M1 polarization of chicken macrophage HD11 can be activated by duck Tembusu virus via MyD88-NF-κB-mediated signaling pathway.

Duck Tembusu virus (DTMUV) has caused significant economic losses to the global duck industry since its outbreak in 2010. The macrophages act as the key immune cell, and its polarization in different functional states is very important for host's immune responses and microbial infections. Avian macrophages are the main target cells of DTMUV, its polarization induced by DTMUV and the underlying mechanisms were explored in this study. Through quantitative real-time PCR, nitrite assay, and flow cytometry analysis, we found that DTMUV caused severe inflammatory responses in chicken macrophage line HD11 by reprogramming the expression of M1- and M2-associated genes, leading to the polarization of HD11 macrophage to M1-type. In term of mechanism, transcriptomics was performed to analyze the M1-type polarization triggered by DTMUV, it was found that most differential genes were implicated in biological processes, and DTMUV infection significantly activated innate immune signaling pathways, including cytokine-cytokine receptor interaction, MAPK signaling pathway. Moreover, transcription factors NF-κB and AP1 also be activated after viral infection. However, further validation analysis by inhibitors and siRNAs of NF-κB and AP1 showed that NF-κB molecule was essential for DTMUV-induced M1 polarization in HD11 cell, but not AP1. Additionally, the inhibiting assays targeting MyD88 and TRIF molecules were conducted to determine their effect on NF-κB and M1-associated genes upregulated by DTMUV. The results showed that although the inhibition of both MyD88 and TRIF significantly downregulated the mRNA level of NF-κB, but the expression of M1-associated genes such as CD86 was lower in MyD88 inhibition group than in the other group, indicating that the role of MyD88 in mediating M1 polarization induced by DTMUV was more important. Overall, these results demonstrated that DTMUV infection induces M1-type polarization in chicken macrophage HD11 through MyD88-NF-κB signaling pathways. This finding will lay the foundation for further study the pathogenesis of DTMUV, and provide new insights into the prevention and control of this disease.

Retrospective Investigation and Genetic Variation Analysis of Chicken Infectious Anemia in Shandong Province, 2020-2022.

Chicken infectious anemia (CIA) is a vertical transmission infectious chicken disease caused by the chicken infectious anemia virus (CAV). The disease can induce stunting and immunosuppression in chicks by infecting bone marrow-derived stem cells, causing huge economic losses for the poultry industry. To determine the prevalence of CIA in Shandong Province, China, 854 suspected CIA samples were collected and analyzed in 13 cities in Shandong from 2020 to 2022. The PCR results showed that a total of 115 CAV were isolated. The CAV-positive rates were 17.21% (26/151) in 2020, 12.23% (35/286) in 2021, and 12.94% (54/417) in 2022, with severe mixed infections. Among them, CAV and fowl adenovirus (FAdV) were the most common, accounting for 40.86%. VP1 gene homology analysis showed that isolated strains shared 96.1-100% homology with the previously reported CAV strains. Genetic variation analysis showed that most of the isolated CAV strains were located in genotype A. These results indicate that CIA infection in Shandong chickens in recent years has been prevalent and mixed infections are common, but there were no significant genetic variations. Our results extend the understanding of the prevalence and genetic evolution of CIA in Shandong Province. They will offer new references for further study of the epidemiology and virus variation and the prevention and control of this disease.

Indirect ELISA Using Multi-Antigenic Dominants of p30, p54 and p72 Recombinant Proteins to Detect Antibodies against African Swine Fever Virus in Pigs.

African swine fever (ASF) caused by ASF virus (ASFV) is a fatal disease in pigs and results in great economic losses. Due to the lack of available vaccines and treatments, serological diagnosis of ASF plays a key role in the surveillance program, but due to the lack of knowledge and the complexity of the ASFV genome, the candidate target viral proteins are still being researched. False negativity is still a big obstacle during the diagnostic process. In this study, the high antigenic viral proteins p30, p54 and p72 were screened to find the antigenic dominant domains and the tandem His-p30-54-72 was derived. An indirect enzyme-linked immunosorbent assay (iELISA) coated with His-p30-54-72 was developed with a cut-off value of 0.371. A total of 192 clinical samples were detected by His-p30-54-72-coated indirect ELISA (iELISA) and commercial ASFV antibody kits. The results showed that the positive rate of His-p30-54-72-coated iELISA was increased by 4.7% and 14.6% compared with a single viral protein-based commercial ASFV antibody kits. These results provide a platform for future ASFV clinical diagnosis and vaccine immune effect evaluation.

Molecular epidemiological survey of porcine epidemic diarrhea in some areas of Shandong and genetic evolutionary analysis of S gene.

Responsible for the acute infectious disease porcine epidemic diarrhea (PED), PED virus (PEDV) induces severe diarrhea and high mortality in infected piglets and thus severely harms the productivity and economic efficiency of pig farms. In our study, we aimed to investigate and analyze the recent status and incidence pattern of PEDV infection in some areas of Shandong Province, China. We collected 176 clinical samples of PED from pig farms in different regions of Shandong Province during 2019-2021. PEDV, TGEV, and PORV were detected using RT-PCR. The full-length sequences of positive PEDV S genes were amplified, the sequences were analyzed with MEGA X and DNAStar, and a histopathological examination of typical PEDV-positive cases was performed. RT-PCR revealed positivity rates of 37.5% (66/176) for PEDV, 6.82% (12/176) for transmissible gastroenteritis virus, and 3.98% (7/176) for pig rotavirus. The test results for the years 2019, 2020, and 2021 were counted separately, PEDV positivity rates for the years were 34.88% (15/43), 39.33% (35/89), and 36.36% (16/44), respectively. Histopathological examination revealed atrophied, broken, and detached duodenal and jejunal intestinal villi, as typical of PED, and severe congestion of the intestinal submucosa. Moreover, the results of our study clearly indicate that the G2 subtype is prevalent as the dominant strain of PEDV in Shandong Province, where its rates of morbidity and mortality continue to be high. Based on a systematic investigation and analysis of PEDV's molecular epidemiology across Shandong Province, our results enrich current epidemiological data regarding PEDV and provide some scientific basis for preventing and controlling the disease.

Epidemiological survey of PRRS and genetic variation analysis of the ORF5 gene in Shandong Province, 2020-2021.

Since the rise of porcine reproductive and respiratory syndrome virus (PRRSV) in China, mutations have occurred regularly. In particular, the emergence of HP-PRRSV has significantly improved the pathogenicity of PRRSV. It has brought huge economic losses to the Chinese pig farming industry. To understand the current prevalence and evolution of PRRSV in Shandong Province, 1,344 samples suspected of having PRRSV were collected from local hog farms of different sizes. Genetic variation in the isolated PRRSV ORF5 gene was analyzed using the RT-PCR method. The results showed that the detection rate of PRRSV in the collected samples was 25.44%. The predominant strain of PRRSV in Shandong Province is still NADC30-like. However, it cannot be ignored that NADC34-like is also starting to become a prevalent strain. Mutations in ORF5 amino acids 13, 151 and neutralizing epitope (aa36-aa52) in some isolates can cause changes in virulence and ability to escape immunity. This study enriches the epidemiological data on PRRSV in Shandong Province, China. It provides an important reference for the development of new vaccines and for the prevention and control of PRRSV.

Serological Surveillance of the H1N1 and H3N2 Swine Influenza A Virus in Chinese Swine between 2016 and 2021.

Background: Swine influenza A virus (IAV-S) is a common cause of respiratory disease in pigs and poses a major public health threat. However, little attention and funding have been given to such studies. The aim of this study was to assess the prevalence of the Eurasian avian-like H1N1 (EA H1N1), 2009 pandemic H1N1 (pdm/09 H1N1), and H3N2 subtype antibodies in unvaccinated swine populations through serological investigations. Such data are helpful in understanding the prevalence of the IAV-S. Methods: A total of 40,343 serum samples from 17 regions in China were examined using hemagglutination inhibition (HI) tests against EA H1N1, pdm/09 H1N1, and H3N2 IAV-S from 2016 to 2021. The results were analyzed based on a reginal distribution, seasonal distribution, and in different breeding stages. Results: A total of 19,682 serum samples out of the 40,343 were positive for IAV-S (48.79%). The positivity rates to the EA H1N1 subtype, pdm/09 H1N1 subtype, and H3N2 subtype were 24.75% (9,986/40,343), 7.94% (3,205/40,343), and 0.06% (24/40,343), respectively. The occurrences of coinfections from two or more subtypes were also detected. In general, the positivity rates of serum samples were related to the regional distribution and feeding stages. Conclusions: The results of this study showed that the anti-EA H1N1 subtype and pdm/09 H1N1 subtype antibodies were readily detected in swine serum samples. The EA H1N1 subtype has become dominant in the pig population. The occurrences of coinfections from two or more subtypes afforded opportunities for their reassortment to produce new viruses. Our findings emphasized the need for continuous surveillance of influenza viruses.

Non-Structural Protein 3 of Duck Tembusu Virus Induces Autophagy via the ERK and PI3K-AKT-mTOR Signaling Pathways.

Despite autophagy's pivotal role in the replication of viruses such as duck Tembusu virus (DTMUV), which has caused massive economic losses to the poultry industry in the world, the specific relationships between DTMUV and cellular autophagy remain largely unknown. In response, we investigated the interactions between autophagy and DTMUV, the effects of the structural and non-structural proteins of DTMUV on autophagy, and the autophagy-related signaling pathways induced by DTMUV. Among the results, DTMUV increased the autophagy flux in duck embryo fibroblasts (DEF) and BHK-21 cells, while autophagy facilitated viral replication. After we pharmacologically induced autophagy with rapamycin (RAPA), the replication of DTMUV increased by 15.23-fold compared with the control group of DEF cells. To identify which DTMUV protein primarily induced autophagy, all three structural proteins and seven non-structural proteins of DTMUV were transfected into cells, and the results showed that non-structural protein 3 (NS3) induced significant autophagy in DEF cells. By means of Western blot, immunofluorescence, and transmission electron microscopy, we confirmed that NS3 protein could significantly induce autophagy and autophagy flux. Furthermore, we showed that NS3 induced autophagy in DEF cells through extracellular signal-regulated kinase 2 (ERK2) and phosphatidylinositol-3-kinase (PI3K)/AKT and the mammalian target of rapamycin (mTOR) signaling pathways using specific inhibitors and RNA interference assays. Finally, autophagy induced by NS3 promoted DTMUV replication. These results provide novel insight into the relationship between DTMUV and autophagy, broadening the current understanding of the molecular pathogenesis of DTMUV.

The diagnosis and molecular epidemiology investigation of avian hepatitis E in Shandong province, China.

BACKGROUND: Avian hepatitis E virus (HEV) is the pathogenic agent of big liver and spleen disease (BLS) and of hepatitis-splenomegaly syndrome (HSS) in chickens, which have caused economic losses to the poultry industry in China. In this study, 18 samples of BLS chickens were collected to reveal the molecular epidemiological characteristics of avian HEV in the province of Shandong, China. RESULTS: Gross and microscopic lesions of clinical samples were observed; then, virology detection and genetic analysis of avian HEV were performed. The results showed that there was significant swelling and rupture in the liver and that the spleen was enlarged. Microscopic lesions demonstrated obvious hemorrhage in the liver, with infiltration of heterophilic granulocytes, lymphocytes, and macrophages, as well as the reduction of lymphocytes in the spleen. Eleven of the 18 samples were positive for avian HEV, with a positive rate of 61.11%. More importantly, all avian HEV-positive samples were mixed infections: among these, the mixed infections of avian HEV and chicken infectious anemia virus (CIAV) and avian HEV and fowl adenovirus (FAdV) were the most common. Furthermore, the genetic evolution analysis showed that all avian HEV strains obtained here did not belong to the reported 4 genotypes, thus constituting a potential novel genotype. CONCLUSIONS: These results of this study further enrich the epidemiological data on avian HEV in Shandong, prove the genetic diversity of avian HEV in China, and uncover the complex mixed infections of avian HEV clinical samples.

First detection and phylogenetic analysis of porcine circovirus 3 in female donkeys with reproductive disorders.

BACKGROUND: PCV3 is a pathogen associated with porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, and cardiac and multiorgan inflammation, which was newly identified in 2016 in sows in USA. Recently, PCV3 has also been identified from several non-porcine species like (cattle, dog, wild boar, deer, mice and ticks). However, PCV3 infection in donkey is not well established. Since 2019, 300 blood samples were collected from female donkey, which was characterized by abortion and sterility, in Liaocheng city of China. RESULTS: In the present study, an investigation of PCV3 in donkey blood samples was undertaken employing by real time PCR. Positive rates of PCV3 in donkeys reach to 21.0 %. In addition, one full-length PCV3 genome sequence was obtained, and it had a highest identity with porcine circovirus 3 PCV3/CN/Nanjing2017 strain and is clustered to PCV3a genotype based on ORF2 sequences. CONCLUSIONS: This is the first report of detection of PCV3 from female donkeys presenting reproductive failure in large-scale donkey farms, China. In addition, the PCV3 strain identified in this study shared the closest relationship with those from porcine, suggesting that PCV3 may be transmitted from pigs to donkeys. Totally, PCV3 infection in donkey should be concerned although the association between it and reproductive failure are not better understood.

Knockout of p53 leads to a significant increase in ALV-J replication.

Avian leukemia is a common malignant disease, and and its regulatory mechanism is complex. As the most extensive tumor suppressor gene in cancer research, p53 can control multiple functions such as that of DNA repair, induction of apoptosis, cell cycle arrest and so on. In view of the diversity associated with varied function of p53, this study analyzed the possible effect of gene on ALV-J replication and its regulatory mechanism. We successfully constructed a p53 knockout DF-1 cell line (p53-KO-DF-1 cells) by using CRISPR-Cas9 system. When ALV-J was co-infected with DF-1 and p53-KO-DF-1 cells, it was found that compared with wild-type DF-1 cells, the viral copy number of p53-KO-DF-1 cells infected with ALV-J increased significantly 48 h after infection, whereas the expression of innate immune factors such as Il-2,TNF- α, IFN- γ and MX1 decreased significantly. Detection of p53-related tumor genes indicated that after p53 deletion, the expression of c-myc, bcl-2, and bak increased significantly, while the expression of p21 and p27 was noted to be decreased. The cell cycle distribution and apoptosis of the 2 cell lines was detected by flow cytometry analysis. The results showed that p53 knockout prevented G0/G1 and G2 M phase arrest induced by ALV-J, and substantially decreased the rate of apoptosis. Overall, the results indicated that p53 gene can effectively inhibits ALV-J replication by regulating important cellular processes, and p53 gene related proteins involved in cell cycle activity may function as the key targets for the prevention and treatment of ALV-J.

Inhibition of Antiviral Innate Immunity by Foot-and-Mouth Disease Virus Lpro through Interaction with the N-Terminal Domain of Swine RNase L.

Foot-and-mouth disease virus (FMDV) is the pathogen of foot-and-mouth disease (FMD), which is a highly contagious disease in cloven-hoofed animals. To survive in the host, FMDV has evolved multiple strategies to antagonize host innate immune responses. In this study, we showed that the leader protease (Lpro) of FMDV, a papain-like proteinase, promoted viral replication by evading the antiviral interferon response through counteracting the 2',5'-oligoadenylate synthetase (OAS)/RNase L system. Specifically, we observed that the titers of Lpro deletion virus were significantly lower than those of wild-type FMDV (FMDV-WT) in cultured cells. Our mechanistic studies demonstrated that Lpro interfered with the OAS/RNase L pathway by interacting with the N-terminal domain of swine RNase L (sRNase L). Remarkably, Lpro of FMDV exhibited species-specific binding to RNase L in that the interaction was observed only in swine cells, not human, monkey, or canine cells. Lastly, we presented evidence that by interacting with sRNase L, FMDV Lpro inhibited cellular apoptosis. Taken together, these results demonstrate a novel mechanism that Lpro utilizes to escape the OAS/RNase L-mediated antiviral defense pathway. IMPORTANCE FMDV is a picornavirus that causes a significant disease in agricultural animals. FMDV has developed diverse strategies to escape the host interferon response. Here, we show that Lpro of FMDV antagonizes the OAS/RNase L pathway, an important interferon effector pathway, by interacting with the N-terminal domain of sRNase L. Interestingly, such a virus-host interaction is species-specific because the interaction is detected only in swine cells, not in human, monkey, or canine cells. Furthermore, Lpro inhibits apoptosis through interacting with sRNase L. This study demonstrates a novel mechanism by which FMDV has evolved to inhibit host innate immune responses.

The construction of recombinant Lactobacillus casei vaccine of PEDV and its immune responses in mice.

BACKGROUND: Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV) characterized by vomiting, diarrhea, anorexia, and dehydration, which have caused huge economic losses around the world. At present, vaccine immunity is still the most effective method to control the spread of PED. In this study, we have constructed a novel recombinant L. casei-OMP16-PEDVS strain expressing PEDVS protein of PEDV and OMP16 protein of Brucella abortus strain. To know the immunogenicity of the recombinant L. casei-OMP16-PEDVS candidate vaccine, it was compared with BL21-OMP16-PEDVS-F, BL21-OMP16-PEDVS, and BL21-PEDVS recombinant protein. RESULTS: The results showed that we could detect higher levels of IgG, neutralizing antibody, IL-4, IL-10, and INF-γ in serum and IgA in feces of L. casei-OMP16-PEDVS immunized mice, which indicated that L. casei-OMP16-PEDVS candidate vaccine could induce higher levels of humoral immunity, cellular immunity, and mucosal immunity. CONCLUSION: Therefore, L. casei-OMP16-PEDVS is a promising candidate vaccine for prophylaxis of PEDV infection.

The role of 14-3-3 proteins in cell signalling pathways and virus infection.

14-3-3 proteins are highly conserved in species ranging from yeast to mammals and regulate numerous signalling pathways via direct interactions with proteins carrying phosphorylated 14-3-3-binding motifs. Recent studies have shown that 14-3-3 proteins can also play a role in viral infections. This review summarizes the biological functions of 14-3-3 proteins in protein trafficking, cell-cycle control, apoptosis, autophagy and other cell signal transduction pathways, as well as the associated mechanisms. Recent findings regarding the role of 14-3-3 proteins in viral infection and innate immunity are also reviewed.

Excessive copper in feed not merely undermines animal health but affects food safety.

BACKGROUND: Blackened intestines in slaughtered pigs have been commonly observed in China in recent years. However, no cause has been reported. OBJECTIVES: We attempted to determine whether the blackening of the pig intestine was related to an excess of copper (Cu) in their feed. METHODS: In this study, we observed and collected porcine intestines in small- and large-scale pig slaughterhouses in Shandong province from May to October 2018. Twelve types of metal ions were detected in the black intestinal samples. RESULTS: The Cu level in the intestine samples was mostly higher than the Chinese national limit for food. Further study showed that Cu supplementation in most commercial porcine feed also exceeded the national standard. An animal model (mouse) that could mimic the intestinal blackening in pigs was established. Compared to control mice, Cu accumulated in the liver and intestines of mice fed an excessive Cu level, confirming the excessive Cu in the feed may be considered the major cause of blackened porcine intestines. Microscopic examination revealed that black intestines had many particles containing Cu in the lamina propria of the intestinal mucosa, and the intestinal mucosal epithelial cells showed degeneration and necrosis. CONCLUSIONS: In conclusion, overuse of Cu in animal feed can lead to animal poisoning and Cu accumulation in animal products. Such overuse not only harms the health of livestock but can also affect public health.

Epidemiological investigation of porcine circovirus type 2 and its coinfection rate in Shandong province in China from 2015 to 2018.

BACKGROUND: Porcine circovirus type 2 (PCV2) is one of the crucial swine viral pathogens, caused porcine circovirus associated diseases (PCVAD). Shandong province is one of the most important pork producing areas and bears a considerable economic loss due to PCVAD. However, there is limited information on epidemiology and coinfection rate of PCV2 with other critical swine diseases in this area, such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Pseudorabies virus (PRV), and porcine epidemic diarrhea virus (PEDV). RESULTS: Overall, 89.59% serum samples and 36.98% tissue samples were positive for PCV2 specified ELISA and PCR positive for PCV2, respectively. The coinfection rates of PCV2 with PRRSV, PRV, CSFV, and PEDV were 26.73%, 18.37%, 13.06%, and 3.47%, respectively. Moreover, genetic characteristic of PCV2 were analyzed based on the cap genes showing that PCV2d is the dominant sub-genotype circulating in the province. CONCLUSIONS: Our findings reveal that PCV2d, as the dominant strain, is prevailing in pig farms in Shandong province at high levels. There was a high frequency of coinfection of PCV2 and PRRSV.

HoBi-like pestivirus infection leads to bovine death and severe respiratory disease in China.

HoBi-like pestivirus is an emerging atypical pestivirus in cattle and small ruminants, causing clinical signs similar to those observed in bovine viral diarrhoea virus infections. Natural infection of HoBi-like pestivirus has been reported in cattle herds and small ruminants in multiple countries in South America, Europe and Asia. However, HoBi-like pestiviruses were only identified from contaminated bovine serum and small ruminants in China. So far, no clinical cases induced by HoBi-like pestivirus infection were reported in Chinese cattle herds. Here, for the first time, we reported natural infection of HoBi-like pestivirus in a cattle herd in China. Sick cattle with severe respiratory and diarrhoea and high fatality rate were found in a beef cattle herd in Shandong province in November 2017. RT-PCR, viral isolation, sequencing and phylogenetic analysis showed that the primary causative agent was HoBi-like pestivirus. The isolated HoBi-like pestivirus strain, SDJN-China-2019, shared 94.1%-97.5% homology with the LV168-20_16RN strain from Brazil in nucleotide of 5'UTR, Npro and E2 while it shared only 88.5%-92.1% homology with Asian HoBi-like virus strain Th/04-Khonkaen. Multiple unique mutations of amino acid were observed in Npro and E2 proteins of SDJN-China-2019, which were different from that of other reference strains. In summary, this study provides the first evidence of HoBi-like pestivirus infection in Chinese cattle herds, raising potential threat to the cattle industry in China.

Quantitative Proteomic Analysis of Duck Embryo Fibroblasts Infected With Novel Duck Reovirus.

The novel duck reovirus (NDRV) can cause hemorrhage and necrosis on the spleen of Pekin ducks; this disease has resulted in great economic losses to the duck industry. However, the molecular pathogenesis of NDRV remains poorly understood. In the current study, the quantitative proteomic analysis of NDRV-infected duck embryo fibroblasts was performed to explore the cellular protein changes in response to viral infection through iTRAQ coupled with the liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method. A total of 6,137 proteins were obtained in cell samples at 24 h post-infection. Of these, 179 differentially expressed proteins (DEPs) were identified (cutoff set to 1.5-fold change), including 89 upregulated and 90 downregulated proteins. Bioinformatics analysis showed that DEPs can be divided into the cellular component, molecular function, and biological process; they were mainly involved in signal transduction, infectious diseases, cell growth and death, and the immune system. The subcellular localization of most proteins was in the cytoplasm. Importantly, the expressions of signal transducer and activator of transcription 1 (STAT1) and various interferon-stimulated genes (ISGs) were upregulated after NDRV infection. The mRNA transcripts of some ISGs were consistent with proteomic data, showing an increased trend. Results of our study suggested that NDRV infection can elicit strong expression changes of cellular proteins and activate the expression of ISGs from the point of quantitative proteomic analysis. The study provides a new insight into the understanding of NDRV pathogenesis.

Porcine circovirus 3 in cattle in Shandong province of China: A retrospective study from 2011 to 2018.

Porcine circovirus type 3 (PCV3) is a new member of Circovirus, which could cause various symptoms in infected pigs. It has been reported in many countries and detected from various animals. This study retrospectively analyzed serum samples that were randomly collected from 1,499 clinically healthy cattle in Shandong province from 2011 to 2018. The PCV3 DNA was detected in 28.95% (434/1,499) of samples. Twenty-seven cap genes of PCV3 were sequenced and compared with seventy reference sequences. They were in several different branches, but all belonged to PCV3b. The results indicated that PCV3 was prevalent in health cattle in Shandong province of China. Though infected cattle did not show any clinical symptoms, they could be a reservoir for the virus and probably transferred them back to pigs.

The tail domain of PRRSV NSP2 plays a key role in aggrephagy by interacting with 14-3-3ε.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most severe swine diseases that affects almost all swine-breeding countries. Nonstructural protein 2 (NSP2) is one of the most important viral proteins in the PRRSV life cycle. Our previous study showed that PRRSV NSP2 could induce the formation of aggresomes. In this study we explored the effects of aggresome formation on cells and found that NSP2 could induce autophagy, which depended on aggresome formation to activate aggrephagy. The transmembrane and tail domains of NSP2 contributed to aggrephagy and the cellular protein 14-3-3ε played an important role in NSP2-induced autophagy by binding the tail domain of NSP2. These findings provide information on the function of the C-terminal domain of NSP2, which will help uncover the function of NSP2 during PRRSV infection.

Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP3 Regulates Claudin 4 To Facilitate the Early Stages of Infection.

Claudins (CLDN) are a family of proteins that represent the most important components of tight junctions, where they establish the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Several types of viruses make full use of CLDN to facilitate entry into cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry. In this study, we found that CLDN4 functions as an anti-PRRSV factor by blocking its absorption during the early stages of infection. The small extracellular loop (ECL2) of CLDN4 restricted the viral particles outside cells by binding to GP3. A novel function of GP3-mediated regulation of CLDN4 transcription was suggested. CLDN4 can be decreased through downregulating the level of CLDN4 transcription by ubiquitinating the transcription factor, SP1. The mechanism by which highly pathogenic PRRSV infects the epithelium was proposed. Importantly, ECL2 was found to block PRRSV absorption and infection and neutralize the virus. A more in-depth understanding of PRRSV infection is described, and novel therapeutic antiviral strategies are discussed.IMPORTANCE In the present study, the role of CLDN4 in PRRSV infection was studied. The results showed that CLDN4 blocked absorption into cells and restricted extracellular viral particles via the interaction between the CLDN4 small extracellular loop, ECL2, and the viral surface protein GP3. GP3 was found to downregulate CLDN4 through ubiquitination of the transcription factor SP1 to facilitate viral entry. The mechanism by which highly pathogenic PRRSV infects the epithelium is suggested. A novel function of GP3 in regulating gene transcription was discovered. Moreover, ECL2 could block PRRSV absorption and infection, as well as neutralizing the virus in the supernatant, which may lead to the development of novel therapeutic antiviral strategies.