Clinical efficacy and pathological outcomes of transanal endoscopic intersphincteric resection for low rectal cancer.

BACKGROUND: Transanal endoscopic intersphincteric resection (ISR) surgery currently lacks sufficient clinical research and reporting. AIM: To investigate the clinical effectiveness of transanal endoscopic ISR, in order to promote the clinical application and development of this technique. METHODS: This study utilized a retrospective case series design. Clinical and pathological data of patients with lower rectal cancer who underwent transanal endoscopic ISR at the First Affiliated Hospital of Xiamen University between May 2018 and May 2023 were included. All patients underwent transanal endoscopic ISR as the surgical approach. We conducted this study to determine the perioperative recovery status, postoperative complications, and pathological specimen characteristics of this group of patients. RESULTS: This study included 45 eligible patients, with no perioperative mortalities. The overall incidence of early complications was 22.22%, with a rate of 4.44% for Clavien-Dindo grade ≥ III events. Two patients (4.4%) developed anastomotic leakage after surgery, including one case of grade A and one case of grade B. Postoperative pathological examination confirmed negative circumferential resection margins and distal resection margins in all patients. The mean distance between the tumor lower margin and distal resection margin was found to be 2.30 ± 0.62 cm. The transanal endoscopic ISR procedure consistently yielded high quality pathological specimens. CONCLUSION: Transanal endoscopic ISR is safe, feasible, and provides a clear anatomical view. It is associated with a low incidence of postoperative complications and favorable pathological outcomes, making it worth further research and application.

Transcriptome and metabolome analysis reveals PRV XJ delgE/gI/TK protects intracranially infected mice from death by regulating the inflammation.

Pseudorabies virus can cause inflammation in the central nervous system and neurological symptoms. To further investigate the protective mechanism of PRV XJ delgE/gI/TK in the central nervous system, an intracranial PRV-infection mice model was developed. The results demonstrated that immunization with PRV XJ delgE/gI/TK successfully prevented death caused by PRV-intracranial infection. Subsequently, the brains were collected for transcriptome and metabolome analysis. GO and KEGG enrichment analysis indicated that the differentially expressed genes were primarily enriched in pathways such as TNF, NOD-like receptor, JAK-STAT, MAPK, IL-17 and apoptosis signaling. Metabolomics analysis revealed that the differential metabolites were mainly associated with pathways such as fatty acid degradation, arachidonic acid metabolism, linoleic acid metabolism and unsaturated fatty acid biosynthesis. The combined analysis of metabolites and differentially expressed genes revealed a strong correlation between the differential metabolites and TNF, PI3K, and MAPK signaling pathways. Anti-inflammatory metabolites have been shown to inhibit the inflammatory response and prevent mouse death caused by PRV infection. Notably, when glutathione was injected intracranially and dihydroartemisinin was injected intraperitoneally, complete protection against PRV-induced death in mice was observed. Moreover, PRV activates the PI3K/AKT signaling pathway. In conclusion, our study demonstrates that PRV XJ delgE/gI/TK can protects intracranially infected mice from death by regulating various metabolites with anti-inflammatory functions post-immunization.

Establishment and application of an indirect ELISA for Getah virus E2 antibody detection.

Getah virus (GETV) is a mosquito-transmitted disease that affects animals, causing fever, aseptic meningitis, and abortion. Its prevalence in China poses risks to both animal health and public well-being. Currently, there is a scarcity of seroepidemiological data on GETV due to the absence of commercial antibody detection kits for pigs. The aim of this study is to develop a rapid, accurate, and sensitive ELISA, providing a reliable tool for GETV seroepidemiology and laying the foundation for future commercial assay development. In this study, we removed specific hydrophobic domains and intracellular structures from E2 proteins and constructed the recombinant plasmid pCold-TF-E2. The recombinant protein was expressed using a prokaryotic expression system, and efficient purification of the rE2 protein was achieved using a nickel affinity column. The purified rE2 protein is suitable for the development of an indirect ELISA (rE2 ELISA). Following the optimization of reaction conditions for the rE2-ELISA, the cut-off value was 0.356. Additionally, the rE2-ELISA method showed a positive rate of 37.1% for IgG antibodies against GETV when testing 986 pig clinical serum samples collected from pigs in Sichuan between May 2022 and September 2022. The rE2-ELISA method displayed a 95.1% overall agreement with VNT, boasting a sensitivity of 98.2% and a specificity of 92.6%. These results indicate that IgG ELISA based on rE2 protein is an efficient and economical method for the detection of GETV antibodies in pigs, facilitating the diagnosis and prevention of GETV.

First report on identification and genomic analysis of a novel porcine circovirus (porcine circovirus 4) in cats.

Porcine circovirus type 4 (PCV4) is an emerging circovirus, which has been detected in domestic pigs across various provinces in China and Korea. In this study, we aimed to investigate whether cats are susceptible to PCV4. For this purpose, we collected 116 cat samples from animal hospitals in Sichuan Province, China, between 2021 and 2022. Using a SYBR Green-based real-time PCR assay, we detected PCV4 in 5 out of the 116 clinical samples, indicating a positive rate of 4.31% (5/116) and confirming the presence of PCV4 in cats from Sichuan Province, China. Moreover, we successfully sequenced and analyzed the complete genome of one PCV4 strain (SCGA-Cat) along with 60 reference sequences deposited in the GenBank database. SCGA-Cat exhibited high nucleotide homology (98.2-99.0%) with PCV4 strains from other species, including dogs, pigs, dairy cows, and fur animals. Notably, the SCGA-Cat strain from cats clustered closely with a PCV4 strain derived from a pig collected in Fujian Province, China. To the best of our knowledge, this study represents the first report on the molecular detection of PCV4 in cats worldwide, which prompted us to understand the genetic diversity and cross-species transmission of the ongoing PCV4 cases. However, further investigations are needed to explore the association between PCV4 infection and clinical syndromes in cats.

Prokaryotic expression of porcine deltacoronavirus S gene truncated segment and establishment of indirect ELISA detection method.

Porcine deltacoronavirus (PDCoV) is an emerging discovered coronavirus that causes significant losses in the global swine industry. This study aimed to establish an indirect ELISA method for detecting PDCoV antibodies using the truncated gene of PDCoV spike protein (S). The purified S protein was used as the coating antigen for the polyclonal antibody. The conditions were optimized to establish an indirect ELISA detection method for PDCoV based on the S protein, which showed good specificity and no cross-reaction with SVV-VP1, ASFV-P72, GETV-E2, PRV-gE, etc. The method has high repeatability, with coefficients of variation within and between batches less than 10%. Compared with the commercial kit, the positive coincidence rate is 86.40%, the negative coincidence rate is 89.43%, and the total coincidence rate is 91.76%. This ELISA can be used for PDCoV serological investigation and antibody evaluation. It can also lay the foundation for further research and development of PDCoV S protein ELISA antibody detection kit.

The first dog-origin porcine circovirus type 4 complete genomic sequence have high homology with that of pig-derived strains.

Introduction: Porcine circovirus 4 (PCV4) was discovered in 2019 and then proved to be pathogenic to piglets. Nevertheless, few studies were currently available about PCV4 infection in species other than pigs and there is no information about the prevalence of PCV4 in dogs. Methods: To fill this gap, 264 dog samples were collected from animal hospitals in the Southwest of China from 2021 to 2022 and screened for PCV4. Moreover, the complete genome of one PCV4 strain (SCABTC-Dog2022) were obtained successfully and shared a high identity (97.9-99.0%) with other PCV4 strains derived from pigs, dairy cows, raccoon dogs and foxes. The SCABTC-Dog2022 were analyzed together with 51 reference sequences. Results and Discussion: The detected results showed a low percentage of PCV-4 DNA (1.14%, 3/264), indicating that PCV4 could be identified in dogs in southwest China. Phylogenetic tree showed that SCABTC-Dog2022 strain derived from dog were clustered in a closed relative and geographically coherent branch with other PCV4 strains collected from four provinces (Sichuan, Fujian, Hunan and Inner Mongolia) of China. To our knowledge, it is the first detection of PCV4 in dogs globally. The association between PCV4 status and clinical syndromes in dogs deserves additional investigations.

First molecular detection and genetic analysis of porcine circovirus 4 in the Southwest of China during 2021-2022.

Porcine circovirus 4 (PCV4) was identified in 2019 as a novel circovirus species and then proved to be pathogenic to piglets. However, there is a lack of its prevalence in the Southwest of China. To investigate whether PCV4 DNA existed in the Southwest of China, 374 samples were collected from diseased pigs during 2021-2022 and detected by a real-time PCR assay. The results showed that the positive rate of PCV4 was 1.34% (5/374) at sample level, and PCV4 was detected in two of 12 cities, demonstrating that PCV4 could be detected in pig farms in the Southwest of China, but its prevalence was low. Furthermore, one PCV4 strain (SC-GA2022ABTC) was sequenced in this study and shared a high identity (98.1-99.7%) with reference strains at the genome level. Combining genetic evolution analysis with amino acid sequence analysis, three genotypes PCV4a, PCV4b, and PCV4c were temporarily identified, and the SC-GA2022ABTC strain belonged to PCV4c with a specific amino acid pattern (239V for Rep protein, 27N, 28R, and 212M for Cap protein). Phylogenetic tree and amino acid alignment showed that PCV4 had an ancient ancestor with mink circovirus. In conclusion, the present study was the first to report the discovery and the evolutionary analysis of the PCV4 genome in pig herds of the Southwest of China and provide insight into the molecular epidemiology of PCV4.

Association Between Prognostic Nutritional Index and Prognosis in Patients With Heart Failure: A Meta-Analysis.

Background: The prognostic nutritional index (PNI) has been proposed as a marker of malnutrition and associated with the prognosis of cardiovascular disease. However, whether PNI can serve as a potential biomarker for the prognosis of heart failure (HF) upon those established risk factors were still controversial. This meta-analysis aimed to generate comprehensive evidence on the prognostic value of PNI in patients with HF. Methods: Multiple databases (PubMed, Embase, the Cochrane Library, and Google Scholar) were searched for related studies up to January 31, 2022. Observational studies accessed associations between PNI levels and the prognosis in patients with HF were included for meta-analysis. The hazard ratios (HRs) and 95% confidence intervals (CI) were calculated. Results: Fourteen studies, comprising 19,605 patients with HF were included for meta-analysis. The median follow-up duration was 18.5 months. Compared with those with higher PNI (normal nutritional status), patients with HF with lower PNI (malnourished) were associated with a higher risk of all-cause mortality (HR 1.53, 95% CI 1.27-1.85) and composite major adverse cardiac outcomes (MACEs; HR 2.26, 95% CI 1.54-3.31) in the multivariable-adjusted model. Furthermore, when PNI was defined as per 1 increment as a continuous metric, higher PNI was associated with a decrease in all-cause mortality (per 1 increment of PNI: HR 0.94, 95% CI 0.88-0.96) and MACEs (per 1 increment of PNI: HR 0.97, 95% CI 0.95-0.98). Conclusions: The PNI can serve as an easily calculated bedside "malnutrition-inflammation" biomarker in HF. Lower PNI was associated with a worse prognosis in patients with HF.

The Immunity Protection of Central Nervous System Induced by Pseudorabies Virus DelgI/gE/TK in Mice.

Based on a variant strain, we constructed a gE/gI/TK-deleted pseudorabies virus (PRV). A total of 18 female mice were randomized to a vaccination group to receive PRV XJ delgE/gI/TK, a vehicle group to receive Dulbecco's modified Eagle's medium, and a mock group to confirm the protection of PRV delgE/gI/TK on the central nervous system in mice. Subsequently, the vaccination and vehicle groups were infected with PRV XJ. The mice in the vehicle group showed more severe neurological symptoms and higher viral loads than those in the vaccination group. The exudation of Evans blue and the expression of tight junction protein showed no difference in all groups. HE staining showed vacuolar neuronal degeneration in the vehicle group brain, but no tissue lesions were observed in the vaccination group. TNF-α, IL-6, and synuclein were upregulated in the brain of mice in the vehicle group, while those were inhibited among mice in the vaccination group. IFN-ß, IFN-γ, ISG15, Mx1, and OAS1 showed no difference in the brain between the vaccination and vehicle groups. In addition, TNF-α and IL-6 were inhibited, and antiviral factors were increased in the intestine of the mice in the vaccination group compared to those in the vehicle group. Our study showed that PRV XJ delgE/gI/TK inhibited neurological damage and the inflammation of the intestine and brain induced by PRV and activated the innate immunity of the intestine.

Venom serine proteinase homolog of the ectoparasitoid Scleroderma guani impairs host phenoloxidase cascade.

The ant-like bethylid ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae) envenomates host to suppress immune response. Yet, the roles of its venom in inhibiting melanization of the host hemolymph have not been fully characterized. Here, we demonstrated that S. guani envenomation induced strong inhibition of melanization of the hemolymph from Tenebrio molitor (Coleoptera: Tenebrionidae), permitting the successful development of parasitoid offspring. To reveal venom component associated with such function, a serine proteinase homolog (SguaSPH) rich in the venom of S. guani was characterized. It was found that one of the catalytic triad residues for serine proteinase is absent in the amino acid sequence of SguaSPH. This venom component was abundantly expressed in venom apparatus and adult stages. By enzymatic assays, SguaSPH displayed low trypsin and no chymotrypsin activity, and was able to inhibit phenoloxidase activity in the hemolymph of Ostrinia furnacalis (Lepidoptera: Crambidae). The findings suggest that SguaSPH is essential for interfering with hemolymph melanization of S. guani envenomated host via phenoloxidase cascade disruption.

Superoxide dismutase from venom of the ectoparasitoid Scleroderma guani inhibits melanization of hemolymph.

Superoxide dismutase (SOD) known as an important antioxidative stress protein has been recently found in venoms of several parasitoid wasps. However, its functions and characteristics as a virulent factor remain scarcely described. Here, we report the characterization of two venomous SOD genes (SguaSOD1 and SguaSOD3) from the ectoparasitoid, Scleroderma guani. The metal binding sites, cysteine amino acid positions and signature sequences of the SOD family were conserved within SguaSOD1 and SguaSOD3. Relatively high levels of their transcripts were observed in pupae followed a decrease in early adults, after which they had the highest transcriptions, indicating that their productions would be regulated in venom apparatus. Although the two genes showed lower expression in venom apparatus compared to head and thorax, the enzymatic assay revealed that SOD indeed had activity in venom. Further, we showed that recombinant SguaSOD3 suppressed melanization of host hemolymph, implying that this protein used as a virulent factor uniquely impacts the prophenoloxidase cascade.

Genome-based identification and analysis of ionotropic receptors in Spodoptera litura.

The ability to sense and recognize various classes of compounds is of particular importance for survival and reproduction of insects. Ionotropic receptor (IR), a sub-family of the ionotropic glutamate receptor family, has been identified as one of crucial chemoreceptor super-families, which mediates the sensing of odors and/or tastants, and serves as non-chemosensory functions. Yet, little is known about IR characteristics, evolution, and functions in Lepidoptera. Here, we identify the IR gene repertoire from a destructive polyphagous pest, Spodoptera litura. The exhaustive analyses with genome and transcriptome data lead to the identification of 45 IR genes, comprising 17 antennal IRs (A-IRs), 8 Lepidoptera-specific IRs (LS-IRs), and 20 divergent IRs (D-IRs). Phylogenetic analysis reveals that S. litura A-IRs generally retain a strict single copy within each orthologous group, and two lineage expansions are observed in the D-IR sub-family including IR100d-h and 100i-o, likely attributed to gene duplications. Results of gene structure analysis classify the SlitIRs into four types: I (intronless), II (1-3 introns), III (5-9 introns), and IV (10-18 introns). Extensive expression profiles demonstrate that the majority of SlitIRs (28/43) are enriched in adult antennae, and some are detected in gustatory-associated tissues like proboscises and legs as well as non-chemosensory organs like abdomens and reproductive tissues of both sexes. These results indicate that SlitIRs have diverse functional roles in olfaction, taste, and reproduction. Together, our study has complemented the information on chemoreceptor genes in S. litura, and meanwhile allows for target experiments to identify potential IR candidates for the control of this pest.

Parasitism and venom of ectoparasitoid Scleroderma guani impairs host cellular immunity.

Venom is a prominently maternal virulent factor utilized by parasitoids to overcome hosts immune defense. With respect to roles of this toxic mixture involved in manipulating hosts immunity, great interest has been mostly restricted to Ichneumonoidea parasitoids associated with polydnavirus (PDV), of which venom is usually considered as a helper component to enhance the role of PDV, and limited Chalcidoidea species. In contrast, little information is available in other parasitoids, especially ectoparasitic species not carrying PDV. The ectoparasitoid Scleroderma guani injects venom into its host, Tenebrio molitor, implying its venom was involved in suppression of hosts immune response for successful parasitism. Thus, we investigated the effects of parasitism and venom of this parasitoid on counteracting the cellular immunity of its host by examining changes of hemocyte counts, and hemocyte spreading and encapsulation ability. Total hemocyte counts were elevated in parasitized and venom-injected pupae. The spreading behavior of both granulocytes and plasmatocytes was impaired by parasitization and venom. High concentration of venom led to more severely increased hemocyte counts and suppression of hemocyte spreading. The ability of hemocyte encapsulation was inhibited by venom in vitro. In addition to immediate effects observed, venom showed persistent interference in hosts cellular immunity. These results indicate that venom alone from S. guani plays a pivotal role in blocking hosts cellular immune response, serving as a regulator that guarantees the successful development of its progenies. The findings provide a foundation for further investigation of the underlying mechanisms in immune inhibitory action of S. guani venom.

Venomics reveals novel ion transport peptide-likes (ITPLs) from the parasitoid wasp Tetrastichus brontispae.

Despite substantial advances in uncovering constituents of parasitoid venoms due to their potential applications as insecticides and pharmaceuticals, most of these studies are primarily restricted to braconid and ichneumonid wasps. Little information is available regarding virulent factors from venom of Eulophidae. In order to provide insight into the venom components of this family and parasitoid venom evolution, a venom protein repertoire (venomics) of the endoparasitoid wasp, Tetrastichus brontispae was deciphered using a proteomic approach. A large number of diverse venom proteins/peptides were identified, including novel proteins and those proteins commonly found in the venoms of other parasitoids such as serine protease, esterase, dipeptidyl peptidase IV, acid phosphatase, major royal jelly protein, superoxide dismutase, and venom allergen 3/5. Three ion transport peptide-likes (ITPLs) were abundantly detected in T. brontispae venom. Of these, two of them are reported as a novel form for the first time, with the characteristics of lengthened amino acid sequences and additional cysteine residues. These venom ITPLs are obviously apart from other general members within the crustacean hyperglycemic hormone/ion transport peptide (CHH/ITP) family. It implies that they would evolve unique functions essential for parasitism success.

Regulatory Network of MicroRNAs, Host Genes, Target Genes and Transcription Factors in Human Esophageal Squamous Cell Carcinoma.

Abnormally expressed microRNAs (miRNAs) and genes have been found to play key roles in esophageal squamous cell carcinoma (ESCC), but little is known about the underlying mechanisms. The aim of this paper was to assess inter-relationships and the regulatory mechanisms of ESCC through a network-based approach. We built three regulatory networks: an abnormally expressed network, a related network and a global network. Unlike previous examples, containing information only on genes or miRNAs, the prime focus was on relationships. It is worth noting that abnormally expressed network emerged as a fault map of ESCC. Theoretically, ESCC might be treated and prevented by correcting the included errors. In addition, the predicted transcription factors (TFs) obtained by the P-match method also warrant further study. Our results may further guide gene therapy researchers in the study of ESCC.

Regulatory network of microRNAs, target genes, transcription factors and host genes in endometrial cancer.

Genes and microRNAs (miRNAs) have important roles in human oncology. However, most of the biological factors are reported in disperse form which makes it hard to discover the pathology. In this study, genes and miRNAs involved in human endometrial cancer(EC) were collected and formed into regulatory networks following their interactive relations, including miRNAs targeting genes, transcription factors (TFs) regulating miRNAs and miRNAs included in their host genes. Networks are constructed hierarchically at three levels: differentially expressed, related and global. Among the three, the differentially expressed network is the most important and fundamental network that contains the key genes and miRNAs in EC. The target genes, TFs and miRNAs are differentially expressed in EC so that any mutation in them may impact on EC development. Some key pathways in networks were highlighted to analyze how they interactively influence other factors and carcinogenesis. Upstream and downstream pathways of the differentially expressed genes and miRNAs were compared and analyzed. The purpose of this study was to partially reveal the deep regulatory mechanisms in EC using a new method that combines comprehensive genes and miRNAs together with their relationships. It may contribute to cancer prevention and gene therapy of EC.

[Studies on neuronal tracing with pseudorabies virus].

With its abilities of trans-synaptic tracing and self-replication and wide host range, pseudorabies virus (PRV) has been applied in the field of neuroanatomy since the 1970s. Four decades of PRV application have made many advances in researches on neuronal tracing with PRV. Mechanism studies focused on investigating infection of primary neurons and tracing direction in secondary neurons, while application studies focused on development of new pathological strains and innovation of tracing techniques. To date, the mechanism and application of viral tracing are not completely figured out yet. Integration of molecular biology technology will improve the efficiency in related researches.

Networks of MicroRNAs and genes in retinoblastomas.

Through years of effort, researchers have made notable progress in gene and microRNA fields about retinoblastoma morbidity. However, experimentally validated data for genes, microRNAs (miRNAs) and transcription factors (TFs) can only be found in a scattered form, which makes it difficult to conclude the relationship between genes and retinoblastoma systematically. In this study, we regarded genes, miRNAs and TFs as elements in the regulatory network and focused on the relationship between pairs of examples. In this way, we paid attention to all the elements macroscopically, instead of only researching one or several. To show regulatory relationships over genes, miRNAs and TFs clearly, we constructed 3 regulatory networks hierarchically, including a differentially expressed network, a related network and a global network, for analysis of similarities and comparison of differences. After construction of the three networks, important pathways were highlighted. We constructed an upstream and downstream element table of differentially expressed genes and miRNAs, in which we found self-adaption relations and circle-regulation. Our study systematically assessed factors in the pathogenesis of retinoblastoma and provided theoretical foundations for gene therapy researchers. In future studies, especial attention should be paid to the highlighted genes and miRNAs.

Systematical analysis of cutaneous squamous cell carcinoma network of microRNAs, transcription factors, and target and host genes.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules found in multicellular eukaryotes which are implicated in development of cancer, including cutaneous squamous cell carcinoma (cSCC). Expression is controlled by transcription factors (TFs) that bind to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to messenger RNA. Interactions result in biological signal control networks. MATERIALS AND METHODS: Molecular components involved in cSCC were here assembled at abnormally expressed, related and global levels. Networks at these three levels were constructed with corresponding biological factors in term of interactions between miRNAs and target genes, TFs and miRNAs, and host genes and miRNAs. Up/down regulation or mutation of the factors were considered in the context of the regulation and significant patterns were extracted. RESULTS: Participants of the networks were evaluated based on their expression and regulation of other factors. Sub-networks with two core TFs, TP53 and EIF2C2, as the centers are identified. These share self-adapt feedback regulation in which a mutual restraint exists. Up or down regulation of certain genes and miRNAs are discussed. Some, for example the expression of MMP13, were in line with expectation while others, including FGFR3, need further investigation of their unexpected behavior. CONCLUSIONS: The present research suggests that dozens of components, miRNAs, TFs, target genes and host genes included, unite as networks through their regulation to function systematically in human cSCC. Networks built under the currently available sources provide critical signal controlling pathways and frequent patterns. Inappropriate controlling signal flow from abnormal expression of key TFs may push the system into an incontrollable situation and therefore contributes to cSCC development.

Molecular and phylogenetic analysis of the porcine kobuvirus VP1 region using infected pigs from Sichuan Province, China.

BACKGROUND: Porcine kobuvirus (PKoV) is a member of the Kobuvirus genus within the Picornaviridae family. PKoV is distributed worldwide with high prevalence in clinically healthy pigs and those with diarrhea. METHODS: Fecal and intestinal samples (n = 163) from pig farms in Sichuan Province, China were obtained to determine the presence of PKoV using reverse transcription polymerase chain reaction assays. Specific primers were used for the amplification of the gene encoding the PKoV VP1 protein sequence. Sequence and phylogenetic analyses were conducted to clarify evolutionary relationships with other PKoV strains. RESULTS: Approximately 53% (87/163) of pigs tested positive for PKoV. PKoV was widespread in asymptomatic pigs and those with diarrhea. A high prevalence of PKoV was observed in pigs younger than 4 weeks and in pigs with diarrhea. Phylogenetic analysis of 36 PKoV VP1 protein sequences showed that Sichuan PKoV strains formed four distinct clusters. Two pigs with diarrhea were found to be co-infected with multiple PKoV strains. Sequence and phylogenetic analyses revealed diversity within the same host and between different hosts. Significant recombination breakpoints were observed between the CHN/SC/31-A1 and CHN/SC/31-A3 strains in the VP1 region, which were isolated from the same sample. CONCLUSION: PKoV was endemic in Sichuan Province regardless of whether pigs were healthy or suffering from diarrhea. Based on our statistical analyses, we suggest that PKoV was the likely causative agent of high-mortality diarrhea in China from 2010. For the first time, we provide evidence for the co-existence of multiple PKoV strains in one pig, and possible recombination events in the VP1 region. Our findings provide further insights into the molecular properties of PKoV, along with its epidemiology.