Tylvalosin tartrate inhibits the replication stage of the porcine reproductive and respiratory syndrome virus life cycle.

The aetiological agent of porcine reproductive and respiratory syndrome, a deadly disease that affects pigs and seriously jeopardises the global swine industry, is a porcine reproductive and respiratory syndrome virus (PRRSV). Tylvalosin tartrate, which is a macrolide antibiotic, is the active ingredient in Aivlosin. In recent years, tylvalosin tartrate has widely been used to control porcine reproductive and respiratory syndrome in swine herds in China. However, whether tylvalosin tartrate has exerts anti-PRRSV effects remains controversial. In the present study, tylvalosin tartrate exhibited no effect on PRRSV susceptibility but suppressed the replication of PRRSV and the activity of infecting Marc-145 cells. Next, the relationship between the replication cycle of PRRSV and the activity of tylvalosin tartrate was further assessed. Tylvalosin tartrate did not affect the attachment and release stages of PRRSV or act during the internalisation stage of the virus in HuN4; however, contrasting effects were noted for strains CH-1a and SDVD-HN21. Tylvalosin tartrate acted on the replication stage of PRRSV and was not strain-specific in the replication stage of the PRRSV life cycle. The study findings provide an initial clarification of the inhibitory effects of tylvalosin tartrate on PRRSV, providing new insights into the treatment of PRRS.

Improving multi-population genomic prediction accuracy using multi-trait GBLUP models which incorporate global or local genetic correlation information.

In the application of genomic prediction, a situation often faced is that there are multiple populations in which genomic prediction (GP) need to be conducted. A common way to handle the multi-population GP is simply to combine the multiple populations into a single population. However, since these populations may be subject to different environments, there may exist genotype-environment interactions which may affect the accuracy of genomic prediction. In this study, we demonstrated that multi-trait genomic best linear unbiased prediction (MTGBLUP) can be used for multi-population genomic prediction, whereby the performances of a trait in different populations are regarded as different traits, and thus multi-population prediction is regarded as multi-trait prediction by employing the between-population genetic correlation. Using real datasets, we proved that MTGBLUP outperformed the conventional multi-population model that simply combines different populations together. We further proposed that MTGBLUP can be improved by partitioning the global between-population genetic correlation into local genetic correlations (LGC). We suggested two LGC models, LGC-model-1 and LGC-model-2, which partition the genome into regions with and without significant LGC (LGC-model-1) or regions with and without strong LGC (LGC-model-2). In analysis of real datasets, we demonstrated that the LGC models could increase universally the prediction accuracy and the relative improvement over MTGBLUP reached up to 163.86% (25.64% on average).

Secondary metabolites of Bacillus subtilis L2 show antiviral activity against pseudorabies virus.

Bacillus subtilis (B. subtilis) is a commercially important probiotic known to produce secondary metabolites with antibacterial, antifungal and anti-inflammatory activities. However, the potential ability of B. subtilis to combat viruses, especially DNA viruses, has not been extensively investigated. In this study, we identified two distinct B. subtilis strains and examined the efficiency of their secondary metabolites against pseudorabies virus (PRV), a swine herpesvirus resulting in economic losses worldwide. We found that treatment with the secondary metabolites of B. subtilis L2, but not the metabolites of B. subtilis V11, significantly inhibited PRV replication in multiple cells. Notably, the antiviral activity of the metabolites of B. subtilis L2 was thermal stable, resistant to protease digestion. Moreover, these metabolites effectively impeded PRV binding, entry and replication. Importantly, oral administration of the metabolites of B. subtilis L2 protected mice from lethal PRV infection, rescuing weight loss and reducing the viral load in vivo. In summary, our results reveal that the metabolites of B. subtilis L2 exhibit anti-PRV activity both in vitro and in vivo, providing a potential candidate for novel antiviral drugs.

NRF1-mediated mitochondrial biogenesis antagonizes innate antiviral immunity.

Mitochondrial biogenesis is the process of generating new mitochondria to maintain cellular homeostasis. Here, we report that viruses exploit mitochondrial biogenesis to antagonize innate antiviral immunity. We found that nuclear respiratory factor-1 (NRF1), a vital transcriptional factor involved in nuclear-mitochondrial interactions, is essential for RNA (VSV) or DNA (HSV-1) virus-induced mitochondrial biogenesis. NRF1 deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in mice. Mechanistically, the inhibition of NRF1-mediated mitochondrial biogenesis aggravated virus-induced mitochondrial damage, promoted the release of mitochondrial DNA (mtDNA), increased the production of mitochondrial reactive oxygen species (mtROS), and activated the innate immune response. Notably, virus-activated kinase TBK1 phosphorylated NRF1 at Ser318 and thereby triggered the inactivation of the NRF1-TFAM axis during HSV-1 infection. A knock-in (KI) strategy that mimicked TBK1-NRF1 signaling revealed that interrupting the TBK1-NRF1 connection ablated mtDNA release and thereby attenuated the HSV-1-induced innate antiviral response. Our study reveals a previously unidentified antiviral mechanism that utilizes a NRF1-mediated negative feedback loop to modulate mitochondrial biogenesis and antagonize innate immune response.

Recombinant porcine interferon cocktail delays the onset and lessens the severity of African swine fever.

African swine fever (ASF) is a highly contagious and deadly disease that affects domestic and wild pigs. No commercial vaccine or antiviral is currently available against ASF. The control of ASF primarily relies on implementing effective biosecurity measures during the breeding process. Here, we evaluated the preventive and therapeutic potential of the interferon (IFN) cocktail (a mixture of recombinant porcine IFN α and γ) on ASF. The IFN cocktail treatment delayed the onset of ASF symptoms and ASF virus (ASFV) replication for approximately one week. However, IFN cocktail treatment could not prevent the death of the pigs. Further analysis showed that IFN cocktail treatment increased the expression of multiple IFN-stimulated genes (ISGs) in porcine peripheral blood mononuclear cells in vivo and in vitro. Additionally, IFN cocktail modulated the expression of pro- and anti-inflammatory cytokines and reduced tissue injury in the ASFV-infected pigs. Collectively, the results suggest that the IFN cocktail restricts the progression of acute ASF by inducing high levels of ISGs, contributing to the pre-establishment of antiviral status, and modulating the balance of pro- and anti-inflammatory mediators to lessen cytokine storm-mediated tissue damage.

Pseudorabies Virus Tegument Protein UL13 Suppresses RLR-Mediated Antiviral Innate Immunity through Regulating Receptor Transcription.

Pseudorabies virus (PRV) has evolved various strategies to escape host antiviral immune responses. However, it remains unclear whether and how PRV-encoded proteins modulate the RIG-I-like receptor (RLR)-mediated signals for immune evasion. Here, we show that the PRV tegument protein UL13 functions as an antagonist of RLR-mediated antiviral responses via suppression of the transcription of RIG-I and MDA5, but not LGP2. UL13 overexpression significantly inhibits both the mRNA and protein levels of RIG-I and MDA5, along with RIG-I- or MDA5-mediated antiviral immune responses, whereas overexpression of RIG-I or MDA5 counteracts such UL13-induced suppression. Mechanistically, UL13 suppresses the expression of RIG-I and MDA5 by inhibiting activation of the transcription factor NF-κB. Consequently, overexpression of p65 promotes the activation of RIG-I and MDA5 promoters. Moreover, deletion of the p65-binding sites in the promoters of RIG-I or MDA5 abolishes the suppression role of UL13. As a result, mutant PRV lacking UL13 elicits stronger host antiviral immune responses than PRV-WT. Hence, our results provide a novel functional role of UL13-induced suppression of host antiviral immunity through modulating receptors' transcription.

Pseudorabies virus tegument protein UL13 recruits RNF5 to inhibit STING-mediated antiviral immunity.

Pseudorabies virus (PRV) has evolved various immune evasion mechanisms that target host antiviral immune responses. However, it is unclear whether and how PRV encoded proteins modulate the cGAS-STING axis for immune evasion. Here, we show that PRV tegument protein UL13 inhibits STING-mediated antiviral signaling via regulation of STING stability. Mechanistically, UL13 interacts with the CDN domain of STING and recruits the E3 ligase RING-finger protein 5 (RNF5) to promote K27-/K29-linked ubiquitination and degradation of STING. Consequently, deficiency of RNF5 enhances host antiviral immune responses triggered by PRV infection. In addition, mutant PRV lacking UL13 impaired in antagonism of STING-mediated production of type I IFNs and shows attenuated pathogenicity in mice. Our findings suggest that PRV UL13 functions as an antagonist of IFN signaling via a novel mechanism by targeting STING to persistently evade host antiviral responses.

Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection.

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus-associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

Longitudinal monitoring of multidrug resistance in Escherichia coli on broiler chicken fattening farms in Shandong, China.

The extensive use of antibiotics has, in recent years, caused antimicrobial resistance and multidrug resistance in Escherichia coli to gradually develop into a worldwide problem. These resistant E. coli could be transmitted to humans through animal products and animal feces in the environment, thereby creating a problem for bacterial treatment for humans and animals and resulting in a public health issue. Monitoring the resistance of E. coli throughout the broiler fattening period is therefore of great significance for both the poultry industry and public health. In this longitudinal study, samples were taken from 6 conventional broiler fattening farms in Shandong Province, China, at 3 different times within 1 fattening period. The overall isolation rate of E. coli was 53.04% (375/707). Antibiotic resistance was very common in the E. coli isolated from these farms, and differed for different antibiotics, with ampicillin having the highest rate (92.86%) and cefoxitin the lowest (10.12%). Multidrug resistance was as high as 91.07%. More importantly, both the resistance rate of E. coli to the different drugs and the detection rate of drug resistance genes increased over time. The mobile colistin resistance (mcr-1) gene was detected in 24.40% of the strains, and these strains often carried other drug resistance genes, such as those conferring aminoglycoside, ß-lactamase, tetracycline, and sulfonamide resistance. Antimicrobial resistance and drug resistance genes in E. coli were least common in the early fattening stage. The individual detection rates of sul1, sul3, aacC4, aphA3, and mcr-1 were significantly lower (P < 0.05) for the early fattening stage than for the middle and late stages. The rational use of antibiotics, in conjunction with the improvement of the breeding environment during the entire broiler fattening cycle, will be helpful in the development of the poultry industry and the protection of public health.

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

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

Application of Bacillus subtilis as a live vaccine vector: A review.

Bacillus subtilis is widely used as a probiotic in various fields as it regulates intestinal flora, improves animal growth performance, enhances body immunity, has short fermentation cycle, and is economic. With the rapid development of DNA recombination technology, B. subtilis has been used as a potential vaccine expression vector for the treatment and prevention of various diseases caused by bacteria, viruses, and parasites as it can effectively trigger an immune response in the body. In this review, we refer to previous literature and provide a comprehensive analysis and overview of the feasibility of using B. subtilis as a vaccine expression vector, with an aim to provide a valuable reference for the establishment of efficient vaccines.

Inhibition of African Swine Fever Virus Replication by Porcine Type I and Type II Interferons.

Interferons (IFNs) are proteins produced by a variety of cells during the process of virus infection. It can activate the transcription of multiple functional genes in cells, regulate the synergistic effect of multiple signaling pathways, and mediate a variety of biological functions such as antiviral activity and immune regulation. The symptoms of hosts infected with African swine fever virus (ASFV) depend on the combined interaction between viruses and the host. However, it is unclear whether IFNs can be used as an emergency preventive treatment for ASFV. This study focused on the use of recombinant porcine IFNs, produced by Escherichia coli, to inhibit the replication of ASFV. The activity of IFN against ASFV was detected using primary alveolar macrophages at different doses through immunofluorescence assays and quantitative real-time PCR. We found that both 1000 and 100 U/mL doses significantly inhibited the replication of ASFV. Meanwhile, we found that IFNs could significantly trigger the production of a variety of IFN-induced genes (IFIT1, IFITM3, Mx-1, OASL, ISG15, PKR, GBP1, Viperin, BST2, IRF-1, and CXCL10) and MHC molecules, which play key roles in resistance to virus infection. Peripheral blood samples were also obtained from surviving pigs treated with IFNs, and the viral load was determined. Consistent with in vitro tests, low-dose (105 U/kg) recombinant porcine IFNs (PoIFN-α and PoIFN-γ) significantly reduced viral load compared to that with high-dose (106 U/kg) treatment. Our results suggest that recombinant porcine IFNs have high antiviral activity against ASFV, providing a new strategy for the prevention of African swine fever.

Negative elongation factor complex enables macrophage inflammatory responses by controlling anti-inflammatory gene expression.

Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report that widespread promoter-proximal RNA polymerase II pausing in resting macrophages is marked by co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.

Natural infection of a variant pseudorabies virus leads to bovine death in China.

Pseudorabies virus (PRV) infects numerous species of domestic and wild animals leading to severe diseases especially in swine and cattle. Since 2011, the variant PRVs were identified in pigs, which were genetically different from classic strains. Although variant PRV infection is widely observed in pigs, there is still no report of variant PRV infection in cattle. Here, we reported a natural infection of variant PRV leading to acute bovine death in Eastern China. Our study suggests that the new variant PRV strains could be a potential threat to cattle industry and possibly to the public health of human.

Naproxen Exhibits Broad Anti-influenza Virus Activity in Mice by Impeding Viral Nucleoprotein Nuclear Export.

Naproxen is a non-steroidal anti-inflammatory drug that has previously been shown to exert antiviral activity against influenza A virus by inhibiting nucleoprotein (NP) binding to RNA. Here, we show that naproxen is a potential broad, multi-mechanistic anti-influenza virus therapeutic, as it inhibits influenza B virus replication both in vivo and in vitro. The anti-influenza B virus activity of naproxen is more efficient than that of the commonly used neuraminidase inhibitor oseltamivir in mice. Furthermore, the NP of influenza B virus (BNP) has a higher binding affinity to naproxen than influenza A virus NP (ANP). Specifically, naproxen targets the NP at residues F209 (BNP) and Y148 (ANP). This interaction antagonizes the nuclear export of NP normally mediated by the host export protein CRM1. This study reveals a crucial mechanism of broad-spectrum anti-influenza virus activity of naproxen, suggesting that the existing drug naproxen may be used as an anti-influenza drug.

CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication.

Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine.

Hes1 attenuates type I IFN responses via VEGF-C and WDFY1.

Induction of type I interferons (IFNs) is critical for eliciting competent immune responses, especially antiviral immunity. However, uncontrolled IFN production contributes to pathogenesis of autoimmune and inflammatory diseases. We found that transcription factor Hes1 suppressed production of type I IFNs and expression of IFN-stimulated genes. Functionally, Hes1-deficient mice displayed a heightened IFN signature in vivo, mounted enhanced resistance against encephalomyocarditis virus infection, and showed signs of exacerbated experimental lupus nephritis. Mechanistically, Hes1 did not suppress IFNs via direct transcriptional repression of IFN-encoding genes. Instead, Hes1 attenuated activation of TLR upstream signaling by inhibition of an adaptor molecule, WDFY1. Genome-wide assessment of Hes1 occupancy revealed that suppression of WDFY1 was secondary to direct binding and thus enhancement of expression of VEGF-C by Hes1, making Vegfc a rare example of an Hes1 positively regulated gene. In summary, these results identified Hes1 as a homeostatic negative regulator of type I IFNs for the maintenance of immune balance in the context of antiviral immunity and autoimmune diseases.

STING directly activates autophagy to tune the innate immune response.

STING (stimulator of interferon genes) is a central molecule that binds to cyclic dinucleotides produced by the cyclic GMP-AMP synthase (cGAS) to activate innate immunity against microbial infection. Here we report that STING harbors classic LC-3 interacting regions (LIRs) and mediates autophagy through its direct interaction with LC3. We observed that poly(dA:dT), cGAMP, and HSV-1 induced STING-dependent autophagy and degradation of STING immediately after TBK1 activation. STING induces non-canonical autophagy that is dependent on ATG5, whereas other autophagy regulators such as Beclin1, Atg9a, ULK1, and p62 are dispensable. LIR mutants of STING abolished its interaction with LC3 and its activation of autophagy. Also, mutants that abolish STING dimerization and cGAMP-binding diminished the STING-LC3 interaction and subsequent autophagy, suggesting that STING activation is indispensable for autophagy induction. Our results thus uncover dual functions of STING in activating the immune response and autophagy, and suggest that STING is involved in ensuring a measured innate immune response.

Outbreak of myelocytomatosis caused by mutational avian leukosis virus subgroup J in China, 2018.

Avian leukosis virus subgroup J (ALV-J) was isolated in meat-type breeder chickens for the first time in 1988 in the United Kingdom. Due to the application of an eradication program, there were fewer reports related to myelocytomatosis or ALV-J in China after 2013. However, there was another breakout almost simultaneously in six provinces of China in February 2018. On-site, 15- to 20-week-old broiler breeder chickens showed depression, paralysis and weight loss. Mortality for certain flocks reached 15%. Sick chickens showed numerous yellow-white neoplasms growing in the sternum, rib and lumbar vertebra and had hepatic and renal metastasis. Histopathological observation showed all neoplasms were myelocytomas, and there were massive myelocyte-like tumour cells in the liver, kidney and bone marrow. To explore the aetiology of this re-outbreak of myelocytomatosis in China, we collected tumour-bearing chickens and isolated six strains of ALV-J (GM0209-1 to -6). Phylogenetic analysis of gp85 and gp37 showed GM0209 strains were clearly distinct from the prototype strain of ADOL-7501, HPRS-103 and NX0101, and there was a mutation, R176G, in the conserved region between hr1 and hr2 regions of gp85, which was not found in other 44 ALV-J strains. The 3'UTR nucleotide sequences of GM0209 isolates showed there was a signature deletion of 11 nt that was also present in 3'UTR sequences of SCDY1 and NHH, two isolates that have a reported association with haemangioma, indicating this deletion could not determine the tumour type induced by ALV-J. Although the eradication program of ALV-J has been successfully applied in China, the outbreak of ALV-J still occurred, and the virus strain spread quickly. Thus, the biocharacteristics and pathogenesis of mutational ALV-J should be further studied.