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PubMed2003

Establishment and characterization of two new pig cell lines for use in virological diagnostic laboratories.

Two pig cell lines derived from kidney and trachea tissues and referred to as newborn swine kidney (NSK) and newborn pig trachea (NPTr) were established following serial culture of primary cells. They were characterized by an epithelial-like morphology, high capacity to replicate and stability of the cell monolayer for several days after seeding. Their modal chromosome number was modified in comparison to that of primary swine cells and they both displayed a transforming potential in vitro and displayed oncogenicity in nude mice. Infection with pig endogenous retroviruses was detected. Almost all the swine viruses tested, i.e., pseudorabies virus, pig parvovirus, hog cholera virus, transmissible gastroenteritis virus of swine, encephalomyocarditis virus, swine vesicular disease virus and the enteroviruses, except pig reproductive respiratory syndrome virus, were capable of replicating in the new cell lines with titres similar to the ones detected in the reference culture systems. Furthermore, all the selected influenza virus sub-types isolated from human, swine and avian species replicated with cytopathic effect in NSK and NPTr cells, whereas, of all the equine influenza viruses tested only the Miami and Suffolk sub-types replicated.

Animals↗
PubMed1999

One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses.

A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5' nuclease activity of the Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan(TM) probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Forster-type energy transfer. During the polymerase extension steps the Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (C(T) value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism 7700 Sequence Detector. After completion of amplification, the C(T) values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus.

Animals↗
PubMed1999

Development of a nested PCR assay for the detection of canine coronavirus.

A diagnostic test for canine coronavirus (CCV) infection based on a nested polymerase chain reaction (n-PCR) assay was developed and tested using the following coronavirus strains: CCV (USDA strain), CCV (45/93, field strain), feline infectious peritonitis virus (FIPV, field strain), transmissible gastroenteritis virus (TGEV, Purdue strain), bovine coronavirus (BCV, 9WBL-77 strain), infectious bronchitis virus (IBV, M-41 strain) and fecal samples of dogs with CCV enteritis. A 230-bp segment of the gene encoding for transmembrane protein M of CCV is the target sequence of the primer. The test described in the present study was able to amplify both CCV and TGEV strains and also gave positive results on fecal samples from CCV infected dogs. n-PCR has a sensitivity as high as isolation on cell cultures, and can therefore be used for the diagnosis of CCV infection in dogs.

Animals↗
PubMed2000

Seroprevalence of porcine respiratory coronavirus in selected Korean pigs.

A total of 446 serum samples from 88 herds in Korea were examined for antibody to porcine respiratory coronavirus (PRCV) using blocking enzyme-linked immunosorbent assay (ELISA). All serum samples were collected from 24- to 26-week-old finishing pigs between December 1998 and June 1999. By ELISA, 237 out of 446 sera tested (53.1%) and 54 out of 88 sampled herds (61.3%) were positive against PRCV. Of 446 sera from 88 herd tested, 185 (41.5%) serum samples from 22 (25%) herds were seronegative against PRCV and transmissible gastroenteritis virus infection. Our data suggested that seropositive herds for PRCV are distributed diffusely throughout South Korea.

Animals↗
PubMed1995

Cloning, sequencing and expression of the S protein gene from two geographically distinct strains of canine coronavirus.

The gene encoding the spike (S) protein from two geographically distinct strains (American and British) of canine coronavirus (CCV) was cloned and sequenced. The nucleotide sequence revealed open reading frames of 1443 or 1453 amino acids, respectively. Structural features include an N-terminal hydrophobic signal sequence, a hydrophilic cysteine-rich cluster near the C-terminus, two heptad repeats and 29 or 33 potential N-glycosylation sites. Pairwise comparisons of S amino acid sequences from these isolates with other CCV strains (Insavc1 and K378) revealed that heterogeneity, found mostly in the form of conservative substitutions, is distributed throughout the canine sequences. However, 5 variable regions could be identified. Similar analysis with feline, porcine, murine, chicken and human coronavirus sequences revealed that the canine sequences are much more closely related to the feline S protein sequence than to the porcine S protein sequences even though they are all from the same antigenic group. Moreover, the sequence similarity between CCV isolates and the feline coronavirus, feline infectious peritonitis virus (FIPV) was comparable. Expression of the CCV or the transmissible gastroenteritis virus (TGEV) S gene using the vaccinia virus system produced a protein of the expected size which could induce extensive syncytia formation in infected canine A72 cells.

Amino Acid Sequence↗
PubMed1999

Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3' untranslated region identifies the virus as a close relative of infectious bronchitis virus.

The 3' end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3' untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3' UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3' UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3' UTRs provide additional evidence that these avian coronaviruses are closely related.

3' Untranslated Regions↗
PubMed2003

Increased litter survival rates, reduced clinical illness and better lactogenic immunity against TGEV in gilts that were primed as neonates with porcine respiratory coronavirus (PRCV).

Establishing immunological memory in female piglets at a young age with PRCV was effective in inducing a secondary immune response to a limiting dose of virulent TGEV given orally 13-18 days prior to farrowing. Subsequently, because of passive antibody transfer, the offspring of these primed gilts were more efficient in surviving a lethal TGEV challenge. An average survival rate of 89% occurred in 6 litters of piglets from primed gilts that were boosted with 2.8 x 10(6) plaque forming units (PFU) of TGEV whereas 76% of the piglets survived in three litters that suckled primed gilts boosted with 3.0 x 10(5)PFU of TGEV. Non-primed gilts given identical pre-farrowing doses of TGEV had litter survival rates of 63 and 55%, respectively. Moreover, both groups of litters from primed gilts suffered less clinical illness (as measured by the extent of weight loss post-challenge) than control litters. Priming of the piglets as neonates and boosting the pregnant gilts produced an anamnestic systemic immune response and correspondingly higher milk titers in the primed gilts compared to control animals. Thus, priming piglets with PRCV was beneficial in providing resistance to TGEV and could be incorporated into a vaccine strategy that yields better protection against TGEV.

Animals↗
PubMed1998

Nucleotide sequence of the inter-structural gene region of feline infectious peritonitis virus.

The sequence of the region located between the S and M glycoprotein genes of the 79-1146 strain of feline infectious peritonitis virus (FIPV) is presented. The inter-structural gene region encodes 3 open reading frames (ORFs), termed ORFs 3a, 3b and 4, with nucleotide sequences conforming to the minimum conserved transcription signal upstream of each. An additional ORF, 3x, partially overlaps the 3' end of ORF 3a. The FIPV interstructural gene region is identical in length when compared to the Insavc-1 strain of canine coronavirus (CCV) but differs from various strains of transmissible gastroenteritis virus (TGEV) by the presence of deletions and insertions. The sizes of ORF 3a and 4 are conserved in FIPV, TGEV and CCV. However, as with CCV, the FIPV ORF 3b is truncated in comparison with TGEV.

Animals↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed1998

Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk.

Protection against enteric infections can be provided by the oral administration of pathogen-neutralizing antibodies. To provide passive immunity, 18 lines of transgenic mice secreting a recombinant monoclonal antibody (Mab) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. The genes encoding a chimeric Mab with the variable modules of the murine TGEV-specific Mab 6A.C3 and the constant modules of a human IgG, isotype Mab were expressed under the control of regulatory sequences derived from the whey acidic protein, which is an abundant milk protein. The Mab 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species, which does not allow the selection of neutralization escape mutants. Antibody expression titers of 10(6) were obtained in the milk of transgenic mice that reduced TGEV infectivity 10(6)-fold. The antibody was synthesized at high levels throughout lactation. Integration of matrix attachment region sequences with the antibody genes led to a 20- to 10,000-fold increase in the antibody titer in 50% of the transgenic animals. Antibody expression levels were transgene copy number independent and related to the site of integration. The generation of transgenic animals producing virus neutralizing antibodies in milk could provide an approach to protection against neonatal infections of the enteric tract.

Animals↗
PubMed2004

A novel sorting signal for intracellular localization is present in the S protein of a porcine coronavirus but absent from severe acute respiratory syndrome-associated coronavirus.

Coronaviruses (CoV) mature by a budding process at intracellular membranes. Here we showed that the major surface protein S of a porcine CoV (transmissible gastroenteritis virus) is not transported to the cell surface but is retained intracellularly. Site-directed mutagenesis indicated that a tyrosine-dependent signal (YXXI) in the cytoplasmic tail is essential for intracellular localization of the S protein. Surface expression of mutant proteins was evident by immunofluorescence analysis and surface biotinylation. Intracellularly retained S proteins only contained endoglycosidase H-sensitive N-glycans, whereas mutant proteins that migrated to the plasma membrane acquired N-linked oligosaccharides of the complex type. Corresponding tyrosine residues are present in the cytoplasmic tails of the S proteins of other animal CoV but not in the tail portion of the S protein of severe acute respiratory syndrome (SARS)-CoV. Changing the SEPV tetrapeptide in the cytoplasmic tail to YEPI resulted in intracellular retention of the S protein of SARS-CoV. As the S proteins of CoV have receptor binding and fusion activities and are the main target of neutralizing antibodies, the differences in the transport behavior of the S proteins suggest different strategies in the virus host interactions between SARS-CoV and other coronaviruses.

Amino Acid Substitution↗
PubMed2002

Coronaviruses from pheasants (Phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys.

Reverse-transcriptase polymerase chain reactions (RT-PCRs) were used to examine RNA extracted from mouth/nasal swabs from pheasants exhibiting signs of respiratory disease. The oligonucleotides used were based on sequences of infectious bronchitis virus (IBV), the coronavirus of domestic fowl. A RT-PCR for the highly conserved region II of the 3' untranslated region of the IBV genome detected a coronavirus in swabs from 18/21 estates. Sequence identity with the corresponding region of IBVs and coronaviruses from turkeys was > 95%. A RT-PCR for part of the S1 region of the spike protein gene was positive with 13/21 of the samples. Sequence analysis of the RT-PCR products derived from nine of the pheasant viruses revealed that some of the viruses differed from each other by approximately 24%, similar to the degree of difference exhibited by different serotypes of IBV. Further analysis of the genome of one of the viruses revealed that it contained genes 3 and 5 that are typical of IBV but absent in both the transmissible gastroenteritis virus and murine hepatitis virus groups of mammalian coronaviruses. The nucleotide sequences of genes 3 and 5 of the pheasant virus had a similar degree of identity (approximately 90%) with those of coronaviruses from turkeys and chickens, as is observed when different serotypes of IBV are compared. This work: (a) confirms that coronaviruses are present in pheasants (indeed, commonly present in pheasants with respiratory disease); (b) demonstrates that their genomes are IBV-like in their organization; and (c) shows that there is sequence heterogeneity within the group of pheasant coronaviruses, especially within the spike protein gene. Furthermore, the gene sequences of the pheasant viruses differed from those of IBV to similar extents as the sequence of one serotype of IBV differs from another. On the genetic evidence to date, there is a remarkably high degree of genetic similarity between the coronaviruses of chickens, turkeys and pheasants.

Animals↗
PubMed2004

Molecular dynamics simulations of various coronavirus main proteinases.

In this study, two homology models (denoted as MproST and MproSH) of main proteinase (Mpro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS-CoV) were constructed based on the crystal structures of Mpro from transmissible gastroenteritis coronavirus (TGEV) (MproT) and human coronavirus HcoV-229E (MproH), respectively. Both MproST and MproSH exhibit similar folds as their respective template proteins. These homology models reveal three distinct functional domains as well as an intervening loop connecting domains II and III as found in both template proteins. A catalytic cleft containing the substrate binding sites S1 and S2 between domains I and II are also observed. S2 undergoes more significant structural fluctuation than S1 during the 400 ps molecular dynamics simulations because it is located at the open mouth of the catalytic cleft, while S1 is situated in the very bottom of this cleft. The thermal unfolding of these proteins begins at domain III, where the structure is least conserved among these proteins. Mpro may still maintain its proteolytic activity while it is partially unfolded. The electrostatic interaction between Arg40 and Asp186 plays an important role in maintaining the structural integrity of both S1 and S2.

Amino Acid Sequence↗
PubMed1998

Interferon-alpha response to swine arterivirus (PoAV), the porcine reproductive and respiratory syndrome virus.

We studied the interferon-alpha (IFN-alpha) system in relation to the porcine arterivirus (PoAV), porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant porcine IFN-alpha inhibited the growth of this virus in alveolar macrophage cultures. When pigs were challenged intranasally with PoAV, their serum contained IFN-alpha in relatively low concentrations on the second day after challenge and up to 5 days at the latest. Most animals had no IFN-alpha in their lung secretions, even though PoAV replicates in the respiratory tract. In vitro, PoAV replicates in alveolar macrophages, but neither these nor peripheral blood mononuclear cells (PBMC) produced IFN-alpha in response to infection. This may be because PoAV suppresses IFN-alpha production. When macrophages treated with PoAV were superinfected with swine transmissible gastroenteritis virus (TGEV), a known good inducer of IFN, no IFN-alpha was detected. This suppressive effect was lost when the virus was inactivated by UV light. Our results suggest that downregulation of IFN-alpha production may play an important part in enabling PoAV to replicate in cell cultures and in pigs.

Administration, Intranasal↗
PubMed1990

Nucleotide sequence of the gene encoding the spike glycoprotein of human coronavirus HCV 229E.

The gene encoding the spike glycoprotein of the human coronavirus HCV 229E has been cloned and sequenced. This analysis predicts an S polypeptide of 1173 amino acids with an Mr of 128,600. The polypeptide has 30 potential N-glycosylation sites. A number of structural features typical of coronavirus S proteins can be recognized, including a signal sequence, a membrane anchor, heptad repeat structures and a carboxy-terminal cysteine cluster. A detailed, computer-aided comparison with the S proteins of infectious bronchitis virus, feline infectious peritonitis virus, transmissible gastroenteritis virus and murine hepatitis virus, strain JHM is presented. We have also done a Northern blot analysis of viral RNAs in HCV 229E-infected cells using synthetic oligonucleotides. On the basis of this analysis, and by analogy to the replication strategy of other coronaviruses, we are able to propose a model for the organization and expression of the HCV 229E genome.

Amino Acid Sequence↗
PubMed1991

Sequence analysis of the turkey enteric coronavirus nucleocapsid and membrane protein genes: a close genomic relationship with bovine coronavirus.

The 3' end of the turkey coronavirus (TCV) genome and the gene encoding the nucleocapsid protein (N) were cloned and sequenced. The gene encoding the membrane protein (M) was obtained by cloning a polymerase chain reaction (PCR)-amplified fragment obtained using bovine coronavirus (BCV)-specific primers. Furthermore, five TCV DNA fragments, obtained by PCR on RNA from clinical specimens and corresponding to either the N terminus of the M protein or the complete M protein were also cloned and sequenced. The sequence revealed a 3' non-coding region of 291 bases, an open reading frame (ORF) encoding the N protein with a predicted size of 448 amino acids, or an Mr of 49K, and an ORF encoding the M protein with a predicted size of 230 amino acids and an Mr of 26K. A third ORF, encoding a hypothetical protein of 207 amino acids with an Mr of 23K was found within the N gene sequence. The amino acid sequences of both the N and M proteins were more than 99% similar to those published for BCV. Extensive similarity was also observed between the amino acid sequences of the TCV N protein and those of murine hepatitis virus (MHV) (70%) and human respiratory coronavirus strain OC43 (HCV-OC43) (98%) and between the amino acid sequences of the predicted M proteins of TCV and MHV (86%). Such striking identity suggests that BCV, TCV and HCV-OC43 must have diverged from each other only recently. A potential N-glycosylation site was found at the N terminus of the TCV M protein and is situated at the same location in BCV, MHV and transmissible gastroenteritis virus.

Amino Acid Sequence↗
PubMed1992

Analysis of a 9.6 kb sequence from the 3' end of canine coronavirus genomic RNA.

We have analysed the organization of the 3' end of the genomic RNA of canine coronavirus (CCV), a virus which has a close antigenic relationship to transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV) and feline infectious peritonitis virus (FIPV). Genomic RNA isolated from CCV strain Insavc-1-infected A72 cells was used to generate a cDNA library. Overlapping clones, spanning approximately 9.6 kb [from the 3' end of the polymerase gene, 1b, to the poly(A) tail] were identified. Sequencing and subsequent analyses revealed 10 open reading frames (ORFs). Three of these code for the major coronavirus structural polypeptides S, M and N; a fourth codes for a small membrane protein, SM, a putative homologue of the IBV structural polypeptide 3c, and five code for polypeptides, designated 1b, 3a, 4, 7a and 7b, homologous to putative non-structural polypeptides encoded in the TGEV or FIPV genomes. An extra ORF which had not hitherto been identified in this antigenic group of coronaviruses was designated 3x. Pairwise alignment of these ORFs with their counterparts in TGEV, PRCV and FIPV revealed high levels of identity and highlighted the close relationship between the members of this group of viruses.

Amino Acid Sequence↗