Porcine Respiratory Coronavirus Infection Research Articles

Porcine lung tissue slices: a culture model for PRCV infection and innate immune response investigations

Respiratory coronaviruses (RCoVs) significantly threaten human health, necessitating the development of an ex vivo respiratory culture system for investigating RCoVs infection. Here, we successfully generated a porcine precision-cut lung slices (PCLSs) culture system, containing all resident lung cell types in their natural arrangement. Next, this culture system was inoculated with a porcine respiratory coronavirus (PRCV), exhibiting clinical features akin to humans who were infected by SARS-CoV-2. The results demonstrated that PRCV efficiently infected and replicated within PCLSs, targeting ciliated cells in the bronchioles, terminal bronchioles, respiratory bronchioles, and pulmonary alveoli. Additionally, through RNA-Seq analysis of the innate immune response in PCLSs following PRCV infection, expression levels of interferons, inflammatory cytokines and IFN stimulated genes were significantly upregulated. This ex vivo model may not only offer new insights into PRCV infection in the porcine respiratory tract but also serve as a valuable tool for studying human respiratory CoVs infection.

SEROPREVALENCE OF PORCINE RESPIRATORY CORONAVIRUS AND TRANSMISSIBLE GASTROENTERITIS VIRUS INFECTIONS ON COMMERCIAL PIG FARMS IN SERBIA

Porcine respiratory coronavirus is an enzootic, viral, respiratory disease of pigs, which manifests with mild clinical signs, but it takes part in the etiopathogenesis of the porcine respiratory disease complex. The virus was first discovered in Belgium in 1984 as a deletion mutant of the transmissible gastroenteritis virus. The two viruses are strongly antigenically related which is why they cross-react in serological tests. In this study, we tested 276 serum samples from different categories of pigs using ELISA test, which allows differentiation between the porcine respiratory coronavirus infection and transmissible gastroenteritis. The seroconversion for coronavirus infection was determined in 80.4% of tested samples. Out of 222 positive samples, 219 samples (98.6%) were positive for porcine respiratory coronavirus antibodies, while 3 (1.01%) samples were positive for transmissible gastroenteritis virus antibodies. Depending on the production category, 97.7% of piglets, 83% of sows, and 35% of gilts tested positive for porcine respiratory coronavirus antibodies. In total, 2.3% of piglets tested positive for transmissible gastroenteritis virus antibodies. Taking into account the characteristics of the ELISA test, its sensitivity and specificity, this result can be considered a false positive, because of a cross-reaction between the porcine respiratory coronavirus antibodies and the transmissible gastroenteritis virus. Specific antibodies in other swine production categories against the transmissible gastroenteritis virus were not determined.

Long-Term Expanding Porcine Airway Organoids Provide Insights into the Pathogenesis and Innate Immunity of Porcine Respiratory Coronavirus Infection.

Respiratory coronaviruses cause serious health threats to humans and animals. Porcine respiratory coronavirus (PRCoV), a natural transmissible gastroenteritis virus (TGEV) mutant with partial spike deletion, causes mild respiratory disease and is an interesting animal respiratory coronavirus model for human respiratory coronaviruses. However, the absence of robust ex vivo models of porcine airway epithelium hinders an understanding of the pathogenesis of PRCoV infection. Here, we generated long-term porcine airway organoids (AOs) derived from basal epithelial cells, which recapitulate the in vivo airway complicated epithelial cellularity. Both 3D and 2D AOs are permissive for PRCoV infection. Unlike TGEV, which established successful infection in both AOs and intestinal organoids, PRCoV was strongly amplified only in AOs, not intestinal organoids. Furthermore, PRCoV infection in AOs mounted vigorous early type I and III interferon (IFN) responses and upregulated the expression of overzealous inflammatory genes, including pattern recognition receptors (PRRs) and proinflammatory cytokines. Collectively, these data demonstrate that stem-derived porcine AOs can serve as a promising disease model for PRCoV infection and provide a valuable tool to study porcine respiratory infection. IMPORTANCE Porcine respiratory CoV (PRCoV), a natural mutant of TGEV, shows striking pathogenetic similarities to human respiratory CoV infection and provides an interesting animal model for human respiratory CoVs, including SARS-CoV-2. The lack of an in vitro model recapitulating the complicated cellularity and structure of the porcine respiratory tract is a major roadblock for the study of PRCoV infection. Here, we developed long-term 3D airway organoids (AOs) and further established 2D AO monolayer cultures. The resultant 3D and 2D AOs are permissive for PRCoV infection. Notably, PRCoV mediated pronounced IFN and inflammatory responses in AOs, which recapitulated the inflammatory responses associated with PRCoV in vivo infection. Therefore, porcine AOs can be utilized to characterize the pathogenesis of PRCoV and, more broadly, can serve as a universal platform for porcine respiratory infection.

Porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections

The innate immune response is critical for host defence against respiratory coronaviruses (CoVs). This study demonstrated that an ongoing respiratory virus infection compromises innate immune responses and affects the pathogenesis of a respiratory CoV co-infection. An innate immunosuppressive respiratory virus infection was established by infecting weaned pigs with porcine reproductive and respiratory syndrome virus (PRRSV); 10 days later, the pigs were exposed to porcine respiratory coronavirus (PRCV). The PRRSV/PRCV dual-infected pigs had reduced weight gains, a higher incidence of fever and more severe pneumonia compared with either single infection. Significant suppression of innate immune responses [reduced alpha interferon (IFN-alpha) levels in the lungs and reduced blood natural killer cell cytotoxicity] by the ongoing PRRSV infection was observed in dual-infected pigs, which coincided with exacerbated pneumonia during early PRCV infection. The subsequent PRCV infection led to enhanced PRRSV replication in the lungs and a trend towards increased serum T-helper type 1 (Th1) (IFN-gamma) but decreased Th2 [interleukin (IL)-4] responses, further exacerbating PRRSV pneumonia. Following PRCV infection, more severe PRRSV-related pulmonary alveolar macrophage (PAM) apoptosis occurred, as determined by an in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay, suggesting increased PRRSV replication in PAMs. Collectively, these observations suggest interactive effects between PRCV and PRRSV via early innate (IFN-alpha) and later adaptive Th1 (IFN-gamma) and Th2 (IL-4) immune responses. These findings imply that an existing immunomodulating respiratory viral co-infection may be a contributing factor to more severe pneumonia in respiratory CoV disease. This study provides new insights into host-pathogen interactions related to co-infection by CoVs and other respiratory viruses.

Lung Cell Tropism and Inflammatory Cytokine-Profile of Porcine Respiratory Coronavirus Infection

Knowledge about porcine respiratory coronavirus (PRCV) tropism was limited to morphological identification of target cells and controversial reports on replication in macrophages. This study aimed to clarify the lung cell tropism of a Belgian PRCV strain and to examine the lung profile of inflammatory cytokines for 15 days after intratracheal PRCV inoculation of gnotobiotic piglets. Until 5 days after inoculation, more than 50% of the PRCV-positive cells were type 2 pneumocytes, 30% bronchiolar epithelial cells, and 10% macrophages, as demonstrated by immunofluorescence stainings with specific cell markers. In vitro, PRCV productively infected primary porcine lung epithelial cells, but not porcine al- veolar macrophages. Bronchoalveolar lavage fluids of PRCV-inoculated pigs contained high levels of interferons (IFN-� , IFN-� ) and interleukin-6, moderate interleukin-12 and low tumour necrosis factor-� levels. Our results indicate that PRCV infects primarily type 2 pneumocytes and induces lower lung cytokine levels, compared to swine influenza virus or lipopolysaccharide-complicated PRCV infections.

Cytokine Responses in Porcine Respiratory Coronavirus-Infected Pigs Treated with Corticosteroids as a Model for Severe Acute Respiratory Syndrome

The effectiveness and potential immunosuppressive effects of anti-inflammatory glucocorticoids in the lungs of severe acute respiratory syndrome (SARS) patients are undefined. We treated porcine respiratory coronavirus (PRCV)-infected conventional pigs with the corticosteroid dexamethasone (DEX) as a model for SARS. Innate and Th1 cytokines in bronchoalveolar lavage (BAL) and serum were elevated in PRCV-infected pigs compared to controls, but were decreased after DEX treatment in the PRCV-infected, DEX-treated (PRCV/DEX) pigs. Although decreased in BAL, Th2 cytokine levels were higher in serum after DEX treatment. Levels of the proinflammatory cytokine interleukin-6 in BAL and serum were decreased in PRCV/DEX pigs early but increased later compared to those in phosphate-buffered saline-treated, PRCV-infected pigs, corresponding to a similar trend for lung lesions. PRCV infection increased T-cell frequencies in BAL, but DEX treatment of PRCV-infected pigs reduced frequencies of T cells; interestingly B and SWC3a(+) (monocytes/macrophages/granulocytes) cell frequencies were increased. DEX reduced numbers of PRCV-stimulated Th1 gamma interferon-secreting cells in spleen, tracheobroncheolar lymph nodes, and blood. Our findings suggest that future glucocorticoid treatment of SARS patients should be reconsidered in the context of potential local immunosuppression of immune responses in lung and systemic Th1 cytokine-biased suppression.

Prevalence and Source of Trypanosome Infections in Field-Captured Vector Flies (Glossina pallidipes) in Southeastern Zambia

We detected transmissible gastroenteritis virus (TGEV) antibodies in pig farms in Tochigi prefecture, although the farms had no past record of TGEV vaccination or TGE. Among the farms, Farm A showed a high antibody incidence. We could not confirm if either TGEV or porcine respiratory coronavirus (PRCV) induced the antibodies, since conventional tests failed to discriminate PRCV from TGEV. Therefore, we conducted virological and serological examinations of this farm for 4 years to establish the etiology - TGEV or PRCV. Although no TGEV was detected, PRCVs were isolated from the nasal samples of pigs. Using a commercial ELISA kit, it was found that the antibodies detected in pigs of all the raising stages and sows were raised against PRCV but not TGEV. The phylogenetic analysis of the nucleotide sequences of the isolates showed that they were closely related to each other, and formed a separate cluster apart from the U.S.A. and European strains. In Cesarean-derived, colostrums-deprived piglets inoculated with a PRCV isolate, no clinical signs were seen, and the viruses were mainly isolated from the nasal samples. Moreover, viral genes were detected from the nasal sample of the contact pig. The result suggested that PRCV infection was located in the nasal cavity of pigs, and horizontal transmission easily occurs. From these results, PRCVs with different origins from the exotic PRCVs might be prevalent in pig farms in Japan.

Virological and Serological Studies of Porcine Respiratory Coronavirus Infection on a Japanese Farm

The prevalence of trypanosome infections in tsetse flies, Glossina pallidipes, collected from Chiawa and Chakwenga in Zambia with endemic trypanosomosis was assessed by polymerase chain reaction (PCR). Out of the 550 G. pallidipes, 58 (10.5%) flies were found to harbor trypanosome DNA. Infection rates of tsetse with Trypanosoma vivax universal, Trypanosoma congolense savannah, T. congolense forest and T. congolense kilifi were 4.2% (23/550), 4.7% (26/550), 1.1% (6/550) and 1.6% (9/550), respectively. To determine the mammalian hosts of T. congolense and T. vivax infections from the tsetse flies, mammalian mitochondrion DNA of blood meal in these flies were analyzed by PCR and subsequent gene sequence analysis of the amplicons. Sequence analysis showed the presence of cytochrome b gene (cyt b) of 7 different mammalian species such as human, elephant, buffalo, goat, warthog, greater kudu and cattle. Goats which were main livestock in these areas were further examined to know the extent of its contribution in spreading the infection. We examined the prevalence of trypanosome infections in the domestic goat population in 6 settlements in Chiawa alone. Of the 86 goats sampled, 4 (4.6%), 5 (5.8%), 4 (4.6%) and 4 (4.6%) were positive for T. vivax universal, T. congolense savannah, forest and kilifi, respectively. These findings showed that the host-source of trypanosome infections in vector fly give a vital information about spread of infection. The result of this study will certainly contribute in elucidating more the epidemiology of trypanosomosis.

Effect of porcine respiratory coronavirus infection on lipopolysaccharide recognition proteins and haptoglobin levels in the lungs

Porcine respiratory coronavirus (PRCV) potentiates respiratory disease and proinflammatory cytokine production in the lungs upon intratracheal inoculation with lipopolysaccharide (LPS) at 1 day of infection. This study aimed to quantify LPS-binding protein (LBP), CD14 and haptoglobin in the lungs throughout a PRCV infection. LBP and CD14 recognize LPS and enhance its endotoxic activity, whereas haptoglobin dampens it. Gnotobiotic pigs were inoculated intratracheally with PRCV ( n = 34) or saline ( n = 5) and euthanized 1–15 days post inoculation (DPI). Virus was detected in the lungs from 1 to 9 DPI. Cell-associated CD14 in lung tissue increased up to 15 times throughout the infection, due to an increase in highly CD14 + monocyte-macrophages from 1 to 12 DPI and CD14 + type 2 pneumocytes from 7 to 9 DPI. LBP and soluble CD14 levels in bronchoalveolar lavage fluids were elevated from 1–12 DPI, with up to 35- and 4-fold increases, respectively. Haptoglobin levels increased significantly (×4.5) at 7 DPI. In addition, we found that PRCV could sensitize the lungs to LPS throughout the infection, but the response to LPS appeared less enhanced at the end of infection (7 DPI). The marked increases in LBP, CD14 and haptoglobin were not correlated with the extent of the LPS response.

Use of Monoclonal Antibodies in Blocking ELISA Detection of Transmissible Gastroenteritis Virus in Faeces of Piglets

SummaryMonoclonal antibodies (mAb) to the transmissible gastroenteritis virus (TGEV) nucleoprotein (N) and membrane protein (M) were prepared and used for the comparative assessment of three blocking ELISA variants to detect TGEV. The competitive blocking ELISA format showed the highest sensitivity, allowing detection of 103 TCID50 TGEV/ml in culture medium. Ninety‐nine porcine field faecal samples obtained from 37 herds affected with diarrhoea were examined, and various TGEV levels were found in nine samples from six herds. However, only in three samples were significant TGEV concentrations demonstrated. The relationship between incidence of TGEV gastroenteritis and the spread of porcine respiratory coronavirus infection in pig farms is discussed.

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.

Risk factors associated with seropositivity to porcine respiratory coronavirus in Danish swine herds

Serological screening of swine herds in 1984 indicated that porcine respiratory coronavirus (PRCV) had been introduced into Denmark. To determine risk factors associated with the introduction of PRCV, a cross-sectional study of 408 Danish swine herds was carried out between May 1985 and June 1986. The association between herd-PRCV serological status and possible risk factors, obtained from a field questionnaire, was assessed by unconditional maximum likelihood logistic regression. An increasing herd size, location in the Jutland peninsula (compared with location on the island of Funen) ( OR = 7.9 in a multivariable logistic regression model not including interaction terms), the presence of a slurry system (i.e. pigs living on a slatted floor) ( OR = 4.6) and purchase of pigs ( OR= 1.7) were significantly ( P < 0.05) associated with seropositivity. Two significant interactions, both involving herd size, were subsequently identified. The PRCV serological status of neighbouring herds was found to be related, and closeness of a seropositive herd was associated with an increased risk of a herd being serologically positive. The results of this study indicate that herd size may be an important determinant of airborne transmission of PRCV infection, and that herd size may modify the effect of other risk factors.

Immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine

Despite the pioneering efforts to identify correlates of passive immunity to transmissible gastroenteritis virus (TGEV), effective vaccines for the control of TGE in suckling pigs have remained elusive. The initial concept of an enteromammary immunologic axis in monogastrics originated from studies of lactogenic immunity to TGEV in swine. These studies revealed that infection of pregnant swine with virulent TGEV stimulated high titers of SIgA antibodies in milk which correlated with protection of suckling pigs against TGE; parenteral or oral inoculation with live attenuated or killed TGEV vaccines induced mainly IgG antibodies in milk which generally provided poor protection to suckling pigs. The recent appearance of PRCV infections in swine and continuing studies of TGEV infections, present a unique model for further studies of mucosal immunity. Research using these viruses has increased our understanding of the various components of the common mucosal immune system and their interactions. Although the most important consideration in designing an effective vaccine for TGEV is the stimulation of GALT through intestinal virus replication, studies addressing the contribution of BALT to immunity to TGEV and PRCV may provide insights for alternative vaccine approaches. The mechanism by which exposure to PRCV elicits a variable degree of immunity to TGEV challenge is unknown. Virus replication in the gut or respiratory tract is a major factor affecting the magnitude of the immune response at the respective site and may be necessary for the recruitment of specific immune cells from other mucosal inductive sites, i.e., GALT to BALT and BALT to GALT migration. Further studies on the induction and immune regulation of specific responses to TGEV and PRCV that affect the distribution patterns of IgM-, IgG- and IgA-antibody-secreting cells (ASC) and T lymphocytes should provide valuable insights for optimizing vaccine regimens to elicit the highest mucosal immune responses and optimal protection against TGEV challenge.

An overview of successful TGEV vaccination strategies and discussion on the interrelationship between TGEV and PRCV.

Porcine respiratory coronavirus (PRCV) is a new variant of TGE with an altered pathogenesis. PRCV multiplies mainly in tonsilar tissues and the respiratory tract. There are no enteric symptoms and in experimentally infected pigs, even the respiratory tract infection is usually asymptomatic. PRCV is spread aerogenically through herds and the significance of PRCV as a pathogen in swine has yet to be determined. Despite the differences in pathogenesis and tissue tropism, the behavior of TGEV and PRCV are closely related antigenically. PRCV induces an antibody response in pigs that cannot be distinguished from TGEV-infected pigs by conventional serological assays. PRCV sensitized animals are not protected from TGEV challenge nor is the milk antibody provided to nursing piglets completely effective in prevention of TGEV infections; thus PRCV is not a good vaccine candidate for TGEV infections. PRCV subclinical infections have led to several reported cases of enzootic TGEV in herds that had been diagnosed as TGEV immune strictly on the basis of serum neutralizing titers which were later found to be due to exposure to PRCV. Vaccination studies conducted with the Ambico, oral modified live TGEV vaccine have led to some startling new results: (1) Use of Ambico TGEV modified live vaccine has been shown to provide complete protection against subsequent PRCV challenge and (2) the effectiveness of TGEV vaccination is actually enhanced by previous exposure to PRCV (3) Weanling pigs which have passively acquired circulating TGEV neutralizing antibodies are protected from subsequent PRCV infections.

Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe.

A plasmid, pG3BS, containing a cDNA clone from the 5' coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.

Infection with porcine respiratory coronavirus does not fully protect pigs against intestinal transmissible gastroenteritis virus

Eight nine-week-old specific-pathogen-free pigs which had been infected with the transmissible gastroenteritis virus (TGEV)-related porcine respiratory coronavirus (PRCV) and four uninfected littermates were challenged with TGEV. The previous PRCV infection failed to protect them against the enteric TGEV infection. Virus excretion in faeces was detected by an ELISA in all the pigs for three to six consecutive days after inoculation. Although little diarrhoea was observed, the infection extended through much of the small intestine of one of the previously infected pigs four days after inoculation. Challenge with TGEV caused a secondary neutralising antibody response. By using a peroxidase conjugate of a monoclonal antibody which recognises a specific antigenic site on TGEV, antibodies against TGEV could be distinguished from antibodies against PRCV in an ELISA blocking test.