Molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (TGEV) and porcine respiratory coronavirus (PRCV) field isolates co-circulating in a swine herd

Summary.  TGEV replicates in intestinal enterocytes and causes diarrhea in young pigs. PRCV, a spike (S) gene deletion mutant of TGEV with an altered respiratory tissue tropism, causes mild or subclinical respiratory infections. Comparisons of TGEV and PRCV strains suggest that tropism and pathogenicity are influenced by the S gene and ORF3, respectively. Recently, outbreaks of TGE of reduced virulence were reported in the field. We investigated a similar suspect TGEV outbreak of reduced virulence in nursery pigs from a swine herd in the Midwest. A TGEV strain (BW021898B) was isolated in swine testicular cells from gut contents of a diarrheic pig and three PRCV strains (BW126, BW154, BW155) were isolated from nasal swabs from normal TGEV-seronegative sentinel pigs in contact with the diarrheic pigs. Sequence analysis of the TGEV isolate in the partial S gene and ORF3/3a and ORF3-1/3b revealed high homology with enteropathogenic TGEV strains. Gnotobiotic pig inoculation and histopathological results revealed that this TGEV isolate retained virulence even though in the field outbreak the diarrheal disease was of reduced severity. Sequence analysis of the S gene deletion region of the three PRCV isolates revealed identical deletions between nt 105–752, which differ from deletions previously reported among PRCV strains. The three PRCV isolates had variable sequence changes in ORF 3/3a and ORF 3-1/3b, affecting the ORF size and amino acid sequence. Thus, sequence analysis and pathogenicity studies indicate that this TGEV isolate resembles other enteropathogenic TGEV strains. Therefore, the reduced severity of TGE observed in this herd may be due to the ongoing PRCV infections, which induce antibodies cross-reactive with TGEV and result in decreased disease severity. The results outlined in this study highlight the need to monitor the molecular epidemiology of TGEV/PRCV strains with sensitive differential diagnostic assays, followed by sequence analysis of the critical regions to identify changes and pathogenicity studies to confirm the disease potential of the TGEV isolates. Received August 23, 1999 Accepted December 19, 1999     

Full genomic sequence analysis of swine genotype 3 hepatitis E virus isolated from Shanghai

The full genomic nucleotide sequence of a previously identified genotype 3 hepatitis E virus (HEV), strain SAAS-JDY5, was obtained using RT-PCR and rapid amplification of cDNA ends (RACE). The genome consisted of 7225 nucleotides, excluding a poly-A tail at the 3′ terminus, and contained three open reading frames (ORFs), ORF-1, ORF-2 and ORF-3, encoding 1702, 660 and 113 amino acids, respectively. Phylogenetic analysis confirmed that SAAS-JDY5 belonged to genotype 3 HEV and was most closely related to the Japanese isolate wbJYG1 (AB222184). SAAS-JDY5 shared approximately 87% nucleotide similarity to human and swine strains from the United States, compared with 74–75% similarity to Asian (genotype 4) and Mexican strains (genotype 2). Alignment of the SAAS-JDY5 genomic sequence with reference sequences of the same genotype revealed one nucleotide substitution and one deletion at positions 5145 and 7189 (3′ UTR), respectively. Moreover, SAAS-JDY5 contained two additional nucleotides (AC) at the very end of the 3′-terminus preceding the poly-A tail of the genome. Comparison of the putative amino acid sequence encoded by the SAAS-JDY5 genome with sequences of other genotype 3 isolates revealed 15 unique amino acid substitutions and one deletion in ORF-1, and three substitutions in ORF-2.     

Respiratory and fecal shedding of Porcine respiratory coronavirus (PRCV) in sentinel weaned pigs and sequence of the partial S-gene of the PRCV isolates

Summary. Porcine respiratory coronavirus (PRCV), a spike (S) gene deletion mutant of Transmissible gastroenteritis virus (TGEV), causes mild or subclinical respiratory infections in pigs. The shedding of PRCV/TGEV was studied at different days post-arrival in fecal and nasal swabs from PRCV/TGEV seronegative sentinel pigs introduced into a PRCV seropositive herd with questionable TGEV serology and diarrhea. Nasal shedding of PRCV was detected in 57% and 63% of samples by nested-RT-PCR and cell culture immunofluorescence (CCIF), respectively. However fecal shedding of PRCV was detected in 37% of the samples by nested-RT-PCR and 19% by CCIF. Four respiratory and 5 fecal PRCV strains were isolated in swine testicle cells including nasal/fecal PRCV pairs (isolated at the same time) from 3 pigs. Comparison of nasal/fecal PRCV pairs from individual pigs revealed different deletions in the spike (S) gene (648 or 681nt) in 2 pairs and a consistent change in nt 790/791 (aa T to V) for all pairs. In preliminary studies, inoculation of gnotobiotic pigs with each plaque-purified pair of the nasal and fecal PRCV isolates, revealed no clinical disease but different tropisms. The nasal isolate was shed both nasally and in feces, but the fecal isolate was shed only marginally in feces, and not nasally. Our results show that nested-RT-PCR was as sensitive as CCIF for PRCV detection in nasal swabs, but was more sensitive than CCIF for PRCV detection in fecal samples; alternatively PRCV shed in feces was more labile with loss of infectivity. The S-gene sequence differences found between the fecal and respiratory PRCV isolates may influence their tissue tropism. These new PRCV isolates should be useful to understand the molecular basis of coronavirus tropism and evolution in infected swine.     

Characterisation of a recent virulent transmissible gastroenteritis virus from Britain with a deleted ORF 3a

Summary.  Analyses of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) isolates have suggested that tropism and pathogenicity are influenced by the spike protein and ORF 3. In general, enteric viruses (TGEV) have been shown to contain intact spike and ORF 3 genes, whilst respiratory isolates (PRCV) have major deletions within both regions. Virulence has been correlated to a functional ORF 3. Here, sequence analysis of a recent isolate of virulent TGEV, revealed a variant with an intact spike gene, but a large deletion in ORF 3a. This suggests that ORF 3a is not essential for enteric virulence. Received September 14, 1998 Accepted December 9, 1998     

Processing and antigenicity of entire and anchor-free spike glycoprotein S of coronavirus TGEV expressed by recombinant baculovirus.

The S gene of transmissible gastroenteritis virus (TGEV) was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) using the transfer plasmid pVL941. Infection of Sf9 insect cells with the recombinant virus resulted in the synthesis of a 175K polypeptide which was able to trimerize and was transported to the cell surface as is the authentic TGEV S protein. Despite the lack of complete carbohydrate processing, the recombinant S protein exhibited antigenic properties similar to TGEV S and induced high levels of neutralizing antibodies in immunized rats. Engineering a deletion (70 amino acids) into the carboxy-terminus containing the membrane anchor of the polypeptide allowed its secretion. The oligomerization process and the antigenic profile of the anchor-free S protein were shown to be partially altered.     

Complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein 3b of US strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus

Transmissible gastroenteritis virus (TGEV) isolates that have been adapted to passage in cell culture maintain their infectivity in vitro but may lose their pathogenicity in vivo. To better understand the genomic mechanisms for viral attenuation, we sequenced the complete genomes of two virulent TGEV strains and their attenuated counterparts: virulent TGEV Miller M6 and attenuated TGEV Miller M60 and virulent TGEV Purdue and attenuated TGEV Purdue P115, together with the ISU-1 strain of porcine respiratory coronavirus (PRCV-ISU-1), a naturally occurring TGEV deletion mutant with an altered respiratory tropism and reduced virulence. Pairwise comparison at both the nucleotide (nt) and amino acid (aa) levels between virulent and attenuated TGEV strains identified a common change in nt 1753 of the spike gene, resulting in a serine to alanine mutation at aa position 585 of the spike proteins of the attenuated TGEV strains. Alanine was also present in this protein in PRCV-ISU-1. Particularly noteworthy, the serine to alanine mutation resides in the region of the major antigenic site A/B (aa 506-706) that elicits neutralizing antibodies and within the domain mediating the cell surface receptor aminopeptidase N binding (aa 522-744). Comparison of the predicted polypeptide products of ORF3b showed significant deletions in the naturally attenuated PRCV-ISU-1 and TGEV Miller M60; these deletions occurred at a common break point, suggesting a related mechanism of recombination that may affect viral virulence or tropism. Sequence comparisons at both genomic and protein levels indicated that PRCV-ISU-1 had a closer relationship with TGEV Miller strains than Purdue strains. Phylogenetic analyses showed that virulence is an evolutionarily labile trait in TGEV and that TGEV strains as a group share a common ancestor with PRCV.     

Intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein expressed by recombinant vaccinia virus.

The Spike (S) protein from a virulent British field isolate of porcine transmissible gastroenteritis virus (TGEV) FS772/70 was constructed from cDNA and inserted into the vaccinia virus (VV) thymidine kinase gene locus under the control of the VV early/late gene P7.5k promoter. Recombinant S protein was synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive glycoprotein with high mannose simple oligosaccharides (gp 190) that underwent post-translational modification to an Endo H-resistant glycoprotein with complex oligosaccharides (gp210). Immunofluorescence analysis demonstrated that the majority of recombinant S protein was retained at the Golgi but some S protein was expressed on the plasma membrane. Monoclonal antibodies (mAbs) raised against native S protein reacted with this recombinant S protein; also, mice infected with the recombinant vaccinia virus (rVV) expressing the S protein induced TGEV neutralizing antibodies. A truncated S protein (S delta) was also expressed in rVV-infected cells by introducing a deletion into the S protein cDNA that removed 292 amino acids from the C-terminus. The S delta protein (gp 170) was shown to be antigenically similar to TGEV S protein by immunofluorescence and immunoprecipitation tests but was retained in the endoplasmic reticulum and not expressed on the cell surface.     

Proteolytic maturation of the 206-kDa nonstructural protein encoded by turnip yellow mosaic virus RNA.

The longest open reading frame of turnip yellow mosaic virus genomic RNA (ORF-206) encodes a 206-kDa nonstructural protein. The most prominent in vitro translation products of ORF-206 are the full-length p206 and a shorter N-coterminal 150-kDa protein. We have confirmed these assignments by immunoprecipitation of in vitro translation products with antisera raised to N-terminal and C-terminal regions encoded by ORF-206. The mechanism by which the 150-kDa protein arises from ORF-206 was investigated by in vitro translation of deletion and substitution derivatives transcribed from pTYMC, a cDNA clone of TYMV RNA. The following observations demonstrate that the 150-kDa protein and a C-terminal 70-kDa protein arise from ORF-206 by autoproteolysis: (1) Two regions encoded by ORF-206 were necessary for the formation of the 150-kDa protein: a domain between amino acids 555 and 1051, postulated to encode a protease, and the region between amino acids 1253 and 1261, thought to constitute the protease recognition and/or cleavage site. (2) Mutants with substitutions between amino acids 1253 and 1261 that produce low levels of the 150-kDa protein in in vitro translations also have high levels of p206 and low levels of the 70-kDa protein. (3) The rate of formation of the 150-kDa protein is dilution insensitive, suggesting that proteolysis occurs mainly in cis.     

Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene

The 3' end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into cDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential, nonoverlapping genes were identified within the 3'-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3'-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.     

Three new isolates of porcine respiratory coronavirus with various pathogenicities and spike (S) gene deletions.

Three new isolates of porcine respiratory coronavirus (PRCV) were isolated and partially characterized. These PRCV isolates showed a selective tropism for respiratory tissue and were antigenically related to transmissible gastroenteritis virus. PCR amplification of the 5' half of the spike (S) genes of the three PRCV isolates indicated that a large deletion, characteristic of PRCV, was present. By using cDNA probes specific for the transmissible gastroenteritis virus S gene, the PCR products were shown to be specific in a Southern blot. The three new PRCV isolates were shown to vary in S gene deletion size. In a separate study, these isolates have also been shown to vary in pathogenicity. These new PRCV isolates should serve as important tools in gaining a better understanding of the pathogenesis of coronavirus infections.     

The nucleotide sequence of the coat protein gene and 3′ untranslated region of papaya ringspot virus type W (Aust)

Summary The nucleotide sequence of the 3 terminal region of the Australian isolate of papaya ringspot virus type W [PRSV-W (Aust)] was determined. An open reading frame (864 bp), encoding the putative coat protein gene, occurs upstream of the putative 3 untranslated region (206 bp) and poly(A) tail. A 23 amino acid sequence was obtained from N-terminal analysis of the coat protein from purified virions. This sequence has 100% homology with a region of the amino acid sequence inferred from the nucleic acid sequence of the coat protein gene. However, this region is 13 amino acids downstream from the N terminus predicted for two American isolates of PRSV. The coat protein gene of PRSV-W (Aust) was shown to have 96.8% and 96.4% nucleotide sequence similarity with American isolates of PRSV-W and PRSV-P, respectively.     

Decorin-Binding Protein of Borrelia burgdorferi Is Encoded within a Two-Gene Operon and Is Protective in the Murine Model of Lyme Borreliosis

Isolated outer membranes of Borrelia burgdorferi were used in immunoblotting experiments with sera from immune mice to identify new putative Lyme disease vaccine candidates. One immunoreactive polypeptide migrated on polyacrylamide gels just proximal to outer surface protein C and comigrated with [³H]palmitate-labeled polypeptides. A degenerate oligonucleotide primer based upon internal amino acid sequence information was used to detect the corresponding gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 24-amino-acid putative signal peptide terminated by LLISC, a probable consensus sequence for lipoprotein modification, and a mature protein of 163 amino acids. Immunoblots of a recombinant fusion protein corresponding to this ORF supported the idea that the encoded protein was a previously reported decorin-binding protein (DBP) of B. burgdorferi N40 (B. P. Guo, S. J. Norris, L. C. Rosenberg, and M. Höök, Infect. Immun. 63:3467–3472, 1995). However, further DNA sequencing revealed the presence of a second ORF, designated ORF-1, whose termination codon was 119 bp upstream of the dbp gene. ORF-1 also encoded a putative lipoprotein with a mature length of 167 amino acids. Northern blots, Southern blots, and primer extension analyses indicated that ORF-1 and dbp comprised a two-gene operon located on the 49-kb linear plasmid. Both proteins, which were 40% identical and 56% similar, partitioned into Triton X-114 detergent extracts of B. burgdorferi isolated outer membranes. Mice infected with B. burgdorferi produced high titers of antibodies against the ORF-1-encoded protein and DBP during both early and later stages of chronic infection. Both DBP and the ORF-1-encoded protein were sensitive to proteinase K treatment of intact borreliae, suggesting that they were surface exposed. In active immunization experiments, 78% of mice immunized with recombinant DBP were immune to challenge. While it is not clear whether the two lipoproteins encoded by the ORF-1-dbp operon have analogous decorin-binding functions in vivo, the combined studies implicate DBP as a new candidate for a human Lyme disease vaccine.     

Differentiation of transmissible gastroenteritis virus from porcine respiratory coronavirus and other antigenically related coronaviruses by using cDNA probes specific for the 5'' region of the S glycoprotein gene.

Two cDNA clones prepared from the virulent Miller strain of transmissible gastroenteritis virus (TGEV) were identified, and their nucleotide sequences were determined. The clones were nonoverlapping and located in the 5' region of the S glycoprotein gene. Their nucleotide and predicted amino acid sequences were compared with published sequences of the attenuated Purdue strain of TGEV and feline infectious peritonitis virus (FIPV). TGEV clone pE21 contained 381 bp of the S glycoprotein gene and had greater than 98% nucleotide and amino acid sequence homology with Purdue TGEV and over 87% nucleotide and amino acid sequence homology with FIPV. TGEV clone pD24 contained 267 bp of the S glycoprotein gene. It had greater than 98% nucleotide and amino acid sequence homology with Purdue TGEV but only 54% nucleotide sequence homology and 24% amino acid sequence homology with FIPV. A probe prepared from pD24 could differentiate TGEV from porcine respiratory coronavirus and other antigenically related coronaviruses, FIPV, feline enteric coronavirus, and canine coronavirus in a dot blot hybridization assay.     

Conserved structures among the nucleocapsid proteins of the paramyxoviridae: complete nucleotide sequence of human parainfluenza virus type 3 NP mRNA

The nucleotide sequence of the mRNA coding for the nucleocapsid protein (NP) of the paramyxovirus, human parainfluenza virus type 3 (PIV-3), has been determined. The NP mRNA was found to contain 1642 bases, excluding poly(A), and encode a protein of 515 amino acids, with a molecular weight of 57,823. Amino acid residues 1 through 420 of PIV-3 NP protein showed extensive sequence homology with the corresponding amino acids of Sendai virus nucleocapsid protein. There was virtually no homology between the last 95 amino acids. Comparison of the NP proteins of PIV-3, Sendai virus, measles virus, and canine distemper virus revealed, from amino acid residues 160 through 390, some conserved areas between the corresponding proteins of these paramyxoviruses. The 5' terminal sequence of PIV-3 NP mRNA (5'-AGGATTAAAG-3') was similar to the conserved sequence (formula; see text) found at the 5' termini of Sendai virus mRNAs. Both PIV-3 NP and Sendai virus mRNAs had a common 3' terminal tetranucleotide (5'-TAAG-3') preceding the poly (A) tail.     

Immunogenicity of the S protein of transmissible gastroenteritis virus expressed in baculovirus

Summary Seven fragments of the spike (S) gene cDNA of transmissible gastroenteritis virus (TGEV), as well as the full length cDNA, were cloned and expressed in baculovirus vectors. Piglets were immunized with cells infected with the recombinant viruses. Each of the recombinants induced TGEV-specific antibodies detected in a fixed cell enzyme immunoassay. The amino terminal half of the S protein, containing all four major antigenic sites (A, B, C and D), and encoded by a 2.2 kb fragment of the S gene, induced virus neutralizing (VN) antibody titers comparable with those induced by the complete S protein. Recombinant proteins lacking the A antigenic site, or with a deletion including the putative receptor binding sites and the D antigenic site, were not capable of inducing levels of VN antibodies similar to those induced by the whole S protein.     

Sequence analysis of the leader RNA of two porcine coronaviruses: Transmissible gastroenteritis virus and porcine respiratory coronavirus

The leader RNA sequence was determined for two pig coronaviruses, tranmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV). Primer extension, of a synthetic oligonucleotide complementary to the 5 end of the nucleoprotein gene of TGEV was used to produce a single-stranded DNA copy of the leader RNA from the nucleoprotein mRNA species from TGEV and PRCV, the sequences of which were determined by Maxam and Gilbert cleavage. Northern blot analysis, using a synthetic oligonucleotide complementary to the leader RNA, showed that the leader RNA sequence was present on all of the subgenomic mRNA species. The porcine coronavirus leader RNA sequences were compared to each other and to published coronavirus leader RNA sequences. Sequence homologies and secondary structure similarities were identified that may play a role in the biological function of these RNA sequences.The nucleotide sequence data reported in this paper have been submitted to the EMBL/Genbank/DDBJ nucleotide sequence databases and have been assigned the accession numbers X52157, X52668.