Clofazimine broadly inhibits coronaviruses including SARS-CoV-2.

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.

A novel strategy for an anti-idiotype vaccine: nanobody mimicking neutralization epitope of porcine circovirus type 2.

Vaccination is the most effective method to protect humans and animals from diseases. Anti-idiotype vaccines are safer due to their absence of pathogens. However, the commercial production of traditional anti-idiotype vaccines using monoclonal and polyclonal antibodies (mAb and pAb) is complex and has a high failure rate. The present study designed a novel, simple, low-cost strategy for developing anti-idiotype vaccines with nanobody technology. We used porcine circovirus type 2 (PCV2) as a viral model, which can result in serious economic loss in the pig industry. The neutralizing mAb-1E7 (Ab1) against PCV2 capsid protein (PCV2-Cap) was immunized in the camel. And 12 nanobodies against mAb-1E7 were screened. Among them, Nb61 (Ab2) targeted the idiotype epitope of mAb-1E7 and blocked mAb-1E7's binding to PCV2-Cap. Additionally, a high-dose Nb61 vaccination can also protect mice and pigs from PCV2 infection. Epitope mapping showed that mAb-1E7 recognized the 75NINDFL80 of PCV2-Cap and 101NYNDFLG107 of Nb61. Subsequently, the mAb-3G4 (Ab3) against Nb61 was produced and can neutralize PCV2 infection in the PK-15 cells. Structure analysis showed that the amino acids of mAb-1E7 and mAb-3G4 respective binding to PCV2-Cap and Nb61 were also similar on the amino acids sequences and spatial conformation. Collectively, our study first provided a strategy for producing nanobody-based anti-idiotype vaccines and identified that anti-idiotype nanobodies could mimic the antigen on amino acids and structures. Importantly, as more and more neutralization mAbs against different pathogens are prepared, anti-idiotype nanobody vaccines can be easily produced against the disease with our strategy, especially for dangerous pathogens.IMPORTANCEAnti-idiotype vaccines utilize idiotype-anti-idiotype network theory, eliminating the need for external antigens as vaccine candidates. Especially for dangerous pathogens, they were safer because they did not contact the live pathogenic microorganisms. However, developing anti-idiotype vaccines with traditional monoclonal and polyclonal antibodies is complex and has a high failure rate. We present a novel, universal, simple, low-cost strategy for producing anti-idiotype vaccines with nanobody technology. Using a neutralization antibody against PCV2-Cap, a nanobody (Ab2) was successfully produced and could mimic the neutralizing epitope of PCV2-Cap. The nanobody can induce protective immune responses against PCV2 infection in mice and pigs. It highlighted that the anti-idiotype vaccine using nanobody has a very good application in the future, especially for dangerous pathogens.

African swine fever virus pS273R antagonizes stress granule formation by cleaving the nucleating protein G3BP1 to facilitate viral replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs are regulated by different stimulators including viral infection, which is involved in the antiviral activity of host cells to limit viral propagation. To survive, several viruses have been reported to execute various strategies, such as modulating SG formation, to create optimal surroundings for viral replication. African swine fever virus (ASFV) is one of the most notorious pathogens in the global pig industry. However, the interplay between ASFV infection and SG formation remains largely unknown. In this study, we found that ASFV infection inhibited SG formation. Through SG inhibitory screening, we found that several ASFV-encoded proteins are involved in inhibition of SG formation. Among them, an ASFV S273R protein (pS273R), the only cysteine protease encoded by the ASFV genome, significantly affected SG formation. ASFV pS273R interacted with G3BP1 (Ras-GTPase-activating protein [SH3 domain] binding protein 1), a vital nucleating protein of SG formation. Furthermore, we found that ASFV pS273R cleaved G3BP1 at the G140-F141 to produce two fragments (G3BP1-N1-140 and G3BP1-C141-456). Interestingly, both the pS273R-cleaved fragments of G3BP1 lost the ability to induce SG formation and antiviral activity. Taken together, our finding reveals that the proteolytic cleavage of G3BP1 by ASFV pS273R is a novel mechanism by which ASFV counteracts host stress and innate antiviral responses.

Structural basis of sphingosine-1-phosphate receptor 1 activation and biased agonism.

Sphingosine-1-phosphate receptor 1 (S1PR1) is a master regulator of lymphocyte egress from the lymph node and an established drug target for multiple sclerosis (MS). Mechanistically, therapeutic S1PR1 modulators activate the receptor yet induce sustained internalization through a potent association with ß-arrestin. However, a structural basis of biased agonism remains elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of Gi-bound S1PR1 in complex with S1P, fingolimod-phosphate (FTY720-P) and siponimod (BAF312). In combination with functional assays and molecular dynamics (MD) studies, we reveal that the ß-arrestin-biased ligands direct a distinct activation path in S1PR1 through the extensive interplay between the PIF and the NPxxY motifs. Specifically, the intermediate flipping of W2696.48 and the retained interaction between F2656.44 and N3077.49 are the key features of the ß-arrestin bias. We further identify ligand-receptor interactions accounting for the S1PR subtype specificity of BAF312. These structural insights provide a rational basis for designing novel signaling-biased S1PR modulators.

Generation of porcine circovirus type 4 virus-like particles and their use to detect serum antibodies.

Porcine circovirus type 4 (PCV4), first identified in 2019 as a newly emerging pathogen, has been found in several provinces of China, as well as in Korea and Thailand. Since PCV4 is not included in immunization programs, epidemiological investigations should be conducted for detection of anti-PCV4 antibodies. Virus-like particles (VLPs) are frequently used for serological analysis of pathogen infections. However, there have been no reports on using PCV4 VLPs for serological investigation of PCV4 infection. In this study, we generated self-assembled PCV4 VLPs using an E. coli expression system, purified them using a two-step process, and used them to develop an indirect ELISA. This ELISA method was found to be highly specific, sensitive, and repeatable, making it suitable for PCV4 antibody detection in serum samples. Finally, the ELISA was used to analyze 422 serum samples collected from across several regions in China, 134 of which tested positive. Thus, the PCV4-VLP-based ELISA can effectively detect antibodies against PCV4 in serum samples, making it a useful tool for PCV4 epidemiology.

SARS-CoV-2 hijacks macropinocytosis to facilitate its entry and promote viral spike-mediated cell-to-cell fusion.

Revealing the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and cell-to-cell spread might provide insights for understanding the underlying mechanisms of viral pathogenesis, tropism, and virulence. The signaling pathways involved in SARS-CoV-2 entry and viral spike-mediated cell-to-cell fusion remain elusive. In the current study, we found that macropinocytosis inhibitors significantly suppressed SARS-CoV-2 infection at both the entry and viral spike-mediated cell-to-cell fusion steps. We demonstrated that SARS-CoV-2 entry required the small GTPase Rac1 and its effector kinase p21-activated kinase 1 by dominant-negative and RNAi assays in human embryonic kidney 293T-angiotensin-converting enzyme 2 cells and that the serine protease transmembrane serine protease 2 reversed the decrease in SARS-CoV-2 entry caused by the macropinocytosis inhibitors. Moreover, in the cell-to-cell fusion assay, we confirmed that macropinocytosis inhibitors significantly decreased viral spike-mediated cell-to-cell fusion. Overall, we provided evidence that SARS-CoV-2 utilizes a macropinocytosis pathway to enter target cells and to efficiently promote viral spike-mediated cell-to-cell fusion.

Inflammation plays a critical role in damage to the bronchiolar epithelium induced by Trueperella pyogenes in vitro and in vivo.

Trueperella pyogenes can cause severe pulmonary disease in swine, but the mechanism of pathogenesis is not well defined. T. pyogenes-induced damage to porcine bronchial epithelial cells (PBECs), porcine precision-cut lung slices (PCLS), and respiratory epithelium of mice remains unknown. In this study, we used T. pyogenes 20121 to infect PBECs in air-liquid interface conditions and porcine PCLS. T. pyogenes could adhere to, colonize, and induce cytotoxic effect on PBECs and the luminal surface of bronchi in PCLS, which damaged the bronchiolar epithelium. Moreover, bronchiolar epithelial cells showed extensive degeneration in the lungs of infected mice. Furthermore, western blot showed that the NOD-like receptor (NLR)/C-terminal caspase recruitment domain (ASC)/caspase-1 axis and nuclear factor-kappa B pathway were involved in inflammation in PCLS and lungs of mice, which also confirms that porcine PCLS provide a platform to analyze the pulmonary immune response. Meanwhile, the levels of p-c-Jun N-terminal kinase, p-extracellular signal-regulated kinase, and p-protein kinase B (AKT) were increased significantly, which indicated the mitogen-activated protein kinase and Akt pathways were also involved in inflammation in T. pyogenes-infected mice. In addition, we used T. pyogenes 20121 to infect tumor necrosis factor-alpha (tnf-α-/-) mice, and the results indicated that apoptosis and injury in respiratory epithelium of infected tnf-α-/- mice were alleviated. Thus, the pro-inflammatory cytokine TNF-α played a role in apoptosis and the respiratory epithelium injury in mouse lungs. Collectively, our study provides insight into the inflammatory injury induced by T. pyogenes and suggests that blocking NLR may be a potential therapeutic strategy against T. pyogenes infection.

Neuropilin-1 Facilitates Pseudorabies Virus Replication and Viral Glycoprotein B Promotes Its Degradation in a Furin-Dependent Manner.

Pseudorabies virus (PRV), which is extremely infectious and can infect numerous mammals, has a risk of spillover into humans. Virus-host interactions determine viral entry and spreading. Here, we showed that neuropilin-1 (NRP1) significantly potentiates PRV infection. Mechanistically, NRP1 promoted PRV attachment and entry, and enhanced cell-to-cell fusion mediated by viral glycoprotein B (gB), gD, gH, and gL. Furthermore, through in vitro coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays, NRP1 was found to physically interact with gB, gD, and gH, and these interactions were C-end Rule (CendR) motif independent, in contrast to currently known viruses. Remarkably, we illustrated that the viral protein gB promotes NRP1 degradation via a lysosome-dependent pathway. We further demonstrate that gB promotes NRP1 degradation in a furin-cleavage-dependent manner. Interestingly, in this study, we generated gB furin cleavage site (FCS)-knockout PRV (Δfurin PRV) and evaluated its pathogenesis; in vivo, we found that Δfurin PRV virulence was significantly attenuated in mice. Together, our findings demonstrated that NRP1 is an important host factor for PRV and that NRP1 may be a potential target for antiviral intervention. IMPORTANCE Recent studies have shown accelerated PRV cross-species spillover and that PRV poses a potential threat to humans. PRV infection in humans always manifests as a high fever, tonic-clonic seizures, and encephalitis. Therefore, understanding the interaction between PRV and host factors may contribute to the development of new antiviral strategies against PRV. NRP1 has been demonstrated to be a receptor for several viruses that harbor CendR, including SARS-CoV-2. However, the relationships between NRP1 and PRV are poorly understood. Here, we found that NRP1 significantly potentiated PRV infection by promoting PRV attachment and enhanced cell-to-cell fusion. For the first time, we demonstrated that gB promotes NRP1 degradation via a lysosome-dependent pathway. Last, in vivo, Δfurin PRV virulence was significantly attenuated in mice. Therefore, NRP1 is an important host factor for PRV, and NRP1 may be a potential target for antiviral drug development.

GrpE and ComD contribute to the adherence, biofilm formation, and pathogenicity of Streptococcus suis.

Streptococcus suis is a major bacterial pathogen of swine and an emerging zoonotic agent that has to date resulted in substantial economic losses to the swine industry worldwide, and can cause persistent infection by forming biofilms. GrpE and histidine protein kinase ComD are important proteins implicated in the pathogenicity of S. suis, although whether they play roles in adhesion and biofilm formation has yet to be sufficiently clarified. In this study, we constructed grpE and comD deletion strains of S. suis by homologous recombination, and examined their cell adhesion and biofilm formation capacities compared with those of the wild-type strain. The pathogenicity of the grpE and comD deletion strains was evaluated using a mouse infection model, which revealed that compared with the wild-type, these deletion strains induced milder symptoms and lower bacteremia, as well as comparatively minor organ (brain, spleen, liver, and lung) lesions, in the infected mice. Moreover, the deletion of grpE and comD significantly reduced the pro-inflammatory cytokine (IL-6, IL-1ß, and TNF-α) induction capacity of S. suis. Collectively, the findings of this study indicate that the GrpE and ComD proteins of Streptococcus suis play key roles in the adherence to PK-15 cells and the formation of biofilms, thereby contributing to the virulence of this pathogen.

Evaluating angiotensin-converting enzyme 2-mediated SARS-CoV-2 entry across species.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents a global threat, and the interaction between the virus and angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. However, the mechanisms underpinning ACE2-mediated viral entry across species remains unclear. Using infection assay, we evaluated SARS-CoV-2 entry mediated by ACE2 of 11 different animal species. We discovered that ACE2 of Rhinolophus sinicus (Chinese rufous horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Capra hircus (goat), and Manis javanica (Malayan pangolin) facilitated SARS-CoV-2 entry into nonsusceptible cells. Moreover, ACE2 of the pangolin also mediated SARS-CoV-2 entry, adding credence to the hypothesis that SARS-CoV-2 may have originated from pangolins. However, the ACE2 proteins of Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), or Mus musculus (house mouse) did not facilitate SARS-CoV-2 entry. In addition, a natural isoform of the ACE2 protein of Macaca mulatta (rhesus monkey) with the Y217N mutation was resistant to SARS-CoV-2 infection, highlighting the possible impact of this ACE2 mutation on SARS-CoV-2 studies in rhesus monkeys. We further demonstrated that the Y217 residue of ACE2 is a critical determinant for the ability of ACE2 to mediate SARS-CoV-2 entry. Overall, these results clarify that SARS-CoV-2 can use the ACE2 receptors of multiple animal species and show that tracking the natural reservoirs and intermediate hosts of SARS-CoV-2 is complex.

Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field.

Phylogenetics, Genomic Recombination, and NSP2 Polymorphic Patterns of Porcine Reproductive and Respiratory Syndrome Virus in China and the United States in 2014-2018.

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen that affects the pig industry, is a highly genetically diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus have not been completely elucidated. In this study, comparative analyses of all available genomic sequences of North American (NA)-type PRRSVs (n = 355, including 138 PRRSV genomes sequenced in this study) in China and the United States during 2014-2018 revealed a high frequency of interlineage recombination hot spots in nonstructural protein 9 (NSP9) and the GP2 to GP3 regions. Lineage 1 (L1) PRRSV was found to be susceptible to recombination among PRRSVs both in China and the United States. The recombinant major parent between the 1991-2013 data and the 2014-2018 data showed a trend from complex to simple. The major recombination pattern changed from an L8 to L1 backbone during 2014-2018 for Chinese PRRSVs, whereas L1 was always the major backbone for US PRRSVs. Intralineage recombination hot spots were not as concentrated as interlineage recombination hot spots. In the two main clades with differential diversity in L1, NADC30-like PRRSVs are undergoing a decrease in population genetic diversity, NADC34-like PRRSVs have been relatively stable in population genetic diversity for years. Systematic analyses of insertion and deletion (indel) polymorphisms of NSP2 divided PRRSVs into 25 patterns, which could generate novel references for the classification of PRRSVs. The results of this study contribute to a deeper understanding of the recombination of PRRSVs and indicate the need for coordinated epidemiological investigations among countries.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases. However, the phylogenetic and genomic recombination properties of the PRRS virus (PRRSV) have not been completely elucidated. In this study, we systematically compared differences in the lineage distribution, recombination, NSP2 polymorphisms, and evolutionary dynamics between North American (NA)-type PRRSVs in China and in the United States. Strikingly, we found high frequency of interlineage recombination hot spots in nonstructural protein 9 (NSP9) and in the GP2 to GP3 region. Also, intralineage recombination hot spots were scattered across the genome between Chinese and US strains. Furthermore, we proposed novel methods based on NSP2 indel patterns for the classification of PRRSVs. Evolutionary dynamics analysis revealed that NADC30-like PRRSVs are undergoing a decrease in population genetic diversity, suggesting that a dominant population may occur and cause an outbreak. Our findings offer important insights into the recombination of PRRSVs and suggest the need for coordinated international epidemiological investigations.

Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR.

Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.

Streptococcus suis Serotype 2 Infection Causes Host Immunomodulation through Induction of Thymic Atrophy.

Streptococcus suis serotype 2 is an important bacterial pathogen of swine and is also an emerging zoonotic agent that may be harmful to human health. Although the virulence genes of S. suis have been extensively studied, the mechanisms by which they damage the central immune organs have rarely been studied. In the current work, we wanted to uncover more details about the impact and mechanisms of S. suis on specific populations of thymic and immune cells in infected mice. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed that S. suis infection induced apoptosis in CD3+, CD14+, and epithelial cells from the thymus. S. suis infection resulted in a rapid depletion of mitochondrial permeability and release of cytochrome c (CytC) and apoptosis-inducing factor (AIF) through upregulation of Bax expression and downregulation of Bcl-xl and Bcl2 expression in thymocytes. Moreover, S. suis infection increased cleavage of caspase-3, caspase-8, and caspase-9. Thus, S. suis induced thymocyte apoptosis through a p53- and caspase-dependent pathway, which led to a decrease of CD3+ cells in the thymus, subsequently decreasing the numbers of CD4+ and CD8+ cells in the peripheral blood. Finally, expression dysregulation of proinflammatory cytokines in the serum, including interleukin 2 (IL-2), IL-6, IL-12 (p70), tumor necrosis factor (TNF), and IL-10, was observed in mice after S. suis type 2 infection. Taken together, these results suggest that S. suis infection can cause atrophy of the thymus and induce apoptosis of thymocytes in mice, thus likely suppressing host immunity.

Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection.

Bcl2-associated athanogene (BAG) 3, which is a chaperone-mediated selective autophagy protein, plays a pivotal role in modulating the life cycle of a wide variety of viruses. Both positive and negative modulations of viruses by BAG3 were reported. However, the effects of BAG3 on pseudorabies virus (PRV) remain unknown. To investigate whether BAG3 could modulate the PRV life cycle during a lytic infection, we first identified PRV protein UL56 (pUL56) as a novel BAG3 interactor by co-immunoprecipitation and co-localization analyses. The overexpression of pUL56 induced a significant degradation of BAG3 at protein level via the lysosome pathway. The C-terminal mutations of 181L/A, 185L/A, or 181L/A-185L/A in pUL56 resulted in a deficiency in pUL56-induced BAG3 degradation. In addition, the pUL56 C-terminal mutants that lost Golgi retention abrogated pUL56-induced BAG3 degradation, which indicates a Golgi retention-dependent manner. Strikingly, BAG3 was not observed to be degraded in either wild-type or UL56-deleted PRV infected cells as compared to mock infected ones, whereas the additional two adjacent BAG3 cleaved products were found in the infected cells in a species-specific manner. Overexpression of BAG3 significantly suppressed PRV proliferation, while knockdown of BAG3 resulted in increased viral yields in HEK293T cells. Thus, these data indicated a negative regulation role of BAG3 during PRV lytic infection. Collectively, our findings revealed a novel molecular mechanism on host protein degradation induced by PRV pUL56. Moreover, we identified BAG3 as a host restricted protein during PRV lytic infection in cells.

Viral Coinfection Replaces Effects of Suilysin on Streptococcus suis Adherence to and Invasion of Respiratory Epithelial Cells Grown under Air-Liquid Interface Conditions.

Streptococcus suis is an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis of S. suis infections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence of S. suis, with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin of S. suis contributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt) S. suis strain and a suilysin-negative S. suis mutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wt S. suis strain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negative S. suis.

Two Residues in NSP9 Contribute to the Enhanced Replication and Pathogenicity of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) possesses greater replicative capacity and pathogenicity than classical PRRSV. However, the factors that lead to enhanced replication and pathogenicity remain unclear. In our study, an alignment of all available full-length sequences of North American-type PRRSVs (n = 204) revealed two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in nonstructural protein 9. Next, a series of mutant viruses with either single or double amino acid replacements were generated from HP-PRRSV HuN4 and classical PRRSV CH-1a infectious cDNA clones. Deletion of either of the amino acids led to a complete loss of virus viability. In both Marc-145 and porcine alveolar macrophages, the replicative efficiencies of mutant viruses based on HuN4 were reduced compared to the parent, whereas the replication level of CH-1a-derived mutant viruses was increased. Plaque growth assays showed clear differences between mutant and parental viruses. In infected piglets, the pathogenicity of HuN4-derived mutant viruses, assessed through clinical symptoms, viral load in sera, histopathology examination, and thymus atrophy, was reduced. Our results indicate that the amino acids at positions 519 and 544 in NSP9 are involved in the replication efficiency of HP-PRRSV and contribute to enhanced pathogenicity. This study is the first to identify specific amino acids involved in PRRSV replication or pathogenicity. These findings will contribute to understanding the molecular mechanisms of PRRSV replication and pathogenicity, leading to better therapeutic and prognostic options to combat the virus.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a significant threat to the global pig industry. Highly pathogenic PRRSV (HP-PRRSV) first emerged in China in 2006 and has subsequently spread across Asia, causing considerable damage to local economies. HP-PRRSV strains possess a greater replication capacity and higher pathogenicity than classical PRRSV strains, although the mechanisms that underlie these characteristics are unclear. In the present study, we identified two mutations in HP-PRRSV strains that distinguish them from classical PRRSV strains. Further experiments that swapped the two mutations in an HP-PRRSV strain and a classical PRRSV strain demonstrated that they are involved in the replication efficiency of the virus and its virulence. Our findings have important implications for understanding the molecular mechanisms of PRRSV replication and pathogenicity and also provide new avenues of research for the study of other viruses.

CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of ≤3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.-Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

A GFP-tagged version of the pseudorabies virus protein UL56 localizes to the Golgi and trans-Golgi network through a predicted C-terminal leucine-rich helix in transfected cells.

BACKGROUND: Pseudorabies virus (PRV) protein UL56 (pUL56) has been implicated in viral dissemination and virulence in vivo. However, the properties of PRV pUL56 remain largely unknown. In the present study, we aim to investigate the subcellular localization of pUL56 and the underlying molecular basis in transfected cells. METHODS: Constructs of N-terminal green fluorescent protein (GFP) fused pUL56 and its truncations were employed for investigating subcellular localization and further identifying amino acids crucial for pUL56 localization in transfected Vero cells. Finally, the identified amino acids were replaced with alanine for confirming if these mutations could impair the specific localization of pUL56. RESULTS: The pUL56 predominantly localized at the Golgi and trans-Golgi network (TGN) through its predicted C-terminal transmembrane helix in transfected Vero cells. A Golgi-associated protein Rab6a, independent of interaction with pUL56, was significantly downregulated by pUL56. Further, we found three truncated pUL56 C-terminal fragments (174-184, 175-185 and 191-195) could restrict GFP in the perinuclear region, respectively. Within these truncations, the 174proline (P), 181leucine (L), 185L and 191L were essential for maintaining perinuclear accumulation, thus suggesting an important role of leucine. Alanine (A) mutagenesis assays were employed to generate a series of pUL56 C-terminal mutants on the basis of leucine. Finally, a pUL56 mutant M10 (174P/A-177L/A-181L/A-185L/A-191L/A-194L/A-195I/A-196-197L/A-200L/A) lost Golgi-TGN localization. Thus, our data revealed that the leucine-rich transmembrane helix was responsible for pUL56 Golgi-TGN localization and retention, probably through specific intracellular membrane insertion. CONCLUSION: Our data indicated that the C-terminal transmembrane helix was responsible for the Golgi-TGN localization of pUL56. In addition, the leucines within C-terminal transmembrane helix were essential for maintaining pUL56 Golgi-TGN retention in cells. Further, the pUL56 can induce downregulation of Golgi-associated protein Rab6a.