Dugbe virus ovarian tumour domain interferes with ubiquitin/ISG15-regulated innate immune cell signalling.

The ovarian tumour (OTU) domain of the nairovirus L protein has been shown to remove ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host cell proteins, which is expected to have multiple effects on cell signalling pathways. We have confirmed that the OTU domain from the L protein of the apathogenic nairovirus Dugbe virus has deubiquitinating and deISGylating activity and shown that, when expressed in cells, it is highly effective at blocking the TNF-α/NF-κB and interferon/JAK/STAT signalling pathways even at low doses. Point mutations of the catalytic site of the OTU [C40A, H151A and a double mutant] both abolished the ability of the OTU domain to deubiquitinate and deISGylate proteins and greatly reduced its effect on cell signalling pathways, confirming that it is this enzymic activity that is responsible for blocking the two signalling pathways. Expression of the inactive mutants at high levels could still block signalling, suggesting that the viral OTU can still bind to its substrate even when mutated at its catalytic site. The nairovirus L protein is a very large protein that is normally confined to the cytoplasm, where the virus replicates. When the OTU domain was prevented from entering the nucleus by expressing it as part of the N-terminal 205 kDa of the viral L protein, it continued to block type I interferon signalling, but no longer blocked the TNF-α-induced activation of NF-κB.

Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence.

The sequence of the replicase gene of porcine epidemic diarrhoea virus (PEDV) has been determined. This completes the sequence of the entire genome of strain CV777, which was found to be 28,033 nucleotides (nt) in length (excluding the poly A-tail). A cloning strategy, which involves primers based on conserved regions in the predicted ORF1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of PEDV. Primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire ORF1 of PEDV. Analysis of the nucleotide sequences revealed a small open reading frame (ORF) located near the 5' end (no 99-137), and two large, slightly overlapping ORFs, ORF1a (nt 297-12650) and ORF1b (nt 12605-20641). The ORF1a and ORF1b sequences overlapped at a potential ribosomal frame shift site. The amino acid sequence analysis suggested the presence of several functional motifs within the putative ORF1 protein. By analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in ORF1a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within ORF1b. Comparative amino acid sequence alignments revealed that PEDV is most closely related to human coronavirus (HCoV)-229E and transmissible gastroenteritis virus (TGEV) and less related to murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). These results thus confirm and extend the findings from sequence analysis of the structural genes of PEDV.

Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis.

Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) contains a tripartite negative-sense RNA genome. The smallest RNA segment, S, encodes the nucleocapsid protein N and a nonstructural protein, NSs, in overlapping reading frames. We have generated a mutant virus lacking NSs, called BUNdelNSs, by reverse genetics. Compared with the wild-type (wt) virus, BUNdelNSs exhibited a smaller plaque size and generated titers of virus approximately 1 log lower. In mammalian cells, the mutant expressed greatly increased levels of N protein; significantly, the marked inhibition of host cell protein synthesis shown by wt virus was considerably impaired by BUNdelNSs. When inoculated by the intracerebral route BUNdelNSs killed BALB/c mice with a slower time course than wt and exhibited a reduced cell-to-cell spread, and titers of virus in the brain were lower. In addition, the abrogation of NSs expression changed Bunyamwera virus from a noninducer to an inducer of an interferon-beta promoter. These results suggest that, although not essential for growth in tissue culture or in mice, the bunyavirus NSs protein has several functions in the virus life cycle and contributes to viral pathogenesis.

The Bunyamwera virus nonstructural protein NSs inhibits viral RNA synthesis in a minireplicon system.

The small (S) genomic segment of Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) encodes the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. In order to elucidate the function of NSs, we established a plasmid-based minireplicon system using mammalian cells that express large amounts of T7 RNA polymerase. Expression of N, the viral polymerase protein (L), and a minireplicon containing a reporter gene was sufficient to reconstitute functional virus nucleocapsids. Coexpression of NSs, however, led to a dose-dependent decrease in reporter activity without affecting expression of controls. The inhibition could not be reversed by overexpression of N, L or the minireplicon, indicating that the NSs effect was not caused by a reduction in virus gene expression. The NSs proteins of two other members of the Bunyavirus genus, Guaroa virus and Lumbo virus, were also inhibitory in our system. The intracellular localisation of Bunyamwera virus NSs was investigated and found to be predominantly cytoplasmic, but intranuclear inclusion was also detected. Taken together, these data suggest that, in mammalian cells, the bunyavirus NSs protein controls the activity of the viral polymerase by a highly conserved mechanism.

Further analysis of the genome of porcine epidemic diarrhoea virus.

We report here the continued determination and analysis of the nucleotide sequence of both wild type (wt) and cell culture adapted (ca) porcine epidemic diarrhoea coronavirus (PEDV). These studies were undertaken with two objectives in mind: the identification of common and divergent features in the genomic sequences of wt and ca PEDV which can explain the differences in virulence of these isolates and the further exploration of the relationship of PEDV to other coronaviruses.

Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs.

We provide the first report, to our knowledge, of a helper-independent system for rescuing a segmented, negative-strand RNA genome virus entirely from cloned cDNAs. Plasmids were constructed containing full-length cDNA copies of the three Bunyamwera bunyavirus RNA genome segments flanked by bacteriophage T7 promoter and hepatitis delta virus ribozyme sequences. When cells expressing both bacteriophage T7 RNA polymerase and recombinant Bunyamwera bunyavirus proteins were transfected with these plasmids, full-length antigenome RNAs were transcribed intracellularly, and these in turn were replicated and packaged into infectious bunyavirus particles. The resulting progeny virus contained specific genetic tags characteristic of the parental cDNA clones. Reassortant viruses containing two genome segments of Bunyamwera bunyavirus and one segment of Maguari bunyavirus were also produced following transfection of appropriate plasmids. This accomplishment will allow the full application of recombinant DNA technology to manipulate the bunyavirus genome.

Identification of proteins specified by porcine epidemic diarrhoea virus.

Up to now, little was known about the proteins of porcine epidemic diarrhoea virus (PEDV). Using (i) metabolic labelling, (ii) antisera directed against synthetic peptides and monoclonal antibodies, and (iii) eukaryotic and prokaryotic expression systems, we have started to identify and characterize these proteins. The nucleocapsid protein (N) of PEDV was identified as a phosphoprotein with a relative mobility (M(r)) of 57 k. No additional phosphoproteins were detected. At least three glycoproteins were virion associated. The 180/200 k band most probably represented the surface glycoprotein (S), whereas the 27 k protein was the membrane protein (M). The 21 k species could not yet be identified. A rabbit anti-M protein antiserum was generated by using synthetic peptides corresponding to the C-terminus of M. The specificity of this serum was reconfirmed by transient expression of the M-gene in Vero cells followed by immunofluorescence. In order to generate antisera against the putative gene products of ORF3 and of the internal N reading frames (I-1, I-2, I-3), additional anti-peptide sera were raised. The putative ORF3 product which had been tagged by a 6 Histidine tail, was expressed in E. coli and purified by nickel chelate affinity chromatography before 2-dimensional polyacrylamide gel electrophoresis and immunostaining with a rabbit anti-peptide serum directed against the N-terminus of the ORF3 product. Transient expression of the I-1 and I-3 reading frames was used to confirm the specificity of the corresponding anti-peptide sera. The immunological tools presented in this paper are now being used to identify the putative corresponding gene products specified by PEDV.

Identification of the membrane protein of porcine epidemic diarrhea virus.

Sequence information on the genome of porcine epidemic diarrhea virus(PEDV) has only recently been determined. In contrast, very little is known about the viral proteins. In the present report we have identified the membrane glycoprotein (M) of PEDV by use of rabbit anti-peptide sera and transient expression of the cloned M gene in Vero cells and by expression in the baculovirus system. The native M protein of PEDV is incorporated into virions, is N-glycosylated, and migrates with a relative mobility (Mr) of 27 k in polyacrylamide gels. In contrast, the M protein synthesized by recombinant baculoviruses migrates with a Mr of 23 k, that is, with identical mobility as the deglycosylated product of PEDV. Thus, it appears that M protein specified by the recombinant baculovirus is poorly, if at all, glycosylated. Using monoclonal antibodies and rabbit and rabbit antipeptide sera specific for the N and C termini of the M protein, we were able to show that a 19 k band detected in PEDV-infected cells but not in virions represented a fragment of M from which the C terminus had been cleaved off. Finally, by electron microscopy and immunogold labelling, the relative orientation of M within the virion envelope was determined as NexoCcyt. In conclusion, all of these data strongly support the hypothesis that PEDV should be classified with the group I coronaviruses.

Sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic ORF.

In order to investigate the genome organization of the porcine epidemic diarrhea virus (PEDV) further, cDNA clones covering the region between the nucleocapsid and the spike (S) protein genes were independently constructed and sequenced for the two virulent isolates Br1/87 and CV777. Of the three major ORFs identified, two were found to encode the major and minor coronavirus membrane proteins M and sM. A potentially single ORF, designated ORF3 according to the pattern of the viral subgenomic mRNAs, could be identified between the S and sM genes. A striking variability, essentially generated by short deletions clustered in a few loci, was observed in the ORF3 of both isolates. The largest predicted polypeptide of 223 amino acids showed homology with polypeptides potentially encoded by other members of the same genetic subset, including two shorter polypeptides of human respiratory virus HCV 229E and one of transmissible gastroenteritis virus TGEV. This homology suggests that the two HCV ORFs may have originated from a single precursor. The function of these polypeptides is not known, but the predicted products of the PEDV ORF3 and related ORFs share features suggestive of a membrane-associated protein.

Sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229E and porcine transmissible gastroenteritis virus.

The nucleotide sequence of 1.7 kbp cDNA, comprising the region nearest the 3' end of the genome of the porcine epidemic diarrhoea virus (PEDV), has been independently determined for two European isolates of PEDV. Almost identical results were obtained for the two isolates, which were derived from cases of PEDV infection in Belgium and Britain in 1977 and 1987, respectively. The sequences contained a 1323 nucleotide (nt) open reading frame (ORF), which showed moderate identity to the nucleocapsid (N) gene of other coronaviruses. The greatest similarity at both the nucleic acid and protein levels was to the human coronavirus 229E. The PEDV N gene was, however, notably larger than that of the human 229E and porcine transmissible gastroenteritis viruses. This reflects the presence of a putative insertion of approximately 135 nt located towards the middle of the N gene. A second 336 nt ORF, which might encode a leucine-rich protein similar to, but shorter than, the bovine coronavirus internal protein was found within the PEDV N gene. Several RNA motifs typical of coronaviruses were also observed. These results confirm the earlier provisional classification of PEDV as a coronavirus.

PCR detection of the sheep-associated agent of malignant catarrhal fever.

From a genomic library previously constructed from a lymphoblastoid cell line (LCL) propagated from a bovine case of sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine herpesvirus-2 (OHV-2), several OHV-2 clones were identified and characterised by hybridisation using probes from the unique region of the Alcelaphine herpesvirus-1 (AVH-1) genome. Nucleotide sequence from one clone was generated and the predicted amino acid sequence was found to contain regions of homology with the 140 and 160 kDa tegument proteins of Epstein-Barr virus and herpesvirus saimiri respectively. Oligonucleotide primers were constructed and a polymerase chain reaction (PCR) test was developed for the detection of OHV-2 viral DNA. Amplified product was identified by restriction with RsaI and BmyI. The primers were highly specific for OHV-2 DNA with a limit of detection of 6.4 pg of genomic DNA derived from the parent LCL. This was estimated to correspond to one diploid bovine cell. The PCR was successfully applied to detect OHV-2 DNA in peripheral blood leucocytes (pbl) from clinical cases of SA-MCF and normal sheep.

Sequence analysis of the nucleocapsid protein gene of porcine epidemic diarrhoea virus.

The nucleotide (nt) sequence of 1.7 kbp cDNA representing the 3' end of the PEDV genome has been determined. Viral RNA was reverse transcribed and the cDNA was amplified by polymerase chain reaction using degenerate primers. The sequences of the primers were based on conserved regions of coronaviral genomes. A 1323 nt open reading frame (ORF) showed good homology to the nucleocapsid (N) gene of other coronaviruses. The greatest homologies at the amino acid and the nt levels were observed with Human Coronavirus 229E. A second 336 nt ORF, which might encode a leucine-rich protein, was found within the N gene. Between the 3' end of the N gene and the poly(A) tail was a sequence of eleven nt, which is conserved among the other sequenced coronaviruses. Finally, a seven base sequence similar to the conserved intergenic sequences was present 5' to the N gene. These results confirm the classification of PEDV as a coronavirus.

The detection of Alcelaphine herpesvirus-1 DNA by in situ hybridization of tissues from rabbits affected with malignant catarrhal fever.

Tissue sections and cultured lymphocytes from rabbits clinically affected following experimental infection with Alcelaphine herpesvirus-1 (AHV-1) were assessed for the presence of viral DNA by in situ hybridization with the cloned major HindII repeat sequence of this virus. Small numbers of virus-infected cells were consistently detected only in submandibular lymph nodes, while other tissues showed no evidence of viral DNA. Virus titration in culture suggested that there were higher titres of virus in the lymph nodes, spleen and lung of infected animals than in the kidney or peripheral blood lymphocytes and confirmed the low level of virus in these animals. Substantially more viral DNA was detected by in situ hybridization in lymphocytes following at least 24 h of culture, suggesting that viral replication is normally repressed by the host.

Recovery of a herpesvirus from a roan antelope (Hippotragus equinus).

A herpesvirus was recovered in culture from the cells of a roan antelope (Hippotragus equinus) following cryopreservation in DMSO and it is thought that the DMSO may have been involved in reactivation. The virus was shown to be antigenically related to alcelaphine herpesvirus-1 (AHV-1) of wildebeest and ovine herpesvirus-2 (OHV-2) of domestic sheep (formerly designated the sheep-associated agent of malignant catarrhal fever (MCF]. Cloned DNA fragments of AHV-1 and OHV-2 cross hybridised with DNA prepared from cells infected with the roan antelope virus and the intensity of reaction suggested that this virus was more closely related to AHV-1 than is OHV-2. The virus represents the third gamma herpesvirus isolated from large African antelope and should be provisionally designated hippotragine herpesvirus-1. On inoculation into rabbits the virus induced malignant catarrhal fever indicating that roan antelope should be considered as a possible source of infection.

The derivation of a restriction endonuclease map for Alcelaphine herpesvirus 1 DNA.

The gammaherpesvirus Alcelaphine herpesvirus 1 (AHV-1), indigenous to the wildebeest (Connochaetes species), is a causative agent of the fatal lymphoproliferative disease malignant catarrhal fever in cattle and deer. The genome of the attenuated WC11 isolate of AHV-1 has previously been shown to possess a region of unique DNA together with multiple direct repeat sequences. Approximately 70% of the genome of WC11 has now been cloned into plasmid or bacteriophage vectors and these clones have been used in hybridisation experiments to construct a restriction endonuclease map of the WC11 unique DNA with respect to BamHI. EcoRI, SalI, SmaI and XhoI. The map allows the size of the unique region of the WC11 genome to be estimated as 130 kbp and thus the entire genome as 155-160 kbp. The results confirm a terminal location for the repeat sequences.

Derivation of a DNA clone corresponding to the viral agent of sheep-associated malignant catarrhal fever.

Malignant catarrhal fever is a fatal lymphoproliferative and degenerative disease of ruminants. One causative agent is the gammaherpesvirus alcelaphine herpesvirus 1 (AHV-1), which produces no disease in its natural host, the wildebeest (Connochaetes species). Epidemiological evidence implicates sheep as the carrier of a similar virus. However, attempts to culture this virus from sheep or from animals affected with sheep-associated malignant catarrhal fever (SA-MCF) have failed. Lymphoblastoid cells have been propagated from cattle, deer and rabbits with SA-MCF. Although these cells show no evidence of viral particles or antigens, hybridisation experiments now show that they contain DNA sequences homologous to those of AHV-1. A genomic library was constructed from one of these lymphoblastoid cell lines and a clone identified which hybridised to cloned AHV-1 DNA. The authors believe that this clone contains part of the SA-MCF viral genome, and that the SA-MCF virus and AHV-1 are closely related gammaherpesviruses.

Preliminary characterization of the alcelaphine herpesvirus 1 genome.

Alcelaphine herpesvirus type 1 (AHV-1) is a causative agent of the fatal lymphoproliferative disease malignant catarrhal fever in deer and cattle. The genomes of the attenuated WC11 isolate and the virulent C500 isolate have been studied. The genome of WC11 comprises a region of unique DNA of approximately 130 kbp, which has a G + C content of 50%, and approximately 30 kbp of additional tandem direct repeat sequences with G + C content of 72%. WC11 possesses a major repeat sequence of 950 bp interspersed with a small number of related sequences of different length; these sequences are probably terminal in location. DNA from the C500 isolate has a similar restriction profile to that of WC11 in the unique region, but only one repeat sequence of 1050 bp is present. We propose, on the basis of biological and structural properties, that AHV-1 be included within the gamma 2 group of herpesviruses of which herpesvirus ateles is the prototype.