Search

Abstract Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine viral infectious diseases worldwide. Vaccination is a key strategy for the control and prevention of PRRS. At present, the NADC30-like PRRSV strain has become the predominant epidemic strain in China, superseding the HP-PRRSV strain. The existing commercial vaccines offer substantial protection against HP-PRRSV, but their efficacy against NADC30-like PRRSV is limited. The development of a novel vaccine that can provide valuable cross-protection against both NADC30-like PRRSV and HP-PRRSV is highly important. In this study, an infectious clone of a commercial MLV vaccine strain, GD (HP-PRRSV), was first generated (named rGD). A recombinant chimeric PRRSV strain, rGD-SX-5U2, was subsequently constructed by using rGD as a backbone and embedding several dominant immune genes, including the NSP2, ORF5, ORF6, and ORF7 genes, from an NADC30-like PRRSV isolate. In vitro experiments demonstrated that chimeric PRRSV rGD-SX-5U2 exhibited high tropism for MARC-145 cells, which is of paramount importance in the production of PRRSV vaccines. Moreover, subsequent in vivo inoculation and challenge experiments demonstrated that rGD-SX-5U2 confers cross-protection against both HP-PRRSV and NADC30-like PRRSV, including an improvement in ADG levels and a reduction in viremia and lung tissue lesions. In conclusion, our research demonstrated that the chimeric PRRSV strain rGD-SX-5U2 is a novel approach that can provide broad-spectrum protection against both HP-PRRSV and NADC30-like PRRSV. This may be a significant improvement over previous MLV vaccinations.

Non-structural protein 2 (NSP2) of PRRSV is highly variable and plays crucial roles in the virus's life cycle. To elucidate the function of NSP2 during PRRSV infection, we identified SH3KBP1 as an NSP2-interacting host protein using mass spectrometry. Exogenous SH3KBP1 expression significantly inhibited PRRSV replication by enhancing IFN-I and related ISGs production. Conversely, SH3KBP1 knockdown promoted viral replication by downregulating IFN-I and ISGs levels. In vivo experiments revealed that Sh3kbp1-/- mice were more susceptible to VSV infection, exhibiting reduced serum IFN-β levels. Further investigation showed that SH3KBP1 enhances RIG-I signal transduction by increasing K63-linked polyubiquitination through interaction with the E3 ubiquitin ligase TRIM25. We also found that PRRSV infection and NSP2 overexpression induce the autophagic degradation of SH3KBP1, counteracting the host's innate immune response. A critical interaction site was identified within the third polyproline-arginine motif in NSP2 (453PVPAPR458). Recombinant PRRSV lacking this motif displayed reduced virulence and decreased SH3KBP1 degradation. This study advances our understanding of how PRRSV interferes with the host immune response and offers valuable insights for developing novel attenuated vaccines against PRRSV.

ABSTRACT Metabolism in host cells can be modulated after viral infection, favoring viral survival or clearance. Here, we report that lipid droplet (LD) synthesis in host cells can be modulated by yin yang 1 (YY1) after porcine reproductive and respiratory syndrome virus (PRRSV) infection, resulting in active antiviral activity. As a ubiquitously distributed transcription factor, there was increased expression of YY1 upon PRRSV infection both in vitro and in vivo. YY1 silencing promoted the replication of PRRSV, whereas YY1 overexpression inhibited PRRSV replication. PRRSV infection led to a marked increase in LDs, while YY1 knockout inhibited LD synthesis, and YY1 overexpression enhanced LD accumulation, indicating that YY1 reprograms PRRSV infection-induced intracellular LD synthesis. We also showed that the viral components do not colocalize with LDs during PRRSV infection, and the effect of exogenously induced LD synthesis on PRRSV replication is nearly lethal. Moreover, we demonstrated that YY1 affects the synthesis of LDs by regulating the expression of lipid metabolism genes. YY1 negatively regulates the expression of fatty acid synthase (FASN) to weaken the fatty acid synthesis pathway and positively regulates the expression of peroxisome proliferator-activated receptor gamma (PPARγ) to promote the synthesis of LDs, thus inhibiting PRRSV replication. These novel findings indicate that YY1 plays a crucial role in regulating PRRSV replication by reprogramming LD synthesis. Therefore, our study provides a novel mechanism of host resistance to PRRSV and suggests potential new antiviral strategies against PRRSV infection.IMPORTANCEPorcine reproductive and respiratory virus (PRRSV) has caused incalculable economic damage to the global pig industry since it was first discovered in the 1980s. However, conventional vaccines do not provide satisfactory protection. It is well known that viruses are parasitic pathogens, and the completion of their replication life cycle is highly dependent on host cells. A better understanding of host resistance to PRRSV infection is essential for developing safe and effective strategies to control PRRSV. Here, we report a crucial host antiviral molecule, yin yang 1 (YY1), which is induced to be expressed upon PRRSV infection and subsequently inhibits virus replication by reprogramming lipid droplet (LD) synthesis through transcriptional regulation. Our work provides a novel antiviral mechanism against PRRSV infection and suggests that targeting YY1 could be a new strategy for controlling PRRSV.

Abstract Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease and one of the major causes of death in the global pig industry. PRRS virus (PRRSV) strains have complex and diverse genetic characteristics and cross-protection between strains is low, which complicates vaccine selection; thus, the current vaccination strategy has been greatly compromised. Therefore, it is necessary to identify effective natural compounds for the clinical treatment of PRRS. A small molecule library composed of 720 natural compounds was screened in vitro, and we found that Sanggenon C (SC) was amongst the most effective natural compound inhibitors of PRRSV infection. Compared with ribavirin, SC more significantly inhibited PRRSV infection at both the gene and protein levels and reduced the viral titres and levels of protein expression and inflammatory cytokine secretion to more effectively protect cells from PRRSV infection and damage. Mechanistically, SC inhibits activation of the NF-κB signalling pathway by promoting TRAF2 expression, thereby reducing PRRSV replication. In conclusion, by screening natural compounds, we found that SC suppresses PRRSV infection by regulating the TRAF2/NF-κB signalling pathway. This study contributes to a deeper understanding of the therapeutic targets and pathogenesis of PRRSV infection. More importantly, our results demonstrate that SC has potential as a candidate for the treatment of PRRS.

Abstract Modern human activity is profoundly changing our relationship with microorganisms with the startling rise in the rate of emerging infectious diseases. Nipah virus together with Ebola virus and SARS-CoV-2 are prominent examples. Since COVID-19 and the West African Ebola virus disease outbreak, different chemical disinfectants have been developed for preventing the direct spread of viruses and their efficacy has also been evaluated. However, there are currently no published efficacy studies for the chemical disinfection of Nipah virus. In this study, the virucidal efficacy of three disinfectants (Micro-Chem Plus detergent disinfectant cleaner, FWD and Medical EtOH) against Nipah virus was evaluated in quantitative suspension tests including. Our results showed that the > 4 log reduction achieved for all products in inactivating Nipah virus in 15 s. Even, 19% ethanol was able to inactivate Nipah virus when applied for at least 8 min contact time. Comparative analysis displayed virucidal efficacy of each of the evaluated disinfectants against SARS-CoV-2, Ebola virus and Nipah virus, with only minor differences in working concentrations and contact times required for complete inactivation. We expect that our study can assist in decontamination in healthcare settings and high level biosafety laboratories and can be beneficial to control for emerging enveloped viruses.

ABSTRACT In the 21st century, several human and swine coronaviruses (CoVs) have emerged suddenly and caused great damage to people's lives and property. The porcine epidemic diarrhea virus (PEDV), leading to enormous economic losses to the pork industry and remains a large challenge. PEDV showed extensive cell tropism, and we cannot ignore the potential risk of cross-species transmission. However, the mechanism of adaptation and cell tropism of PEDV remains largely unknown and in vitro isolation of PEDV remains a huge challenge, which seriously impedes the development of vaccines. In this study, we confirmed that the spike (S) protein determines the adaptability of PEDV to monkey Vero cells and LLC-PK1 porcine cells, and isolated exchange of S1 and S2 subunits of adaptive strains did not make PEDV adapt to cells. Further, we found that the cellular adaptability of rCH/SX/2016-SHNXP depends on S1 and the first half of S2 (S3), and the 803L and 976H of the S2 subunit are critical for rCH/SX/2016-S1HNXP+S3HNXP adaptation to Vero cells. These findings highlight the decisive role of PEDV S protein in cell tropism and the potential role of coronaviruses S protein in cross-species transmissibility. Besides, our work also provides some different insight into finding PEDV receptors and developing PEDV and other coronaviruses vaccines. IMPORTANCE CoVs can spill from an animal reservoir into a naive host to cause diseases in humans or domestic animals. PEDV results in high mortality in piglets, which has caused immense economic losses in the pork industry. Virus isolation is the first step in studying viral pathogenesis and developing effective vaccines. However, the molecular mechanism of PEDV cell tropism is largely unknown, and isolation of endemic PEDV strains remains a major challenge. This study confirmed that the S gene is the decisive gene of PEDV adaptability to monkey Vero cells and porcine LLC-PK1 cells by the PEDV reverse genetics system. Isolated exchange of S1 and S2 of adaptive strains did not make PEDV adapt to cells, and the 803L and 976H of S2 subunit are critical for rCH/SX/2016-S1HNXP+S3HNXP adaptation to Vero cells. These results illustrate the decisive role of PEDV S protein in cell tropism and highlight the potential role of coronaviruses S protein in cross-species transmissibility. Besides, our finding also provides some unique insight into identifying PEDV functional receptors and has guiding significance for developing PEDV and other coronavirus vaccines.

In this study, the authors present a genomic surveillance of avian influenza genomes sampled from live poultry markets in China. They report that a number of variants have emerged since 2016 that pose an increased risk to humans. They highlight the importance of continuous genome surveillance of circulating influenza strains.

Rotavirus (RV) is a non–enveloped icosahedral virus with an 11–segment double–stranded RNA genome, belonging to the family of rotaviruses. RV is one of the pathogens causing diarrhea in infants and young animals, and it induces the production of type I interferons (IFNs), which can trigger antiviral function by inducing the production of interferon–stimulated genes (ISGs). Although IFITM3, an ISG localizing to late endosomes, can limit many viral infections, whether or not it restricts the infection of RV is still unknown. Therefore, we attempted to determine whether IFITM3 also restricts RV infection by using over–expression and knockout cell strains. It was found that IFITM3–expressing cell strains were less susceptible to RV infection, as the replication of RV in over–expressing cells was significantly less than in control group cells. Correspondingly, IFITM3–knockout cells were significantly susceptible compared to the normal cells. Furthermore, the IFN–induced antiviral effect was significantly attenuated in the absence of IFITM3, and IFITM3 delayed RV escape from endosomes in the presence of IFITM3, suggesting that endogenous IFITM3 is of great importance in type I IFN–mediated antiviral responses and may restrict infection by affecting the function of the late endosomal compartment. In conclusion, these data provide the first evidence that IFITM3 limits RV infection in vitro and delays RV escape from late endosomes into the cytoplasm.

Abstract Background Porcine epidemic diarrhea virus (PEDV), which is characterized by severe watery diarrhea, vomiting, dehydration and a high mortality rate in piglets, leads to enormous economic losses to the pork industry and remains a large challenge worldwide. Thus, a rapid and reliable method is required for epidemiological investigations and to evaluate the effect of immunization. However, the current diagnostic methods for PEDV are time-consuming and very expensive and rarely meet the requirements for clinical application. Nanobodies have been used in the clinic to overcome these problems because of the advantages of their easy expression and high level of stability. In the present work, a novel biotinylated nanobody-based blocking ELISA (bELISA) was developed to detect anti-PEDV antibodies in clinical pig serum. Results Using phage display technology and periplasmic extraction ELISA (PE-ELISA), anti-PEDV N protein nanobodies from three strains of PEDV were successfully isolated after three consecutive rounds of bio-panning from a high quality phage display VHH library. Then, purified Nb2-Avi-tag fusion protein was biotinylated in vitro. A novel bELISA was subsequently developed for the first time with biotinylated Nb2. The cutoff value for bELISA was 29.27%. One hundred and fifty clinical serum samples were tested by both newly developed bELISA and commercial kits. The sensitivity and specificity of bELISA were 100% and 93.18%, respectively, and the coincidence rate between the two methods was 94%. Conclusions In brief, bELISA is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PEDV vaccines efficacy and indirect diagnosis of PEDV infection.

The pandemic due to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has emerged as a serious global public health issue. Besides the high transmission rate from individual to individual, indirect transmission from inanimate objects or surfaces poses a more significant threat. Since the start of the outbreak, the importance of respiratory protection, social distancing, and chemical disinfection to prevent the spread of the virus has been the prime focus for infection control. Health regulatory organizations have produced guidelines for the formulation and application of chemical disinfectants to manufacturing industries and the public. On the other hand, extensive literature on the virucidal efficacy testing of microbicides for SARS-CoV-2 has been published over the past year and a half. This review summarizes the studies on the most common chemical disinfectants and their virucidal efficacy against SARS-CoV-2, including the type and concentration of the chemical disinfectant, the formulation, the presence of excipients, the exposure time, and other critical factors that determine the effectiveness of chemical disinfectants. In this review, we also critically appraise these disinfectants and conduct a discussion on the role they can play in the COVID-19 pandemic.

Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in West Africa have revealed the lack of investigation on this notorious virus.

Abstract Porcine reproductive and respiratory syndrome virus(PRRSV) is an important immunosuppressive virus which can suppresses infected cells proliferation. In this work, we examined PRRSV ability to manipulate cell cycle progression of MARC-145 cells and explored the potential molecular mechanisms. The results showed that PRRSV infection imposed a growth-inhibitory effect on MARC-145 cells by inducing cell cycle arrest at G2/M phase. This arrest was due to the significant decrease of Cdc2-cyclinB1 complex activity in PRRSV-infected cells and the activity reduction was a result of Cdc2 Tyr15 phosphorylation and the accumulation of Cdc2 and cyclinB1 in the nucleus. Not only elevated Wee1 and Myt1 expression and inactivated Cdc25C, but also increase of p21 and 14–3-3σ in a p53-dependent manner caused the inhibitory Tyr15 phosphorylation of Cdc2. PRRSV infection also activated Chk1. Our data suggest PRRSV infection induces G2/M arrest via various molecular regulatory mechanisms. These results provide a new insights for PRRSV pathogenesis.

Mosquitoes are medically important arthropod vectors that harbor a variety of viruses. Geography and climate are known to be associated with variations in mosquito density, species and viromes. Our study investigated the dynamic changes in mosquito populations, species compositions and viromes in a regularly disinfected environment in Wuhan, China, during 2020. Traps were set in different mosquito habitats, including an urban residential area, two hospitals, a scenic area and a pig farm in a rural region between April and October of 2020. The collected mosquitoes were subjected to morphological identification, RT-qPCR and metagenomic sequencing. A total of 2345 adult mosquitoes were collected. Culex mosquitoes were dominant in both urban regions (90.32%, 1538/1703) and the pig farm (54.98%, 353/642). In RT-qPCR screening, the prevalence of Banna virus was 15% and 3% in mosquitoes from the urban area and the pig farm, respectively, whereas no Japanese encephalitis virus was detected. Culex viromes showed dynamic changes during the collection period. Several mosquito-specific viruses, such as Culex flavivirus, Alphamesonivirus 1, Hubei mosquito virus 2 and Hubei mosquito virus 4, showed seasonal changes and unimodal increases or declines. Other mosquito-specific viruses, such as Wuhan mosquito virus 6, Hubei virga-like virus 2 and Zhejiang mosquito virus 3, were stable in all collected Culex and are potential members of the core viromes. This study improves understanding of the dynamic composition of mosquito species and the viromes that they carry, and provides useful information for guiding mosquito control and mosquito-borne disease prevention strategies.

Given that ebolavirus causes severe and frequently lethal disease, its rapid and accurate detection using available and validated methods is essential for controlling infection. Real-time reverse-transcription PCR (RT-PCR) has proven to be an invaluable tool for ebolaviruses diagnostics. Many assays with different targets have been developed, but they have not been externally compared or validated, and limits of detection are not uniformly reported. Here we compared and evaluated the sensitivity, reproducibility and specificity of 23 in-house assays under the same conditions. Our results showed that these assays were highly gene- and species- specific when evaluated using in vitro RNA transcripts and viral RNA, and the potential limits of detection were uniformly reported ranging from 102 to 106 in vitro synthesized RNA transcripts copies perμL and 1–100 TCID50/mL. The comparison of these assays indicated that those targeting the more conservative NP gene could be the better option for EVD case definition and quantitative measurement because of its higher sensitivity for the same species. Our analysis could contribute to the standardization of ebolavirus detection and quantification assays, which can offer a better understanding of the meaning of results across laboratories and time points, as well as make them easy to implement, especially under outbreak conditions.

Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV) could lead to pandemic diseases and huge financial losses to the swine industry worldwide. Curcumin, a natural compound, has been reported to serve as an entry inhibitor of hepatitis C virus, chikungunya virus and vesicular stomatitis virus. In this study, we investigated the potential effect of curcumin on early stages of PRRSV infection. Results Curcumin inhibited infection of Marc-145 cells and porcine alveolar macrophages (PAMs) by four different genotype 2 PRRSV strains, but had no effect on the levels of major PRRSV receptor proteins on Marc-145 cells and PAMs or on PRRSV binding to Marc-145 cells. However, curcumin did block two steps of the PRRSV infection process: virus internalization and virus-mediated cell fusion. Conclusions Our results suggested that an inhibition of genotype 2 PRRSV infection by curcumin is virus strain-independent, and mainly inhibited by virus internalization and cell fusion mediated by virus. Collectively, these results demonstrate that curcumin holds promise as a new anti-PRRSV drug.

Porcine reproductive and respiratory syndrome (PRRS) is a leading disease in pig industry worldwide and can result in serious economic losses each year. The PRRS epidemic situation in China has been very complicated since the unprecedented large-scale highly pathogenic PRRS (HP-PRRS) outbreaks in 2006. And now the HP-PRRS virus (HP-PRRSV) and classical North American type PRRSV strains have coexisted in China. Rapid differential detection of the two strains of PRRSV is very important for effective PRRS control. The real-time RT-PCR for simultaneous detection and differentiation of HP-PRRSV and PRRSV by using both SYBR Green and TaqMan probes was developed and validated. Both assays can be used for rapid detection and strain-specific identification of HP-PRRSV and PRRSV. However, the TaqMan probe method had the highest detection rate whereas the conventional RT-PCR was the lowest. The real-time RT-PCR developed based on SYBR Green and TaqMan probe could be used for simultaneous detection and differentiation of HP-PRRSV and PRRSV in China, which will benefit much the PRRS control and research.

BACKGROUND: As an important factor affecting meat quality, intramuscular fat (IMF) content is a topic of worldwide concern. Emerging evidences indicate that microRNAs play important roles in adipocyte differentiation. However, miRNAome has neither been studied during porcine intramuscular preadipocyte differentiation, nor compared with subcutaneous preadipocytes. The objectives of this study were to identify porcine miRNAs involved in adipogenesis in primary preadipocytes, and to determine whether intramuscular and subcutaneous adipocytes differ in the expression and regulation of miRNAs. RESULTS: miRNAomes in primary intramuscular and subcutaneous adipocytes during differentiation were first sequenced using the Solexa deep sequencing method. The sequences and relative expression levels of 224 known (98.2% in miRbase 18.0) and 280 potential porcine miRNAs were identified. Fifty-four of them changed in similar pattern between intramuscular vascular stem cells (IVSC) and subcutaneous vascular stem cells (SVSC) differentiation, such as miR-210, miR-10b and miR-99a. Expression levels of 10 miRNAs were reversely up-or down-regulated between IVSC and SVSC differentiation, 19 were up-or down-regulated only during IVSC differentiation and 55 only during SVSC differentiation. Additionally, 30 miRNAs showed fat-depot specific expression pattern (24 in cells of intramuscular origin and 6 in cells of subcutaneous origin). These adipogenesis-related miRNAs mainly functioned by targeting similar pathways in adipogenesis, obesity and syndrome. CONCLUSION: Comparison of miRNAomes in IVSC and SVSC during differentiation revealed that many different miRNAs are involved in adipogenesis, and they regulate SVSC and IVSC differentiation through similar pathways. These miRNAs may serve as biomarkers or targets for enhancing IMF content, and uncovering their function in IMF development will be of great value in the near future.

Understanding the dynamics of muscle transcriptome during development and between breeds differing in muscle growth is necessary to uncover the complex mechanism underlying muscle development. Herein, we present the first transcriptome-wide longissimus dorsi muscle development research concerning Lantang (LT, obese) and Landrace (LR, lean) pig breeds during 10 time-points from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) using Solexa/Illumina's Genome Analyzer. The data demonstrated that myogenesis was almost completed before 77 dpc, but the muscle phenotypes were still changed from 77 dpc to 28 dpn. Comparative analysis of the two breeds suggested that myogenesis started earlier but progressed more slowly in LT than in LR, the stages ranging from 49 dpc to 77 dpc are critical for formation of different muscle phenotypes. 595 differentially expressed myogenesis genes were identified, and their roles in myogenesis were discussed. Furthermore, GSK3B, IKBKB, ACVR1, ITGA and STMN1 might contribute to later myogenesis and more muscle fibers in LR than LT. Some myogenesis inhibitors (ID1, ID2, CABIN1, MSTN, SMAD4, CTNNA1, NOTCH2, GPC3 and HMOX1) were higher expressed in LT than in LR, which might contribute to more slow muscle differentiation in LT than in LR. We also identified several genes which might contribute to intramuscular adipose differentiation. Most important, we further proposed a novel model in which MyoD and MEF2A controls the balance between intramuscular adipogenesis and myogenesis by regulating CEBP family; Myf5 and MEF2C are essential during the whole myogenesis process while MEF2D affects muscle growth and maturation. The MRFs and MEF2 families are also critical for the phenotypic differences between the two pig breeds. Overall, this study contributes to elucidating the mechanism underlying muscle development, which could provide valuable information for pig meat quality improvement. The raw data have been submitted to Gene Expression Omnibus (GEO) under series GSE25406.

Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we report on the first genome-wide host transcriptional responses to classical North American type PRRSV (N-PRRSV) strain CH 1a infection using Solexa/Illumina's digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results suggest that N-PRRSV appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processes. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS.