Environmental stability of porcine respiratory coronavirus in aquatic environments.

Coronaviruses (CoVs) are a family of viruses that are best known as the causative agents of human diseases like the common cold, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) and COVID-19. CoVs spread by human-to-human transmission via droplets or direct contact. There is, however, concern about potential waterborne transmission of SARS-CoV-2, the virus responsible for COVID-19, as it has been found in wastewater facilities and rivers. To date, little is known about the stability of SARS-CoV-2 or any other free coronavirus in aquatic environments. The inactivation of terrestrial CoVs in seawater is rarely studied. Here, we use a porcine respiratory coronavirus (PRCV) that is commonly found in animal husbandry as a surrogate to study the stability of CoVs in natural water. A series of experiments were conducted in which PRCV (strain 91V44) was added to filtered and unfiltered fresh- and saltwater taken from the river Scheldt and the North Sea. Virus titres were then measured by TCID50-assays using swine testicle cell cultures after various incubation times. The results show that viral inactivation of PRCV in filtered seawater can be rapid, with an observed 99% decline in the viral load after just two days, which may depend on temperature and the total suspended matter concentration. PRCV degraded much slower in filtered water from the river Scheldt, taking over 15 days to decline by 99%, which was somewhat faster than the PBS control treatment (T99 = 19.2 days). Overall, the results suggest that terrestrial CoVs are not likely to accumulate in marine environments. Studies into potential interactions with exudates (proteases, nucleases) from the microbial food web are, however, recommended.

Comparative Pathogenesis of Bovine and Porcine Respiratory Coronaviruses in the Animal Host Species and SARS-CoV-2 in Humans.

Discovery of bats with severe acute respiratory syndrome (SARS)-related coronaviruses (CoVs) raised the specter of potential future outbreaks of zoonotic SARS-CoV-like disease in humans, which largely went unheeded. Nevertheless, the novel SARS-CoV-2 of bat ancestral origin emerged to infect humans in Wuhan, China, in late 2019 and then became a global pandemic. Less than 5 months after its emergence, millions of people worldwide have been infected asymptomatically or symptomatically and at least 360,000 have died. Coronavirus disease 2019 (COVID-19) in severely affected patients includes atypical pneumonia characterized by a dry cough, persistent fever, and progressive dyspnea and hypoxia, sometimes accompanied by diarrhea and often followed by multiple organ failure, especially of the respiratory and cardiovascular systems. In this minireview, we focus on two endemic respiratory CoV infections of livestock: bovine coronavirus (BCoV) and porcine respiratory coronavirus (PRCV). Both animal respiratory CoVs share some common features with SARS-CoV and SARS-CoV-2. BCoV has a broad host range including wild ruminants and a zoonotic potential. BCoV also has a dual tropism for the respiratory and gastrointestinal tracts. These aspects, their interspecies transmission, and certain factors that impact disease severity in cattle parallel related facets of SARS-CoV or SARS-CoV-2 in humans. PRCV has a tissue tropism for the upper and lower respiratory tracts and a cellular tropism for type 1 and 2 pneumocytes in lung but is generally a mild infection unless complicated by other exacerbating factors, such as bacterial or viral coinfections and immunosuppression (corticosteroids).

Porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs.

We performed a comprehensive analysis of innate and adaptive immune responses in dual-virus infected pigs to understand whether a pre-existing immunomodulatory respiratory viral infection affects the overall immunity to a subsequent porcine respiratory coronavirus (PRCV) infection in pigs. Pigs were either mock-infected or infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus known to cause immunosuppressive respiratory disease, and then pigs were co-infected with PRCV, which normally causes subclinical respiratory infection. We collected samples for six independent experiments from 178 pigs that were also used for pathological studies. We detected a significant reduction in innate NK-cell-mediated cytotoxic function in PRRSV-infected pigs, which was synergistically further decreased in pigs co-infected with PRCV. Subsequently, in association with clinical signs we observed elevated levels of proinflammatory (IL-6), Th-1 (IL-12), and regulatory (IL-10 and TGF-ß) cytokines. Increased frequencies of CD4CD8 double-positive T lymphocytes and myeloid cells, in addition to the elevated Th-1 and proinflammatory cytokines in dual-infected pigs, contributed to the severity of lung disease in pigs. The results of our study clarify how each virus modulates the host innate and adaptive immune responses, leading to inflammatory reactions and lung pathology. Thus measurements of cytokines and frequencies of immune cells may serve as indicators of the progression of respiratory viral co-infections, and provide more definitive approaches for treatment.

Virological and serological studies of porcine respiratory coronavirus infection on a Japanese farm.

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.

Detection of respiratory pathogens in air samples from acutely infected pigs.

Pathogens causing significant respiratory disease in growing pigs include Porcine reproductive and respiratory syndrome virus, Porcine circovirus 2, swine influenza virus, porcine respiratory coronavirus, Mycoplasma hyopneumoniae, and Bordetella bronchiseptica. The objective of this research was to characterize the respiratory excretion of these pathogens by acutely infected pigs. Pigs were inoculated under experimental conditions with 1 pathogen. Samples were collected from the upper respiratory tract and exhaled air. All pathogens were detected in swabs of the upper respiratory tract, but only M. hyopneumoniae and B. bronchiseptica were detected in expired air from individually sampled, acutely infected pigs. These findings suggest either that the acutely infected pigs did not aerosolize the viruses or that the quantity of virus excreted was below the detection threshold of current sampling or assay systems, or both, at the individual-pig level.

Altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus.

The pathogenesis and optimal treatments for severe acute respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of porcine respiratory coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] [n = 41], PRCV/DEX [n = 41], mock/PBS [n = 23], and mock/DEX [n = 25]) were inoculated intranasally and intratracheally with the ISU-1 strain of PRCV (1 x 10(7) PFU) or cell culture medium. DEX was administered (once daily, 2 mg/kg of body weight/day, intramuscularly) from postinoculation day (PID) 1 to 6. In PRCV/DEX pigs, significantly milder pneumonia, fewer PRCV-positive cells, and lower viral RNA titers were present in lungs early at PID 2; however, at PID 4, 10, and 21, severe bronchointerstitial pneumonia, significantly higher numbers of PRCV-positive cells, and higher viral RNA titers were observed compared to results for PRCV/PBS pigs. Significantly lower numbers of CD2(+), CD3(+), CD4(+), and CD8(+) T cells were also observed in lungs of PRCV/DEX pigs than in those of PRCV/PBS pigs at PID 8 and 10, coincident with fewer gamma interferon (IFN-gamma)-secreting cells in the tracheobronchial lymph nodes as determined by enzyme-linked immunospot assay. Our results confirm that DEX treatment alleviates PRCV pneumonia early (PID 2) in the infection but continued use through PID 6 exacerbates later stages of infection (PID 4, 10, and 21), possibly by decreasing cellular immune responses in the lungs (IFN-gamma-secreting T cells), thereby creating an environment for more-extensive viral replication. These data have potential implications for corticosteroid use with SARS-CoV patients and suggest a precaution against prolonged use based on their unproven efficacy in humans, including possible detrimental secondary effects.

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.