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In Vietnam, diarrhea, especially persistent diarrhea, is one of the most common diseases in children, while a significant proportion of cases are negative with pathogens; thus, there is an urgent need to understand gut bacterial dysbiosis. In this study, bacteria in the fecal samples of five healthy and ten diarrheal children were separated from other residues, then adopted to extract their metagenomic DNA for evaluating their diversity based on V3 and V6–V8 regions and the 16S rRNA gene by PCR-RFLP and PCR-DGGE. As a result, bacterial metagenomic DNAs with high quality, quantity and diversity were successfully extracted using a GeneJET kit and a chemical protocol. A sequence analysis of 73 representative DNA fragments from gels indicated a remarkable bacterial dysbiosis in all groups of diarrhea. Viral diarrhea was characterized by extremely reduced bacterial diversity with the blossom of Bifidobacterium and Streptococcus. Streptococcus was also the most abundant in persistent diarrhea. Beneficial bacteria that may play a role in the self- rebalance in intestinal bacterial communities, such as Bifidobacterium, Lactobacillus, and Enterococcus, were seen in all diarrheal groups, while Bacteroides and Akkermansia muciniphila were seen in the healthy group but absent in the diarrheal groups. This study provides additional evidence for a relationship between intestinal bacterial dysbiosis and diarrhea in children, emphasizing an increase in Streptococcus. [ABSTRACT FROM AUTHOR]

This article discusses the mechanisms by which enteric viruses, such as rotavirus, norovirus, sapovirus, astrovirus, adenovirus, and poliovirus, induce diarrhea. The viruses disrupt intestinal homeostasis by infecting specific cells and causing structural changes, impairing nutrient absorption and leading to malabsorptive diarrhea. The enteric nervous system is also involved in diarrhea development, as viral infections activate serotonin secretion, leading to increased intestinal motility and fluid secretion. Viroporins, which are virally encoded proteins, play a role in manipulating cellular calcium signaling and facilitating viral replication, contributing to diarrhea. The article concludes by highlighting the importance of understanding these mechanisms for the development of antivirals and vaccines to reduce infection-associated diarrheal illness. [Extracted from the article]

The Porcine epidemic diarrhea virus (PEDV) presents a substantial risk to the domestic pig industry, resulting in extensive and fatal viral diarrhea among piglets. Recognizing the mucosal stimulation triggered by PEDV and harnessing the regulatory impact of lactobacilli on intestinal function, we have developed a lactobacillus-based vaccine that is carefully designed to elicit a strong mucosal immune response. Through bioinformatics analysis, we examined PEDV S proteins to identify B-cell linear epitopes that meet the criteria of being non-toxic, soluble, antigenic, and capable of neutralizing the virus. In this study, a genetically modified strain of Lactobacillus mucosae G01 (L.mucosae G01) was created by utilizing the S layer protein (SLP) as a scaffold for surface presentation. Chimeric immunodominant epitopes with neutralizing activity were incorporated at various sites on SLP. The successful expression of SLP chimeric immunodominant epitope 1 on the surface of L.mucosae G01 was confirmed through indirect immunofluorescence and transmission electron microscopy, revealing the formation of a transparent membrane. The findings demonstrate that the oral administration of L.mucosae G01, which expresses the SLP chimeric immunodominant gene epitope1, induces the production of secreted IgA in the intestine and feces of mice. Additionally, there is an elevation in IgG levels in the serum. Moreover, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 are significantly increased compared to the negative control group. These results suggest that L. mucosae G01 has the ability to deliver exogenous antigens and elicit a specific mucosal immune response against PEDV. This investigation presents new possibilities for immunoprophylaxis against PEDV-induced diarrhea. [ABSTRACT FROM AUTHOR]

The Porcine epidemic diarrhea virus (PEDV) presents a substantial risk to the domestic pig industry, resulting in extensive and fatal viral diarrhea among piglets. Recognizing the mucosal stimulation triggered by PEDV and harnessing the regulatory impact of lactobacilli on intestinal function, we have developed a lactobacillus-based vaccine that is carefully designed to elicit a strong mucosal immune response. Through bioinformatics analysis, we examined PEDV S proteins to identify B-cell linear epitopes that meet the criteria of being non-toxic, soluble, antigenic, and capable of neutralizing the virus. In this study, a genetically modified strain of Lactobacillus mucosae G01 (L.mucosae G01) was created by utilizing the S layer protein (SLP) as a scaffold for surface presentation. Chimeric immunodominant epitopes with neutralizing activity were incorporated at various sites on SLP. The successful expression of SLP chimeric immunodominant epitope 1 on the surface of L.mucosae G01 was confirmed through indirect immunofluorescence and transmission electron microscopy, revealing the formation of a transparent membrane. The findings demonstrate that the oral administration of L.mucosae G01, which expresses the SLP chimeric immunodominant gene epitope1, induces the production of secreted IgA in the intestine and feces of mice. Additionally, there is an elevation in IgG levels in the serum. Moreover, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 are significantly increased compared to the negative control group. These results suggest that L. mucosae G01 has the ability to deliver exogenous antigens and elicit a specific mucosal immune response against PEDV. This investigation presents new possibilities for immunoprophylaxis against PEDV-induced diarrhea. [ABSTRACT FROM AUTHOR]

Norovirus (NoV) genogroup II, polymerase type P31, capsid genotype 4, Sydney_2012 variant (GII.P31/GII.4_Sydney_2012) has been circulating at high levels for over a decade, raising the question of whether this strain is undergoing molecular alterations without demonstrating a substantial phylogenetic difference. Here, we applied next-generation sequencing to learn more about the genetic diversity of 14 GII.P31/GII.4_Sydney_2012 strains that caused epidemics in a specific region of Japan, with 12 from Kyoto and 2 from Shizuoka, between 2012 and 2022, with an emphasis on amino acid (aa) differences in all three ORFs. We found numerous notable aa alterations in antigenic locations in the capsid region (ORF2) as well as in other ORFs. In all three ORFs, earlier strains (2013–2016) remained phylogenetically distinct from later strains (2019–2022). This research is expected to shed light on the evolutionary properties of dominating GII.P31/GII.4_Sydney_2012 strains, which could provide useful information for viral diarrhea prevention and treatment. [ABSTRACT FROM AUTHOR]

Human astrovirus (HAstV) is a known cause of viral gastroenteritis in children worldwide, but HAstV can cause also severe and systemic infections in immunocompromised patients. There are three clades of HAstV: classical, MLB, and VA/HMO. While all three clades are found in gastrointestinal samples, HAstV-VA/HMO is the main clade associated with meningitis and encephalitis in immunocompromised patients. To understand how the HAstV-VA/HMO can infect the central nervous system, we investigated its sequence-divergent capsid spike, which functions in cell attachment and may influence viral tropism. Here we report the high-resolution crystal structures of the HAstV-VA1 capsid spike from strains isolated from patients with gastrointestinal and neuronal disease. The HAstV-VA1 spike forms a dimer and shares a core beta-barrel structure with other astrovirus capsid spikes but is otherwise strikingly different, suggesting that HAstV-VA1 may utilize a different cell receptor, and an infection competition assay supports this hypothesis. Furthermore, by mapping the capsid protease cleavage site onto the structure, the maturation and assembly of the HAstV-VA1 capsid is revealed. Finally, comparison of gastrointestinal and neuronal HAstV-VA1 sequences, structures, and antigenicity suggests that neuronal HAstV-VA1 strains may have acquired immune escape mutations. Overall, our studies on the HAstV-VA1 capsid spike lay a foundation to further investigate the biology of HAstV-VA/HMO and to develop vaccines and therapeutics targeting it. Author summary: Human astroviruses, well-established as a leading cause of viral diarrhea, are increasingly associated with viral encephalitis in immunocompromised patients, with the sequence-divergent VA1 strains being the most common in these cases. Our study presents the high-resolution capsid spike structures from VA1 strains isolated from patients with both gastrointestinal and neuronal manifestations. Compared to the classical human astrovirus capsid spike structure, the VA1 capsid spike structure has striking differences in size, shape, and surface features, suggesting that the divergent VA1 strains may have evolved a different mechanism to attach to host cells, which in turn may influence their ability to infect the central nervous system. On the other hand, comparison of the gastrointestinal and neuronal VA1 spike structures reveals few differences. Instead, sequence and antigenic studies suggest that neuronal VA1 strains isolated from immunocompromised patients may have acquired mutations to escape immunoglobulin therapy. Altogether, this work provides a structural basis to investigate the mechanism of infection by the divergent human astrovirus VA1 strains and to develop tailored diagnostics, vaccines, and therapeutics against them. [ABSTRACT FROM AUTHOR]

Background: Porcine epidemic diarrhea (PED) is an infectious disease of the digestive tract caused by the porcine epidemic diarrhea virus (PEDV), characterized by vomiting, severe diarrhea, and high mortality rates in piglets. In recent years, the distribution of this disease in China has remarkably increased, and its pathogenicity has also increased. PEDV has been identified as the main cause of viral diarrhea in piglets. This study aimed to understand the genetic evolution and diversity of PEDV to provide a theoretical basis for the development of new vaccines and the prevention and treatment of PED. Methods: A PEDV strain was isolated from the small intestine of a diarrheal piglet using Vero cells. The virus was identified using reverse transcription-polymerase chain reaction (RT-PCR), indirect immunofluorescence assay (IFA), and transmission electron microscopy. The whole genome sequence was sequenced, phylogenetic analysis was conducted using MEGA (version 7.0), and recombination analysis was performed using RDP4 and SimPlot. The S protein amino acid sequence was aligned using Cluster X (version 2.0), and the S protein was modeled using SWISS-MODEL to compare differences in structure and antigenicity. Finally, the piglets were inoculated with PEDV to evaluate its pathogenicity in newborn piglets. Result: PEDV strain CH/HLJ/18 was isolated. CH/HLJ/18 shared 89.4–99.2% homology with 52 reference strains of PEDV belonging to the GII-a subgroup. It was a recombinant strain of PEDV BJ-2011-1 and PEDV CH_hubei_2016 with a breakpoint located in ORF1b. Unique amino acid deletions and mutations were observed in the CH/HLJ/18 S protein. The piglets then developed severe watery diarrhea and died within 7 d of inoculation with CH/HLJ/18, suggesting that CH/HLJ/18 was highly pathogenic to newborn piglets. Conclusion: A highly pathogenic recombinant PEDV GII-a strain, CH/HLJ/18, was identified in China, with unique deletion and mutation of amino acids in the S protein that may lead to changes in protein structure and antigenicity. These results will be crucial for understanding the prevalence and variation of PEDV and for preventing and controlling PED. [ABSTRACT FROM AUTHOR]

It is estimated that more than 4% of the causes of inpatient deaths in 2017 were due to diarrheal diseases. The current study is aimed to provide pooled prevalence of microorganisms causing diarrhea among Sudanese as well as determine any socio-cultural risk factors associated. A systematic review of the literature was conducted and regulated in accordance to PRISMA. After abstract and full text screening Twenty-one research articles were recruited to the study. Among witch eighteen research articles determined prevalence of causative microorganisms of Diarrhea; eight research articles determining prevalence of causative microorganisms of Diarrhea were conducted in Khartoum State, five in Gezira State, two in White Nile State, one in Kordofan State while two studies were conducted in several States. Moreover, majority of studies were concerned of prevalence among children while two studies were toward general population as well as mothers of children. The pooled prevalence of viral diarrhea in less than five years old children was 22.90% [15.37, 30.43] among more than 14 thousands' participants, the pooled prevalence of parasitic diarrhea was 31.40% [19.53, 43.27] among participants from different age groups while the pooled prevalence of bacterial diarrhea was 36.20% [14.00, 58.40]. No associated risk factors were able to be synthesized from included studies. [ABSTRACT FROM AUTHOR]

Human sapovirus (HuSaV) is a common cause of gastroenteritis worldwide and is responsible for approximately 4% of acute gastroenteritis episodes in Europe. As reported with norovirus, patients with immunocompromised states are at increased risk of developing HuSaV infection, which can lead to persistent diarrhea and chronic viral shedding in some individuals. Chronic infections are incompletely investigated in these patients, and, due to the lack of specific treatment for HuSaV infection, different clinical approaches were carried out in order to provide further evidence on clinical evolution of these patients with different treatments. In this retrospective study, we report five immunocompromised pediatric patients with recurrent diarrhea caused by HuSaV and long-term viral shedding. Stool samples were analyzed by real-time PCR and tested for enteropathogenic viruses and bacteria and protozoa. Among transplant recipients, reduction of immunosuppressant therapy led to clinical improvement and relief of symptoms, maintaining a balance between managing the infection and preventing graft rejection. Nitazoxanide for 14 days was only used in one of these patients, showing to be an effective therapy to achieve reduction in time to resolution of symptoms. Neither nitazoxanide nor modification of immunosuppressant therapy could avoid recurrences. Further investigations are needed to develop new approaches that can both clear the infection and avoid persistent diarrhea in these patients. [ABSTRACT FROM AUTHOR]

Lacticaseibacillus is one of the predominant microorganisms in gut from human and animal, and the lacticaseibacillus have effective applications against the viral diarrhea of piglets in the farm. However, the function and the concrete cell single pathways of the active ingredient from lacticaseibacillus was not clear within anti-infection in the postbiotics research. Here, we compared the biological function of extracellular polysaccharides (EPS) purified from lacticaseibacillus casei (L. casei) and gene editing lacticaseibacillus casei with the CRISPER-Cas9 technology, which were with the ability of antioxidation and anti-inflammation, and the EPS could also inhibit the ROS production within the Porcine Small Intestinal Epithelial Cells-J2 (IPEC-J2). Interestingly, we found that both of EPS and genome editing lacticaseibacillus casei could specifically target the IFN-λ expression in the IPEC-J2, which was beneficial against the PEDV infection in the virus replication and production with the qRT-PCR and indirect immunofluorescence methods. Finally, the STAT3 cell single pathway was stimulated to transcribe IFN-λ with the EPS to elucidate the detailed mechanism of activating type III IFN signals receptor of IL-10R2, which play the function between anti-inflammation and anti-virus in the PEDV infection. Taken together, our research linked a postbiotics of EPS with the antiviral infection of PEDV, which suggest that the lacticaseibacillus itself still have displayed the potential immunomodulatory activities, and highlight the immunomodulatory potential of EPS-producing microbes. [ABSTRACT FROM AUTHOR]

Transmissible gastroenteritis virus (TGEV) is an important swine enteric coronavirus causing viral diarrhea in pigs of all ages. Currently, the development of antiviral agents targeting host proteins to combat viral infection has received great attention. The heat shock protein 90 (HSP90) is a critical host factor and has important regulatory effects on the infection of various viruses. However, its roles in porcine coronavirus infection remain unclear. In this study, the effect of HSP90 on TGEV infection was evaluated. In addition, the influence of its inhibitor VER-82576 on proinflammatory cytokine (IL-6, IL-12, TNF-α, CXCL10, and CXCL11) production induced by TGEV infection was further analyzed. The results showed that the knockdown of HSP90AB1 and HSP90 inhibitor VER-82576 treatment resulted in a reduction in TGEV M gene mRNA levels, the N protein level, and virus titers in a dose-dependent manner, while the knockdown of HSP90AA1 and KW-2478 treatment had no significant effect on TGEV infection. A time-of-addition assay indicated that the inhibitory effect of VER-82576 on TGEV infection mainly occurred at the early stage of viral replication. Moreover, the TGEV-induced upregulation of proinflammatory cytokine (IL-6, IL-12, TNF-α, CXCL10, and CXCL11) expression was significantly inhibited by VER-82576. In summary, these findings indicated that HSP90AB1 is a host factor enhancing TGEV infection, and the HSP90 inhibitor VER-82576 could reduce TGEV infection and proinflammatory cytokine production, providing a new perspective for TGEV antiviral drug target design. [ABSTRACT FROM AUTHOR]

Enteric viruses like canine coronavirus (CCoV), canine parvovirus (CPV), and canine rotavirus (CRV) cause high morbidity and mortality in dogs, especially in young canines and hence pose great economic losses to dog breeders worldwide. This creates immense psychological distress and trauma for dog owners. CCoV is mistakenly diagnosed as CPV in many cases by clinicians though CCoV causes less severe disease than CPV. Sometimes both CCoV and CPV co-exist in a patient causing severe clinical disease and even death in dogs. In such cases, clinicians are uncertain about the prognosis. CRV is detected in several countries but no research has been carried out on it in Pakistan yet. The objective of this research was to observe if CCoV, CPV and CRV co-exist in diarrheic dogs. A total of 384 stool samples of dogs, that were reported at different clinics in Lahore, with the complaint of diarrhea, were collected and rendered for rapid detection of the viruses using immuno-chromatographic test kits followed by PCR-confirmation of the positive samples. The individual prevalence of CCoV, CPV and CRV turned out to be 21.6, 26.3 and 0% respectively. The prevalence of CCoV and CPV co-infection was 3.4%. Male German shepherd dogs under the age of 1 year are highly prone to get viral diarrhea in July and August. [ABSTRACT FROM AUTHOR]

Simple Summary: Porcine rotavirus (PoRV) is a major pathogen causing dehydrating diarrhea and fatality in newborn piglets, resulting in substantial economic losses in the swine industry worldwide. Therefore, the development of a safe and effective medication for treating PoRV infection is imperative. This study provides the first report on the anti-PoRV activity of Portulaca oleracea L. (POL). Our findings demonstrate that POL-P has antiviral activity against PoRV infection in vitro, and thus might be developed into a novel antiviral agent to control PoRV in pig farms. Diarrhea is one of the most common causes of death in young piglets. Porcine rotavirus (PoRV) belongs to the genus Rotavirus within the family Reoviridae, and is considered to be the primary pathogen causing diarrhea in piglets. Portulaca oleracea L. (POL) has been reported to alleviate diarrhea and viral infections. However, the antiviral effect of Portulaca oleracea L. polysaccharide (POL-P), an active component of POL, on PoRV infection remains unclear. This study demonstrated that the safe concentration range of POL-P in IPEC-J2 cells is 0–400 μg/mL. POL-P (400 μg/mL) effectively inhibits PoRV infection in IPEC-J2 cells, reducing the expression of rotavirus VP6 protein, mRNA and virus titer. Furthermore, on the basis of viral life cycle analysis, we showed that POL-P can decrease the expression of PoRV VP6 protein, mRNA, and virus titer during the internalization and replication stages of PoRV. POL-P exerts antiviral effects by increasing IFN-α expression and decreasing the expression levels of TNF-α, IL-6, and IL-10 inflammatory factors. Overall, our study found that POL-P is a promising candidate for anti-PoRV drugs. [ABSTRACT FROM AUTHOR]

Background and Aim: To meet domestic demand for meat, Indonesia imports live cattle from Australia, which have nonbovine viral diarrhea (BVD)-free status. The consequence of importing live cattle from Australia is potentially introducing a novel BVD variant to Indonesia. Therefore, detecting BVD early and determining the agent's characteristics and clinical symptoms are necessary. This study aimed to detect and characterize clinical symptoms of bovine viral diarrhea (BVD) and highlight the importance of farm management as a risk factor for the spread of BVD. This study aimed to provide information about the effectiveness of preventive measures against BVD in Australian-imported cattle at the Tanjung Priok Seaport Agricultural Quarantine. Bovine viral diarrhea is among the most common diarrheal diseases found in feedlots and is a severe health and economic problem in cattle. Materials and Methods: All cattle in a selected feedlot were examined for clinical symptoms on their first day of arrival. The sampling criteria included age, body weight, body temperature (BT), animal breath (AB), pulse (PL), conjunctivitis (CJ), hyperlacrimation (HL), hypersalivation (HS), DR, fever, limping leg (LL), emaciation, stomatitis (ST), weakness (WK), and coronitis (CR). In addition, 64 blood samples were taken from cattle that exhibited clinical symptoms of BVD. On the 3rd day of arrival, a blood sample showing positive clinical symptoms was examined using antigen (Ag)-capture enzymelinked immunosorbent assay (ELISA). The data from these clinical symptoms were analyzed alongside the laboratory results using multidimensional scale analysis, heatmap distribution, and principal component analysis (PCA). Furthermore, the positive serum samples obtained from the Ag-capture ELISA underwent a nested multiplex polymerase chain reaction and molecular detection and genetic characterization of BVDV based on the 5' untranslated region of the viral genome, followed by sequence and phylogenetic tree analyses. Results: Using PCA, 12 clinical symptom characteristics of BVD were determined from 13 clinical symptoms synergized with five cattle positive for Ag-capture ELISA. The clinical symptoms included internal factors such as physiological conditions of CJ, HL, HS, DR, BT, LL, loss of appetite, ST, WK, CR, AB, and PL. The screening test showed that five samples tested positive for the BVD Ag, while 59 tested negative. Phylogenetic tree analysis using a 360-nucleotide portion of the NS5B gene showed that Sample B23F5R had a distinct path compared to the other two samples in the phylogenetic diagram. The profile of sample B23F5R was closely related to BVDV reference subgenotype 1-a group (NCBI, access no. LC068605), with a homology percentage of 92.36%. Furthermore, this sample was similar to the BVDV reference 1-a, Strain 12, identified in Japan. The other two samples, B13F5R and A13F5R, showed close resemblance to the BVDV reference subgenotype 1-a that had been previously identified in Indonesia (NCBI, access no. MK411755), with homology percentages of 97.81% and 97.75%, respectively. Conclusion: The BVDV-1a strain is the main subtype present in beef cattle imported from Australia to West Java, Indonesia. The characteristics of clinical symptoms associated with BVD infection comprised 12 symptoms synergized with the positive sample in the PCA. The present results can facilitate the development of preventive and control measures for BVD circulation in Indonesia. [ABSTRACT FROM AUTHOR]

Background Antibiotics are commonly overused for diarrheal illness in many low- and middle-income countries, partly due to a lack of diagnostics to identify viral cases, in which antibiotics are not beneficial. This study aimed to develop clinical prediction models to predict risk of viral-only diarrhea across all ages, using routinely collected demographic and clinical variables. Methods We used a derivation dataset from 10 hospitals across Bangladesh and a separate validation dataset from the icddr,b Dhaka Hospital. The primary outcome was viral-only etiology determined by stool quantitative polymerase chain reaction. Multivariable logistic regression models were fit and externally validated; discrimination was quantified using area under the receiver operating characteristic curve (AUC) and calibration assessed using calibration plots. Results Viral-only diarrhea was common in all age groups (<1 year, 41.4%; 18–55 years, 17.7%). A forward stepwise model had AUC of 0.82 (95% confidence interval [CI],.80–.84) while a simplified model with age, abdominal pain, and bloody stool had AUC of 0.81 (95% CI,.78–.82). In external validation, the models performed adequately although less robustly (AUC, 0.72 [95% CI,.70–.74]). Conclusions Prediction models consisting of 3 routinely collected variables can accurately predict viral-only diarrhea in patients of all ages in Bangladesh and may help support efforts to reduce inappropriate antibiotic use. [ABSTRACT FROM AUTHOR]

Simple Summary: Porcine epidemic diarrhea virus (PEDV), porcine bocavirus (PBoV), and porcine rotavirus (PoRV) are associated with porcine viral diarrhea. The three viruses are often comorbid in mixed infection; therefore, it is necessary to develop a specific and rapid method for simultaneous detection. In this study, a multiple detection method, loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD), was established to detect three pathogens in 125 field samples. The consistency between the real-time fluorescence quantitative PCR (rt-qPCR) and the triplex LAMP–LFD assay was over 99%. The assay has been proven to be highly specific and sensitive. Porcine epidemic diarrhea virus (PEDV), porcine bocavirus (PBoV), and porcine rotavirus (PoRV) are associated with porcine viral diarrhea. In this study, triplex loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD) was established for the simultaneous detection of PEDV, PoRV, and PBoV. The PEDV-gp6, PoRV-vp6, and PBoV-vp1 genes were selected to design LAMP primers. The amplification could be carried out at 64 °C using a miniature metal bath within 30 min. The triplex LAMP–LFD assay exhibited no cross-reactions with other porcine pathogens. The limits of detection (LODs) of PEDV, PoRV, and PBoV were 2.40 × 101 copies/μL, 2.89 × 101 copies/μL, and 2.52 × 101 copies/μL, respectively. The consistency between rt-qPCR and the triplex LAMP–LFD was over 99% in field samples testing. In general, the triplex LAMP–LFD assay was suitable for the rapid and simultaneous detection of the three viruses in the field. [ABSTRACT FROM AUTHOR]

Porcine viral diarrhea is very common in clinical practice and has caused huge losses to the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important pathogens of porcine viral diarrhea. Co-infection situations among these three viruses in clinics are common, which increases the difficulty of differential diagnosis. Currently, polymerase chain reaction (PCR) is commonly used to detect pathogens. TaqMan real-time PCR is more sensitive than conventional PCR and has better specificity and accuracy. In this study, a triplex real-time RT-PCR assay based on TaqMan probes was developed for differential detection of PEDV, PoRV, and PDCoV. The triplex real-time RT-PCR assay developed in this study could not detect unrelated pathogens and showed satisfactory specificity, sensitivity, repeatability, and reproducibility with a limit of detection (LOD) of 6.0 × 101 copies/μL. Sixteen clinical samples were used to compare the results of the commercial RT-PCR kit and the triplex RT-PCR for PEDV, PoRV, and PDCoV detection, and the results were completely consistent. A total of 112 piglet diarrhea samples collected from Jiangsu province were next used to study the local prevalence of PEDV, PoRV, and PDCoV. The positive rates of PEDV, PoRV, and PDCoV detected by the triplex real-time RT-PCR were 51.79% (58/112), 59.82% (67/112), and 2.68% (3/112), respectively. The co-infections of PEDV and PoRV were frequent (26/112, 23.21%), followed by the co-infections of PDCoV and PoRV (2/112, 1.79%). This study established a useful tool for simultaneous differentiation of PEDV, PoRV, and PDCoV in practice and provided valuable information on the prevalence of these diarrhea viral pathogens in Jiangsu province. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of bifidobacterium triple viable bacteria combined with recombinant human interferon α-1b injection on neutrophil rod nucleus, lobular nucleus and serum myocardial zymogram in children patients with viral diarrhea.Methods Ninety-three children patients with viral diarrhea in this hospital from April 2017 to December 2019 were retrospectively collected as the study subjects and divided into two groups according to the treatment regimen.The two groups were given the routine treatment. On this basis, 46 cases treated with bifidobacterium triple viable bacteria served as the control group, and 47 cases treated by bifidobacterium triple viable bacteria combined with recombinant human interferon α-1b injection as the observation group.The clinical efficacy, symptom relief time, neutrophil rod nucleus, lobular nucleus, serum inflammatory cytokines (IL-6, IL-8, TNF-α), myocardial zymogram [creatine kinase (CK), creatine kinase isoenzyme (CK-MB), hydroxybutyrate dehydrogenase (HBDH), lactate dehydrogenase (LDH)] and immune function before and after treatment were compared between the two groups.Results The total effective rate in the observation group was 95.74% (45/47), which was higher than 80.43% (37/46) in the control group, and the difference was statistically significant (P <0.05).The antiemetic, antidiarrheal and antipyretic time of the observation group was shorter than that of the control group (P <0.05).The count of neutrophil rod nucleus and lobulated nucleus after 1, 3 d treatment in the observation group was lower than that in the control group, and the differences were statistically significant (P <0.05).The serum inflammatory factors IL-6, IL-8 and TNF-α levels after 1, 3 d treatment in the observation group was lower than those in the control group (P <0.05).Serum CK, CK-MB, LDH and HBDH levels after 1, 3 d treatment in the observation group were lower than those in the control group, and the differences were statistically significant (P <0.05).The CD4+ and CD4+/CD8+ levels after 1, 3 d treatment in the observation group were higher than those in the control group, the CD8+ level was lower than that in the control group, and the differences were statistically significant(P <0.05).Conclusion Bifidobacterium triple viable bacteria combined with recombinant human interferon α-1b is conducive to enhance the treatment effect and shorten the symptom improvement time in the children patients with viral diarrhea by reducing the level of myocardial zymogram and regulating immune inflammatory response. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) remains one of the major causative microorganisms of viral diarrhea in piglets worldwide, with no approved drugs for treatment. We identified a natural molecule, flavonol, which is widely found in tea, vegetables and herbs. Subsequently, the antiviral activity of compound flavonol was evaluated in Vero cells and IPEC-J2 cells, and its anti-PEDV mechanism was analyzed by molecular docking and molecular dynamics. The results showed that flavonol could effectively inhibit viral progeny production, RNA synthesis and protein expression of PEDV strains in a dose-dependent manner. When flavonol was added simultaneously with viral infection in Vero cells, it demonstrated potent anti-PEDV activity by affecting the viral attachment and internalization phases. Similarly, in IPEC-J2 cells, flavonol effectively inhibited PEDV infection at different stages of infection, except for the release phase. Moreover, flavonol mainly interacts with PEDV Mpro through hydrogen bonds and hydrophobic forces, and the complex formed by it has high stability. Importantly, flavonol also showed broad-spectrum activity against other porcine enteric coronaviruses such as TGEV and PDCoV in vitro. These findings suggest that flavonol may exert antiviral effects by interacting with viral Mpro, thereby affecting viral replication. This means that flavonol is expected to become a potential drug to prevent or treat porcine enteric coronavirus. • PEDV induces distinct morphological changes in both Vero cells and IPEC-J2 cells. • Flavonol primarily disrupts the initial stage of PEDV infection in Vero cells. • Flavonol prevents the advancement of multiple life cycle phases of PEDV propagation in IPEC-J2 cells. • Flavonol exhibits a wide-ranging efficacy against porcine enteric coronavirus activity. [ABSTRACT FROM AUTHOR]

Background: Human norovirus (HuNoV) is the leading viral cause of diarrhea, with GII.4 as the predominant genotype of HuNoV outbreaks globally. However, new genogroup variants emerge periodically, complicating the development of anti-HuNoV vaccines; other prophylactic or therapeutic medications specifically for HuNoV disease are lacking. Passive immunization using oral anti-HuNoV antibodies may be a rational alternative. Here, we explore the feasibility of using avian immunoglobulins (IgY) for preventing HuNoV infection in vitro in a human intestinal enteroid (HIE) model. Methods: Hens were immunized with virus-like particles (VLP) of a GII.4 HuNoV strain (GII.4/CHDC2094/1974/US) by intramuscular injection. The resulting IgY was evaluated for inhibition of binding to histo-blood group antigens (HBGA) and viral neutralization against representative GII.4 and GII.6 clinical isolates, using an HIE model. Results: IgY titers were detected by three weeks following initial immunization, persisting at levels of 1:221 (1:2,097,152) from 9 weeks to 23 weeks. Anti-HuNoV IgY significantly (p < 0.05) blocked VLP adhesion to HBGA up to 1:12,048 dilution (0.005 mg/mL), and significantly (p < 0.05) inhibited replication of HuNoV GII.4[P16] Sydney 2012 in HIEs up to 1:128 dilution (0.08 mg/mL). Neutralization was not detected against genotype GII.6. Conclusions: We demonstrate the feasibility of IgY for preventing infection of HIE by HuNoV GII.4. Clinical preparations should cover multiple circulating HuNoV genotypes for comprehensive effects. Plans for animal studies are underway. [ABSTRACT FROM AUTHOR]

Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex TaqMan probe-based real-time PCR for the rapid simultaneous detection and differentiation of PEDV subtypes G1 and G2, RVA, and RVC. This test is highly sensitive, as the detection limits were 20 and 100 copies/μL for the G1 and G2 subtypes of PEDV, respectively, and 50 copies/μL for RVA and RVC, respectively. Eighty-eight swine clinical samples were used to evaluate this new test. The results were 100% in concordance with the standard methods. Since reassortment between porcine and human rotaviruses has been reported, this multiplex test not only provides a basis for the management of swine diarrheal viruses, but also has the potential to impact public health as well. [ABSTRACT FROM AUTHOR]

Porcine bocavirus (PBoV) was first identified in Sweden in 2009. Due to its association with healthy as well as diseased pigs, its role in clinical disease has not been reported yet. In the present study, bocavirus was identified from the intestinal content of a 30-day-old piglet and its whole genome was constructed and phylogenetic analysis was carried on. The pathogenesis of bocavirus was investigated following orogastric inoculation of the colostrum-deprived newborn piglet with bacteria free intestinal content. The bocavirus-inoculated piglets developed diarrhea, shed virus in the rectal swabs from 18 h post inoculation and developed macroscopic and microscopic lesions in small intestine with virus confirmed by conventional PCR. This study experimentally confirmed pathogenicity and characterized bocavirus as the etiological agent of diarrhea in the colostrum-deprived newborn piglets. On phylogenetic analysis, it was observed that this virus has long evolutionary history with subsequent mutation as well as better host adaptation. This study highlights the importance of identifying bocavirus as the etiological agent of viral diarrhea that could threaten livestock, public health as well as economic loss. • A porcine bocavirus (PBoV/HB/30/2018) associated with diarrhea was discovered in China. • Pathogenicity of PBoV/HB/30/2018 was evaluated in colostrum-deprived newborn piglets. • Complete genome of PBoV/HB/30/2018 was sequenced and assembled. • The origin and phylogeny of PBoV/HB/30/2018 were investigated. [ABSTRACT FROM AUTHOR]

Comprehensive data on bacterial and viral pathogens of diarrhea and studies applying culture-independent methods for examining antibiotic resistance in wastewater are lacking. This study aimed to simultaneously quantify antibiotic resistance genes (ARGs), class 1 integron-integrase (int1), bacterial and viral pathogens of diarrhea, 16S rRNA, and other indicators using a high-throughput quantitative PCR (HT-qPCR) system. Thirty-six grab wastewater samples from a wastewater treatment plant in Japan, collected three times a month between August 2022 and July 2023, were centrifuged, followed by nucleic acid extraction, reverse transcription, and HT-qPCR. Fourteen targets were included, and HT-qPCR was performed on the Biomark X9™ System (Standard BioTools). For all qPCR assays, R2 was ≥0.978 and the efficiencies ranged from 90.5% to 117.7%, exhibiting high performance. Of the 36 samples, 20 (56%) were positive for Norovirus genogroup II (NoV-GII), whereas Salmonella spp. and Campylobacter jejuni were detected in 24 (67%) and Campylobacter coli in 13 (36%) samples, with mean concentrations ranging from 3.2 ± 0.8 to 4.7 ± 0.3 log 10 copies/L. NoV-GII detection ratios and concentrations were higher in winter and spring. None of the pathogens of diarrhea correlated with acute gastroenteritis cases, except for NoV-GII, suggesting the need for data on specific bacterial infections to validate bacterial wastewater-based epidemiology (WBE). All samples tested positive for sul1 , int1 , and bla CTX-M , irrespective of season. The less explored bla NDM-1 showed a wide prevalence (>83%) and consistent abundance ranging from 4.3 ± 1.0 to 4.9 ± 0.2 log 10 copies/L in all seasons. sul1 was the predominant ARG, whereas absolute abundances of 16S rRNA, int1 , and bla CTX-M varied seasonally. int1 was significantly correlated with bla CTX-M in autumn and spring, whereas it showed no correlation with bla NDM-1 , questioning the applicability of int1 as a sole indicator of overall resistance determinants. This study exhibited that the HT-qPCR system is pivotal for WBE. [Display omitted] • HT-qPCR system successfully analyzed pathogens and ARGs in a single run. • NoV-GII concentration was linked with AGE cases but not bacterial pathogens. • Specific bacterial infection data could be useful for validation of bacterial WBE. • sul1 , bla CTX-M , an ESBL, and the less explored bla NDM-1 were widely prevalent. • int1 correlated with bla CTX-M in some seasons and did not correlate with bla NDM-1. [ABSTRACT FROM AUTHOR]

Canine parvovirus (CPV) is the main cause of viral diarrhea in dogs. CPV became a global disease in 1978 and was endemic all over the world. CPV-2 was the first strain to be identified, but with genetic mutations, new genotypes such as CPV-2a/2b/2c/new-2a/new-2b have emerged. In this study, 128 fecal samples of stray dogs suspected of CPV-2 infection were collected from January to March 2021 in Shanghai, China. All samples were screened by PCR and further analyzed by VP2 gene. The positive rate of CPV-2 was 9.4% (12/128), of which 6 CPV-2 isolates were successfully isolated. Phylogenetic tree analysis showed that 4 isolates were CPV-2c genotype and 2 were new-CPV-2b genotype. VP-2 is a key protein that determines the antigenic properties, host range and receptor binding of cpv-2. The results of VP2 amino acid sequence analysis in this study showed that the CPV-2c isolated strain was the same as the previous strains reported in China, including F267Y, Y324I, Q370R and A5G mutations in addition to the typical N426E mutations. Similarly, in addition to the conventional N426D, S297A, F267Y and Y324I mutations, the new CPV-2b isolate also had a new mutation of T440A. This study further confirmed the prevalence of CPV-2c and new-CPV-2b in Shanghai, and also found a new mutation site of new-CPV-2c, which provided a theoretical basis for further enriching the epidemiological data of CPV-2 in Shanghai, as well as the development of vaccines and the prevention and control of the disease. • High prevalence of CPV-2 in stray dogs. • CPV-2c and new-CPV-2b co-exist and circulate in Stray dogs locally in Shanghai, China. • Current Vaccines may not be able to prevent the CPV prevalence by the mutated strains. [ABSTRACT FROM AUTHOR]

In this report, a multiplex PCR method was developed for the detection of three diarrhea-associated viruses in mink, including circovirus (MCV), bocavirus (MBoV), and enteritis virus (MEV). Three compatible sets of primers specific for each virus were designed respectively based on their conserved sequences. After optimization of the crucial factors such as primer concentration and annealing temperature in single and multiple amplification, three specific fragments were simultaneously amplified with the highest sensitivity and specificity in one PCR reaction. The fragments amplified were 259 bp (MCV)，455 bp (MBoV) and 671 bp (MEV). The sensibility of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There were no cross-reactions with some relevant pathogens like mink coronavirus, canine distemper virus, and aleutian mink disease virus. In our study we analyzed viral DNA in mink fecal samples by multiplex PCR assay from China, which revealed the occurrence of MCV, MBoV, and MEV as 3.1 %, 5.7 %, and 9.8 %, respectively. The testing results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100 %. These results indicated that the method could provide technical support for rapid detection of the three diarrhea-associated viruses, and epidemiological investigation of mink viral diarrhea. ● A multiplex PCR assay was developed to simultaneously detect the DNA of MCV, MBoV, and MEV. ● The sensitivity of multiplex PCR for MCV, MBoV, and MEV was 1,020 copies, 1,020 copies, and 858 copies, respectively. ● The assay was specific for detecting one or more of the viruses. [ABSTRACT FROM AUTHOR]

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/μL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi. • The quadruplex RT-qPCR method shows high specificity and efficiency, enabling simultaneous lab diagnosis of TGEV, PEDV, PDCoV, and PoRVA. It reduces detection costs by 75% and boosts efficiency by 300%. • The developed quadruplex RT-qPCR kit is comparable to the commercial kit from GM Biotechnology INT, implying its commercial potential. PEDV, PoRVA, and PEDV/PoRVA co-infections are the main causes of pig diarrhea in Guangxi, with TGEV and PDCoV detection rates being secondary factors during 2022-2023. [ABSTRACT FROM AUTHOR]

Viral diarrhea is the predominant digestive tract sickness in piglings, resulting in substantial profit losses in the porcine industry. Porcine rotavirus A (PoRVA) and porcine epidemic diarrhea virus (PEDV) are the main causes of grave gastroenteritis and massive dysentery, especially in piglets. PoRVA and PEDV have high transmissibility, exhibit similar clinical symptoms, and frequently co-occur. Therefore, to avoid financial losses, a quick, highly efficient, objective diagnostic test for the prevention and detection of these diseases is required. Enzymatic recombinase amplification (ERA) is a novel technology based on isothermal nucleic acid amplification. It demonstrates high sensitivity and excellent specificity, with a short processing time and easy operability, compared with other in vitro nucleic acid amplification technologies. In this study, a dual ERA method to detect and distinguish between PEDV and PoRVA nucleic acids was established. The method shows high sensitivity, as the detection limits were 101 copies/μL for both viruses. To test the usefulness of this method in clinical settings, we tested 64 swine clinical samples. Our results were 100% matched with those acquired using a commercially available kit. Therefore, we have successfully developed a dual diagnostic ERA nucleic acids method for detecting and distinguishing between PEDV and PoRVA. • The reaction principle of ERA and its current application are introduced. • Almost all currently circulating strains of PEDV and PoRVA can be detected. • ERA has the strengths of simple operation, outstanding sensitivity and strong specificity. [ABSTRACT FROM AUTHOR]

Rotavirus (RV) infections are the most important viral cause of diarrhea in piglets in Switzerland and are thought to cause substantial economic losses to the pig industry. However, no data are available on the occurrence and dynamics of the main porcine RV species, namely RVA, RVB, and RVC, and the diversity of the circulating strains. We therefore tested fecal samples from a cross-sectional (n = 95) and a longitudinal (n = 48) study for RVA, RVB, and RVC by real-time RT-PCR and compared the results of the cross-sectional study to postmortem findings. In addition, eight samples were fully genotyped by using next-generation sequencing. In the cross-sectional study, triple RV infections significantly correlated with diarrhea and wasting and were most frequent in the weaned age group. In the longitudinal study, the shedding of RV peaked one week after weaning and decreased thereafter. Here, mainly double infections were seen, and only a few animals showed diarrhea. The full-genome sequencing revealed a genotype pattern similar to other European countries and, importantly, co-infection by up to four RVA strains. Our results imply that the weaning of piglets may trigger not only RV shedding but facilitate co-infection of multiple RV species and strains in the same host. [ABSTRACT FROM AUTHOR]

Background: Viral infections are mostly highly contagious and may cause widespread health problems. Some studies reported that the dietary fiber (DF) may be effective in reducing the complications of viral infections in intensive care unit (ICU) patients. The present review study aimed to investigate the effect of DF on common complications in critically ill patients with viral infections. Methods: A literature review was conducted for the published papers in English from January 2001 to July 2021 using related keywords. Studies with clinical trial or case‐control design described the effects of fiber intake on the complications of viral infections in patients admitted to the ICU were collected. Results: DF may reduce the mortality rate of viral infections through modulating inflammatory processes. A higher intake of DF intake may improve hyperglycemia and impaired glucose tolerance in patients with viral infections. A high‐fiber formula in enteral nutrition was reported to reduce the risk of diarrhea in patients with viral infections. Conclusion: DF may reduce the complications of viral infections such as inflammation, diarrhea, hyperglycemia, and mortality in critically ill patients. Future longitudinal studies on the amount and type of DF are warranted. [ABSTRACT FROM AUTHOR]

Background: Diarrheal illness is a leading cause of antibiotic use for children in low- and middle-income countries. Determination of diarrhea etiology at the point-of-care without reliance on laboratory testing has the potential to reduce inappropriate antibiotic use. Methods: This prospective observational study aimed to develop and externally validate the accuracy of a mobile software application (‘App’) for the prediction of viral-only etiology of acute diarrhea in children 0–59 months in Bangladesh and Mali. The App used a previously derived and internally validated model consisting of patient- specific (‘present patient’) clinical variables (age, blood in stool, vomiting, breastfeeding status, and mid- upper arm circumference) as well as location- specific viral diarrhea seasonality curves. The performance of additional models using the ‘present patient’ data combined with other external data sources including location-specific climate, data, recent patient data, and historical population- based prevalence were also evaluated in secondary analysis. Diarrhea etiology was determined with TaqMan Array Card using episode-specific attributable fraction (AFe) >0.5. Results: Of 302 children with acute diarrhea enrolled, 199 had etiologies above the AFe threshold. Viral- only pathogens were detected in 22% of patients in Mali and 63% in Bangladesh. Rotavirus was the most common pathogen detected (16% Mali; 60% Bangladesh). The present patient+ viral seasonality model had an AUC of 0.754 (0.665–0.843) for the sites combined, with calibration-in-the-large α = −0.393 (−0.455––0.331) and calibration slope β = 1.287 (1.207–1.367). By site, the present patient+recent patient model performed best in Mali with an AUC of 0.783 (0.705–0.86); the present patient+ viral seasonality model performed best in Bangladesh with AUC 0.710 (0.595–0.825). Conclusions: The App accurately identified children with high likelihood of viral-only diarrhea etiology. Further studies to evaluate the App’s potential use in diagnostic and antimicrobial stewardship are underway. Funding: Funding for this study was provided through grants from the Bill and Melinda GatesFoundation (OPP1198876) and the National Institute of Allergy and Infectious Diseases (R01AI135114). Several investigators were also partially supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK116163). This investigation was also supported by the University of Utah Population Health Research (PHR) Foundation, with funding in part from the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR002538. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in the study design, data collection, data analysis, interpretation of data, or in the writing or decision to submit the manuscript for publication. [ABSTRACT FROM AUTHOR]

Almost all of the deaths happening under the age of 5 occur in the developed countries of Africa and Asia. This study included children admitted to the surgical care, aged 6 months to 5 years, who suffered from acute gastroenteritis and received treatment at Samawah, Iraq, from December 2018 to December 2019. Test results detected different types of rotaviruses, adenoviruses, astroviruses using ELISA. 56.6% of the infections were attributed to a viral pathogen. The main cause was attributed to rotavirus and adenovirus. The causative agents of diarrheal diseases in 28.1% of cases are rotaviruses, in 17.05% - adenoviruses, in 11.43% - astroviruses. Viral mono-infections are detected more often than mixed infections. Viral intestinal infections are characterized by seasonality and rise in the cold season, with a peak incidence of rotavirus infection in April, adenovirus infection in November, and astrovirus infection in December. [ABSTRACT FROM AUTHOR]

7‐Chloro‐2‐(propylthio)thiazolo[5,4‐d]pyrimidine 4 was prepared and used as a precursor for synthesizing new derivatives of thiazolo[5,4‐d]pyrimidine through the nucleophilic displacement of the chlorine atom by different types of amino derivatives. Reaction of compound 4 with primary and secondary amines produced the corresponding substituted amino derivatives. Reaction of 4 with hydrazine hydrate then followed by condensation with aromatic aldehydes afforded hydrazone derivatives. Reaction of 4 with hydrazine hydrate then followed by reaction with isothiocyanates gave the disubstituted thiosemicarbazides. Finally, treatment of 4 with hydrazine derivatives afforded the novel disubstituted hydrazine derivatives. Preliminary antiviral screening on the bovine viral diarrhea virus (BVDV) was carried out, which was classified as a member of the same family of Hepatitis C virus. The results indicated that some of the tested compounds exhibited anti‐BVDV activity which may be suitable for a new lead molecule to design more potent anti‐BVDV agents. [ABSTRACT FROM AUTHOR]

Thiosemicarbazones which previously characterized as a new class that inhibit anti-bovine viral diarrhea virus (BVDV) prompted us for synthesizing a series of new thiosemicarbazone derivatives. Thus, in this study, we reported the synthesis and antiviral evaluation of a series of 5-(arylazo)salicylaldehyde thiosemicarbazone derivatives for their expected antiviral activity. The desired products were synthesized from the condensation of 5-(arylazo)salicylaldehyde derivatives with N-(4)-substituted thiosemicarbazide derivatives. Antiviral screening was performed to test the anti-bovine viral diarrhea virus properties. From the obtained results, compounds 5, 23 and 24 showed highly selective activity against BVDV by blocking the viral RNA synthesis in cell culture. The BVDV in antiviral drug studies is valuable surrogate for the hepatitis C virus (HCV); so, the above results provided a novel candidate for the development of anti-HCV agents. [ABSTRACT FROM AUTHOR]

BACKGROUND & OBJECTIVE: As Rotavirus leads to significant morbidity in children 2 months to 5 years, this study was done to observethe effect of one dose of nitazoxanide on the total duration of rotavirus diarrhea.The primary objective was to evaluate thereduction inthe mean duration of viral diarrhea in children after administration of first dose of Nitazoxanide. METHODOLOGY: This descriptive study was done in the Department of Pediatric Medicine Unit I, Mayo Hospital, Lahore fromJan 2016 to June 2016 after approval from the ethical review committee. Sample size calculated was 175 using WHO sample size calculator. Patients of 4-5 years were given 200 mg nitazoxanide twice daily for 3 days; 100 mg nitazoxanide was given twice daily to 12-47 months age group, and patient of <12 months age were given 7.5 mg/kgtwice day along with routine care and were followed 12 hourly till the passage of normally formed stools. Duration of diarrhea was recorded. RESULTS: Out of 175 cases, 58.9%(n=103) were between 2-36 months age while 41.2%(n=72) were 37-60 months age, mean+SD was calculated as 35.06+13.34 months, meanreduction in duration of rotavirus diarrhea in children after administration of first dose of Nitazoxanide was recorded as 32.85+2.32 hours. CONCLUSION: With the use of nitazoxanide along with routine care for rotavirus diarrhea children, the mean duration of rotavirus diarrhea was reported to be less than 1.5 days. [ABSTRACT FROM AUTHOR]

Introduction: acute diarrhea in children under five years is a public health problem in developing countries and particularly in malaria-endemic areas where both diseases co-exist. The present study examined the etiology of childhood diarrhea and its comorbidity with malaria in a rural area of Burkina Faso. Methods: conventional culture techniques, direct stools examination, and viruses´ detection by rapid tests were performed on the fresh stools and microscopy was used to diagnose malaria. Some risk factors were also assessed. Results: on a total of 191 samples collected, at least one pathogen was identified in 89 cases (46.6%). The proportions of pathogens found on the 89 positive stool samples were parasites 51.69% (46 cases), viruses 39.33% (35 cases), and bacteria 14.61% (13 cases), respectively. The relationship between malaria and infectious diarrhea was significant in viral and parasites causes (p=0.005 and 0.043 respectively). Fever, vomiting and abdominal pain were the major symptoms associated with diarrhea, with 71.51%, 31.72% and 23.66% respectively. The highest viral diarrhea prevalence was reported during the dry season (OR=5.29, 95% CI: 1.74 - 16.07, p=0.001) while parasite diarrhea was more encountered during the rainy season (OR=0.41, 95% CI: 0.33 - 0.87, p=0.011). Conclusion: Giardia spp and rotavirus were the leading cause of acute diarrhea in Nanoro, Burkina Faso with a predominance of rotavirus in children less than 2 years. Parasite and viral diarrhea were the most pathogens associated with malaria. However, the high rate of negative stool samples suggests the need to determine other enteric microorganisms. [ABSTRACT FROM AUTHOR]

Background: Diarrhea is one of the leading causes of childhood morbidity and mortality in lower- and middle-income countries. In such settings, access to laboratory diagnostics are often limited, and decisions for use of antimicrobials often empiric. Clinical predictors are a potential non-laboratory method to more accurately assess diarrheal etiology, the knowledge of which could improve management of pediatric diarrhea. Methods: We used clinical and quantitative molecular etiologic data from the Global Enteric Multicenter Study (GEMS), a prospective, case-control study, to develop predictive models for the etiology of diarrhea. Using random forests, we screened the available variables and then assessed the performance of predictions from random forest regression models and logistic regression models using 5-fold cross-validation. Results: We identified 1049 cases where a virus was the only etiology, and developed predictive models against 2317 cases where the etiology was known but non-viral (bacterial, protozoal, or mixed). Variables predictive of a viral etiology included lower age, a dry and cold season, increased height-for-age z-score (HAZ), lack of bloody diarrhea, and presence of vomiting. Cross-validation suggests an AUC of 0.825 can be achieved with a parsimonious model of 5 variables, achieving a specificity of 0.85, a sensitivity of 0.59, a NPV of 0.82 and a PPV of 0.64. Conclusion: Predictors of the etiology of pediatric diarrhea can be used by providers in low-resources setting to inform clinical decision-making. The use of non-laboratory methods to diagnose viral causes of diarrhea could be a step towards reducing inappropriate antibiotic prescription worldwide. Author summary: Diarrhea is one of the leading causes of death in young children worldwide. In low-resource settings, diarrhea testing is not available or too expensive, and the decision to prescribe antibiotics is often made without testing. Using clinical information to predict which cases are caused by viruses, and thus wouldn't need antibiotics, would help to improve appropriate use of antibiotics. We used data from a large study of childhood diarrhea, paired with advanced statistical methods including machine learning, to come up with the top clinical factors that could predict a viral cause of diarrhea. We compared 1049 cases where a virus was the only cause, with 2317 cases where the cause was known but not a virus. We found that a lower age, dry and cold season, nutritional status defined by increased height, lack of blood diarrhea, and vomiting, were the clinical factors most predictive of whether the diarrhea was caused by a virus. We found that, using just those 5 factors, we were able to predict a viral cause with good accuracy. Our findings can be used by doctors to guide the appropriate use of antibiotics for diarrhea in children. [ABSTRACT FROM AUTHOR]

Although porcine deltacoronavirus (PDCoV) is a significant pandemic threat in the swine population and has caused significant economic losses, information regarding the immune response in conventionally weaned pigs infected with PDCoV is scarce. Hence, the immune response in conventionally weaned pigs infected with PDCoV was assessed after challenge and rechallenge. After the first challenge, obvious diarrhea and viral shedding developed successively in all pigs in the four inoculation dose groups from 3 to 14 days postinfection (dpi), and all pigs recovered (no clinical symptoms or viral shedding) by 21 dpi. All pigs in the four groups exhibited significantly increased PDCoV-specific IgG, IgA and virus-neutralizing (VN) antibody (Ab) titers and IFN-γ levels in the serum after the first challenge. All pigs were completely protected against rechallenge at 21 dpi. The serum levels of PDCoV-specific IgG, IgA, and VN Abs increased further after rechallenge. Notably, the IFN-γ level declined continuously after 7 dpi. In addition, the levels of PDCoV-specific IgG, IgA and VN Abs in saliva increased significantly after rechallenge and correlated well with the serum Ab titers. Furthermore, the appearance of clinical symptoms of PDCoV infection in conventionally weaned pigs was delayed with reduced inoculation doses. In summary, the data presented here offer important reference information for future PDCoV animal infection and vaccine-induced immunoprotection experiments. [ABSTRACT FROM AUTHOR]

Rotavirus infection is the most common cause of viral diarrhea in infants and young children but uncommon and usually asymptomatic in adults. In the winter of 2017–2018, a large-scale outbreak of rotavirus in both children and adults was reported in Thailand. The current study focused on the prevalence, genotyping, and molecular characterization of rotavirus infections in Thai adults from July 2016 to December 2019. In 2,598 stool samples collected from adult residents of Bangkok (aged #x2265; 15 years) with acute gastroenteritis, rotavirus was detected via real-time RT-PCR analysis of the VP6 gene. G, P and I genotypes were determined by direct sequencing of VP7, VP4, and VP6 genes, respectively. Our results showed 8.7% (226/2,598) of stool samples were positive for rotavirus. The incidence of rotavirus was high during the winter season of 2017–2018 (17.7%) compared to another studied periods (4.5% between July 2016- October 2017 and 2.8% between March 2018- December 2019). Nucleotide sequencing of VP7 and VP4 revealed G3P[8] as the predominant strain (33.2%,75/226), followed by G9P[8] (17.3%,39/226), and G2P[4] (15.0%,34/226). Uncommon G and P combinations were additionally detected at low frequencies. VP6 sequencing was conducted to discriminate I genotype between the Wa and DS-1 genogroup. The unusual DS-1-like G3P[8] strain was most prevalent amomg rotavirus strains detected in this study (29.6%, 67/226), and the corresponding VP7 sequences showed high nucleotide identity with unusual DS-1-like globally circulating strains. Our study demonstrates that rotavirus outbreaks in adults are attributable not only to high prevalence of RV infection but also the unusual DS-like genogroup. The collective findings reinforce the importance of investigating rotavirus diagnosis in adults suffering from acute gastroenteritis and taking appropriate preventive measures. [ABSTRACT FROM AUTHOR]

Objective: To analyze the clinical prognosis and risk factors of viral diarrhea in children. Mrthods: 200 cases of pediatric viral diarrhea admitted to our hospital from January 2018 to December 2019 were retrospectively analyzed. The clinical characteristics of pediatric viral diarrhea were analyzed. According to the disease recovery group, 128 patients with recovery were used as the experimental group, and 72 patients without recovery were used as the reference group. Logistic analysis was used to analyze the risk factors for the recovery of children with viral diarrhea. Results: Of the 200 patients, 121 were infected with HAstV, accounting for 60.50%; 36 were infected with HAD, accounting for 18.00%; 15 were infected with HUCV, accounting for 7.50%; 10 were infected with HRV, accounting for 5.00%; 18 were infected with infection, accounting for 9.00%. The clinical symptoms of patients with HAstV type viral diarrhea are mainly respiratory symptoms, fever and vomiting; the clinical symptoms of patients with HAD type viral diarrhea are mainly fever and vomiting; the clinical symptoms of patients with HUCV type viral diarrhea are mainly respiratory symptoms and fever; HRV The clinical symptoms of patients with type viral diarrhea are mainly fever and vomiting; the clinical symptoms of patients with mixed infection type are mainly respiratory symptoms, fever and vomiting. Age, CNS damage, myocardial damage, and whether diarrhea has been contacted in the past 1 week are risk factors for disease recovery in children with viral diarrhea, P<0.05. Conclusion: Different types of viral diarrhea in children have different clinical symptoms. The prognosis of children with viral diarrhea is closely related to age, central nervous systemdamage, myocardial damage, and whether or not diarrhea has been exposed for nearly 1 week. It should cause clinical attention and attention. [ABSTRACT FROM AUTHOR]

Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the main pathogens causing viral diarrhea in pig, mixed infections of these two viruses are very common in intensive pig rearing. However, there is a lack of a method to simultaneously detect and distinguish PEDV and TGEV in preclinical levels. In this study, we aimed to establish a dual ultrasensitive nanoparticle DNA probe-based PCR assay (dual UNDP-PCR) based on functionalized magnetic bead enrichment and specific nano-technology amplification for simultaneous detection and distinguish diagnosis of PEDV and TGEV. The detection limit of dual UNDP-PCR for single or multiple infections of PEDV and TGEV is 25 copies/g, which is 400 times more sensitive than the currently known duplex RT-PCR, showing better specificity and sensitivity without cross-reaction with other viruses. For pre-clinical fecal samples, the dual UNDP-PCR showed a markedly higher positive detection rate (52.08%) than conventional duplex RT-PCR (13.21%), can rapidly and accurately identify targeted pathogens whenever simple virus infection or co-infection. In summary, this study provides a technique for detecting and distinguishing PEDV and TGEV in preclinical levels, which is high sensitivity, specificity, repeatability, low cost and broad application prospect. [ABSTRACT FROM AUTHOR]

Objective: To analyze the etiology characteristics and the clinical significance of serum procalcitonin (PCT), cluster of differentiation 64 (CD64) and soluble triggering receptors expressed on myeloid cells-1 (sTREM-1) in preschool children with bacterial diarrhea. Methods: 214 children with infectious diarrhea who were admitted to Dongguan People's Hospital Affiliated to Southern Medical University from January 2017 to March 2018 were selected as research subjects. It included bacterial diarrhea (bacterial group, n=101) and viral diarrhea (viral group, n=113), and 113 healthy school age children who were admitted to hospital during the same period were selected as control group. The types of pathogens in the bacterial group were counted, and the differences of serum PCT, CD64 and sTREM-1 levels between the two groups of diarrhea children and the control group were compared, and the positive rates of three serological indicators and the diagnostic efficacy of the three indicators for children with bacterial diarrhea were also compared. Results: Among the 101 Bacterial diarrhea in children, 213 bacterial strains were cultured in feces. The positive rates of Escherichia coli, Shigella and Salmonella were the highest, accounting for 41.78%, 23.01% and 15.96% respectively. The levels and positive rates of serum PCT, CD64 index and sTREM-1 in bacterial group were higher than those in viral group and control group, and the sTREM-1 level in viral group was higher than those in control group (P<0.05). The sensitivity, specificity, positive predictive value and negative predictive value of the three indexes were compared, the differences were not statistically significant (P>0.05). Conclusion: The main pathogens in preschool children with bacterial diarrhea are Gram-negative bacteria.The PCT, CD64 index and sTREM-1 could be used as early sensitive indicators for judging bacterial diarrhea. [ABSTRACT FROM AUTHOR]

Objective: To analyze the etiology characteristics and the clinical significance of serum procalcitonin (PCT), cluster of differentiation 64 (CD64) and soluble triggering receptors expressed on myeloid cells-1 (sTREM-1) in preschool children with bacterial diarrhea. Methods: 214 children with infectious diarrhea who were admitted to Dongguan People's Hospital Affiliated to Southern Medical University from January 2017 to March 2018 were selected as research subjects. It included bacterial diarrhea (bacterial group, n=101) and viral diarrhea (viral group, n=113), and 113 healthy school age children who were admitted to hospital during the same period were selected as control group. The types of pathogens in the bacterial group were counted, and the differences of serum PCT, CD64 and sTREM-1 levels between the two groups of diarrhea children and the control group were compared, and the positive rates of three serological indicators and the diagnostic efficacy of the three indicators for children with bacterial diarrhea were also compared. Results: Among the 101 Bacterial diarrhea in children, 213 bacterial strains were cultured in feces. The positive rates of Escherichia coli, Shigella and Salmonella were the highest, accounting for 41.78%, 23.01% and 15.96% respectively. The levels and positive rates of serum PCT, CD64 index and sTREM-1 in bacterial group were higher than those in viral group and control group, and the sTREM-1 level in viral group was higher than those in control group (P<0.05). The sensitivity, specificity, positive predictive value and negative predictive value of the three indexes were compared, the differences were not statistically significant (P>0.05). Conclusion: The main pathogens in preschool children with bacterial diarrhea are Gram-negative bacteria. The PCT, CD64 index and sTREM-1 could be used as early sensitive indicators for judging bacterial diarrhea. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the effect of bovine colostrum (BC) on the treatment of children with acute diarrhea attending the outpatient clinic.Methods: This double-blind randomized controlled trial was conducted on 160 children with diarrhea; 80 cases were randomly treated with BC group and 80 cases randomly received placebo (placebo group). All cases were investigated for bacterial causes of diarrhea (Salmonella spp, Shigella spp, diarrheagenic E. coli (DEC), Campylobacter spp., and Vibrio cholerae) as well as for Rotavirus antigen in stool.Results: After 48 h, the BC group had a significantly lower frequency of vomiting, diarrhea and Vesikari scoring compared with the placebo group (p = 0.000, p = 0.000, p = 0.000, respectively), whether it was due to Rotavirus or E. coli infection.Conclusions: BC is effective in the treatment of acute diarrhea and can be considered as adjuvant therapy in both viral and bacterial diarrhea to prevent diarrhea-related complications. [ABSTRACT FROM AUTHOR]

Aim: The research aimed to study the effect of interferon (IFN)-based drugs on the behavior of immunological parameters in calves during the specific prevention of associative infections. Materials and Methods: The object of research was 45 black motley cows and their calves from birth to 2 months of life. Serum and colostrum samples were screened for antibodies against Rotavirus, diarrhea, and coronavirus using serological methods. The testing was performed before vaccination, 40 days before calving, 20 days before calving, and before calving. Colostrum samples were taken during the first milk yield. Serum samples from calves were drawn before colostrum feeding as well as at 7, 14, and 21 days, and 1 and 2 months of age. To measure the level of immunoglobulins A, M, and G, additional serum samples were collected from calves at 25, 35, 65, and 75 days after birth. Results: Giving pregnant cows, an IFN-based drug at a dose of 1 ml/kg 48 h before vaccination results in the development and accumulation of antibodies to Rotavirus, coronavirus, and viral diarrhea (VD) in the colostrum, with a titer of 7.6±0.3 log2, 5.8±0.34 log2, and 4.4±0.18 log2, respectively. It indicates an increase in the antigenic activity of the multivalent vaccine. Conclusion: IFN-based drugs enhance the protective effect of vaccination against associative infections in the newborn calves. They stimulate a rise in the titer of antibodies to Rotavirus, coronavirus, VD, and mucosal disease complex as well as an increase in immunoglobulins A, M, and G. [ABSTRACT FROM AUTHOR]

Background: In China, large-scale outbreaks of severe diarrhea caused by viruses have occurred in pigs since late 2010. To investigate the prevalence and genetic evolution of diarrhea-associated viruses responsible for the outbreaks, a total of 2987 field diarrheal samples collected from 168 pig farms in five provinces in Southern China during 2012–2018 were tested. Results: Porcine epidemic diarrhea virus (PEDV) was most frequently detected virus with prevalence rates between 50.21 and 62.10% in samples, and 96.43% (162/168) in premises, respectively. Porcine deltacoronavirus (PDCoV) was the second prevalent virus with prevalence rates ranging from 19.62 to 29.19% in samples, and 70.24% (118/168) in premises, respectively. Both transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) were detected at low prevalence rates of < 3% in samples and 10.12% in premises. In this study, we identified a newly emerged swine acute diarrhea syndrome coronavirus (SADS-CoV) in diarrheal samples of piglets from Fujian province in Southern China, and the prevalence rate of SADS-CoV was 10.29% (7/68). Co-infections of these diarrhea-associated viruses were common. The most frequent co-infection was PEDV with PDCoV, with an average detection rate of 12.72% (380/2987, ranging from 8.26–17.33%). Phylogenetic analysis revealed that PEDVs circulating in Southern China during the last 7 years were clustered with the variant strains of PEDV in genotype IIa. The most frequent mutations were present in the collagenase equivalent (COE) and epitope regions of the spike gene of the PEDVs currently circulating in the field. Genetic relationships of PDCoVs were closely related with Chinese strains, other than those present in the USA, South Korea, Thailand and Lao's public. Conclusions: The findings of this study indicated that variant PEDV, PDCoV, and SADS-CoV were leading etiologic agents of porcine diarrhea, and either mono-infections or co-infections of pathogenic enteric CoVs were common in pigs in Southern China during 2012–2018. Thus, significant attention should be paid in order to effectively prevent and control porcine viral diarrhea. [ABSTRACT FROM AUTHOR]

Rotavirus B (RVB) is a causative agent leading to acute viral gastroenteritis diarrhea in both children and young animals, and has been commonly detected in piglets. In order to determine the causative agent of diarrheal outbreak occurring in December 2022 in piglets from a pig herd in Luoyang, Henan province of China, four common viral pathogens causing piglet diarrhea-three coronaviruses and rotavirus A (RVA) were first tested and found negative, therefore metagenomic sequencing was performed to explore other potential pathogens in the diarrheal samples. Unexpectedly, the most abundant viral reads mapped to RVB, and were de novo assembled to complete 11 viral gene segments. Sequence comparisons revealed that 5 gene segments encoding VP1, VP2, VP3, NSP3 and NSP4 of RVB strain designated as HNLY-2022 are most closely related to RVB strains derived from herbivores with low nucleotide similarities of 65.7–75.3%, and the remaining segments were relatively close to porcine RVB strains with the VP4 gene segment showing very low nucleotide identity (65.0%) with reference strains, indicating HNLY-2022 is a new reassortant RVB strain. Based on the previously proposed genotype classification criterion, the genotype constellation of RVB strain HNLY-2022 is G6-P[6]-I4-R6-C6-M6-A7-N5-T7-E5-H4 with more than half of the genotypes (P[6], R6, C6, M6, T7 and E5) newly reported. Therefore, the new reassortant RVB strain is the likely causative agent for the diarrheal outbreak of piglets occurred in China and more epidemiological studies should be conducted to monitor the spread of this newly identified porcine RVB strain. • Outbreak of piglet diarrhea occurred in Henan province of China was associated with a new rotavirus B. • HNLY-2022 is a reassortant RVB strain with the gene segments probably arose from pigs and herbivores. • The genotypes P[6], R6, C6, M6, T7 and E5 of RVB were firstly identified. [ABSTRACT FROM AUTHOR]

An efficient procedure for the synthesis of 5-(aryldiazo)salicylaldehyde thiosemicarbazone derivatives 1-25 was achieved via the condensation of 5-(aryldiazo)salicylaldehyde derivatives I–V with N-(4)-substituted thiosemicarbazide derivatives. Antiviral activity of the synthesized compounds was carried out. From the obtained results, it was noticed that compound 11 has a strong antiviral activity. [ABSTRACT FROM AUTHOR]

To prevent porcine viral diarrhea, a vaccine combination that can provide good antibody levels needs to be determined. In this study, we screened 30 pregnant sows divided into six experimental groups, namely, five immunized groups and one control group, to investigate the antibody level differences of different vaccine combinations on porcine epidemic diarrhea (PED), porcine transmissible gastroenteritis (TGE), porcine rotavirus (PoR) IgG, and PED IgA. The antibody level was detected by ELISA. Results showed that the antibody levels of PED and TGE IgG in serum and PED IgA in breast milk of the "PT+PT*" vaccine combination group were higher than those of the other groups, and vaccine combination including "PTR" could stimulate the sows to produce PoR IgG antibody. These findings revealed that the vaccine combination of "PT+PT*" is optimal for preventing porcine viral diarrhea, and "PTR+PT*" may be an alternative option in areas under PoRV infection risk. This study suggested that pig farms should select suitable immunization on the basis of the local epidemic situation of porcine viral diarrhea. [ABSTRACT FROM AUTHOR]

• A low-cost and sensitive real-time PCR assay for enteric disease-associated porcine viruses was developed. • This assay could be used for the detection of one or more diarrhea-causing pig viruses in one reaction. • Diagnostic assay is suitable for epidemiological survey of viral diarrhea infection in high-throughput clinical samples. In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube. Five distinct melt peaks were obtained with different melting temperature (Tm) value corresponding to each of the five viruses. This method was highly sensitive to detect and distinguish TGEV, RVA, RVC, PEDV and PCV2 with the limits of detection ranging from 5 to 50 copies/μL. The intra-assay and inter-assay reproducibility were good with coefficient of variation of Tm and cycle threshold values less than 0.32% and 2.86%, respectively. Testing of 90 field samples by the single and multiplex real-time PCR assays demonstrated a concordance of 91.1%. Thus, the EvaGreen multiplex real-time PCR is a rapid, sensitive and low-cost diagnostic tool for differential detection and routine surveillance of TGEV, RVA, RVC, PEDV and PCV2 in pigs. [ABSTRACT FROM AUTHOR]