Novel interacting proteins identified by tandem affinity purification and mass spectrometry associated with IFITM3 protein during PDCoV infection.

Interferon-induced transmembrane 3 (IFITM3) is a membrane-associated protein that exhibits antiviral activities against a wide range of viruses through interactions with other cellular and viral proteins. However, knowledge of the mechanisms of IFITM3 in Porcine deltacoronavirus (PDCoV) infection has been lacking. In this study, we demonstrate that IFN-α treatment induces the upregulation of IFITM3 activity and thus attenuates PDCoV infection. PDCoV replication is inhibited in a dose-dependent manner by IFITM3 overexpression. To clarify the novel roles of IFITM3 during PDCoV infection, proteins that interact with IFITM3 were screened by TAP/MS in an ST cell line stably expressing IFITM3 via a lentivirus. We identified known and novel candidate IFITM3-binding proteins and analyzed the protein complexes using GO annotation, KEGG pathway analysis, and protein interaction network analysis. A total of 362 cellular proteins associate with IFITM3 during the first 24 h post-infection. Of these proteins, the relationship between IFITM3 and Rab9a was evaluated by immunofluorescence colocalization analysis using confocal microscopy. IFITM3 partially colocalized with Rab9a and Rab9a exhibited enhanced colocalization following PDCoV infection. We also demonstrated that IFITM3 interacts specifically with Rab9a. Our results considerably expand the protein networks of IFITM3, suggesting that IFITM3 participates in multiple cellular processes during PDCoV infection.

Identification and integrated analysis of lncRNAs and miRNAs in IPEC-J2 cells provide novel insight into the regulation of the innate immune response by PDCoV infection.

BACKGROUND: Noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), are pivotal regulators involved in the pathogenic mechanism of multiple coronaviruses. Porcine deltacoronavirus (PDCoV) has evolved multiple strategies to escape the innate immune response of host cells, but whether ncRNAs are involved in this process during PDCoV infection is still unknown. RESULTS: In this study, the expression profiles of miRNAs, lncRNAs and mRNAs in IPEC-J2 cells infected with PDCoV at 0, 12 and 24 hours postinfection (hpi) were identified through small RNA and RNA sequencing. The differentially expressed miRNAs (DEmiRNAs), lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) were screened from the comparison group of IPEC-J2 cells at 0 and 12 hpi as well as the comparison group of IPEC-J2 cells at 12 and 24 hpi. The target genes of these DEncRNAs were predicted. The bioinformatics analysis of the target genes revealed multiple significantly enriched functions and pathways. Among them, the genes that were associated with innate immunity were specifically screened. The expression of innate immunity-related ncRNAs and mRNAs was validated by RT-qPCR. Competing endogenous RNA (ceRNA) regulatory networks among innate immunity-related ncRNAs and their target mRNAs were established. Moreover, we found that the replication of PDCoV was significantly inhibited by two innate immunity-related miRNAs, ssc-miR-30c-3p and ssc-miR-374b-3p, in IPEC-J2 cells. CONCLUSIONS: This study provides a data platform to conduct studies of the pathogenic mechanism of PDCoV from a new perspective and will be helpful for further elucidation of the functional role of ncRNAs involved in PDCoV escaping the innate immune response.

Epidemiology of Porcine Pseudorabies from 2010 to 2018 in Tianjin, China.

Pseudorabies (PR), the causative agent of Aujeszky's disease, has rapidly increased in recent years and has caused significant economic losses. To understand the seroprevalence and epidemiological characteristics of PR in Tianjin, China, a total of 23,627 blood and 1,093 tissue samples were collected from 228 pig farms during January 2010 to December 2018. The Pseudorabies virus (PRV) glycoprotein E (gE) antibody was tested by enzyme-linked immunosorbent assay (ELISA), and wild-type PRV (WT PRV) was detected by gE-polymerase chain reaction (PCR). Macroscopic and microscopic lesions were observed in tissue samples. The results showed that 46.70% of the serum samples and 49.76% of pig farms were seropositive for PRV gE antibody based on the ELISA results, and 13.54% of the tissue samples were positive for WT PRV detected by PCR. The positive rate of serum samples increased rapidly after 2011 and reached 62.40% in 2013. Although it gradually decreased from 2014 to 2018, the positive rate of serum samples remained at a high level. The positive rate of pig farms showed the same trend. Moreover, after 2011, the detection rate of WT PRV was increased rapidly and was significantly higher than in 2010 and 2011. Macroscopic and microscopic lesions were observed in various tissues during histopathological examination. Based on univariate analysis, the increased risk of seropositivity was associated with the immune status and infection in sows and fattening pigs. These findings demonstrate that PR was prevalent in the region of Tianjin, China. These epidemiological data can assist in the control of PR.

Isolation, identification and genetic characterization analysis of a fowl aviadenovirus serotype 4 strain from Tianjin, China.

A fowl aviadenovirus serotype 4 (FAdV-4), Y17215-1, was isolated from the liver of chickens with Hydropericardium-hepatitissyndrome(HHS) in a chicken farm of Tianjin, China. Obvious cytopathic effects were observed in the infected chicken liver hepatocellular carcinoma cell line (LMH cells) at 24 h post infection (hpi), which consisted of enlarger and rounder shape of cells. The typical and specific green fluorescence was observed by indirect immunofluorescence assay (IFA). Tissue Culture Infectious Dose50 (TCID50) of it measured after five stable passage in LMH cells reached 106.5TCID50/0.1 mL. The strain was inoculated through allantoic membrane of 10-day specific pathogen free(SPF) Chick embryos, the thicker allantoic membranes were observed at 120 hpi. 7-day-old SPF chickens were inoculated with the strain via intramuscular (i.m.) or intranasal (i.n.) injection which resulted in 100% mortality of test chickens. Additionally, the sickness and death of cohabitation chickens in the test group were observed which indicated that the virus can infect healthy chickens by horizontal transmission. The sick chickens showed depression, anorexia and diarrhea with green watery feces. Y17215-1-inoculated chickens mainly presented swollen liver with blood spot, and the enhancement of effusion or yellow gel like effusion that were observed in the pericardium through necropsy. Histopathological examination showed focal necrosis of hepatocytes and characteristic eosinophilic inclusion bodies in the cytoplasm. The results showed that the Y17215-1 isolate had high pathogenicity to SPF chickens. The phylogenetic analysis of the major structural proteins including hexon, fiber-1 and fiber-2 revealed that Y17215-1 strain belongs to C species of fowl aviadenovirus of aviadenovirus family, and has high homology with other Chinese strains isolated in recent years, but was distinct from ON1、MX-SHP95、KR5 and other foreign isolates. This study laid a foundation for further study of epidemiological investigation, pathogenic mechanism as well as the diagnosis and control technology of FAdV-4.

Transcriptome analysis of PK-15 cells in innate immune response to porcine deltacoronavirus infection.

Porcine deltacoronavirus (PDCoV) is a newly emerged swine enteropathogenic coronavirus affecting pigs of all ages and causing diarrhea problems. Research findings indicate that PDCoV has evolved strategies to escape innate immune response in host cells, but mechanism of PDCoV in innate immune modulation is not well understood. In this study, we report our findings on identifying the alterations of host cell innate immune response affected by PDCoV infection and exploring the gene expression profiles of PK-15 cells at 0, 24, and 36 h PDCoV post infection by RNA sequencing. A total of 3,762 and 560 differentially expressed genes (DEGs) were screened by comparison of uninfected PK-15 cells and infected PK-15 cells at 24 h post infection (hpi) (INF_24h versus NC), and also comparison of infected PK-15 cells between 24 and 36 hpi (INF_36h versus INF_24h), which included 156 and 23 porcine innate immune-related genes in the DEGs of INF_24h versus NC and INF_36h versus INF_24h, respectively. Gene Ontology function classification and Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment analysis were performed based on the DEGs that exhibited the same expression tendencies with most of the innate immune-associated genes among these PK-15 cell samples described above. The enrichment results indicated that extensive gene functions and signaling pathways including innate immune-associated functions and pathways were affected by PDCoV infection. Particularly, 4 of 5 innate immune signaling pathways, which were primarily affected by PDCoV, played important roles in I-IFN's antiviral function in innate immune response. Additionally, 16 of the host cell endogenous miRNAs were predicted as potential contributors to the modulation of innate immune response affected by PDCoV. Our research findings indicated that the innate immune-associated genes and signaling pathways in PK-15 cells could be modified by the infection of PDCoV, which provides a fundamental foundation for further studies to better understand the mechanism of PDCoV infections, so as to effectively control and prevent PDCoV-induced swine diarrheal disease outbreaks.

Novel triple-reassortant H1N1 swine influenza viruses in pigs in Tianjin, Northern China.

Pigs are susceptible to both human and avian influenza viruses and therefore have been proposed to be mixing vessels for the generation of pandemic influenza viruses through reassortment. In this study, for the first time, we report the isolation and genetic analyses of three novel triple-reassortant H1N1 swine influenza viruses from pigs in Tianjin, Northern China. Phylogenetic analysis showed that these novel viruses contained genes from the 2009 pandemic H1N1 (PB2, PB1, PA and NP), Eurasian swine (HA, NA and M) and triple-reassortant swine (NS) lineages. This indicated that the reassortment among the 2009 pandemic H1N1, Eurasian swine and triple-reassortant swine influenza viruses had taken place in pigs in Tianjin and resulted in the generation of new viruses. Furthermore, three human-like H1N1, two classical swine H1N1 and two Eurasian swine H1N1 viruses were also isolated during the swine influenza virus surveillance from 2009 to 2013, which indicated that multiple genetic lineages of swine H1N1 viruses were co-circulating in the swine population in Tianjin, China. The emergence of novel triple-reassortant H1N1 swine influenza viruses may be a potential threat to human health and emphasizes the importance of further continuous surveillance.

Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals.

During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health.

[Expression of TLR3 and IL-8, Eotaxin in the airway smooth muscle cells induced by PolyI:C].

AIM: to observe the effect of PolyI:C on the expression of TLR3 and inflammation cytokine in the airway smooth muscle cells ASMCs of rat, to investigate relationship between the expression of Toll-like receptor 3 (TLR3) in the airway smooth muscle cells and airway inflammation. METHODS: airway smooth muscle cells were derived from rat airway tissue and were cultured in vitro, ASMCs were divided into 2 groups: control group and PolyI:C group, PolyI:C group was divided into the different time spots. The mRNA expression of TLR3 was measured by RT-PCR. The level of NF-kappaB was measured by Western blot. The concertration of IL-8 and Eotaxin in ASMCs supernatant were determined by ELISA. RESULTS: the expression of TLR3 mRNA and NF-kappaB protein in the ASMCs in PolyI:C group was significantly higher than those in control group(P<0.05), the concentration of IL-8 and Eotaxin in the ASMCs in PolyI:C group was significantly higher compared to control group (P<0.05); and have time dependent manner. CONCLUSION: poly I:C promoted the expression of TLR3 in ASMCs, activated NF-kappaB, incresead the concentration of IL-8 and Eotaxin, involved in airway inflammation of asthma.

[Detection of molecular markers of amantadine resistance in swine influenza viruses by pyrosequencing].

OBJECTIVE: To evaluate the resistance against the adamantine of multi-genotype (H1N1, H3N2 and H9N2) swine influenza viruses isolated from China in recent years by pyrosequencing. METHODS: Mutation in one of five key amino acid residues (positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses, leading to resistance against the adamantine class of anti-influenza drugs. The residues L26, V27, A30, S31, and G34 in the M2 protein were targeted for pyrosequencing,and 10 swine influenza viruses obtained from China during 2004 to 2008 were used to perform the amantadine resistance analysis. RESULTS: All 5 H1N1 swine influenza viruses were adamantine resistance, three mutations were founded in these isolates, namely V27T, V27I and S31N. Other five isolates, including four H3N2 and one H9N2 swine influenza virus, were proved to be sensitive to amantadine. CONCLUSION: Pyrosequencing technology based on the M2 gene can be used to determine the amantadine resistance for multi-genotype swine influenza viruses.

SARS-associated coronavirus transmitted from human to pig.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was isolated from a pig during a survey for possible routes of viral transmission after a SARS epidemic. Sequence and epidemiology analyses suggested that the pig was infected by a SARS-CoV of human origin.