MARCH8 inhibits pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in the trans-Golgi network.

The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack MARCH8 to promote virus replication, highlighting its dual role in the viral lifecycle. Most studies on MARCH8 have focused on RNA viruses, leaving its role in DNA viruses largely unexplored. Pseudorabies virus (PRV) is a large DNA virus that poses a potential threat to humans. In this study, we found that MARCH8 inhibited PRV replication at the cell-to-cell fusion stage. Interestingly, our findings proved that MARCH8 blocks gB cleavage by recruiting furin but this activity does not inhibit viral infection in vitro. Furthermore, we confirmed that MARCH8 inhibits cell-to-cell fusion independent of its E3 ubiquitin ligase activity but dependent on the interaction with the cell-to-cell fusion complex (gB, gD, gH, and gL). Finally, we discovered that the distribution of the cell-to-cell fusion complex is significantly altered and trapped within the trans-Golgi network. Overall, our results indicate that human MARCH8 acts as a potent antiviral host factor against PRV via trapping the cell-to-cell fusion complex in the trans-Golgi network.

MARCH1 and MARCH2 inhibit pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in trans-Golgi network.

The membrane-associated RING-CH (MARCH) family of proteins are members of the E3 ubiquitin ligase family and are essential for a variety of biological functions. Currently, MARCH proteins are discovered to execute antiviral functions by directly triggering viral protein degradation or blocking the furin cleavage of viral class I fusion proteins. Here, we report a novel antiviral mechanism of MARCH1 and MARCH2 (MARCH1/2) in the replication of Pseudorabies virus (PRV), a member of the Herpesviridae family. We discovered MARCH1/2 restrict PRV replication at the cell-to-cell fusion step. Furthermore, MARCH1/2 block gB cleavage, and this is dependent on their E3 ligase activity. Interestingly, the blocking of gB cleavage by MARCH1/2 does not contribute to their antiviral activity in vitro. We discovered that MARCH1/2 are associated with the cell-to-cell fusion complex of gB, gD, gH, and gL and trap these viral proteins in the trans-Golgi network (TGN) rather than degrading them. Overall, we conclude that MARCH1/2 inhibit PRV by trapping the viral cell-to-cell fusion complex in TGN.

Minor envelope proteins from GP2a to GP4 contribute to the spread pattern and yield of type 2 PRRSV in MARC-145 cells.

In China, porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are widely used. These vaccines, which contain inactivated and live attenuated vaccines (LAVs), are produced by MARC-145 cells derived from the monkey kidney cell line. However, some PRRSV strains in MARC-145 cells have a low yield. Here, we used two type 2 PRRSV strains (CH-1R and HuN4) to identify the genes responsible for virus yield in MARC-145 cells. Our findings indicate that the two viruses have different spread patterns, which ultimately determine their yield. By replacing the viral envelope genes with a reverse genetics system, we discovered that the minor envelope proteins, from GP2a to GP4, play a crucial role in determining the spread pattern and yield of type 2 PRRSV in MARC-145 cells. The cell-free transmission pattern of type 2 PRRSV appears to be more efficient than the cell-to-cell transmission pattern. Overall, these findings suggest that GP2a to GP4 contributes to the spread pattern and yield of type 2 PRRSV.

Novel technologies are turning a dream into reality: conditionally replicating viruses as vaccines.

Conditionally replicating viruses (CRVs) are a type of virus with one or more essential gene functions that are impaired resulting in the disruption of viral genome replication, protein synthesis, or virus particle assembly. CRVs can replicate only if the deficient essential genes are supplied. CRVs are widely used in biomedical research, particularly as vaccines. Traditionally, CRVs are generated by creating complementary cell lines that provide the impaired genes. With the development of biotechnology, novel techniques have been invented to generate CRVs, such as targeted protein degradation (TPD) technologies and premature termination codon (PTC) read-through technologies. The advantages and disadvantages of these novel technologies are discussed. Finally, we provide perspectives on what challenges need to be overcome for CRVs to reach the market.

Protective efficacy of a candidate live attenuated vaccine derived from the SD-R strain of lineage 1 porcine reproductive and respiratory syndrome virus against a lethal challenge with HP-PRRSV HuN4 in piglets.

IMPORTANCE: Both highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and NADC30-like PRRSV have caused tremendous economic losses to the Chinese pig industry. In this study, a good challenge model was established to evaluate the protection afforded by the candidate SD-R vaccine against infection with a representative HP-PRRSV strain (HuN4). The control piglets in the challenge experiment displayed obvious clinical symptoms of PRRSV infection, with a mortality rate up to 40%. In contrast, all the piglets in the vaccinated challenged group survived, and only some pigs had transient fever. The daily gain of SD-R immunized group piglets was significantly increased, and the pathological changes were significantly reduced. In addition, the viral replication levels in the serum of the immunized group were significantly lower than those of the challenged control group. The live attenuated vaccine SD-R strain can provide protection against HP-PRRSV challenge, indicating that the SD-R strain is a promising vaccine candidate for use in the swine industry.

Genomic characteristics of a novel emerging PRRSV branch in sublineage 8.7 in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused serious economic losses to the pig industry worldwide. During the continuous monitoring of PRRSV, a new PRRSV strain type with novel characteristics was first identified in three different regions of Shandong Province. These strains presented a novel deletion pattern (1 + 8 + 1) in the NSP2 region and belonged to a new branch in sublineage 8.7 based on the ORF5 gene phylogenetic tree. To further study the genomic characteristics of the new-branch PRRSV, we selected a sample from each of the three farms for whole-genome sequencing and sequence analysis. Based on the phylogenetic analysis of the whole genome, these strains formed a new independent branch in sublineage 8.7, which showed a close relationship with HP-PRRSV and intermediate PRRSV according to nucleotide and amino acid homology but displayed a completely different deletion pattern in NSP2. Recombinant analysis showed that these strains presented similar recombination patterns, all of which involved recombination with QYYZ in the ORF3 region. Furthermore, we found that the new-branch PRRSV retained highly consistent nucleotides at positions 117-120 (AGTA) of a quite conserved motif in the 3'-UTR; showed similar deletion patterns in the 5'-UTR, 3'-UTR and NSP2; retained characteristics consistent with intermediate PRRSV and exhibited a gradual evolution trend. The above results showed that the new-branch PRRSV strains may have the same origin and be similar to HP-PPRSV also evolved from intermediate PRRSV, but are distinct strains that evolved simultaneously with HP-PRRSV. They persist in some parts of China through rapid evolution, recombine with other strains and have the potential to become epidemic strains. The monitoring and biological characteristics of these strains should be further studied.

New Characterization of Multi-Drug Resistance of Streptococcus suis and Biofilm Formation from Swine in Heilongjiang Province of China.

The aim of this study was to investigate the antimicrobial resistance profiles and genotypes of Streptococcus suis in Heilongjiang Province, China. A total of 29 S. suis were isolated from 332 samples collected from 6 pig farms. The results showed that serotypes 2, 4 and 9 were prevalent, and all the clinical isolates were resistant to at least two antibacterial drugs. The most resisted drugs were macrolides, and the least resisted drugs were fluoroquinolones. Resistant genes ermB and aph (3')-IIIa were highly distributed among the isolates, with the detection rates of 79.31% and 75.86%. The formation of biofilm could be observed in all the isolated S. suis, among which D-1, LL-1 and LL-3 strains formed stronger biofilm structure than other strains. The results indicate that S. suis in Heilongjiang Province presents a multi-drug resistance to commonly used antimicrobial drugs, which was caused by the same target gene, the dissemination of drug resistance genes, and bacterial biofilm.

A novel viral vaccine platform based on engineered transfer RNA.

In recent years, an increasing number of emerging and remerging virus outbreaks have occurred and the rapid development of vaccines against these viruses has been crucial. Controlling the replication of premature termination codon (PTC)-containing viruses is a promising approach to generate live but replication-defective viruses that can be used for potent vaccines. Here, we used anticodon-engineered transfer RNAs (ACE-tRNAs) as powerful precision switches to control the replication of PTC-containing viruses. We showed that ACE-tRNAs display higher potency of reading through PTCs than genetic code expansion (GCE) technology. Interestingly, ACE-tRNA has a site preference that may influence its read-through efficacy. We further attempted to use ACE-tRNAs as a novel viral vaccine platform. Using a human immunodeficiency virus type 1 (HIV-1) pseudotyped virus as an RNA virus model, we found that ACE-tRNAs display high potency for read-through viral PTCs and precisely control their production. Pseudorabies virus (PRV), a herpesvirus, was used as a DNA virus model. We found that ACE-tRNAs display high potency for reading through viral PTCs and precisely controlling PTC-containing virus replication. In addition, PTC-engineered PRV completely attenuated and lost virulence in mice in vivo, and immunization with PRV containing a PTC elicited a robust immune response and provided complete protection against wild-type PRV challenge. Overall, replication-controllable PTC-containing viruses based on ACE-tRNAs provide a new strategy to rapidly attenuate virus infection and prime robust immune responses. This technology can be used as a platform for rapidly developing viral vaccines in the future.

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.

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.

Recombinant characteristics, pathogenicity, and viral shedding of a novel PRRSV variant derived from twice inter-lineage recombination.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a significant threat to the global pig industry. In this study, a novel recombinant PRRSV, SD043, was isolated from a pig farm experiencing disease in 2019. Phylogenetic analysis revealed that SD043 belonged to lineage 1 of PRRSV-2 while recombination analyses revealed that it is a recombinant virus from lineage 1 and lineage 8 strains. Based on further analysis, SD043 underwent recombination twice. Pathogenicity studies revealed that SD043 causes mild clinical symptoms, thymus atrophy, and severe histopathological lesions in the lungs. Notably, virus shedding in SD043-infected piglets was detectable at 10 days post-inoculation with a high viral load in the respiratory or digestive tract, indicating that the recombinant PRRSV appears to shed higher numbers of virus. Furthermore, genomic surveillance based on all available PRRSVs circulating in Shandong province revealed an increasing increase in recombinant PRRSV since 2015, with the recombinant pattern (between lineages 1 and 8) being the same as that of SD043. These findings enable a better understanding of the process of twice recombination and virus shedding of recombinant PRRSV and can strengthen the prevention and control of the PRRSV epidemic.

Characterization of Two Immunodominant Antigenic Peptides in NSP2 of PRRSV-2 and Generation of a Marker PRRSV Strain Based on the Peptides.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread disease with great economic importance in the pig industry. Although vaccines against the PRRS virus (PRRSV) have been employed for more than 20 years, differentiating infected from vaccinated animals remains challenging. In this study, all 907 non-structural protein 2 (NSP2) full-length sequences of PRRSV-2 available from GenBank were aligned. Two peptides, at positions 562-627 (m1B) and 749-813 (m2B) of NSP2, were selected, and their potential for use in differential diagnosis was assessed. Both m1B and m2B were recognized by PRRSV-positive pig serum in peptide-coated enzyme-linked immunosorbent assays. Further epitope identification yielded five overlapping short peptides for the immunodominant regions of m1B and m2B. Using the infectious clone of PRRSV HuN4-F112 as a template, the deletion mutants, rHuN4-F112-m1B, rHuN4-F112-m2B, and rHuN4-F112-C5-m1B-m2B, were generated and successfully rescued in Marc-145 cells. Growth kinetics revealed that the deletion of m1B and m2B did not significantly affect virus replication. Hence, m1B and m2B show potential as molecular markers for developing a PRRSV vaccine.

CRISPR-Cas13d Exhibits Robust Antiviral Activity Against Seneca Valley Virus.

In recent years, Seneca Valley virus (SVV) as a newly identified pathogen of porcine vesicular disease spread quickly and has posed a potential threat to the swine industry in several countries resulting in economic losses. Considering the evolution of SVV, attention should be given to controlling SVV epidemics. So far there are no commercial vaccines or drugs available to combat SVV. Therefore, development of strategies for preventing and controlling SVV infection should be taken into account. In the current study, we evaluated whether the CRISPR-Cas13d system could be used as a powerful tool against SVV infection. Besides, selected crRNAs showed different capacity against SVV infection. Our study suggests the CRISPR-Cas13d system significantly inhibited SVV replication and exhibited potent anti-SVV activity. This knowledge may provide a novel alternative strategy to control epidemics of SVV in the future.

Generation of Premature Termination Codon (PTC)-Harboring Pseudorabies Virus (PRV) via Genetic Code Expansion Technology.

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.

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.

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.

Evasion of Antiviral Innate Immunity by Porcine Reproductive and Respiratory Syndrome Virus.

Innate immunity is the front line for antiviral immune responses and bridges adaptive immunity against viral infections. However, various viruses have evolved many strategies to evade host innate immunity. A typical virus is the porcine reproductive and respiratory syndrome virus (PRRSV), one of the most globally devastating viruses threatening the swine industry worldwide. PRRSV engages several strategies to evade the porcine innate immune responses. This review focus on the underlying mechanisms employed by PRRSV to evade pattern recognition receptors signaling pathways, type I interferon (IFN-α/ß) receptor (IFNAR)-JAK-STAT signaling pathway, and interferon-stimulated genes. Deciphering the antiviral immune evasion mechanisms by PRRSV will enhance our understanding of PRRSV's pathogenesis and help us to develop more effective methods to control and eliminate PRRSV.

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.

Identification of interaction domains in the pseudorabies virus ribonucleotide reductase large and small subunits.

Alphaherpesviral ribonucleotide reductase (RNR) is composed of large (pUL39, RR1) and small (pUL40, RR2) subunits. This enzyme can catalyze conversion of ribonucleotide to deoxynucleotide diphosphates that are further phosphorylated into deoxynucleotide triphosphate (dNTPs). The dNTPs are substrates for de novo viral DNA synthesis in infected host cells. The enzymatic activity of RNR depends on association between RR1 and RR2. However, the molecular basis underlying alphaherpesviral RNR complex formation is still largely unknown. In the current study, we investigated the pseudorabies virus (PRV) RNR interaction domains in pUL39 and pUL40. The interaction of pUL39 and pUL40 was identified by co-immunoprecipitation (co-IP) and colocalization analyses. Furthermore, the interaction amino acid (aa) domains in pUL39 and pUL40 were mapped using a series of truncated proteins. Consequently, the 90-210 aa in pUL39 was identified to be responsible for the interaction with pUL40. In turn, the 66-152, 218-258 and 280-303 aa in pUL40 could interact with pUL39, respectively. Deletion of 90-210 aa in pUL39 completely abrogated the interaction with pUL40. Deletion of 66-152, 218-258 and 280-303 aa in pUL40 remarkably weakened the interaction with pUL39, whereas a weak interaction could still be observed. Amino acid sequence alignments showed that the interaction domains identified in PRV pUL39/pUL40 were relatively non-conserved among the selected RNR subunits in alphaherpesviruses HSV1, HSV2, HHV3(VZV), BHV1, EHV1 and DEV. However, they were relatively conserved among PRV, HSV1 and HSV2. Collectively, our findings provided some molecular targets for inhibition of pUL39-pUL40 interaction to antagonize viral replication in PRV infected hosts.