Coinfection with porcine epidemic diarrhea virus and Clostridium perfringens type A enhances disease severity in weaned pigs.

Clostridium perfringens is a constituent of the normal gut microbiome in pigs; however, it can potentially cause pre- and post-weaning diarrhea. Nevertheless, the importance of this bacterium as a primary pathogen of diarrhea in piglets needs to be better understood, and the epidemiology of C. perfringens in Korean pig populations is unknown. To study the prevalence and typing of C. perfringens, 203 fecal samples were collected from diarrheal piglets on 61 swine farms during 2021-2022 and examined for the presence of C. perfringens and enteric viruses, including porcine epidemic diarrhea virus (PEDV). We determined that the most frequently identified type of C. perfringens was C. perfringens type A (CPA; 64/203, 31.5%). Among the CPA infections, single infections with CPA (30/64, 46.9%) and coinfections with CPA and PEDV (29/64, 45.3%) were the most common in diarrheal samples. Furthermore, we conducted animal experiments to investigate the clinical outcome of single infections and coinfections with highly pathogenic (HP)-PEDV and CPA in weaned piglets. The pigs infected with HP-PEDV or CPA alone showed mild or no diarrhea, and none of them died. However, animals that were co-inoculated with HP-PEDV and CPA showed more-severe diarrheal signs than those of the singly infected pigs. Additionally, CPA promoted PEDV replication in coinfected piglets, with high viral titers in the feces. A histopathological examination revealed more-severe villous atrophy in the small intestine of coinfected pigs than in singly infected pigs. This indicates a synergistic effect of PEDV and CPA coinfection on clinical disease in weaned piglets.

Oral administration of Lactic acid bacteria inhibits PEDV infection in young piglets.

Since the emergence of the highly pathogenic porcine epidemic diarrhea virus (PEDV) strain in 2010, the prevention of porcine epidemic diarrhea (PED) in pig farms remains problematic. To find the reasons behind the high mortality in young piglets, the relative mRNA expression of inflammation-related factors in infected pigs of different ages as well as uninfected pigs were detected by RT-qPCR. The results showed that the mRNA expression of these factors including IL-6 and TNF-α was more increased in infected younger piglets than infected older pigs. To clarify the relationship between these inflammation related factors, the pairwise linear correlation between the relative expression of these factors were analyzed and showed as network mapping with different correlation coefficients. A strong positive correlation was observed between the expression of various factors in 1-week-old piglets. Combined with the difference in mortality of PEDV infection in pigs of different ages, we hypothesized that lactic acid bacteria (LAB) could inhibit PEDV infection in newborn piglets, and an in vivo experiment was carried out. The results of survival rate and wet/dry ratio showed that LAB alleviated PEDV indued mortality and diarrhea. The detection of viral copies and tissue section staining showed less observed viruses in LAB treated pig. RT-qPCR results of gene expression in intestines showed that LAB modulated the gene expression of various host barrier genes, indicating that LAB is potential to inhibit PEDV infection by regulating the host intestinal barrier. However, to use LAB as therapy, how to improve the efficiency on inhibiting PEDV infection needs further studies.

Experimental Infection of Pigs with Recent European Porcine Epidemic Diarrhea Viruses.

Porcine epidemic diarrhea virus (PEDV), belonging to the genus Alphacoronavirus, can cause serious disease in pigs of all ages, especially in suckling pigs. Differences in virulence have been observed between various strains of this virus. In this study, four pigs were inoculated with PEDV from Germany (intestine/intestinal content collected from pigs in 2016) and four pigs with PEDV from Italy (intestine/intestinal material collected from pigs in 2016). The pigs were re-inoculated with the same virus on multiple occasions to create a more robust infection and enhance the antibody responses. The clinical signs and pathological changes observed were generally mild. Two distinct peaks of virus excretion were seen in the group of pigs inoculated with the PEDV from Germany, while only one strong peak was seen for the group of pigs that received the virus from Italy. Seroconversion was seen by days 18 and 10 post-inoculation with PEDV in all surviving pigs from the groups that received the inoculums from Germany and Italy, respectively. Attempts to infect pigs with a swine enteric coronavirus (SeCoV) from Slovakia were unsuccessful, and no signs of infection were observed in the inoculated animals.

Comparison of pathogenicity of porcine deltacoronavirus CZ2020 from cell culture and intestinal contents in 27-day-old piglets.

Porcine deltacoronavirus (PDCoV) is an emenging swine enteropathogenic coronavirus that can cause high mortality rate. It affects pigs of all ages, but most several in neonatal piglets. Little is known regarding the pathogenicity of PDCoV against 27-day-old piglets. In this study, 27-day-old piglets were experimentally infected with PDCoV CZ2020 from cell culture, the challenged piglets do not have obvious symptoms from 1 to 7 days post-challenge (DPC), while viral shedding was detected in rectal swab at 1 DPC. Tissues of small intestines displayed slight macroscopic and microscopic lesions with no viral antigen detection. On the other hand, 27-day-old piglets were infected with PDCoV from intestinal contents, the piglets developed mild to severe diarrhea, shedding increasing from 2 to 7 DPC, and developed macroscopic and microscopic lesions in small intestines with clear viral antigen confirmed by immunohistochemistry staining. Indicating the small intestine was still the major target organ in PDCoV-challenged pigs at the age of 27-day-old. Diarrhea caused by PDCoV from intestinal contents in 27-day-old piglets is less reported. Thus, our results might provide new insights into the pathogenesis of PDCoV.

The gut microbiome: a missing link in understanding the gastrointestinal manifestations of COVID-19?

Coronavirus disease 2019 (COVID-19), which is caused by infection with SARS-CoV-2, presents with a broad constellation of both respiratory and nonrespiratory symptoms, although it is primarily considered a respiratory disease. Gastrointestinal symptoms-including nausea, abdominal pain, vomiting, and diarrhea-rank chief among these. When coupled with the presence of viral RNA in fecal samples, the presence of gastrointestinal symptoms raises relevant questions regarding whether SARS-CoV-2 can productively infect the upper or lower gastrointestinal tract. Despite the well-documented prevalence of gastrointestinal symptoms and the high rate of SARS-CoV-2 fecal RNA shedding, the biological, clinical, and epidemiological relevance of these findings is unclear. Furthermore, the isolation of replication-competent virus from fecal samples has not been reproducibly and rigorously demonstrated. Although SARS-CoV-2 shedding likely occurs in a high proportion of patients, gastrointestinal symptoms affect only a subset of individuals. Herein, we summarize what is known about gastrointestinal symptoms and fecal viral shedding in COVID-19, explore the role of the gut microbiome in other respiratory diseases, speculate on the role of the gut microbiota in COVID-19, and discuss potential future directions. Taking these concepts together, we propose that studying gut microbiota perturbations in COVID-19 will enhance our understanding of the symptomology and pathophysiology of this novel devastating disease.

Early clinical and CT features of COVID-19 and community-acquired pneumonia from a fever observation ward in Ningbo, China.

INTRODUCTION: We aimed to compare the early clinical manifestations, laboratory results and chest computed tomography (CT) images of COVID-19 patients with those of other community-acquired pneumonia (CAP) patients to differentiate CAP from COVID-19 before reverse transcription-polymerase chain reaction results are obtained. METHODS: The clinical and laboratory data and chest CT images of 51 patients were assessed in a fever observation ward for evidence of COVID-19 between January and February 2020. RESULTS: 24 patients had laboratory-confirmed COVID-19, whereas 27 individuals had negative results. No statistical difference in clinical features was found between COVID-19 and CAP patients, except for diarrhoea. There was a significant difference in lymphocyte and eosinophil counts between COVID-19 and CAP patients. In total, 22 (91.67%) COVID-19 patients had bilateral involvement and multiple lesions according to their lung CT images; the left lower lobe (87.50%) and right lower lobe (95.83%) were affected most often, and all lesions were located in the peripheral zones of the lung. The most common CT feature of COVID-19 was ground-glass opacity, found in 95.83% of patients, compared to 66.67% of CAP patients. CONCLUSION: Diarrhoea, lymphocyte counts, eosinophil counts and CT findings (e.g. ground-glass opacity) could help to distinguish COVID-19 from CAP at an early stage of infection, based on findings from our fever observation ward.

An emerging novel bovine coronavirus with a 4-amino-acid insertion in the receptor-binding domain of the hemagglutinin-esterase gene.

The hemagglutinin-esterase (HE) protein of betacoronavirus lineage A is a secondary receptor in the infection process and is involved in the emergence of new betacoronavirus genotypes with altered host specificity and tissue tropism. We previously reported a novel recombinant bovine coronavirus (BCoV) strain that was circulating in dairy cattle in China, but this virus was not successfully isolated, and the genetic characteristics of BCoV are still largely unknown. In this study, 20 diarrheic faecal samples were collected from a farm in Liaoning province that had an outbreak of calf diarrhea (≤ 3 months of age) in November 2018, and all of the samples tested positive for BCoV by RT-PCR. In addition, a BCoV strain with a recombinant HE (designated as SWUN/A1/2018) and another BCoV strain with a recombinant HE containing an insertion (designated as SWUN/A10/2018) were successfully isolated in cell culture (TCID50: 104.25/mL and 104.73/mL, respectively). Unexpectedly, we identified the emergence of a novel BCoV variant characterized by a 12-nt bovine gene insertion in the receptor-binding domain in a natural recombinant HE gene, suggesting a novel evolutionary pattern in BCoV.

Diarrhea Is Associated With Prolonged Symptoms and Viral Carriage in Corona Virus Disease 2019.

BACKGROUND & AIMS: We compared clinical, laboratory, radiological, and outcome features of patients with SARS-CoV-2 infection (COVID-19) with pneumonia, with vs without diarrhea. METHODS: We performed a retrospective, single-center analysis of 84 patients with SARS-CoV-2 pneumonia in Wuhan Union Hospital, China, from January 19 through February 7, 2020. Cases were confirmed by real-time reverse-transcriptase PCR of nasal and pharyngeal swab specimens for SARS-CoV-2 RNA. Blood samples were analyzed for white blood cell count, lymphocyte count, alanine aminotransferase, creatine kinase, lactate dehydrogenase, D-dimer, C-reactive protein, and in some cases, immunoglobulins, complement, lymphocyte subsets, and cytokines. Virus RNA was detected in stool samples by real-time PCR. RESULTS: Of the 84 patients with SARS-CoV-2 pneumonia, 26 (31%) had diarrhea. The duration of fever and dyspnea in patients with diarrhea was significantly longer than those without diarrhea (all P < .05). Stool samples from a higher proportion of patients with diarrhea tested positive for virus RNA (69%) than from patients without diarrhea (17%) (P < .001). As of February 19, a lower proportion of patients with diarrhea had a negative result from the latest throat swab for SARS-CoV-2 (77%) than patients without diarrhea (97%) (P = .010), during these patients' hospitalization. Of 76 patients with a negative result from their latest throat swab test during hospitalization, a significantly higher proportion of patients with diarrhea had a positive result from the retest for SARS-CoV-2 in stool (45%) than patients without diarrhea (20%) (P = .039). CONCLUSIONS: At a single center in Wuhan, China, 31% of patients with SARS-CoV-2 pneumonia had diarrhea. A significantly higher proportion of patients with diarrhea have virus RNA in stool than patients without diarrhea. Elimination of SARS-CoV-2 from stool takes longer than elimination from the nose and throat.

Inactivating Three Interferon Antagonists Attenuates Pathogenesis of an Enteric Coronavirus.

Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.

Diarrhea During COVID-19 Infection: Pathogenesis, Epidemiology, Prevention, and Management.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) pandemic is a worldwide emergency. An increasing number of diarrhea cases is reported. Here we investigate the epidemiology, clinical presentation, molecular mechanisms, management, and prevention of SARS-CoV-2 associated diarrhea. We searched on PubMed, EMBASE, and Web of Science up to March 2020 to identify studies documenting diarrhea and mechanism of intestinal inflammation in patients with confirmed diagnosis of SARS-CoV-2 infection. Clinical studies show an incidence rate of diarrhea ranging from 2% to 50% of cases. It may precede or trail respiratory symptoms. A pooled analysis revealed an overall percentage of diarrhea onset of 10.4%. SARS-CoV uses the angiotensin-converting enzyme 2 (ACE2) and the serine protease TMPRSS2 for S protein priming. ACE2 and TMPRSS2 are not only expressed in lung, but also in the small intestinal epithelia. ACE2 is expressed furthermore in the upper esophagus, liver, and colon. SARS-CoV-2 binding affinity to ACE2 is significantly higher (10-20 times) compared with SARS-CoV. Several reports indicate viral RNA shedding in stool detectable longer time period than in nasopharyngeal swabs. Current treatment is supportive, but several options appear promising and are the subject of investigation. Diarrhea is a frequent presenting symptom in patients infected with SARS-CoV-2. Increasing evidence indicates possible fecal oral transmission, indicating the need for a rapid and effective modification of the screening and diagnostic algorithms. The optimal methods to prevent, manage, and treat diarrhea in COVID-19 infected patients are subjects of intensive research.