Background paper. The appearance of the porcine respiratory coronavirus has created new problems and perspectives.
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Animals↗
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Induction of milk IgA antibodies by porcine respiratory coronavirus infection.
An ELISA was developed to examine the prevalence of TGEV-specific immunoglobulin (Ig)A in the milk of sows, infected in the field with PRCV or with TGEV. It was shown that previous PRCV-infections can induce the secretion of IgA antibodies in the milk. However, only 9 out of 28 PRCV-infected sows had IgA in their milk whereas 11 TGEV-infected sows all secreted IgA. On farms where a reinfection with PRCV occurred, the number of IgA-secreting sows increased from 2 to 11 on a total of 13 sows. This showed that the presence of IgA antibodies in the milk may depend upon the occurrence of reinfection with PRCV. As demonstrated by density gradient analysis, the milk IgA induced by PRCV was 11S secretory IgA and had the capacity to neutralize TGEV.
Animals↗
Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization.
Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes, were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods.
Animals↗
Establishment of transformed swine fibroblast cell lines using SV40 large T antigen.
Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.
Animals↗
Significance of lysosomes in the morphogenesis of coronaviruses.
Virus-containing electron-dense membrane-bound cytoplasmic bodies are described in tracheal epithelial cells of chickens infected with Infectious Bronchitis Virus and in intestinal epithelial cells of swine infected with Porcine Epidemic Diarrhea Virus. Using silver-methenamine staining, phosphotungstic acid staining and acid phosphatase enzyme cytochemical staining of ultra-thin sections, these bodies were shown to be virus-containing secondary lysosomes and residual bodies. The accumulation of viral particles in the lysosomes is suggested to possibly represent an intracellular defense mechanism. However, no morphological alterations were found indicating a destruction of the viruses by the lytic lysosomal enzymes.
Animals↗
An immunoelectron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, CV 777, and several coronaviruses.
A possible antigenic relationship between the porcine enteropathogenic coronavirus-like agent (CVLA) and 6 known coronaviruses was examined by immunoelectron microscopy (IEM) and by immunofluorescence (IF). CVLA did not show cross reactivity with infectious bronchitis virus, transmissible gastroenteritis virus (TGEV), canine coronavirus (CCV) hemagglutinating encephalomyelitis virus (HEV), neonatal calf diarrhea coronavirus (NCDCV) or feline infectious peritonitis virus (FIPV). Antigenic relationship was detected by IEM between TGEV and CCV, NCDCV and HEV and by IF between TGEV and CCV, TGEV and FIPV, HEV and NCDCV.
Animals↗
Molecular cloning and sequence determination of the peplomer protein gene of feline infectious peritonitis virus type I.
cDNA clones spanning the entire region of the peplomer (S) gene of feline infectious peritonitis virus (FIPV) type I strain KU-2 were obtained and their complete nucleotide sequences were determined. A long open reading frame (ORF) encoding 1464 amino acid residues was found in the gene, which was 12 residues longer than the ORF of the FIPV type II strain 79-1146. The sequences of FIPV type I and mainly -tPV type II were compared. The homologies at the N- (amino acid residues 1-693) and C- (residues 694-1464) terminal halves were 29.8 and 60.7%, respectively. This was much lower than that between FIPV type II and other antigenically related coronaviruses, such as transmissible gastroenteritis virus of swine and canine coronavirus. This supported the serological relatedness of the viruses and confirmed that the peplomer protein of FIPV type I has distinct structural features that differ from those of antigenically related viruses.
Amino Acid Sequence↗
Characteristics of the porcine kidney cell line IB-RS-2 clone D10 (IB-RS-2 D10) which is free of hog cholera virus.
A Brazilian stock of clone C17 of the IB-RS-2 porcine kidney cell line which was contaminated with hog cholera virus (HCV) was cloned. One clone designated IB-RS-2 D10 was determined to be free of HCV, 20 other viruses, and Mycoplasma. IB-RS-2 D10 cells possessed the same viral susceptibility pattern as the contaminated parent cells to the viruses of foot-and-mouth disease, swine vesicular disease, vesicular exanthema of swine, transmissible gastroenteritis, and several other viruses. The IB-RS-2 D10 cells had a median chromosome count of 34, were morphologically epithelioid cells, and were resistant to HCV infection. Freedom from HCV affords advantages for vaccine production and avoids laboratory contamination.
Animals↗
Respiratory and fecal shedding of porcine respiratory coronavirus (PRCV) in sentinel weaned pigs and sequence of the partial S-gene of the PRCV isolates.
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 681 nt) 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.
Animals↗
Testing the hypothesis of a recombinant origin of the SARS-associated coronavirus.
The origin of severe acute respiratory syndrome-associated corona-virus (SARS-CoV) is still a matter of speculation, although more than one year has passed since the onset of the SARS outbreak. In this study, we implemented a 3-step strategy to test the intriguing hypothesis that SARS-CoV might have been derived from a recombinant virus. First, we blasted the whole SARS-CoV genome against a virus database to search viruses of interest. Second, we employed 7 recombination detection techniques well documented in successfully detecting recombination events to explore the presence of recombination in SARS-CoV genome. Finally, we conducted phylogenetic analyses to further explore whether recombination has indeed occurred in the course of coronaviruses history predating the emergence of SARS-CoV. Surprisingly, we found that 7 putative recombination regions, located in Replicase 1ab and Spike protein, exist between SARS-CoV and other 6 coronaviruses: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), bovine coronavirus (BCoV), human coronavirus 229E (HCoV), murine hepatitis virus (MHV), and avian infectious bronchitis virus (IBV). Thus, our analyses substantiate the presence of recombination events in history that led to the SARS-CoV genome. Like the other coronaviruses used in the analysis, SARS-CoV is also a mosaic structure.
Computational Biology↗
SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses.
Coronaviruses are etiologic agents of respiratory and enteric diseases in humans and in animals. In this study, a one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on SYBR Green chemistry and degenerate primers was developed for the generic detection of coronaviruses. The primers, designed in the open reading frame 1b, enabled the detection of 32 animal coronaviruses including strains of canine coronavirus, feline coronavirus, transmissible gastroenteritis virus (TGEV), bovine coronavirus (BCoV), murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). A specific amplification was also observed with the human coronaviruses (HCoV) HCoV-NL63, HCoV-OC43, HCoV-229E and severe acute respiratory syndrome coronavirus (SARS-CoV). The real-time RT-PCR detected down to 10 cRNA copies from TGEV, BCoV, SARS-CoV and IBV. In addition, the assay exhibited a high sensitivity and specificity on clinical samples from different animal species. The developed assay represents a potential tool for laboratory diagnostics and for detecting still uncharacterized coronaviruses.
Animals↗
Design and construction of African swine fever virus chimeras incorporating foreign viral epitopes.
In the present work we have studied the feasibility of introducing foreign epitopes into the African swine fever virus (ASFV) particle by genetic manipulation of the virus. For this purpose, we developed specific transfer vectors containing the gene encoding for the highly antigenic structural ASFV protein p54 in which foreign sequences were introduced. DNA sequences encoding continuous linear epitopes, the antigenic site A from foot-and-mouth disease virus (FMDV) VP1 protein and the DA3 antigenic determinant from transmissible gastroenteritis coronavirus (TGEV) nucleoprotein N, were separately cloned into the p54 gene, in a region encoding a non-essential domain of the protein. Chimeric p54 genes were inserted by homologous recombination into the thymidine kinase (TK) locus of ASFV genome. The resulting recombinant viruses efficiently expressed both chimeric proteins under transcriptional control of the p54 promoter, and the chimeric gene products were recognized by antibodies to both p54 and foreign epitopes. The modified p54 proteins were also found in the viral particles and complemented the function of the wild-type p54, since deletion of the p54 gene from recombinant viruses did not affected virus replication in Vero cells. This work demonstrates for the first time the feasibility of incorporating foreign amino acid sequences (up to 18 residues) into a protein component of the ASFV particle without affecting virus viability.
African Swine Fever Virus↗
Feline and canine coronaviruses are released from the basolateral side of polarized epithelial LLC-PK1 cells expressing the recombinant feline aminopeptidase-N cDNA.
In this study feline (FECV and FIPV) and canine (CCoV) coronavirus entry into and release from polarized porcine epithelial LLC-PK1 cells, stably expressing the recombinant feline aminopeptidase-N cDNA, were investigated. Virus entry appeared to occur preferentially through the apical membrane, similar to the entry of the related porcine coronavirus transmissible gastroenteritis virus (TGEV) into these cells. However, whereas TGEV is released apically, feline and canine coronaviruses were found to be released from the basolateral side of the epithelial cells. These observations indicate that local infections as caused by TGEV, FECV and CCoV do not strictly correlate with apical release, as suggested by earlier work.
Aminopeptidases↗
Sialic acids as receptor determinants for coronaviruses.
Among coronaviruses, several members are able to interact with sialic acids. For bovine coronavirus (BCoV) and related viruses, binding to cell surface components containing N-acetyl-9- O-acetylneuraminic acid is essential for initiation of an infection. These viruses resemble influenza C viruses because they share not only the receptor determinant, but also the presence of an acetylesterase that releases the 9- O-acetyl group from sialic acid and thus abolishes the ability of the respective sialoglycoconjugate to function as a receptor for BCoV. As in the case of influenza viruses, the receptor-destroying enzyme of BCoV is believed to facilitate the spread of virus infection by removing receptor determinants from the surface of infected cells and by preventing the formation of virus aggregates. Another coronavirus, porcine transmissible gastroenteritis virus (TGEV) preferentially recognizes N-glycolylneuraminic acid. TGEV does not contain a receptor-destroying enzyme and does not depend on the sialic acid binding activity for infection of cultured cells. However, binding to sialic acids is required for the enteropathogenicity of TGEV. Interaction with sialoglycoconjugates may help the virus to pass through the sialic acid-rich mucus layer that covers the viral target cells in the epithelium of the small intestine. We discuss that the BCoV group of viruses may have evolved from a TGEV-like ancestor by acquiring an acetylesterase gene through heterologous recombination.
Acetylation↗
The tissue distribution of spontaneous cell-mediated cytotoxicity effector lymphocytes in swine.
Peripheral blood lymphocytes, intraepithelial lymphocytes from the small intestine and lymphocytes from the thymus, spleen, mesenteric lymph nodes and Peyer's patches from 5 young adult pigs were used as effector cells in a spontaneous cell-mediated cytotoxicity chromium release assay against PK-15 cells persistently infected with transmissible gastroenteritis virus as targets. Both peripheral blood and intraepithelial lymphocytes caused marked specific chromium release, while the lymphocytes from the remaining tissues were inactive in spontaneous cell-mediated cytotoxicity.
Animals↗
The participation of antibody in spontaneous and antibody-dependent cell-mediated cytotoxicity in the pig.
Treatment of porcine lymphocytes with trypsin reduced their spontaneous cell-mediated cytotoxicity (SCMC) activity against target cells persistently infected with transmissible gastroenteritis virus (PK15-TGE cells), but had no effect on antibody-dependent cell-mediated cytotoxicity (ADCC). SCMC activity was partially restored to trypsin-treated lymphocytes by incubation in RPMI-1640 medium or in medium containing F(ab')2 fragments of rabbit anti-porcine immunoglobulin, but not by brief incubation in autologous serum. F(ab')2 fragments of anti-porcine immunoglobulin did not block the SCMC reaction, but ADCC was greatly reduced by this reagent. Thus SCMC and ADCC mediated by porcine lymphocytes against PK15-TGE target cells clearly involved two distinct mechanisms in terms of antibody participation and sensitivity to trypsin.
Animals↗
Sequence analysis of the nucleocapsid protein gene of human coronavirus 229E.
Human coronaviruses are important human pathogens and have also been implicated in multiple sclerosis. To further understand the molecular biology of human coronavirus 229E (HCV-229E), molecular cloning and sequence analysis of the viral RNA have been initiated. Following established protocols, the 3'-terminal 1732 nucleotides of the genome were sequenced. A large open reading frame encodes a 389 amino acid protein of 43,366 Da, which is presumably the nucleocapsid protein. The predicted protein is similar in size, chemical properties, and amino acid sequence to the nucleocapsid proteins of other coronaviruses. This is especially evident when the sequence is compared with that of the antigenically related porcine transmissible gastroenteritis virus (TGEV), with which a region of 46% amino acid sequence homology was found. Hydropathy profiles revealed the existence of several conserved domains which could have functional significance. An intergenic consensus sequence precedes the 5'-end of the proposed nucleocapsid protein gene. The consensus sequence is present in other coronaviruses and has been proposed as the site of binding of the leader sequence for mRNA transcriptional start. This region was also examined by primer extension analysis of mRNAs, which identified a 60-nucleotide leader sequence. The 3'-noncoding region of the genome contains an 11-nucleotide sequence, which is relatively conserved throughout the Coronavirus family and lends support to the theory that this region is important for the replication of negative-strand RNA.
Amino Acid Sequence↗
Sequence analysis of the membrane protein gene of human coronavirus 229E.
Human coronaviruses (HCV) are ubiquitous pathogens which cause respiratory, gastrointestinal, and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the complete nucleotide sequence of the membrane protein (M) gene was determined from cloned cDNA. The open reading frame is preceded by a consensus transcriptional initiation sequence UCUAAACU, identical to the one found upstream of the N gene. The M gene encodes a 225-amino acid polypeptide with a molecular weight (MW) of 25,822, slightly higher than the apparent MW of 19,000-22,000 observed for the unprocessed M protein obtained after in vitro translation and immunoprecipitation. The M amino acid sequence presents a significant degree of homology (38%) with its counterpart of transmissible gastroenteritis coronavirus (TGEV). The M protein of HCV-229E is highly hydrophobic and its hydropathicity profile shows a transmembranous region composed of three major hydrophobic domains characteristic of a typical coronavirus M protein. About 10% (20 amino acids) of the HCV-229E M protein constitutes a hydrophilic and probably external portion. One N-glycosylation and three potential O-glycosylation sites are found in this exposed domain.
Amino Acid Sequence↗