Genomic organisation of a virulent Taiwanese strain of transmissible gastroenteritis virus.

Transmissible gastroenteritis (TGE) infection causes 65% of infectious piglet diarrhoea in Taiwan. A virulent Taiwanese strain, TFI, of transmissible gastroenteritis virus (TGEV) from a field outbreak was isolated in cell culture and plaque purified. Phenotypic differences were observed in the ability of TFI to infect certain cell lines. TGEV strains TLM-83 (PRCV Belgium), TO-163 (TGEV Japan) and Purdue-115 (TGEV USA) infected both ST (swine testis) and RPTG (pig kidney) cell lines whereas TFI infected ST but not RPTG cells. To investigate this phenotypic variation cDNA was generated from TFI genomic and amplified by PCR with oligonucleotides derived from published TGEV sequence data. An 8.4kb cDNA derived from the 3'-end of the TFI genome was sequenced. Eight ORFs, corresponding to the three structural protein genes, four potential genes and the 3'-end of an incomplete ORF whose amino acid sequence corresponded to the carboxyl end of the 1b subunit of the polymerase gene, were identified on the TFI sequence. The overall sequence similarity of TFI with the other TGEV strains was over 97%. However, several deletions, insertions and point mutations were found on the TFI sequence when compared with other TGEV strains. The TFI S protein was found to contain 1449 amino acids, as also identified for the FS772/70 and Miller TGEV strains, but two amino acids longer than the Purdue S protein. The TFI ORF-3a gene encodes 72 amino acids, however, a 37 nucleotide deletion was found 16 nucleotides downstream of the TFI ORF-3a stop codon.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of PCR genome mapping for the characterisation of TGEV strains.

Previous studies on different transmissible gastroenteritis virus (TGEV) strains, including porcine respiratory coronavirus (PRCV), have identified regions within the genome that are polymorphic as regards insertions and deletions. For example the 672 base deletion within the S gene and multiple deletions 5', within and 3' of the ORF-3a gene were detected in strains of PRCV. The presence of deletions may be associated with a change in the virulence, attenuation or tissue tropism of the isolate. The Nouzilly (188-SG) TGEV vaccine strain was attenuated by passage of a cell culture adapted virulent isolate D-52 188 times through swine testis cells after treatment with gastric juice. PCR amplification with oligonucleotides, corresponding to known TGEV sequences, were used to analyse D-52 and 188-SG for genetic variation. Results with several pairs of oligonucleotides within the first 1565 nucleotides of the S gene did not identify a deletion within this region of the genome from either strain. However, oligonucleotides directed against the ORF-3a/3b region detected a deletion of about 250 nucleotides within the 188-SG genome but not in the D-52 genome. Since all the attenuated TGEV strains so far sequenced, PRCV, Miller SP and 188-SG, contained deletions within the ORF-3a/3b, it would suggest that this region of the TGEV genome is involved in regulating viral virulence.

Control of TGEV mRNA transcription.

Coronavirus proteins are translated from a nested set of subgenomic mRNAs which have common 3' termini with unique 5' extensions. Evidence suggests that coronavirus mRNAs are generated by a mechanism of leader primed transcription. Leader RNA binds to consensus sequences upstream of each gene on full length negative strand viral RNA and transcription proceeds to the 5' end of the negative strand to produce the nested set of mRNAs. Even though this gives rise to polycistronic mRNA species only the 5' extension of each mRNA is translated to give the viral proteins. The leader RNA for TGEV is about 90 nucleotides long and contains the sequence which recognises the leader binding sites on the negative strand RNA. Evidence suggests that the length of the leader binding sequence may be involved in transcriptional control of individual mRNAs. In order to investigate this a virus specific mRNA isolation method was developed to measure the relative amounts of mRNAs synthesised during an infection of LLC-PK1 cells with TGEV (strain FS772/70). Thus the relative quantity of each mRNA can be determined and correlated with the variation in size of the leader binding site.

Two monoclonal antibodies (CC17, CC29) recognizing an antigen (Bo5) on bovine T lymphocytes, analogous to human CD5.

Two new monoclonal antibodies (CC17 and CC29) raised against bovine thymocytes are described. The antibodies, both of which were IgG1, recognize a molecule of approximately 67,000 molecular weight on bovine T cells. They react T cells in peripheral blood, the lymph node paracortex and the periateriolar lymphoid sheath in the spleen. Both the cortex and medulla of the thymus are stained but the medulla reacts more intensely. They do not stain B cells in peripheral blood, the ileal Peyer's patch, the cortex or the primary follicles in lymph nodes. No activity was found on cells outside the lymphoid system, i.e. monocytes, alveolar macrophages or endothelial and epithelial tissue. The antigen recognized is considered to be the bovine homologue of CD5 (T1) in humans and Lyt1 in mice. The mAbs appear to be particularly useful for detecting cells in the peripheral blood of young calves which are of the T cell lineage but do not express BoT2 or the mature pan T cell antigen recognized by mAb IL-A27 and may thus allow identification of a population of bovine lymphocytes previously described as null cells.

Isolation and characterization of two group A rotaviruses with unusual genome profiles.

Analysis of the genomic dsRNA of rotaviruses isolated from calves with subclinical infections has revealed eight calves excreting group A viruses with unusual genome profiles. Two of these virus strains, C7-176 and C7-183, were grown and cloned by plaque purification in cell culture. Examination of their genome profiles after cloning showed that the unusual pattern had been retained. They were without RNA segment 11 but had an extra band (6a) migrating between segments 6 and 7. However, they contained the triplet of segments 7, 8 and 9, of similar size, which is characteristic of group A rotaviruses. The number and mol. wt. of the intracellular polypeptides induced by these viruses were similar to those of the bovine group A rotavirus UK strain. Analysis of the RNA transcripts produced by the transcription in vitro of purified C7-183 virus showed that segment 6a produced a large RNA transcript of corresponding size. After isolation, this transcript was translated in a rabbit reticulocyte lysate preparation and yielded a single polypeptide, vp11, equivalent to the product of segment 11 of rotaviruses with typical genome profiles.

Pathogenesis and epidemiology of bovine virus diarrhoea virus infection of cattle.

An outline of the clinical diseases that arise following BVDV infection is given. Isolates of BVDV can be separated into two forms non-cytopathic and cytopathic depending on their effect on cell cultures. In utero infection of the foetus with non-cytopathic virus may result in a number of syndromes, such as abortions, stillbirths or weak calves. It may also result in the birth of calves with a persistent viraemia and these animals may later develop mucosal disease as a result of superinfection with a "homologous" cytopathic strain of BVDV. Acute infection of seronegative animals in usually mild and subclinical. A chronic disease also occurs and this can be protracted with progressive wastage and diarrhoea. This condition has not yet been well defined but it is suggested that it may be the result of superinfection of a viraemic animal with a "heterologous" cytopathic strain of virus. The maintenance of non-cytopathic virus within the cattle population can be either by the slow spread following acute infections of seronegative animals or, more importantly, by spread from persistently viraemic cattle. The cytopathic virus is usually found in association with cases of mucosal disease and may be maintained in the population only by continually arising, possibly by mutation, from the non-cytopathic virus. It is recommended that persistently viraemic animals are eliminated from the herd to avoid in utero infections and the possibility of subsequent mucosal disease.

Characterisation of rotavirus isolates from sub-clinically infected calves by genome profile analysis.

Rotaviruses isolated from 43 sub-clinically infected calves from a single farm were analysed by genome profile analysis. The isolates showed genomic variation and eight different profiles were observed, including one which was atypical for Group A rotaviruses. The 3' terminal labelling method for the analysis of genome profiles used in this study required only 1 ng of viral RNA, an increase of 1000-fold in sensitivity over ethidium bromide staining for detecting all rotavirus genome segments. However, dual infections involving two rotaviruses with distinct profiles could not be detected if the concentrations of the viruses differed by greater than 10-fold.

Variation in the intracellular polypeptide profiles from different isolates of bovine virus diarrhoea virus.

Variation of the intracellular polypeptides induced in calf testis cells by 5 cloned isolates of bovine virus diarrhoea virus (BVDV) was examined. Three of the isolates were cytopathic (NADL, C 2415 and Pe 515 c) and two were non-cytopathic (C 1226 and Pe 515 nc) in these cells. The isolates Pe 515 c and Pe 515 nc were both isolated from an animal with clinical signs of mucosal disease. In cells infected with NADL, 8 virus specific proteins (vp 1 to vp 8) with molecular weights ranging from 120,000 (vp 1) to 23,000 (vp 8) were detected. Isolates C 2415 and Pe 515 c gave a similar array of polypeptides to NADL, but the 3 cytopathic isolates could be distinguished by the variation in the molecular weights of some of the proteins. The non-cytopathic isolates could also be distinguished from each other by this type of molecular variation; however, one feature that characterised these strains, when compared to the cytopathic isolates, was the absence of vp 2. Comparison of the polypeptides induced by Pe 515 c and Pe 515 nc showed that apart from the lack of vp 2 in the Pe 515 nc virus profile, the molecular weights of the other viral proteins were similar. This supports serological evidence that for mucosal disease to occur the pair of cytopathic and non-cytopathic viruses must be closely related. Four of the polypeptides induced by Pe 515 c were shown to be glycoproteins.

Variation in virulence of bovine rotaviruses.

Forty-six gnotobiotic calves aged less than 16 days or 42-116 days were infected with three strains of bovine rotavirus designated C3-160, CP-1 and PP-1. Each virus was passaged and cloned in cell culture (cloned viruses) but CP-1 and PP-1 were also used before culture (faecal viruses). Infection of calves aged less than 16 days with faecal or cloned CP-1 caused disease whereas cloned C3-160 and faecal or cloned PP-1 caused subclinical infections. The clinical signs of disease were change in faecal colour to pale yellow or cream, increase of 2- to 7-fold in the volume of faecal output and, usually, anorexia. With the virulent CP-1 virus and the avirulent C3-160, similar amounts of virus were excreted in the faeces for 4-6 days. Infection of calves aged 56-116 days with faecal CP-1 produced disease of similar severity to that seen in calves aged 7-10 days infected with the same virus. No differences in clinical signs, virus excretion or levels of convalescent antibody were seen between the two groups. With cloned CP-1, 5 of 8 older calves developed disease but 3 showed only mild signs of infection. It was concluded that two strains of rotavirus caused sub-clinical infections in young calves while a third was virulent in calves up to at least 116 days of age.

Comparison of ureaplasmas from sheep and goats with Ureaplasma diversum and U. urealyticum.

Ureaplasmas isolated from sheep and goats were compared by immunofluorescence with antisera prepared in calves and by PAGE of polypeptides labelled by growth in the presence of [35S]methionine. The ovine and caprine strains constituted two groups defined by serology and polypeptide composition that were not related to the animal species from which they originated. Strains representing these two groups were compared with Ureaplasma urealyticum (human isolates) and U. diversum (bovine isolates). They could not be classified with either but were more similar to the U. diversum strains.

Comparison of mycoplasmatales virus MV-Lg-pS2-L172 with plasmavirus MV-L2 and the other mycoplasma viruses.

Mycoplasma virus MV-Lg-pS2-L172 was sensitive to heat (56 degrees C/30 minutes), Nonidet-P40 and ether. In these respects it resembled Plasmavirus MB-L2. However, it differed from MV-L2 (and the other mycoplasma viruses, MV-L1, MV-L3 and BN1 virus) in reciprocal plaque inhibition and serum neutralization tests (MV-L2 only). By plaque formation on host lawns resistant to the different mycoplasma viruses, including MV-Lg-pS2-L172, this latter virus was shown to be distinct from the other viruses, including MV-L2. Both MV-Lg-pS2-L172 and MV-L2 possessed one polypeptide band (out of 10) that was not common to the heterologous virus.

Effect of sulphydryl reagents on the biological activities, polypeptide composition and morphology of haemagglutinating encephalomyelitis virus.

The effect of sulphydryl reagents on haemagglutinating encephalomyelitis virus (HEV), a coronavirus of pigs, was investigated. Using increasing concentrations of dithiothreitol (DTT), 50% of the virus infectivity and haemagglutination (HA) activity could be removed by i.5 mM and 4 to 5 mM respectively. The effect of DTT concentrations on the polypeptide composition was also examined. Of the three external glycoproteins gp 125 was found to be the most susceptible, 50% being removed by incubation of the virus with 5 to 6 mM-DTT. Of the other two glycoproteins gp 180 was unaffected by DDT concentrations up to 100 mM and the amount of gp 100 gradually declined at concentrations above 20 mM. The rates of removal of the virus HA activity and gp 125 suggested that this polypeptide was an essential part of the virus haemagglutinin. The lack of evidence for any interpeptide disulphide bonds suggested that the loss of these glycoproteins was due to an alteration in their conformation brought about by the cleavage of intrapeptide disulphide bonds. The loss of protein from the surface of the virus resulted in a change in the virus morphology with the appearance of thin fibrous projections instead of the characteristic petal-like coronavirus projections.

Isolation of subviral components from transmissible gastroenteritis virus.

Exposure of purified transmissible gastroenteritis virus, a porcine coronavirus, to non-ionic detergents resulted in the removal of the surface projections and greater than 98% of the virus lipid. Virus RNA was associated with a subviral particle which had a sedimentation coefficient of 650S, compared with 495S for the intact virion, and which banded in Cs2SO4 gradients at 1-295 g/ml. Negatively stained preparations of subviral particles were shown by electron microscopy to contain spherical particles of 60 to 70 nm diam., similar in appearance to those derived from oncornaviruses. Polyacrylamide gel electrophoresis of the polypeptides from isolated subviral particles showed that these structures contained three of the four major virus structural proteins, the arginine-rich polypeptide VP2 and the two membrane glycopolypeptides VP2 and 4. The detergent-liberated surface projections, composed of a single species of sulphated glycopolypeptide, VPI, were isolated by rate-zonal centrifugation through sucrose gradients followed by precipitation with ammonium sulphate in the presence of bovine serum albumin.

The polypeptide structure of transmissible gastroenteritis virus.

The polypeptides of purified preparations of the coronavirus responsible for transmissible gastroenteritis of pigs have beem examined by polyacrylamide gel electrophoresis. Four major polypeptides, VPI (mol. wt. 200000), VP2 (50 000), VP3 (30000) and VP4 (28500) and two minor polypeptides, VPIa (105000) and VPIb (80500) have been reproducibly demonstrated in the virion, of which VPI, VP3 and VP4 contain carbohydrate. Treatment of the virion with the proteolytic enzyme bromelain removes the surface projections and VPI, thus identifying this glycopolypeptide as the major structural component of the projection.

Characterization of Mycoplasmatales virus-laidlawii 3.

Purified preparations of Mycoplasmatales virus-laidlawii 3 were negatively stained and studied by electron microscopy. They were seen to consist of uniform sized particles having a polyhedral head, 57 nm by 61 nm, and a short tail, 25 nm long, joined to the head at one vertex by a collar. The particles were shown to have buoyant densities of 1-477 g/ml in CsC1, 1-32 g/ml and 1-26 g/ml in potassium tartrate and a sedimentation coefficient, S20,W, OF 290 +/- 13. They are composed of 35-2% double-stranded DNA and five structural polypeptides with approximate mol. wt. of 172000, 81000 73000, 68000 and 43000. The classification of the virus from its morphology and chemical properties is discussed.

The influence of pH on the growth and stability of transmissible gastroenteritis virus in vitro.

The influence of pH on the growth of transmissible gastroenteritis virus (TGEV) in adult pig thyroid cell culture, and on the stability of the virus was studied. At pH 7.2 and 100 fold higher than those at pH 8.0. The adsorption, penetration and uncoating steps of the viral replicative cycle were shown to be unaffected by pH variation. Synthesis of TGEV RNA during the first 12 hours post infection was found to be unaffected by pH variation between the range 6.5-8.0. After 12 hours breakdown of this RNA appeared to occur in cultures held at pH 7.2 and 8.0 but not at pH 6.5. When incubated at 37 degrees C for 24 hours the virus infectivity was found to be least affected by pH 6.5 but when kept at 4 degrees C for the same length of time, the virus infectivity remained constant between pH 5.0 and pH 8.0.

A new general method for the assessment of the molecular-weight distribution of polydisperse preparations. Its application to an intestinal epithelial glycoprotein and two dextran samples, and comparison with a monodisperse glycoprotein.

A specimen of intestinal glycoprotein isolated from the pig and two samples of dextran, all of which are polydisperse (that is, the preparations may be regarded as consisting of a continuous distribution of molecular weights), have been examined in the ultracentrifuge under meniscus-depletion conditions at equilibrium. They are compared with each other and with a glycoprotein from Cysticercus tenuicollis cyst fluid which is almost monodisperse. The quantity c(-(1/3)) (c=concentration) is plotted against xi (the reduced radius); this plot is linear when the molecular-weight distribution approximates to the ;most probable', i.e. when M(n):M(w):M(z): M((z+1))....... is as 1:2:3:4: etc. The use of this plot, and related procedures, to evaluate qualitatively and semi-quantitatively molecular-weight distribution functions where they can be realistically approximated to Schulz distributions is discussed. The theoretical basis is given in an Appendix.