Virome characterization of game animals in China reveals a spectrum of emerging pathogens.

Game animals are wildlife species traded and consumed as food and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1,941 game animals, representing 18 species and five mammalian orders, sampled across China. From this, we identified 102 mammalian-infecting viruses, with 65 described for the first time. Twenty-one viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high-risk viruses. We inferred the transmission of bat-associated coronavirus from bats to civets, as well as cross-species jumps of coronaviruses from bats to hedgehogs, from birds to porcupines, and from dogs to raccoon dogs. Of note, we identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence.

Transmission of HCoV-OC43 to pet hamsters.

Coronaviruses (CoVs) are a significant group of pathogens that pose a serious threat to both human and animal health, with some being zoonotic and displaying frequent cross-species transmission. Human CoV-OC43 (HCoV-OC43) is one of the four common human CoVs that can cause seasonal mild to moderate respiratory diseases in humans. In this study, we identified HCoV-OC43 for the first time in two asymptomatic pet hamsters, which share a high similarity with the human-derived HCoV-OC43 strain, suggesting potential cross-species transmission of HCoV-OC43 to pet hamsters. The finding emphasizes the need to strengthen pathogen monitoring of livestock and pets in close contact with humans to provide early warning of public safety.

Emergence and adaptive evolution of influenza D virus.

As a novel member of the Orthomyxoviridae, influenza D virus (IDV) was firstly isolated from swine. However, cattle were found to serve as its primary reservoir. The study of IDV emergence can shed light into the dynamics of zoonotic infections and interspecies transmission. Although there is an increasing number of strains and sequenced IDV strains, their origin, epidemiology and evolutionary dynamics remain unclear. In this study, we reconstruct the diversity and evolutionary dynamics of IDVs. Molecular detection of swine tissue samples shows that six IDV positive samples were identified in the Eastern China. Phylogenetic analyses suggest three major IDV lineages designated as D/Japan, D/OK and D/660 as well as intermediate lineages. IDVs show strong association with geographical location indicating a high level of local transmission, which suggests IDVs tend to establish a local lineage of in situ evolution. In addition, the D/OK lineage widely circulates in swine in Eastern China, and all of the Chinese virus isolates form a distinct sub-clade (D/China sub-lineage). Furthermore, we identified important amino acids in the HEF gene under positive selection that might affect its receptor binding cavity relevant for its broader cell tropism. The combined results highlight that more attention should be paid to the potential threat of IDV to livestock and farming in China.

Etiology and genetic evolution of canine coronavirus circulating in five provinces of China, during 2018-2019.

As the outbreaks of COVID-19 in worldwide, coronavirus has once again caught the attention of people. Canine coronavirus is widespread among dog population, and sometimes causes even fatal cases. Here, to characterize the prevalence and evolution of current circulating canine coronavirus (CCoV) strains in China, we collected 213 fecal samples from diarrheic pet dogs between 2018 and 2019. Of the 213 samples, we found 51 (23.94%) were positive for CCoV. Co-infection with canine parvovirus (CPV), canine astrovirus (CaAstV), canine kobuvirus (CaKV), Torque teno canis virus (TTCaV) were ubiquitous existed. Mixed infection of different CCoV subtypes exists extensively. Considering the limited sequences data in recent years, we sequenced 7 nearly complete genomes and 10 complete spike gene. Phylogenetic analysis of spike gene revealed a new subtype CCoV-II Variant and CCoV-IIa was the most prevalent subtype currently circulating. Moreover, we identified strain B906_ZJ_2019 shared 93.24% nucleotide identifies with previous strain A76, and both of them clustered with CCoV-II Variant, which were not well clustered with the known subtypes. Recombination analysis of B906_ZJ_2019 indicated that strain B906_ZJ_2019 may a recombinant variant between CCoV-I and CCoV-II, which is consistent with strain A76. Furthermore, amino acid variations widely existed among current CCoV-IIa strains circulating in China and the classic CCoV-IIa strains, in spite of the unknown functions. In a word, we report a useful information as to the etiology and evolution of canine coronavirus in China based on the available sequences, which is urgent for the devise of future effective disease prevention and control strategies.

Extensive serum biomarker analysis in patients with nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a fast-growing cancer characterized by high occurrences of nodal and distant metastases and poor prognosis. It is therefore important to identify new serum biomarkers for the early diagnosis and prognostic prediction of this disease. The present study identifies biomarkers in NPC patient serum using a solid-phase antibody array detecting the expression profiles of 174 cytokines in a single experiment. ELISA was performed to validate the array results. The levels of TIMP-2, SELL, CCL24, MMP-1, MMP-3, IGF-I and IL-8 were significantly higher in serum from NPC patients, while the levels of MSP-alpha and HCC-4 were lower. Furthermore, the validation results were identical to those obtained from the antibody array. These results indicate that these cytokines might serve as novel biomarkers for the diagnosis and prognostic prediction of NPC.

Toll-like receptor 5-mediated IL-17C expression in intestinal epithelial cells enhances epithelial host defense against F4+ ETEC infection.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of post-weaning diarrhea (PWD) in piglets. The IL-17 cytokine family is well known to play important roles in the host defense against bacterial infections at the mucosa. Previously, we reported the potential role of IL-17A in clearing an ETEC infection in piglets. IL-17C, another member of the IL-17 family, is highly expressed in the intestinal epithelium, however, its role during an ETEC infection is still unclear. In this study, we demonstrate that F4+ ETEC induce IL-17C mRNA and protein expression in intestinal tissues as well as in porcine intestinal epithelial cells (IPEC-J2). This IL-17C production is largely dependent on TLR5 signaling in IPEC-J2 cells. Both F4+ ETEC infection and exogenous IL-17C increased the expression of antimicrobial peptides and tight junction proteins, such as porcine beta-defensin (pBD)-2, claudin-1, claudin-2 and occludin in IPEC-J2 cells. Taken together, our data demonstrate that TLR5-mediated IL-17C expression in intestinal epithelial cells enhances mucosal host defense responses in a unique autocrine/paracrine manner in the intestinal epithelium against ETEC infection.

Uric acid regulates hepatic steatosis and insulin resistance through the NLRP3 inflammasome-dependent mechanism.

BACKGROUND & AIMS: Hyperuricemia significantly increases risk of non-alcoholic fatty liver disease (NAFLD) and insulin resistance. However, the mechanisms responsible for this association are as yet unclear. This study aimed to investigate the effects and underlying mechanisms of uric acid on development of NAFLD and insulin resistance. METHODS: We initially analyzed the impact of uric acid on the development of hepatic steatosis and insulin resistance in mice and in two cell models, HepG2 and L02. Subsequently, we studied the role of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in uric acid-induced fat accumulation and insulin signaling impairment. RESULTS: We found that uric acid directly induces hepatocyte fat accumulation, insulin resistance, and insulin signaling impairment both in vivo and in vitro. We also found that uric acid-induced NLRP3 inflammasome activation, whereas lowering uric acid by allopurinol inhibited NLRP3 inflammasome activation in a high fat diet mouse model of NAFLD. Moreover, knocking down NLRP3 expression significantly attenuated uric acid-induced fat accumulation both in HepG2 cells and L02 cells. Knocking down NLRP3 expression also rescued uric acid-induced insulin signaling impairment in both cell types. CONCLUSIONS: Uric acid regulates hepatic steatosis and insulin resistance through the NLRP3 inflammasome. Uric acid may be a new therapeutic target for NAFLD and insulin resistance.

To Save Pangolins: A Nutritional Perspective.

Pangolins are one of the world's most trafficked mammals. Since pangolins are highly adapted to ants and termites, they are important for controlling forest termite infestations. In addition to their ecological value, pangolins have economic and medicinal value. Currently, poaching and habitat destruction have radically reduced the number of pangolins, and Manis pentadactyla, Manis javanica, and Manis culionensis are now considered the most threatened pangolin species. In addition to the control of hunting and illegal trade, ex situ breeding is also a useful conservation method. However, many technical obstacles still limit the success of ex situ pangolin breeding. The special feeding traits of pangolins require a diet that meets nutritional and ethological needs. Based on the existing literature and practical experience, this review aims to compare the natural diet and successful diet in the human care of pangolins, to outline the key factors of successful ex situ maintenance from a dietary perspective, and the strategies to improve their conservation success in animal care centers and in the wild. The type of food used in successful pangolin protection agencies is quite variable in nutritional composition. In the diet of pangolins in the wild, the nutrient profile of different species of termites and ants and even the same species of termites and ants but different types (queens, soldiers, etc.) also displays differences. The crude protein content of some ants is higher than that of other foods, such as eggs, milk, and common cat food. The mineral and vitamin concentrations of ants also exceed many common food items, such as oil, meat, and eggs. However, not much is known about the bioavailability of minerals from ants and termites. Based on comparisons between foods, it is clear that the main difference between diets in the wild and in human care of pangolins is that the latter contains fewer insects and vitamins, such as vitamin E, vitamin A, and vitamin B2, and more carbohydrates and non-protein substances than the former. Although many successful dietary formulae have been developed, the pangolin's nutritional needs are still less well studied. A diet with the nutrient concentrations observed in the wild may add to successful ex situ conservation.

Effects of lipoteichoic and arachidonic acids on the immune-regulatory mechanism of bovine mammary epithelial cells using multi-omics analysis.

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows. It mainly utilizes the properties of its pathogenic factor, lipoteichoic acid (LTA), to elicit a host-cell inflammatory response and evade the host-cell immune response. Arachidonic acid (AA) has a regulatory role in the inflammatory response, cell metabolism, and apoptosis. The study aimed to establish a cell model by determining the optimal concentration of LTA and AA for cell induction using the Cell Counting Kit-8 assay and the quantitative polymerase chain reaction of interleukin (IL)-1ß, IL-2, and IL-6. MAC-T cells were planted in 36 10-cm2 culture dishes at a density of 1 × 107 cells per dish. They were treated with LTA for 24 h to constitute the LTA group and with AA for 12 h to constitute the AA group. The cells were pretreated with LTA for 24 h followed by treatment with AA for 12 h to constitute the LTA + AA group. Using proteomic, transcriptomic, and metabolomic analyses, this study determined that LTA can regulate the expression of Actin Related protein 2/3 complex (ARPC)3, ARPC4, Charged Multivesicular Body Protein 3, protein kinase cGMP-dependent, NF-κB Inhibitor Alpha,and other genes to affect cellular metabolism, immune regulation and promote apoptosis. In contrast, AA was observed to regulate the expression of genes such as ARPC3, ARPC4, Charged Multivesicular Body Protein 3, Laminin Gamma 1, Insulin Receptor, Filamin B, and Casein Kinase 1 Epsilon to inhibit cellular apoptosis and promote immune regulation, which provides a theoretical basis for future studies.

Emergence and Characterization of a Novel Reassortant Canine Influenza Virus Isolated from Cats.

Cats are susceptible to a wide range of influenza A viruses (IAV). Furthermore, cats can serve as an intermediate host, and transfer avian influenza virus (AIV) H7N2 to a veterinarian. In this report, a novel reassortant influenza virus, designated A/feline/Jiangsu/HWT/2017 (H3N2), and abbreviated as FIV-HWT-2017, was isolated from nasal swab of a symptomatic cat in Jiangsu province, China. Sequence analysis indicated that, whilst the other seven genes were most similar to the avian-origin canine influenza viruses (CIV H3N2) isolated in China, the NS gene was more closely related to the circulating human influenza virus (H3N2) in the region. Therefore, FIV-HWT-2017 is a reassortant virus. In addition, some mutations were identified, and they were similar to a distinctive CIV H3N2 clade. Whether these cats were infected with the reassortant virus was unknown, however, this random isolation of a reassortant virus indicated that domestic or stray cats were "mixing vessel" for IAV cannot be ruled out. An enhanced surveillance for novel influenza virus should include pet and stray cats.

One-step triplex reverse-transcription PCR detection of porcine epidemic diarrhea virus, porcine sapelovirus, and porcine sapovirus.

Swine diarrhea can be caused by multiple agents, including porcine epidemic diarrhea virus (PEDV), porcine sapelovirus (PSV), and porcine sapovirus (SaV). We designed a one-step triplex reverse-transcription PCR (RT-PCR) detection method including 3 pairs of primers that focused on the S1 gene of PEDV, a conserved gene of PSV, and the VP1 gene of SaV. The optimal concentrations of upstream and downstream primers in the triplex RT-PCR were 0.24 µM for PEDV, 0.15 µM for PSV, and 0.2 µM for SaV, and the optimal annealing temperature was 55.5°C. Triplex RT-PCR assessment of 402 piglet diarrhea samples was compared with conventional individual RT-PCR. Concordance rates in both tests for individual viruses were 100%, 97.6%, and 94.4% for PEDV, PSV, and SaV, respectively. PEDV, PSV, and SaV were detected in 57.2%, 10.4%, and 9.0% of the samples, respectively. The high sensitivity and specificity of this triplex RT-PCR-based detection method for PEDV, PSV, and SaV could allow rapid detection and analysis of mixed infections by these 3 viruses.

Isolation and characterization of Chinese porcine epidemic diarrhea virus with novel mutations and deletions in the S gene.

Porcine epidemic diarrhea (PEDV) has raised growing concerns in the pig-breeding industry because it has caused significant economic losses. To better understand the molecular epidemiology and genetic diversity of PEDV field isolates, in this study, the complete spike (S) and ORF3 genes of 17 PEDV variants in Zhejiang, China during 2014 to 2017, were characterized and analyzed. Phylogenetic analysis based on the S gene and ORF3 gene of these 17 novel PEDV strains and PEDV reference strains indicated that all the PEDV strains fell into two groups designated G1 and G2. Notably, the strains identified in 2014-2015 were in G2, while the other five strains identified from 2016 to 2017 were in G1. Sequencing and phylogenetic analyses showed that recently prevalent Chinese PEDV field strains shared higher identities with United States strains than with South Korean strains. Compared with classical vaccine strains, a series of deletions and frequently occurring mutations were observed in the receptor binding domains of our PEDV strains. Besides, we successfully isolated and reported the genetic characterization two novel PEDV strains, PEDV-LA1 and PEDV-LY4-98, found on the Chinese mainland, which had significant variations in the S gene. Meanwhile, the virulence of the new mutants may be changed, the PEDV-LY4-98 strain, which has multiple mutations in the signal peptide-encoding fragment of the S gene showed delayed cytopathic effects and smaller plaque size compared with strain PEDV-LA1, which lacks these mutations. Three unique amino acid substitutions (L7, G8, and V9) were identified in the SP-encoding fragment of the S1 N-terminal domain of the PEDV-LY4-98 S protein compared with the S proteins of all the previous PEDV strains. The animal experiment revealed that these two novel strains were high pathogenic to neonatal pigs. Whether these amino acids substitutions and the N-glycosylation site substitutions influence the antigenicity and pathogenicity of PEDV remains to be investigated. Meanwhile, amino acid substitutions in the neutralizing epitopes may have conferred the capacity for immune evasion in these PEDV field strains. This study improves our understanding of ongoing PEDV outbreaks in China, and it will guide further efforts to develop effective measures to control this virus.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus isolates in Eastern China.

INTRODUCTION: Porcine epidemic diarrhea virus (PEDV) is one of the most common viral pathogens causing swine diarrhea. METHODOLOGY: We performed a genetic evolution analysis of the S1 gene of endemic PEDV strains in Eastern China. The S1 genes of 37 PEDV-positive samples were amplified and sequenced, and compared to the standard CV777 strain, 120 nucleotides were found to have mutations. RESULTS: The nucleotide and deduced amino acid homologies between the sequences and those of the CV777 strain were 90%-91% and 88.2%-90%, respectively, and their homologies to the vaccine strain were 88.6%-89.7% and 86.2%-87.8%, respectively. Genetic evolution and variation analyses indicated that the 37 PEDV strains belonged to genogroup 2-1, while the CV777 strain, vaccine strain, and earlier Chinese strains all belonged to genogroup 1-1. CONCLUSIONS: The newly emerged clinical PEDV strains indicate that the PEDV CV777 vaccine currently used in China may not fully protect pigs from infection with recent epidemic strains, and will require the development of new vaccine strains.

Identification of host cellular proteins LAGE3 and IGFBP6 that interact with orf virus protein ORFV024.

OBJECTIVE: Orf virus (ORFV) is the pathogen causing contagious pustular dermatitis in goats, sheep and herdsmen. Evidence has confirmed that ORFV can be used as a preventive and therapeutic immunomodulatory agent in several animal models. Our previous data demonstrated that ORFV024 is able to inhibit activation of the NF-κB signaling pathway and act as an important modulator for early immune responses against viral infection. However, the molecular mechanism by which ORFV024 exerting biological function remains unclear. In the present study, we explored and analyzed the function of host cellular proteins that interact with ORFV024. METHODS: The yeast two-hybrid (Y2H) assay was performed to screen proteins interacting with ORFV024 using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). Two of the screened proteins were further confirmed by confocal microscopy, His-tag pull-down assay and CO-Immunoprecipitation (CO-IP) assay. In addition, the ORFV024 interaction network was constructed using the STRING database. RESULTS: In this study, 11 ovine cellular proteins were found to interact with ORFV024. In view of the importance of LAGE3 and IGFBP6 in the ORFV024 functional analysis, we further constructed LAGE3 and IGFBP6 interaction networks. The interactions between ORFV024 and LAGE3 or IGFBP6 were confirmed by confocal microscopy, LAGE3 was further confirmed in the His-tag pull-down assay and CO-IP assay. CONCLUSIONS: Our findings indicate that ORFV024 can interact with ovine cellular proteins LAGE3 and IGFBP6.

[The ground reflectance spectrum retrieval from ETM images].

Retrieval of ground reflectance spectrum from satellite sensor digital count requires knowledge of the atmospheric conditions. Images of spectral radiance from ground-atmosphere system recorded by the multi-spectral imager ETM which boarded Landsat-7 sensor can retrieve the ground reflectivity spectrum. The uncertainty of reflectance spectrum retrieval is no more than 17% at the band 1 of ETM, and less than 10% at the band 2 and 3 of ETM. It is superior to those arithmetics widely used at present. Retrieval of ground radiance spectrum from ground-atmosphere system can be used to synthesize the sRGB true color image, but the definition is not excellent. And it was proved that the color of the images can not reflect the actual nature of earth objects before being adjusted. And the accuracy of interpretation based on true color synthesized images is superior to those based on the source images. So the precision of such reflectance spectrum retrieval is not as good as expected if applied to the true color photography on the ground.

Transcriptomic profiles of human foreskin fibroblast cells in response to orf virus.

Orf virus has been utilized as a safe and efficient viral vector against not only diverse infectious diseases, but also against tumors. However, the nature of the genes triggered by the vector in human cells is poorly characterized. Using RNA sequencing technology, we compared specific changes in the transcriptomic profiles in human foreskin fibroblast cells following infection by the orf virus. The results indicated that orf virus upregulates or downregulates expression of a variety of genes, including genes involved in antiviral immune response, apoptosis, cell cycle and a series of signaling pathways, such as the IFN and p53-signaling pathways. The orf virus stimulates or inhibits immune gene expression such as chemokines, chemokine receptors, cytokines, cytokine receptors, and molecules involved in antigen uptake and processing after infection. Expression of pro-apoptotic genes increased at 8 hours post-infection. The p53 signaling pathway was activated to induce apoptosis at the same time. However, the cell cycle program was promoted after infection, which may be due to the immunomodulatory genes of the orf virus. This presents the first description of transcription profile changes in human foreskin fibroblast cells after orf virus infection and provides an in-depth analysis of the interaction between the host and orf virus. These data offer new insights into the understanding of the mechanisms of infection by orf virus and identify potential targets for future studies.

[Study of fluorescence spectra of starch suspension].

To achieve detection, monitoring and automation of starch graft copolymerzation, the investigation was carried out according to the luminescence of free radical produced in the reaction. The investigation on the fluorescence spectra of starch-water suspension excited by ultraviolet light and its characteristics contributes to the study of the characteristics and mechanisms of free radical coming into being, emitting and disappearing. The experimental results indicate that starch-water suspension can emit fluorescence, whose peak wavelength is about 339 nm. Furthermore, the physical mechanism of fluorescence emission of starch is analyzed. It is considered that this fluorescence comes from the transition from nonbonding electrons in the hetero-atom (O) of the functional group (C--O--C) called ether linkage to the antibonding orbital sigma*.

Probiotic supplementation does not improve eradication rate of Helicobacter pylori infection compared to placebo based on standard therapy: a meta-analysis.

This meta-analysis included eligible randomized controlled trials (RCTs) with the aim of determining whether probiotic supplementation can improve H. pylori eradication rates. PUBMED, EBSCO, Web of Science, and Ovid databases were searched. We included RCTs that investigated the effect of combining probiotics, with or without a placebo, with standard therapy. A total of 21 RCTs that reported standard therapy plus probiotics were included. Compared to the placebo group, the probiotics group was 1.21(OR 1.21, 95% CI: 0.86, 1.69) and 1.28 (OR 1.28, 95% CI: 0.88, 1.86) times more likely to achieve eradication of H. pylori infection in intent-to-treat (ITT) analysis and per protocol (PP) analysis, respectively. Probiotics with triple therapy plus a 14-day course of treatment did not improve the eradication of H. pylori infection (OR 1.44, 95% CI: 0.87, 2.39) compared to the placebo. Moreover, the placebo plus standard therapy did not improve eradication rates compared to standard therapy alone (P = 0.816). However, probiotics did improve the adverse effects of diarrhea and nausea. These pooled data suggest that the use of probiotics plus standard therapy does not improve the eradication rate of H. pylori infection compared to the placebo.