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Diarrhea outbreaks in piglets on pig farms are commonly attributed to porcine epidemic diarrhea virus (PEDV) infection. This research analyzed the S gene prevalence variation and recombination patterns in PEDV GII strains. Throughout the previous two years, 172 clinical samples of piglet diarrhea have been collected, from which 24 PEDV isolates have been isolated. Analysis of the evolutionary relationships among all 24 S genes revealed that 21 were most closely related to strains within the GII-a subgroup. The 2 isolates grouped into one clade with the GII-b subgroup. According to the mutation analysis of the amino acids (aa) that encode the S protein, 43 of the common aa in strains of the GII subtype were found to have undergone a change in polarity or charge, and 36 of these aa had a mutation frequency of more than 90%. Three different aa mutation sites were identified as exclusive to GII-a subtype strains. The genomes of three PEDV isolates were sequenced, and the resulting range in genome length was 28,035−28,041 nt. The results of recombination analysis showed that the SD1 isolate is a novel strain recombinant from the foreign S-INDEL strain and a domestic GII subtype strain. Based on the findings, the PEDV GII-a strain has been the most circulating strain in several parts of China during the previous two years. Our study reveals for the first time the unique change of aa mutations in the S protein of the GII-a subtype strain and the new characteristics of the recombination of foreign strains and domestic GII subtype strains, indicating that it is crucial to monitor the epidemic dynamics of PEDV promptly to prevent and control the occurrence of PED effectively. [ABSTRACT FROM AUTHOR]

Since 2018, the outbreak and prevalence of the African swine fever virus (ASFV) in China have caused huge economic losses. Less virulent ASFVs emerged in 2020, which led to difficulties and challenges for early diagnosis and control of African swine fever (ASF) in China. An effective method of monitoring ASFV antibodies and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, ASFV p17 was successfully expressed in CHO cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on purified p17 was established and optimized. The monoclonal antibody (mAb) against p17 recognized a conservative linear epitope (3TETSPLLSH11) and exhibited specific reactivity, which was conducive to the identification of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing p17. The ELISA method efficiently detected clinical ASFV infection and effectively monitored the antibody levels in vivo after recombinant PRRSV live vector virus expressing p17 vaccination. Overall, the determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of the ASFV antigen protein. The indirect ELISA method and mAb against ASFV p17 revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) is an important viral disease, causing significant economic losses to the swine industry worldwide. Atypical cases caused by highly pathogenic PRRS virus (HP-PRRSV) emerged in 2006 in China. The vaccine strain HuN4-F112 has been developed from the wild-type HP-PRRSV HuN4 through repeated passages on MARC-145 cells. However, the mechanisms of attenuation have yet to be defined. Previous studies have shown that the vaccine strain HuN4-F112 could not effectively replicate in porcine alveolar macrophages (PAMs). In the present study, a series of chimeric and mutant PRRSVs were constructed to investigate regions associated with the virus attenuation. Firstly, the corresponding genome regions (ORF1a, ORF1b and ORFs 2-7) were exchanged between two infectious clones of HuN4 and HuN4-F112, and then the influence of small regions in ORF1a and ORF2-7 was evaluated, then influence of specific amino acids on NSP2 was tested. NSP2 was determined to be the key gene that regulated infection efficiency on PAMs, and amino acids at 893 and 979 of NSP2 were the key amino acids. The results of in vivo study indicated that NSP2 was not only important for infection efficiency in vitro, but also influenced the virulence, which was indicated by the results of survival rate, temperature, viremia, lung score and tissue score. [ABSTRACT FROM AUTHOR]

• CVC1302, a complex of PRRs agonist, induced improved type I immunity. • CVC1302 in oil formulation elicited marked repositioning of cDC2s to the interfollicular region (IFR) of the lymph node. • CVC1302 in oil formulation led to increased number of CXCL10-producing monocytes (Mo) in the IFR and attract antigen-specific CD4+ T cells. Multifunctional CD4+ T helper 1 (Th1) cells, producing IFN-γ, TNF-α and IL-2, define a correlate of vaccine-mediated protection against intracellular infection. In our previous study, we found that CVC1302 in oil formulation promoted the differentiation of IFN-γ+/TNF-α+/IL-2+Th1 cells. In order to extend the application of CVC1302 in oil formulation, this study aimed to elucidate the mechanism of action in improving the Th1 immune response. Considering the signals required for the differentiation of CD4+ T cells to Th1 cells, we detected the distribution of innate immune cells and the model antigen OVA-FITC in lymph node (LN), as well as the quantity of cytokines produced by the innate immune cells. The results of these experiments show that, cDC2 and OVA-FITC localized to interfollicular region (IFR) of the draining lymph nodes, inflammatory monocytes localized to both IFR and T cell zone, which mainly infiltrate from the blood. In this inflammatory niche within LN, CD4+ T cells were attracted into IFR by CXCL10, secreted by inflammatory monocytes, then activated by cDC2, secreting IL-12. Above all, CVC1302 in oil formulation, on the one hand, targeted antigen and inflammatory monocytes into the LN IFR in order to attract CD4+ T cells, on the other hand, targeted cDC2 to produce IL-12 in order to promote optimal Th1 differentiation. The new finding will provide a blueprint for application of immunopotentiators in optimal formulations. [ABSTRACT FROM AUTHOR]

Abstract: A colloid gold strip (CGS) test for detecting antibodies to H5 subtype Avian Influenza Virus (AIV) was developed. The test is based on membrane chromatography and uses colloidal gold conjugated with inactivated AIV-H5N1 as the tracer. On the test strip, a baculovirus expressed haemagglutinin (HA) protein was used as the capture complex at the “test line”, and anti-HA monoclonal antibody was used as the capture antibody at the “control line”. The assembled test strip was housed in a plastic case. Compared with the hemagglutination inhibition test, the sensitivity and specificity of the CGS test were 88.8% and 97.6%, respectively, in detecting H5N1 antibodies in 830 serum samples from vaccinated chickens. Because it is rapid and does not require specialized equipment or technicians, the CGS test should be useful for detecting H5N1 antibodies in the field. [Copyright &y& Elsevier]

Porcine reproductive and respiratory syndrome (PRRS) is one of the major drivers of economic loss in the swine industry worldwide. In commercial pig production, vaccination is the first option in an attempt to control infectious diseases. Pigs are therefore often immunized with different vaccines, and almost all of them are delivered via the intramuscular (IM) route. However, the IM injection may result in physical damage, stress reactions, and is labor demanding. An alternative route is urgently needed to reduce the disadvantages of conventional vaccination. In this study, a needle-free intradermal (ID) delivery system was evaluated for delivering a live PRRS vaccine as compared with the traditional needle-syringe method. Fifty-two 4-week-old piglets were divided into six groups: piglets in groups A-C were immunized using ID delivery system with 104, 105 and 106 TCID 50 of PRRS candidate vaccine strain rHN-NP49, respectively; piglets in group D were immunized IM with 105 TCID 50 of rHN-NP49; and group E and F were used as challenge and control groups, respectively. At 28 days post vaccination, piglets in group A to E were challenged with a lethal dose of highly-pathogenic PRRSV. Similar results were found in viremia and antibody response among the ID and IM groups during the immunization stage. After challenge, similar results were found in average body weight gain, viral shedding, serum viral load, and clinical score among the immunization groups, with a higher protection ratio in the ID group compared with IM group with the same immunization dose. These results demonstrated that the ID delivery system could provide similar or even better protection compared with IM route, and could be an effective route for PRRS vaccination. [ABSTRACT FROM AUTHOR]

Tripartite motif protein 21 (TRIM21) is an E3 ubiquitin ligase and cytosolic antibody receptor of the TRIM family. Previous reports have indicated that TRIM21 plays an important role during viral infection. This study aimed at examining the role of TRIM21 in the replication of porcine epidemic diarrhea virus (PEDV) and showed that TRIM21 inhibits PEDV proliferation by targeting and degrading the nucleocapsid (N) protein through the proteasomal pathway. Furthermore, the endogenous expression of TRIM21 was found to be downregulated by PEDV infection in Vero and LLC-PK1 cells. Overexpression of TRIM21 inhibited PEDV replication, whereas knockdown of TRIM21 increased viral titers and N protein levels. TRIM21 was found to interact and colocalize with the N protein, and the TRIM21-mediated antiviral effect was dependent on its ubiquitin ligase activity, which engages in polyubiquitination and degradation of the N protein in a proteasome-dependent manner. Taken together, these findings provide information about the role of TRIM21 in PEDV proliferation and increase our understanding of host-virus interactions. [ABSTRACT FROM AUTHOR]

• rPRRSV-E2 immunized piglets developed high levels of antibodies against HP-PRRSV and CSFV. • Immunized pigs were well protected from the challenge of HP-PRRSV or CSFV at 20 weeks and 24 weeks post vaccination. • The immune duration of rPRRSV-E2 is at least 5 months against both HP-PRRSV and CSFV. Classical swine fever virus (CSFV) and Porcine reproductive and respiratory syndrome virus (PRRSV) are both important pathogens which seriously harm the economic swine industry worldwide. We have previously demonstrated that rPRRSV-E2 is a promising live, virus-vectored vaccine that provides 100% protection against highly pathogenic PRRSV (HP-PRRSV) and CSFV. Here, we evaluated the duration of immunity (DOI) of the vaccine strain, rPRRSV-E2. Vaccine or cell culture medium was administered to piglets at 4 weeks of age. All immunized piglets developed high levels of antibodies, which could maintain for up to 23 weeks, against PRRSV and CSFV. All immunized pigs were well protected from the challenge of HP-PRRSV or CSFV at 20 weeks and 24 weeks post vaccination. The vaccine protection rate was still 100% at 24 weeks after immunization. The immune efficacy results showed that the immune duration of rPRRSV-E2 could be up to 5 months. [ABSTRACT FROM AUTHOR]

Background: Porcine epidemic diarrhea virus (PEDV) infection causes an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets. During PEDV infection, the spike protein (S) is a major virion structural protein interacting with receptors and inducing neutralizing antibodies. However, the neutralizing B-cell epitopes within PEDV S protein have not been well studied. Methods: To accurately identify the important immunodominant region of S1, the purified truncated S1 proteins (SA, SB, SC, SD and SE) were used to immunize BALB/c mice to prepare polyclonal antibodies. The antisera titers were determined by indirect ELISA, western blot and IFA after four immunizations to find the important immunodominant region of S1, and then purified the immunodominant region of S1 protein and immunized mice to generate the special antibodies, and then used recombinant peptides to determine the B-cell epitopes of monoclonal antibodies. Results: Five antisera of recombinant proteins of the spike protein region of PEDV were generated and we found that only the polyclonal antibody against part of the S1 region (signed as SE protein, residues 666–789) could recognize the native PEDV. Purified SE protein was used to immunize BALB/c mice and generate mAb 2E10. Pepscan of the SE protein demonstrated that SE16 (722SSTFNSTREL731) is the minimal linear epitope required for reactivity with the mAb 2E10. Further investigation indicated that the epitope SE16 was localized on the surface of PEDV S protein in the 3D structure. Conclusions: A mAb 2E10 that is specifically bound to PEDV was generated and identified a specific linear B-cell epitope (SE16, 722SSTFNSTREL731) of the mAb. The epitope region of PEDV S1 localized in the different regions in comparison with the earlier identified epitopes. These findings enhance the understanding of the PEDV spike protein structure for vaccine design and provide a potential use for developing diagnostic methods to detect PEDV. [ABSTRACT FROM AUTHOR]

• Gal-1 overexpression strongly inhibits PRRSV replication in multiple strains. • Gal-1 knockdown or knockout facilitates PRRSV replication. • Interaction between Gal-1 and Nsp11 might regulate IFN and ISG activation. • Gal-1 exhibits antiviral activity against EAV, PEDV, JEV, PRV, and CSFV. • Gal-1 is potentially efficacious for applications to control PRRSV infection. Porcine reproductive and respiratory syndrome virus (PRRSV) causes great economic losses to the swine industry globally; however, effective control measures for this virus are limited. Here, we screened a porcine alveolar macrophage (PAM) cDNA library with a yeast two-hybrid system to reveal that galectin-1 (Gal-1), an endogenous innate immune protein encoded by LGALS1 , interacts with nonstructural protein 11 (Nsp11) of PRRSV. Western blotting and viral titer assays indicated that Gal-1 overexpression suppressed replication in multiple PRRSV strains (P < 0.001), whereas Gal-1 knockdown or knockout increased viral titer and nucleocapsid protein expression. The Gal-1-specific anti-PRRSV effect was associated with the endoribonuclease domain of Nsp11 through inactivation of interferon-antagonist function and stimulation of interferon-stimulated gene expression. Additionally, Gal-1 interacted with PRRSV E protein but not with PRRSV glycoproteins, and recombinant Gal-1 treatment inhibited PRRSV in PAMs and MARC-145 cells. Furthermore, Gal-1 inhibited replication in multiple viruses, including equine arteritis virus, porcine epidemic diarrhea virus, pseudorabies virus, Japanese encephalitis virus, and classical swine fever virus, suggesting its potential broad application for antiviral strategies. Our findings provide insight into the important role of Gal-1 in PRRSV pathogenesis and its potential use as a novel therapeutic target against PRRSV infection. [ABSTRACT FROM AUTHOR]

• ASFV p17 was successfully expressed in CHO cells using a suspension culture system. • 1B4 recognized a conservative linear epitope (8LLSHNLSTREGIK20) among genotype i and genotype II ASFV strains. • 1B4 effectively recognized the ectopically expressed p17 and foreign gene expression in recombinant PRRSV expressing ASFV p17. • 1B4 can be a useful tool to evaluate the expression of p17 in the recombinant virus, making it meaningful for future vaccine research on ASF. As a highly pathogenic large DNA virus, African swine fever virus (ASFV) has huge particles and numerous encoded proteins. At present, few of the existing studies on ASFV proteins have investigated the function of p17. Specific antibodies against p17 to promote the development of prevention techniques against African swine fever (ASF) are urgently needed. Herein, we successfully expressed ASFV p17 in CHO cells using a suspension culture system and generated a monoclonal antibody (mAb) against p17. The mAb recognized a novel linear epitope (8LLSHNLSTREGIK20) and exhibited specific reactivity, which was conducive to the identification of ectopically expressed p17, the recombinant porcine reproductive and respiratory syndrome virus expressing p17, and the ASFV-SY18. The epitope was conservative among genotype I and genotype II ASFV strains. Overall, the mAb against p17 revealed efficient detection and promising application prospects, making it a useful tool for future vaccine research on ASF. Determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of ASFV antigen protein. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea (PED) is an acute, highly infectious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which seriously endangers the healthy development of the pig industry. PEDV N protein is the most abundant viral structural protein, which can be combined with viral genomic RNA to form ribonucleoprotein complexes, thereby participating in the transcription and replication of the virus. However, how PEDV hijacks the host transcription translation system to promote viral proliferation remains unclear. In this study, we found that there is an interaction between PEDV N, polyadenylate-binding protein cytoplasmic 1 (PABPC1) and eukaryotic initiation factor 4F (eIF4F) proteins through coimmunoprecipitation, GST pulldown and fluorescence microscopy experiments. PABPC1 could bind to the poly(A) tail of the mRNA, and eIF4F could bind to the 5' end cap structure of the mRNA, so the interaction of PABPC1 and eIF4F could facilitate mRNA forming a circular shape to promote translation to the proteins. To further explore the effect of N protein capture protein translation element PABPC1 and eIF4F on PEDV replication, we overexpressed PABPC1, eIF4F (containing eIF4A, eIF4E and eIF4G) separately on Vero cells and LLC-PK1 cells, and we found that the PABPC1 and eIF4F protein could promote PEDV replication. Taken together, our data suggested that PEDV N protein promoted cyclization of viral mRNA carried by N protein through binding with PABPC1 and eIF4F proteins, thus promoting viral transcription and facilitating viral replication. • PABPC1 and eIF4F interacted with the PEDV N protein. • PABPC1 or eIF4F overexpression promoted PEDV replication. • PABPC1 or eIF4F knockdown inhibited PEDV replication in both Vero cells and LLC-PK1 cells. [ABSTRACT FROM AUTHOR]

• Bone marrow stromal cell antigen 2 (BST2) can inhibit viral replication. • BST2 exhibits significant anti-PRRSV activity. • BST2 expression is up-regulated during the early phase of infection. • BST2 restricts the expression of viral proteins. Porcine reproductive and respiratory syndrome virus (PRRSV) has seriously affected the viability of swine industries worldwide, and effective measures to control PRRSV are urgently required. Understanding the mechanisms of action of antiviral proteins is crucial for developing antiviral strategies. Interferon-induced bone marrow stromal cell antigen 2 (BST2) can inhibit the replication of various viruses via different pathways. However, little is known about the effects of BST2 on PRRSV. Therefore, this study aimed to evaluate whether the interferon-induced BST2 can inhibit PRRSV replication. We used western blotting and RT-qPCR techniques to analyze the effect of BST2 overexpression and knockdown on PRRSV replication. Overexpression of BST2 inhibited the replication of PRRSV, whereas knockdown of BST2 by small interfering RNA promoted PRRSV replication. Additionally, the expression of BST2 was upregulated during the early phase of PRRSV infection in porcine alveolar macrophages. Analysis of PRRSV proteins showed that BST2 restricted the expression of several non-structural viral proteins. BST2 downregulated the expression of Nsp12 through a proteasome-dependent pathway and downregulated the expression and transcription of E protein. These findings demonstrate the potential of BST2 as a critical regulator of PRRSV replication. [ABSTRACT FROM AUTHOR]

Abstract In recent years, the outbreaks of porcine epidemic diarrhea (PED) caused by the highly virulent porcine epidemic diarrhea virus (PEDV) variants occurred frequently in China, resulting in severe economic impacts to the pork industry. In this study, we selected and analyzed the genetic evolution of 15 PEDV representative strains that were identified in fecal samples of diarrheic piglets in 10 provinces and cities during 2011–2017. The phylogenetic analysis indicated that all the 15 PEDV isolates clustered into G2 genotype associated with the current circulating strains. Compared with the genome of the prototype strain CV777, these strains had 103–120 amino acid mutations in their S proteins, most of which were in the N terminal domain of S1 (S1-NTD). We also found 37 common mutations in all these 15 strains, although these strains shared 96.9–99.7% nucleotide homology and 96.3–99.8% amino acid homology in the S protein compared with the other original pandemic strains. Computational analysis showed that these mutations may lead to remarkable changes in the conformational structure and asparagine (N)-linked glycosylation sites of S1-NTD, which may be associated with the altered pathogenicity of these variant PEDV strains. We evaluated the pathogenicity of the PEDV strain FJzz1 in piglets through oral and intramuscular infection routes. Compared with oral infection, intramuscular infection could also cause typical clinical signs but with a slightly delayed onset, confirming that the variant PEDV isolate FJzz1 was highly pathogenic to suckling piglets. In conclusion, we analyzed the genetic variation and pathogenicity of the emerging PEDV isolates of China, indicating that G2 variant PEDV strains as the main prevalent strains that may mutate continually. This study shows the necessity of monitoring the molecular epidemiology and the etiological characteristics of the epidemic PEDV isolates, which may help better control the PED outbreaks. Highlights • G2 variant PEDV strains as the main prevalent strains continue to mutate along with time. • The isolated strains were prone to mutate in the S protein with many common amino acid mutations. • These mutations may lead to remarkable changes in the conformational structure of S1-NTD and the pathogenicity of PEDV. • Both oral and intramuscular routes caused typical clinical signs to piglets. [ABSTRACT FROM AUTHOR]

Different strains of porcine reproductive and respiratory syndrome virus (PRRSV) have emerged and circulated in different regions of mainland China since 1996, particularly after 2006. In 2012, NADC30-like PRRSV was first isolated in Henan Province. By 2016, it had spread to most provinces in China. In the present study, the whole genomes (excluding the poly(A) tails) of 13 newly emerged NADC30-like PRRSV strains were sequenced and analyzed. Furthermore, the pathogenicity of SD53-1603, one of the 13 PRRSV strains, was assessed. Phylogenetic analysis showed that these 13 newly emerged NADC30-like PRRSV strains, together with some reference strains, formed a new subgroup (subgroup 5), characterized by a predicted 131-amino-acid deletion in the nonstructural protein (NSP) 2. However, low levels of whole-genome similarity and a wide variety of recombination patterns complicated the classification of the NADC30-like PRRSV isolates. Interestingly, almost all of the recombination breakpoints found in these 13 PRRSV isolates and other NADC30-like PRRSV isolates occurred in genes encoding NSPs and/or minor structural proteins. In addition, piglets infected with the newly emerged NADC30-like strain SD53-1603 displayed clear clinical respiratory symptoms and underwent typical pathological changes. The findings may be useful for elucidating the characteristics and epidemic status of NADC30-like PRRSV in China. [ABSTRACT FROM AUTHOR]

Since late 2011, a pseudorabies virus (PRV) variant with increased virulence in old pigs had caused major disease outbreaks and great economic losses to the pig industry in China. The gene mutations that contributed to the increased virulence were unclear. To study the basis of the enhanced pathogenicity, an infectious bacterial artificial chromosome (BAC) clone consisting of the complete genome of the PRV variant was developed. Using homologous recombination and Cre/LoxP recombination, the recombinant virus rJS-2012-BAC carrying a BAC insertion downstream of the open reading frame (ORF) of gG was constructed. The circular genome of rJS-2012-BAC was extracted from infected Vero cells and transformed into Escherichia coli DH10B, generating the BAC clone pBAC-JS2012. The loxP sites flanking the BAC vector were used to excise the BAC sequences using Cre recombinase. The reconstituted BAC-excision virus, vJS2012 L, from pBAC-JS2012 exhibited similar biological properties to the wild-type virulent strain JS-2012. To manipulate the BAC clone pBAC-JS2012, the galK selection system was adopted to delete the gI/gE gene from pBAC-JS2012 in E. coli and to generate the gI/gE-deleted virus vJS2012-ΔgE/gI. The BAC clone, pBAC-JS2012, retained the same level of virulence as its parent strain and was easily manipulated using a galK system which would facilitate the study of the enhanced pathogenicity of the PRV variant as well as other studies on PRV. [ABSTRACT FROM AUTHOR]

Porcine galectin-3 (GAL3) is a 29-kDa protein encoded by a single gene, LGALS3 , located on chromosome 1. Here, using a yeast two-hybrid screen of a cDNA library from porcine alveolar macrophage cells (PAMs), we report for the first time that GAL3 interacts with nonstructural protein 12 (Nsp12) of the porcine reproductive and respiratory syndrome virus (PRRSV). Although extensive research has focused on porcine reproductive and respiratory syndrome (PRRS), little is known about the pathogen and host interactions involving individual nonstructural viral proteins, especially Nsp12. Here, we showed that GAL3 interacted with viral Nsp12 following co-transfection of HEK293 cells with GAL3- and Nsp12-expressing plasmids. Additionally, we observed that PPRSV infection led to reduced GAL3 levels during the late phase of infection in both MARC-145 cells and PAMs. Importantly, GAL3 overexpression significantly suppressed the replication of both type 1 and 2 PRRSV strains, whereas knockout of endogenous LGALS3 in MARC-145 cells significantly increased viral titer and expression of the nucleocapsid protein. These results strongly support a direct inhibitory effect of GAL3 on PRRSV replication, which might contribute to an overall antiviral effect. Furthermore, our findings provide insights into the molecular basis of the role Nsp12 plays in PRRSV pathogenesis. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) contribute to gene regulation at the post-transcriptional level and are capable of mRNA silencing by binding to target sites exhibiting high degrees of complementarity. Therefore, cloning host miRNA-recognition sequences into the genome of RNA viruses represents a rational strategy for manipulating viral replication. Here, we performed deep sequencing to obtain small-RNA (sRNA)-expression profiles from in vitro-cultured MARC-145 cells post infection with porcine reproductive and respiratory syndrome virus (PRRSV) and chose six candidate miRNAs of different abundance (miR-21, miR-140-3p, miR-185, miR-26a, miR-505, and miR-199a) for further study. Based on the full-length cDNA clone p7USC, we constructed a number of PRRSV mutants that provided complementary base-pairing target sites for the miRNAs in 3′ untranslated regions. Our results showed that all low- and moderate- abundant miRNA-target mutants showed similar growth properties, whereas the highest-abundant miRNA-target mutant blocked both viral transcription and replication. Discontinuous mutations in high-abundant miRNA-target sites subsequently recovered viral viability and propagation. These results demonstrated the copy number of endogenous miRNAs and the extent of sRNA complementarity were key factors to silence potential mRNA expression/translation, thereby determining PRRSV viability. Interestingly, the mutant containing miR-140-target sites (v140-t) showed strong suppression of viral replication from P1 to P3 in vitro, as shown by virus titer, plaque morphology, and qRT-PCR assays. To assess genetic stability, sequencing of v140-t (P1, P3, P5 and P10) revealed spontaneous mutations preferentially located among several nucleotides near the 3′ end of the insertion region and corresponding to the “seed region” of miR-140-3p, explaining the induced viral repression and the direction of virus evolution. This approach provided a general silencing strategy for limiting PRRSV replication by endogenous miRNAs in MARC-145 cells. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) and classical swine fever (CSF) are economically significant diseases that affect the swine industry worldwide. However, the current vaccination strategy, which uses two single live attenuated vaccines, can result in interference for each other. In addition, the universally used CSFV vaccine C-strain does not allow for differentiation of infected and vaccinated animals. In this study, rPRRSV-E2, PRRS virus (PRRSV) expressing CSF virus (CSFV) E2, was constructed by reverse genetics. The E2 gene of CSFV was inserted between ORF1b and ORF2 in the genome of the PRRS vaccine virus, HuN4-F112. A copy of transcriptional regulatory sequence 6 was inserted at the 3′ terminal of the exogenous gene to produce CSFV E2 as a unique subgenomic mRNA transcript. The rPRRSV-E2 was stable for at least 25 serial cell passages. Single-shot intramuscular immunization of rPRRSV-E2 into pigs induced PRRSV-specific and CSFV-specific antibodies and fully protected pigs from lethal challenge with highly-pathogenic PRRSV and CSFV. These results demonstrate that a novel strategy for recombinant PRRSV production is effective, and suggest that rPRRSV-E2 is a promising live, virus-vectored vaccine against PRRS and a marker vaccine against CSF. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens that has caused tremendous economic losses to the swine industry worldwide. Although extensive research has been focused on PRRSV, little is known about the structure and biological functions of individual nonstructural viral proteins, especially the nonstructural protein 12 (Nsp12). In this study, we generated and identified the monoclonal antibody (mAb) against PRRSV Nsp12. Six strains of hybridoma cells named 2B10, 2B12, 5E1, 5G6, 5E7, and 8B2 secreting anti-Nsp12 mAbs were obtained by the hybridoma technique. All the mAbs were specifically reacted with PRRSV by indirect immunofluorescence assay and four of them (2B12, 5E1, 5G6, and 5E7) were specifically reacted by Western blot. Furthermore, the 5E7 specifically recognized multiple type 2 PRRSV strains, including highly pathogenic and classical PRRSV strains, but not type 1 PRRSV strain. Taken together, the mAbs against Nsp12 provide a valuable tool to specifically recognize type 2 PRRSV as a diagnostic reagent and study the biological function of Nsp12 in the future. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) exploits host metabolic pathways to support its replication. To improve the understanding of lipid metabolic changes that could occur upon IAV infection, a comprehensive analysis of lipid metabolites in A549 cells infected with the avian H9N2 virus at the different time points was performed. It was found that H9N2 infection could largely promote the level of lipid metabolites. Further, these metabolites were mainly included in glycerophospholipids (GPs), sphingolipids (SPs), glycerolipids (GLs), fatty acids (FAs), sterollipids (STs), triglycerides (TGs), and prenol lipids (PRs). Specifically, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these metabolites were mainly associated with the glycerphospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and autophagy. Furthermore, it is interesting to note that these metabolites, including FFA(19:1), PE(P-17:0_20:3), PE(P-18:1_20:2), LPC(14:0/0:0), PE(O-18:0_20:3), and MGDG(16:0_18:1), are upregulated and shared in the top 10 at 12 h, 24 h, 36 h, and 48 h after H9N2 infection, indicative of the possibility of acting as biomarkers for the diagnosis in the lung infected with influenza virus. These pathways and altered metabolites could provide new understandings about biological characteristics and pathogenicity of influenza virus and have the potential to serve as biomarkers for influenza. • A comprehensive analysis of lipid metabolism in response to H9N2 infection is analyzed. • H9N2 infection largely leads to the upregulation of lipid metabolites. • FFA(19:1), PE(P-17:0_20:3), PE(P-18:1_20:2), LPC(14:0/0:0) and so on are always upregulated throughout the process. • KEGG pathways show that glycerphospholipid metabolism, GPI-anchor biosynthesis, and autophagy are the main pathways. [ABSTRACT FROM AUTHOR]

Although several studies have investigated the functions of influenza PA-X, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on virus replication, pathogenicity and host response remains unclear. Herein, we generated two mutated viruses expressing a full-length or deficient PA-X protein based on the A/California/04/2009 (H1N1) virus that expresses a truncated PA-X to understand three different expressions of PA-X protein on virus replication, pathogenicity and host immune responses. The results showed that expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X form. Furthermore, the full-length PA-X expression exhibited a greater effect on virus pathogenicity than the truncated PA-X form. Our results provide novel insights of PA-X on viral replication, pathogenicity and host immune responses. [ABSTRACT FROM AUTHOR]

Canine kobuvirus (CaKVs) was a newly described virus detected in dogs in the US and Italy. To learn more about CaKVs, 5 of 106 fecal samples from diarrhea dogs were positive with CaKVs in China, and the full genome of CaKVs strain CH-1 isolated from dog with diarrhea was sequenced. The genome consists of 8186 nucleotides, excluding the 3′ poly (A) tail, and an open reading frame that maps between nucleotide positions 601 and 7943 which encodes a 2446 amino acid polyprotein. Based on the complete amino acid sequence of polyprotein, phylogenetic analysis showed that CH-1 was grouped along with other canine kobuvirus strains detected in the USA (US-PC0082, AN211D). [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) has recently caused high mortality in suckling piglets with subsequent large economic losses to the swine industry. Many intracellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, are activated by viral infection. The PI3K/Akt pathway is an important cellular pathway that has been shown to be required for virus replication. In the present study, we found that the PEDV JS-2013 strain activated Akt in Vero cells at early (5–15 min) and late stages (8–10 h) of infection. Inhibiting PI3K, an upstream activator of Akt, enhanced PEDV replication. Inhibiting GSK-3α/β, one of the downstream effectors of PI3K/Akt pathway and regulated by Akt during PEDV infected Vero cells, also enhanced PEDV replication. Collectively, our data suggest that PI3K/Akt/GSK-3α/β signaling pathway is activated by PEDV and functions in inhibiting PEDV replication. [ABSTRACT FROM AUTHOR]

Giardia duodenalis is an important intestinal protozoan parasite, infections of which are frequently seen in cattle and cause intermittent diarrhea and weight loss in young animals around the world. The release of neutrophil extracellular traps (NETs) is an effector mechanism of neutrophils to fight against invading pathogens including parasites. In this present study, we aimed to investigate the effect of Giardia duodenalis trophozoites on bovine NETs formation, and to further examine its basic characteristics and molecular mechanisms. Scanning electron microscopy analyses displayed that Giardia duodenalis trophozoites exposure triggered NET-like filamentary structures released by bovine polymorphonuclear leukocytes (PMNs), and many trophozoites were entrapped within these structures. Immunofluorescence analyses illustrated that these structures were mainly composed of DNA, histones, Myeloperoxidase (MPO) and neutrophil elastase (NE), which confirmed the classical characteristics of NETs. NETs quantification showed that Giardia duodenalis trophozoites significantly increased NETs formation, and it is in a dose-dependent manner from 4:1–1:2 ratio of PMN: trophozoites. Furthermore, pharmacological inhibitory experiment indicated that P2X1 receptor and PAD4 were essential for Giardia duodenalis trophozoites-triggered NETs formation. Additionally, LC3B-based immunostaining analyses revealed that autophagy and NETs formation occurred simultaneously in Giardia duodenalis trophozoites-exposed bovine PMN, imply that autophagy may play a key role in Giardia duodenalis trophozoites-triggered bovine NETs. In summary, these findings suggest that NETs formation might have a crucial role in innate host defense against Giardia duodenalis trophozoites. Hence, we call for future molecular investigations not only on Giardia duodenalis trophozoites-triggered NETs but also on its potential role in giardiasis-related pathology in vivo. • Giardia duodenalis triggered bovine neutrophil extracellular trap (NET) formation. • Giardia duodenalis increased bovine NET formation dose-dependently. • Giardia duodenalis -triggered NET formation was dependent on P2X1 receptor and PAD4 enzyme. • Autophagy and NET formation occurred simultaneously in Giardia duodenalis -exposed bovine PMN. [ABSTRACT FROM AUTHOR]

KLF16, a member of KLFs (Krüppel-like factors), contributes to the progression of a variety of cancer types. There is, however, still uncertain regarding the role of KLF16 in viral replication and the signaling mechanism of type I IFN. It was discovered that KLF16 inhibited the replication of porcine epidemic diarrhea virus (PEDV) through the type I IFN signaling pathway. Besides, it can also be found that the expression of KLF16 was down-regulated after PEDV infection of LLC-PK1 cells. Furthermore, overexpression of KLF16 inhibited the replication of PEDV in Vero cells as well as LLC-PK1 cells, whereas the replication of PEDV was promoted by the knockdown of KLF16. KLF16 up-regulated the expression of interferons (IFNs) via the TRAF6-pTBK1-pIRF3 pathway with the aim of promoting the host antiviral innate immune response. In addition, the obtained findings proved that KLF16 plays a novel role in antiviral action, thereby offering novel possibilities for preventing and controlling PEDV. • KLF16 was downregulated in porcine epidemic diarrhea virus (PEDV)-infected LLC-PK1 cells. • KLF16 overexpression inhibited PEDV proliferation in both Vero cells and LLC-PK1 cells. • KLF16 silencing promoted PEDV replication. • KLF16 inhibited PEDV replication by activating type-I IFN signaling via the TRAF6-pTBK1-pIRF3 pathway. [ABSTRACT FROM AUTHOR]

• A distinct cellular proteomic response to H9N2 infection is analyzed through a 4D label free proteomic method. • Endogenous expression of ZCCHC3 is induced by H9N2 infection. • ZCCHC3 exerts antiviral effect on H9N2 infection. • ZCCHC3 promotes IFN-β signaling and downstream gene transcrption. • Viral NS1 protein counteracts the effect of ZCCHC3. Influenza a virus exploits host machinery to benefit its replication in host cells. Knowledge of host factors reveals the complicated interaction and provides potential targets for antiviral treatment. Here, instead of the traditional proteomic analysis, we employed a 4D label free proteomic method to identify cellular factors in A549 cells treated with avian H9N2 virus. We observed that 425 proteins were upregulated and 502 proteins were downregulated. Western blotting and quantitative real-time PCR results showed that the zinc-finger CCHC-type containing protein 3 (ZCCHC3) levels were markedly induced by H9N2 infection. Transient expression assay showed that ZCCHC3 expression decreased NP protein levels and viral titers, whereas knockdown of ZCCHC3 enhanced viral growth. Specifically, ZCCHC3 promoted the expression of IFN-β, leading to the increased transcription of IFN downstream antiviral factors. Surprisingly, viral NS1 protein was able to antagonize the antiviral effect of ZCCHC3 by downregulating IFN-β. Eventually, we observed that chicken finger CCCH-type containing protein 3, named ZC3H3, could also suppress the replication of H9N2 virus and the coronavirus-infectious bronchitis virus (IBV) in DF-1 cells. Together, our results showed the cellular proteomic response to H9N2 infection and identified ZCCHC3 as a novel antiviral factor against H9N2 infection, contributing to the understanding of host-virus interaction. [ABSTRACT FROM AUTHOR]

Autophagy-related 4B (ATG4B) is found to exert a vital function in viral replication, although the mechanism through which ATG4B activates type-I IFN signaling to hinder viral replication remains to be explained, so far. The current work revealed that ATG4B was downregulated in porcine epidemic diarrhea virus (PEDV)-infected LLC-PK1 cells. In addition, ATG4B overexpression inhibited PEDV replication in both Vero cells and LLC-PK1 cells. On the contrary, ATG4B knockdown facilitated PEDV replication. Moreover, ATG4B was observed to hinder PEDV replication by activating type-I IFN signaling. Further detailed analysis revealed that the ATG4B protein targeted and upregulated the TRAF3 protein to induce IFN expression via the TRAF3-pTBK1-pIRF3 pathway. The above data revealed a novel mechanism underlying the ATG4B-mediated viral restriction, thereby providing novel possibilities for preventing and controlling PEDV. • ATG4B was downregulated in porcine epidemic diarrhea virus (PEDV)-infected LLC-PK1 cells. • ATG4B overexpression inhibited PEDV proliferation in both Vero cells and LLC-PK1 cells. • ATG4B knockdown promoted PEDV replication. • ATG4B hindered PEDV replication by activating type-I IFN signaling via the TRAF3-pTBK1-pIRF3 pathway. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) can impact viral infections by binding to sequences with partial complementarity on viral RNA transcripts, usually resulting in the repression of virus replication. In the present study, we identified a potential binding site for miR-130 in the 5′ untranslated region (bps 155-162) of the porcine reproductive and respiratory syndrome virus (PRRSV) genome. We found that the delivery of multiple miR-130 family mimics, especially miR-130b, resulted in inhibition of PRRSV replication in vitro. miR-130 was effective in inhibiting the replication of multiple type 2 PRRSV strains, but not against vSHE, a classical type 1 strain. miR-130 over-expression did not induce IFN-α or TNF-α expression in either uninfected or PRRSV-infected porcine alveolar macrophages. Results from luciferase reporter assays indicated that miR-130 directly targeted the PRRSV 5′ UTR. Intranasal inoculation of piglets with miR-130b exhibited antiviral activity in vivo and partially protected piglets from an otherwise lethal challenge with HP-PRRSV strain vJX143. Overall, these results demonstrate the importance of the miR-130 family in modulating PRRSV replication and also provide a scientific basis for using cellular miRNAs in anti-PRRSV therapies. [ABSTRACT FROM AUTHOR]

Emerging pseudorabies virus (PRV) variant have led to pseudorabies outbreaks in Chinese pig farms. The commercially available PRV vaccine provides poor protection against the PRV variant. In this study, a gE/gI deleted PRV strain JS-2012-△gE/gI was generated from a PRV variant strain using homologous DNA recombination. Compared to the parental strain JS-2012, JS-2012-△gE/gI grew slowly and showed small plaque morphology on Vero cells. The safety and immunological efficacy of JS-2012-△gE/gI was evaluated as a vaccine candidate. JS-2012-△gE/gI was avirulent to suckling piglets, but was able to provide full protection for young piglets against challenge with both the classical virulent PRV and the emerging PRV variant. After sows were vaccinated with the gE/gI-deleted strain, their suckling offspring were resistant to an otherwise lethal challenge with the classical and the variant PRVs. Piglets inoculated with JS-2012-△gE/gI did not develop PRV-specific gE-ELISA antibodies. Thus, JS-2012-△gE/gI appears to be a promising marker vaccine candidate to control PRV variant circulating in pig farms in China. [ABSTRACT FROM AUTHOR]

The lapinized attenuated vaccine against classical swine fever (CSF) has been used in China for over half a century and has generally prevented large-scale outbreaks in recent years. However, since late 2014, a large number of new cases of CSF were detected in many immunized pig farms in China. Several of these CSV viruses were isolated and characterized. Phylogenetic and genomic sequence analyses indicate that these new isolates, as well as some reference isolates, form a new subgenotype named 2.1d, and share several consistent molecular characteristics. Since these new isolates emerged in disparate geographic regions within 5 months, this suggests that these isolates may be widespread. Given that current vaccines do not appear to provide effective protection against this new subgenotype, further investigation of these strains is urgently needed. [ABSTRACT FROM AUTHOR]

• A full- length infectious clone of NADC30-like strain was constructed successfully. • The rescued virus, rZJqz, was indistinguishable from their parental virus vZJqz21 in virological characteristics. • rZJqz retained the similar pathogenicity and the typical clinical symptoms. • These findings represent a useful tool for the further development of a new broad-spectrum vaccine. NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV), which is highly homologous to the NADC30 strain isolated in the United States. The NADC30-like PRRSV was first reported in 2014 in China, where it spread and gradually caused an epidemic. Currently, growing research has shown that NADC30-like strains have greater propensity to recombine with other PRRSV strains, particularly the PPRSV vaccine virus used clinically, making the prevention and control of PRRSV highly complex. To carry out an in-depth molecular biology and virulence analysis, a full-length infectious clone of the NADC30-like strain was successfully constructed and rescued by reverse genetic manipulation. The rescued virus, rZJqz, was indistinguishable from its parental virus, ZJqz21, based on virological characteristics. Further animal experiments demonstrated that rZJqz retained similar pathogenicity and induced the typical clinical symptoms and viral shedding observed in the ZJqz21 challenge model. Together, these results provide a useful tool for further study of the biological characteristics and pathogenicity of NADC30-like strains. Moreover, these findings also provide a solid foundation for studying the recombination of different PRRSVs and developing new and effective universal vaccines in the future. [ABSTRACT FROM AUTHOR]

• ZCCHC3 overexpression inhibits PRV proliferation. • ZCCHC3 knockdown facilitates PRV proliferation. • ZCCHC3 positively regulates IFN-β expression. • PRV infection reduces endogenous ZCCHC3 expression. • PRV UL13 and UL24 proteins suppress the expression of ZCCHC3. Zinc finger CCHC-type containing protein 3 (ZCCHC3) acts as an antiviral factor that interacts with RIG-I and cGAS to modulate innate signaling against viral infections. Here, we investigated the role of porcine ZCCHC3 during pseudorabies virus (PRV) proliferation. We found that porcine ZCCHC3 plays an inhibitory role in the proliferation of PRV by regulating cellular innate immune responses. Further, overexpression of ZCCHC3 inhibited gB protein levels and viral titers, whereas knockdown of ZCCHC3 promoted viral growth. ZCCHC3 overexpression increased IFN-β expression to upregulate downstream gene expression, thus leading to the suppression of viral replication. However, PRV infection reduced the endogenous expression of ZCCHC3 in permissive cells. Importantly, PRV-encoded UL13 and UL24 proteins were identified to inhibit the expression of ZCCHC3, thus antagonizing its antiviral effect. Collectively, our data underscore the important role of ZCCHC3 against PRV infection and promote understandings of viral proteins in PRV pathogenesis. [ABSTRACT FROM AUTHOR]

ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for virus viability, but all serine mutant clones produced infectious progeny viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30. [ABSTRACT FROM AUTHOR]

The N gene of porcine epidemic diarrhea virus (PEDV) was amplified by RT-PCR using specific primers, and inserted into the expression vector pCold-I to construct a recombinant plasmid pCold-I-N. The recombinant plasmid was expressed in Escherichia coli BL21 (DE3) under IPTG induction. Then, female BALB/c mice were immunized with the purified recombinant N protein and one strain of hybridoma cells named 2B8 secreting anti-N protein monoclonal antibodies (MAb) was obtained by hybridoma technique. The MAb was specifically reacted with PEDV and identified by Western blot and indirect immunofluorescence assays. This work indicated that the MAb would be a valuable tool as a specific diagnostic reagent for PEDV epidemiological surveys and diagnosis in the future. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play important roles in viral infections, especially by modulating the expression of cellular factors essential to viral replication or the host innate immune response to infection. To identify host miRNAs important to controlling porcine reproductive and respiratory syndrome virus (PRRSV) infection, we screened 15 miRNAs that were previously implicated in innate immunity or antiviral functions. Over-expression of the miR-26 family strongly inhibited PRRSV replication in vitro, as shown by virus titer assays, Western blotting, and qRT-PCR assays. MiR-26a inhibited the replication of both type 1 and type 2 PRRSV strains. Mutating the seed region of miR-26 restored viral titers. Luciferase reporters showed that miR-26a does not target the PRRSV genome directly but instead affects the expression of type I interferon and the IFN-stimulated genes MX1 and ISG15 during PRRSV infection. These results demonstrate the important role of miR-26a in modulating PRRSV infection and also support the possibility of using host miR-26a to achieve RNAi-mediated antiviral therapeutic strategies. [ABSTRACT FROM AUTHOR]

The sterile alpha motif and HD domain 1 (SAMHD1) protein is a novel innate immunity restriction factor that inhibits HIV-1 infection in myeloid cells. Here, we cloned the full-length SAMHD1 complementary DNA (cDNA) from porcine peripheral blood lymphocytes. The porcine SAMHD1 cDNA was of 3951 bp with an open reading frame of 1884 bp, encoding a polypeptide of 627 amino acids. Porcine SAMHD1 mRNA was detected in all swine tissues examined, with the higher expression in the tonsil, lung, liver, and lymph node tissues. The SAMHD1 protein was localized to the nucleus. Overexpression of SAMHD1 blocked the proliferation of HuN4, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV), in MARC-145 cells, by inhibiting the synthesis of the HuN4 complement RNA. The antiviral effects of the simian SAMHD1 protein were nearly equivalent to those of porcine SAMHD1 in the HuN4-infected MARC-145 cells. Phosphorylation analysis of SAMHD1 showed that overexpressed SAMHD1 protein was in primarily an unphosphorylated state. SAMHD1 overexpression increased the transcript abundance of IFN-stimulated genes ISG15 and ISG56. The mRNA levels of SAMHD1 and ISGs were significantly increased in porcine alveolar macrophages infected with HP-PRRSV. SAMHD1 protein level was also elevated, and the protein was not phosphorylated during infection. Collectively, our data indicate that SAMHDI inhibits HP-PRRSV proliferation through inhibiting the replication of HP-PRRSV. SAMHD1 might be the protein participating in the IFN signaling and is thus an important immunoregulatory protein in innate immunity. [ABSTRACT FROM AUTHOR]

The factors that determine the transcription-regulating sequence (TRS) activity of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unclear. In this study, the effect of mutagenesis of conserved C nucleotides at positions 5 and 6 in the leader TRS (TRS-L) and/or canonical body TRS7 (TRS-B) on the synthesis of subgenomic (sg) mRNA and virus infectivity was investigated in the context of a type 2 PRRSV infectious cDNA clone. The results showed that a double C mutation in the leader TRS completely abolished sg mRNAs synthesis and virus infectivity, but a single C mutation did not. A single C or double C mutation in TRS-B or/and TRS-B impaired or abolished the corresponding sg mRNA synthesis. Introduction of identical mutations in the leader and body TRSs partially restored sg mRNA7.1 and/or sg mRNA7.2 transcription, indicating that the base-pairing interaction between sense TRS-L and cTRS-B is a crucial factor influencing sg mRNA synthesis. Analysis of the mRNA leader-body junctions of mutants provided evidence for a mechanism of discontinuous minus-strand transcription. This study also showed that mutational inactivation of TRS-B or TRS-B did not affect the production of infectious progeny virus, and the sg mRNA formed from each of them could express N protein. However, TRS-B plays more important roles than TRS-B in maintaining the growth characteristic of type 2 PRRSV. These results provide more insight into the molecular mechanism of genome expression and subgenomic mRNA transcription of PRRSV. [ABSTRACT FROM AUTHOR]

• PRV pUL16 localized to the mitochondria, nuclei and cytoplasm. • PRV pUL16 weakened the inhibition of LRPPRC for the growth of PRV. • LRPPRC influenced STING-mediated activation of NF-κB. Pseudorabies is caused by pseudorabies virus (PRV), a member of the Herpesvirus family, and has caused tremendous damage to the pig industry. Protein unique lone 16 (pUL16) is a conserved envelope protein in all herpesviruses, that is known to play an important role in several aspects, including virus diffusion in cells and virulence in mice. It has been shown that the pUL16 can interact with the virus proteins UL11, UL49, UL21, gD, and gE. However, the research to date on pUL16 has only focused on etiology, without discussing the possible cellular pathways involved in PRV infection. Leucine-rich PPR motif-containing protein (LRPPRC) is a multifunctional cellular protein that participates in various cellular processes, such as RNA processing, splicing, stabilization, editing, translation, and energy metabolism. This was the first caspase-independent apoptosis protein to be identified. In this study, immune precipitation and mass spectrometry was performed to define the function of the pUL16 in PRV infection to study the possible cellular pathways in which pUL16 may participate. It was found that LRPRRC could interact with PRV pUL16, which may indicate that UL16 is involved in a redox reaction or cellular apoptosis. This is the first study of the interaction between pUL16 and host proteins, which has positive significance to gain a further understanding of the pUL16. [ABSTRACT FROM AUTHOR]

Abstract: Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that is causing massive economic loss in the Chinese duck industry. To obtain a live vaccine candidate against the disease, the DTMUV isolate FX2010 was passaged serially in chicken embryo fibroblasts (CEFs). Characterization of FX2010-180P revealed that it was unable to replicate efficiently in chicken embryonated eggs, nor intranasally infect mice or shelducks at high doses of 5.5log10 tissue culture infectious doses (TCID50). FX2010-180P did not induce clinical symptoms, or pathological lesions in ducks at a dose of 5.5log10 TCID50. The attenuation of FX2010-180P was due to 19 amino acid changes and 15 synonymous mutations. Importantly, FX2010-180P elicited good immune responses in ducks inoculated at low doses (3.5log10 TCID50) and provided complete protection against challenge with a virulent strain. These results indicate that FX2010-180P is a promising candidate live vaccine for prevention of duck Tembusu viral disease. [Copyright &y& Elsevier]

The 3′ untranslated region (3′ UTR), including the poly (A) tail, reportedly plays an important role in arterivirus replication, but the roles of the cis-acting elements present in the 3′ UTR of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unknown. In the present study, PCR-based mutagenic analysis was conducted on the 3′ UTR of PRRSV infectious full-length cDNA clone pAPRRS to investigate the structure and function of the conserved terminal nucleotides between the poly (A) tail and the 3′ UTR region. Our findings indicated that the conservation of the primary sequence of the 3′ terminal nucleotides, rather than the surrounding secondary structure, was vital for viral replication and infectivity. Four nucleotides (nt) (5′-AAUU-3′) at the 3′ proximal end of the 3′ UTR and the dinucleotide 5′-AU-3′ exerted an important regulatory effect on viral viability. Of the five 3′-terminal nucleotides of the 3′ UTR (5′-AACCA-3′), at least three, including the last dinucleotide (5′-CA-3′), were essential for maintaining viral infectivity. Taken together, the 3′-terminal conserved sequence plays a critical role in PRRSV replication and may function as a contact site for specific assembly of the replication complex. [ABSTRACT FROM AUTHOR]

Abstract: Goose parvovirus (GPV), newcastle disease virus (NDV), goose herpesvirus (GHV) and goose adenovirus (GAV) are considered collectively to be four of the most important and widespread viruses of geese. Because all of these viruses cause similar pathological changes, histological differentiation among these viruses is difficult. A reliable, specific and sensitive multiplex PCR (mPCR) assay was developed for the combined detection of GPV, NDV, GHV and GAV in clinical samples of geese. Using the mPCR technique, single infections with GPV (28/76; 36.8%), NDV (9/76; 11.8%), GHV (3/76; 3.9%) and GAV (12/76; 15.8%) were identified in the samples; co-infections with GAV and either GPV or NDV (31.6%; 24/76) were also identified with this approach. The results for all of the samples tested were the same in both the uPCR and mPCR systems. The mPCR approach is considered to be useful for routine molecular diagnosis and epidemiological applications in geese. [Copyright &y& Elsevier]

Highly pathogenic porcine reproductive and respiratory syndrome virus PRRSV (HP-PRRSV) was one of the most devastating diseases of the pig industry, among various strategies, vaccination was one of the most useful tools for PRRS control. Attenuated live vaccine was used worldwide, however, the genetic basis of HP-PRRSV virulence change during attenuation remain to be determined. Here, to identify virulence associated regions of HP-PRRSV during attenuation in vitro, six full-length infectious cDNA clones with interchanges of 5′UTR + ORF1a, ORF1b, and ORF2-7 + 3′UTR regions between HP-PRRSV strain HuN4-F5 and its attenuated vaccine strain HuN4-F112 were generated, and chimeric viruses were rescued. Piglets were inoculated with chimeric viruses and their parental viruses, and rectal temperature were recorded daily, and serum were collected for future experiments. Our results showed that ORF1a played an important role on virus replication, cytokine response and lung damage, the exchange of ORF1b and ORF2-7 in different backbone led to different exhibition on virus replication in vivo/vitro and cytokine response. Among 9 PRRSV attenuated series, consistent amino acid changes during PRRSV attenuation were found in NSP4, NSP9, GP2, E, GP3 and GP4. Our study provides a fundamental data for the investigation of PRRSV attenuation, the different results of the virulence change among different studies indicated that different mechanisms might be used during PRRSV virulence enhancement in vivo and attenuation in vitro. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol-3-kinase (PI3K)/Akt is an important cellular pathway that has been shown to participate in various replication steps of multiple viruses. In the present study, we compared the phosphorylation status of Akt during infection of MARC-145 cells and porcine alveolar macrophages (PAMs) with highly pathogenic PRRSV (HP-PRRSV) strain HuN4. We observed that biphasic activation of Akt was induced in at both the early stage (5, 15 and 30 min postinfection) and the late stage (12 and 24 h postinfection) of HP-PRRSV infection of MARC-145 cells, while an early-phase activation of Akt was found exclusively in virus-infected PAMs in vitro. Analysis with the PI3K-specific inhibitor LY294002 confirmed that PI3K acted as the upstream activator for the virus-induced activation of Akt. UV-irradiation-inactivated virus still induced the early event in PAMs but not in MARC-145 cells, suggesting that different mechanisms are employed for the early-stage induction of phosphorylated Akt within different cell cultures. We further demonstrated that FoxO1 and Bad, which serve as downstream targets of Akt, were phosphorylated in virus-infected MARC-145 cells. Moreover, the suppression of phosphorylated Akt with LY294002 significantly inhibited the virus-induced cytopathic effect (CPE) on MARC-145 cells, but it had a negligible effect on virus propagation. Collectively, our data provide new evidence of a novel role for the PI3K/Akt pathway in PRRSV infection of MARC-145 cells. [ABSTRACT FROM AUTHOR]

Abstract: The 5′ untranslated region (UTR) is believed to be vital for the replication of porcine reproductive and respiratory syndrome virus (PRRSV), yet its functional mechanism remains largely unknown. In this study, to define the cis-acting elements for viral replication and infectivity, The 5′ UTR swapping chimeric clones pTLV8 and pSHSP5 were constructed based on two different genotypes full-length infectious cDNA clone pAPRRS and pSHE backbones. Between them, vTLV8 could be rescued from pTLV8 and had similar virological properties to vAPRRS, including phenotypic characteristic and RNA synthesis level. However, pSHSP5 exhibited no evidence of infectivity. Taken together, the results presented here demonstrate that only the 5′ UTR of type 1 PRRSV did not affect the infectivity and replication of type 2 PRRSV in vitro. The 5′ UTR of type 2 PRRSV could be functionally replaced by its counterpart from type 1. [Copyright &y& Elsevier]

SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China. [ABSTRACT FROM AUTHOR]