigger at 5 h p.i. and showed doughnut-like forms at 6 h p.i. In transfected Vero cells, the UL3 protein localized exclusively in the nucleoplasm and specifically in the nucleolus. Five deletion mutants of the UL3 protein were constructed for transfection assays and the results showed that the region containing amino acids 100–164 was important for nucleolar localization. Moreover, green fluorescent protein (GFP)-targetting experiments showed that the region containing amino acids 100–164 was able to transport non-nucleolar GFP to the nucleolus as a fusion protein.

Equine herpesvirus type 4 (EHV-4) is a major respiratory pathogen of horses. Unlike most other members of the Alphaherpesvirinae, EHV-4 was regarded as non-neurotropic. Here, neural and lymphoid tissues of 17 horses have been analysed post-mortem. EHV-4 DNA was detected in 11 cases (65%) by PCR, exclusively in the trigeminal ganglia. In order to define the transcriptional activity, RNA preparations of 10 EHV-4 DNA-positive ganglia were investigated by nested RT–PCR. EHV-4-specific transcripts derived from genes 63 [herpes simplex virus type 1 (HSV-1) ICP0 gene homologue] and 64 (HSV-1 ICP4 gene homologue) were detected in six trigeminal ganglia. In one other case, only gene 64-specific transcripts were present. All of the transcripts proved to be antisense orientated when a strand-specific RT–PCR was applied. Type-specific primers for gene 33 (encoding glycoprotein B) served to detect transcripts of an acute EHV-4-infection, which were found in only one of the six ganglia positive for gene 63- and gene 64-specific transcripts. Overall, these studies clearly demonstrate that EHV-4 is latent in trigeminal ganglia.

The 24 kbp KpnI restriction fragment A from the unique long genome region of infectious laryngotracheitis virus (ILTV, gallid herpesvirus-1) has been sequenced. The analysed region contains 14 open reading frames sharing homology with conserved alphaherpesvirus genes. Arrangement of the UL6 to UL20 homologues of ILTV is almost identical to that found in the herpes simplex virus type 1 genome. As in other herpesviruses the UL15 gene consists of two exons and is expressed from a spliced mRNA. However, the UL16 gene, which is usually localized within the intron sequence of UL15, is not conserved at this position of the ILTV genome. Another unique feature is the absence of any putative N-glycosylation motifs within the deduced ILTV UL10 gene product, which is the homologue of the conserved herpesvirus glycoprotein M. After preparation of a monospecific antiserum, two distinct UL10 proteins with apparent molecular masses of 36 and 31 kDa were identified in ILTV-infected cells as well as in purified virions. None of these UL10 gene products is modified by N- or O-linked glycosylation. Isolation of a green fluorescent protein-expressing UL10 deletion mutant of ILTV revealed that this gene is not required for virus replication in cell culture.

Glycoprotein B (gB, gpUL55) is the major antigen for the induction of neutralizing antibodies against human cytomegalovirus (HCMV), making it an attractive molecule for active and passive immunoprophylaxis. The region between aa 552 and 635 of HCMV gB (termed AD-1) has been identified as the immunodominant target for the humoral immune response following natural infection. AD-1 represents a complex domain which requires a minimal continuous sequence of more than 70 aa for antibody binding. Neutralizing as well as non-neutralizing antibodies can bind to AD-1 in a competitive fashion. The fine specificity of AD-1-binding monoclonal antibodies (MAbs) and affinity-purified human polyclonal antibodies was analysed by using recombinant proteins containing single amino acid substitutions spanning the entire AD-1 domain. Our results revealed that all MAbs had individual patterns of binding to the mutant proteins indicating the presence of a considerable number of distinct antibody-binding sites on AD-1. The neutralization capacity of antibodies could not be predicted from their binding pattern to AD-1 mutant proteins. Polyclonal human antibodies purified from different convalescent sera showed identical binding patterns to the mutant proteins suggesting that the combined antibody specificities present in human sera are comparable between individuals. Neutralization capacities of polyclonal human AD-1 antibodies did not exceed 50% indicating that, during natural infection, a considerable proportion of non-neutralizing antibodies are induced and thus might provide an effective mechanism to evade complete virus neutralization.

Epstein–Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes. In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated. LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation. However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier et al., Journal of General Virology 77, 2807–2818, 1996). In this study a deletion mutation was introduced into the LMP2 gene using an E. coli mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells. In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported. Careful quantification of B cell transformation using the EGFP+LMP2− recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2− virus to immortalize primary human B cells when compared to that of wild-type virus.

Human herpesvirus-8 (HHV-8), a gammaherpesvirus that is thought to be the viral aetiologic agent of Kaposi’s sarcoma and primary effusion lymphoma, encodes a homologue to cellular interferon regulatory factors (IRFs). The HHV-8 IRF homologue (vIRF; ORF K9) has previously been shown to inhibit gene induction by interferons and IRF-1 and to transform NIH3T3 cells or Rat-1 cells. Additionally, expression of antisense to vIRF in BCBL-1 cells results in the repression of certain HHV-8 genes, suggesting that vIRF may also positively regulate gene expression. We demonstrate that vIRF activates transcription when directed to DNA by the GAL4 DNA-binding domain. GAL-vIRF truncation constructs that individually are incapable of activating transcription can cooperate in transactivation when coexpressed in HeLa cells, suggesting that multiple regions of vIRF are involved in transactivation. These studies broaden the potential mechanisms of action of vIRF to include transcriptional activation as well as transcriptional repression.

A putative non-hr origin of DNA replication was identified in the Spodoptera littoralis multinucleocapsid nucleopolyhedrovirus (SpliNPV) genome by transient replication assays. The putative SpliNPV ori was mapped to the PstI-J fragment between 75·1–77·9 map units in the SpliNPV genome. While the DNA sequence of the putative SpliNPV ori aligned with regions within the non-hr oris of Autographa californica, Orgyia pseudotsugata and Spodoptera exigua multinucleocapsid nucleopolyhedroviruses, it has limited DNA sequence identity with these elements. The sequence of the putative SpliNPV non-hr ori fragment contains a unique distribution of imperfect palindromes, multiple direct repeats and putative transcription factor-binding sites. Transient expression assays indicated that the putative SpliNPV ori fragment repressed SpliNPV lef-3 promoter-mediated luciferase reporter gene expression. However, the putative SpliNPV ori fragment itself was capable of directing luciferase expression in the absence of a recognizable baculovirus promoter element in an orientation-independent fashion, suggesting that DNA sequence motifs within its sequence can activate transcription. Gel mobility shift analyses confirmed that proteins within nuclear extracts from both uninfected and virus-infected cells bound with specificity to the putative SpliNPV ori fragment.