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.

The N-glycosylation at positions 652 and 661 of viral spike protein negatively modulates porcine deltacoronavirus entry.

N-glycosylation is a highly conserved glycan modification that plays crucial roles in various physiological processes, including protein folding, trafficking, and signal transduction. Porcine deltacoronavirus (PDCoV) poses a newly emerging threat to the global porcine industry. The spike protein of PDCoV exhibits a high level of N-glycosylation; however, its role in viral infection remains poorly understood. In this study, we applied a lentivirus-based entry reporter system to investigate the role of N-glycosylation on the viral spike protein during PDCoV entry stage. Our findings demonstrate that N-glycosylation at positions 652 and 661 of the viral spike protein significantly reduces the infectivity of PDCoV pseudotyped virus. Overall, our results unveil a novel function of N-glycosylation in PDCoV infection, highlighting its potential for facilitating the development of antiviral strategies.

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.

Reviving Bistable Perception in Patients With Depression by Decreasing the Overestimation of Prior Precision.

Slower perceptual alternations, a notable perceptual effect observed in psychiatric disorders, can be alleviated by antidepressant therapies that affect serotonin levels in the brain. While these phenomena have been well documented, the underlying neurocognitive mechanisms remain to be elucidated. Our study bridges this gap by employing a computational cognitive approach within a Bayesian predictive coding framework to explore these mechanisms in depression. We fitted a prediction error (PE) model to behavioral data from a binocular rivalry task, uncovering that significantly higher initial prior precision and lower PE led to a slower switch rate in patients with depression. Furthermore, serotonin-targeting antidepressant treatments significantly decreased the prior precision and increased PE, both of which were predictive of improvements in the perceptual alternation rate of depression patients. These findings indicated that the substantially slower perception switch rate in patients with depression was caused by the greater reliance on top-down priors and that serotonin treatment's efficacy was in its recalibration of these priors and enhancement of PE. Our study not only elucidates the cognitive underpinnings of depression, but also suggests computational modeling as a potent tool for integrating cognitive science with clinical psychology, advancing our understanding and treatment of cognitive impairments in depression.

Identification of a linear B-cell epitope on the "puff" loop of the Senecavirus A VP2 protein involved in receptor binding.

Senecavirus A (SVA) is an important emerging swine pathogen that causes vesicular lesions in swine and acute death in newborn piglets. VP2 plays a significant role in the production of antibodies, which can be used in development of diagnostic tools and vaccines. Herein, the aim of the current study was to identify B-cell epitopes (BCEs) of SVA for generation of epitope-based SVA marker vaccine. Three monoclonal antibodies (mAbs), named 2E4, 1B8, and 2C7, against the SVA VP2 protein were obtained, and two novel linear BCEs, 177SLGTYYR183 and 266SPYFNGL272, were identified by peptide scanning. The epitope 177SLGTYYR183 was recognized by the mAb 1B8 and was fully exposed on the VP2 surface, and alanine scanning analysis revealed that it contained a high continuity of key amino acids. Importantly, we confirmed that 177SLGTYYR183 locates on "the puff" region within the VP2 EF loop, and contains three key amino acid residues involved in receptor binding. Moreover, a single mutation, Y182A, blocked the interaction of the mutant virus with the mAb 1B8, indicating that this mutation is the pivotal point for antibody recognition. In summary, the BCEs that identified in this study could be used to develop diagnostic tools and an epitope-based SVA marker vaccine.

Pseudorabies virus gM and its homologous proteins in herpesviruses induce mitochondria-related apoptosis involved in viral pathogenicity.

Apoptosis is a critical host antiviral defense mechanism. But many viruses have evolved multiple strategies to manipulate apoptosis and escape host antiviral immune responses. Herpesvirus infection regulated apoptosis; however, the underlying molecular mechanisms have not yet been fully elucidated. Hence, the present study aimed to study the relationship between herpesvirus infection and apoptosis in vitro and in vivo using the pseudorabies virus (PRV) as the model virus. We found that mitochondria-dependent apoptosis was induced by PRV gM, a late protein encoded by PRV UL10, a virulence-related gene involved in enhancing PRV pathogenicity. Mechanistically, gM competitively combines with BCL-XL to disrupt the BCL-XL-BAK complex, resulting in BCL-2-antagonistic killer (BAK) oligomerization and BCL-2-associated X (BAX) activation, which destroys the mitochondrial membrane potential and activates caspase-3/7 to trigger apoptosis. Interestingly, similar apoptotic mechanisms were observed in other herpesviruses (Herpes Simplex Virus-1 [HSV-1], human cytomegalovirus [HCMV], Equine herpesvirus-1 [EHV-1], and varicella-zoster virus [VZV]) driven by PRV gM homologs. Compared with their parental viruses, the pathogenicity of PRV-ΔUL10 or HSV-1-ΔUL10 in mice was reduced with lower apoptosis and viral replication, illustrating that UL10 is a key virulence-related gene in PRV and HSV-1. Consistently, caspase-3 deletion also diminished the replication and pathogenicity of PRV and HSV-1 in vitro and in mice, suggesting that caspase-3-mediated apoptosis is closely related to the replication and pathogenicity of PRV and HSV-1. Overall, our findings firstly reveal the mechanism by which PRV gM and its homologs in several herpesviruses regulate apoptosis to enhance the viral replication and pathogenicity, and the relationship between gM-mediated apoptosis and herpesvirus pathogenicity suggests a promising approach for developing attenuated live vaccines and therapy for herpesvirus-related diseases.

QueryTrack: Joint-Modality Query Fusion Network for RGBT Tracking.

Existing RGB-Thermal trackers usually treat intra-modal feature extraction and inter-modal feature fusion as two separate processes, therefore the mutual promotion of extraction and fusion is neglected. Then, the complementary advantages of RGB-T fusion are not fully exploited, and the independent feature extraction is not adaptive to modal quality fluctuation during tracking. To address the limitations, we design a joint-modality query fusion network, in which the intra-modal feature extraction and the inter-modal fusion are coupled together and promote each other via joint-modality queries. The queries are initialized based on the multimodal features of the current frame, making the subsequent fusion adaptive to modal quality fluctuation during tracking. Then the joint-modality query fusion (JQF) utilizes the queries to interact with RGB-T features, allowing the intra-modal enhancement and the inter-modal interactions to be unified for mutual promotion. In this way, JQF can distinguish and enhance the complementary modality features, while filtering out redundant information. For real-time tracking, we propose regional cross-attention for cross-modal interactions to reduce computational cost. Our end-to-end tracker sets a new state-of-the-art performance on multiple RGBT tracking benchmarks including LasHeR, VTUAV, RGBT234 and GTOT, while running at a real-time speed.

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.

Human MARCH1, 2, and 8 block Ebola virus envelope glycoprotein cleavage via targeting furin P domain.

Membrane-associated RING-CH (MARCH) family proteins were recently reported to inhibit viral replication through multiple modes. Previous work showed that human MARCH8 blocked Ebola virus (EBOV) glycoprotein (GP) maturation. Our study here demonstrates that human MARCH1 and MARCH2 share a similar pattern to MARCH8 in restricting EBOV GP-pseudotyped viral infection. Human MARCH1 and MARCH2 retain EBOV GP at the trans-Golgi network, reduce its cell surface display, and impair EBOV GP-pseudotyped virions infectivity. Furthermore, we uncover that the host proprotein convertase furin could interact with human MARCH1/2 and EBOV GP intracellularly. Importantly, the furin P domain is verified to be recognized by MARCH1/2/8, which is critical for their blocking activities. Besides, bovine MARCH2 and murine MARCH1 also impair EBOV GP proteolytic processing. Altogether, our findings confirm that MARCH1/2 proteins of different mammalian origins showed a relatively conserved feature in blocking EBOV GP cleavage, which could provide clues for subsequent MARCHs antiviral studies and may facilitate the development of novel strategies to antagonize enveloped virus infection.

Prevalence and genetic evolution of porcine reproductive and respiratory syndrome virus in commercial fattening pig farms in China.

BACKGROUND: To investigate the prevalence and evolution of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) at commercial fattening pig farms, a total of 1397 clinical samples were collected from a single fattening cycle at seven pig farms in five provinces of China from 2020 to 2021. RESULTS: The RT‒PCR results revealed that PRRSV was present on all seven farms, and the percentage of PRRSV-positive individuals was 17.54-53.33%. A total of 344 partial NSP2 gene sequences and 334 complete ORF5 gene sequences were obtained from the positive samples. The statistical results showed that PRRSV-2 was present on all seven commercial fattening farms, and PRRSV-1 was present on only one commercial fattening farm. A total of six PRRSV-2 subtypes were detected, and five of the seven farms had two or more PRRSV-2 subtypes. L1.8 (L1C) PRRSV was the dominant epidemic strain on five of the seven pig farms. Sequence analysis of L1.8 (L1C) PRRSV from different commercial fattening pig farms revealed that its consistency across farms varied substantially. The amino acid alignment results demonstrated that there were 131 aa discontinuous deletions in NSP2 between different L1.8 (L1C) PRRSV strains and that the GP5 mutation in L1.8 (L1C) PRRSV was mainly concentrated in the peptide signal region and T-cell epitopes. Selection pressure analysis of GP5 revealed that the use of the PRRSV MLV vaccine had no significant episodic diversifying effect on L1.8 (L1C) PRRSV. CONCLUSION: PRRSV infection is common at commercial fattening pig farms in China, and the percentage of positive individuals is high. There are multiple PRRSV subtypes of infection at commercial fattening pig farms in China. L1.8 (L1C) is the main circulating PRRSV strain on commercial fattening pig farms. L1.8 (L1C) PRRSV detected at different commercial fattening pig farms exhibited substantial differences in consistency but similar molecular characteristics. The pressure on the GP5 of L1.8 (L1C) PRRSV may not be directly related to the use of the vaccines.

Improved detection sensitivity of anti-PRV variant antibodies through preparation of anti-gB and anti-gE monoclonal antibodies and development of blocking ELISAs.

Since 2011, PRV has resurged in China and is characterized by a mutated strain with significant alterations in antigenicity and virulence. Therefore, we hypothesized that antibody detection kits based on classic PRV strains may have limitations in detecting PRV variants. For more sensitive antibody detection of PRV variants, two MABs targeting the gB and gE proteins were developed. IFA revealed that these MABs exhibited strong reactivity toward both classic and variant PRV strains. MAB-gE recognizes a novel conserved linear B-cell epitope (41PSAEVWD47), while MAB-gB recognizes a conformational B-cell epitope. The binding of both MABs was effectively inhibited in the PRV-positive pig blood samples. Accordingly, we established blocking-ELISAs to detect anti-PRV gB and gE antibodies, which achieved higher sensitivity than commercial kits. Moreover, the clinical serum samples results of our method and that of IFA were in high agreement, and our test results had a higher coincidence rate than that of a commercial kit. Assessing antibody levels by our methods at various times following immunization and challenge accurately reflected the trend of antibody-level changes and revealed the conversion to positive antibody status before the commercial kit. Our method is crucial for monitoring PRV infections, assessing immune responses, and controlling disease.

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.

The emerging roles of MARCH8 in viral infections: A double-edged Sword.

The host cell membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, regulates intracellular turnover of many transmembrane proteins and shows potent antiviral activities. Generally, 2 antiviral modes are performed by MARCH8. On the one hand, MARCH8 catalyzes viral envelope glycoproteins (VEGs) ubiquitination and thus leads to their intracellular degradation, which is the cytoplasmic tail (CT)-dependent (CTD) mode. On the other hand, MARCH8 traps VEGs at some intracellular compartments (such as the trans-Golgi network, TGN) but without inducing their degradation, which is the cytoplasmic tail-independent (CTI) mode, by which MARCH8 hijacks furin, a cellular proprotein convertase, to block VEGs cleavage. In addition, the MARCH8 C-terminal tyrosine-based motif (TBM) 222YxxL225 also plays a key role in its CTI antiviral effects. In contrast to its antiviral potency, MARCH8 is occasionally hijacked by some viruses and bacteria to enhance their invasion, indicating a duplex role of MARCH8 in host pathogenic infections. This review summarizes MARCH8's antiviral roles and how viruses evade its restriction, shedding light on novel antiviral therapeutic avenues.

Protective Efficacy of a Candidate Live-Attenuated Vaccine Derived from the SD-R Strain against NADC34-like Porcine Reproductive and Respiratory Syndrome Virus.

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) strains were first detected in China in 2017 and became major circulating strains in 2021. Our previous study showed that the live-attenuated vaccine candidate SD-R strain could provide broad cross-protection against different NADC30-like PRRSVs (sublineage 1.8). However, the protective effect of SD-R against NADC34-like PRRSV is unclear. Here, a novel NADC34-like PRRSV, LNTZJ1341-2012, was isolated from a pig farm experiencing disease in 2020. Sequence analysis revealed that LNTZJ1341-2012 belonged to PRRSV-2 sublineage 1.5, exhibited the same Nsp2 amino-acid deletion characteristics as IA/2014/NADC34, and had not recombined with other strains. Additionally, a good challenge model was established to evaluate the protection afforded by the candidate SD-R vaccine against infection with a representative NADC34-like strain (LNTZJ1341-2012). The control piglets in the challenge experiment displayed clinical signs typical of PRRSV infection, including transient fever, high viremia, mild clinical symptoms, and histopathological changes in the lungs and submaxillary lymph nodes. In contrast, SD-R vaccination significantly reduced serum and lung tissue viral loads, and vaccinated piglets did not show any clinical symptoms or histopathological changes. Our results demonstrated that LNTZJ1341-2012 is a mildly virulent NADC34-like PRRSV and that the live-attenuated vaccine SD-R can prevent the onset of clinical signs upon challenge with the NADC34-like PRRSV LNTZJ1341-2012 strain, indicating that SD-R is a promising vaccine candidate for the swine industry.

Corrigendum: The critical role of PARPs in regulating innate immune responses.

[This corrects the article DOI: 10.3389/fimmu.2021.712556.].

A Shadow Imaging Bilinear Model and Three-Branch Residual Network for Shadow Removal.

The current shadow removal pipeline relies on the detected shadow masks, which have limitations for penumbras and tiny shadows, and results in an excessively long pipeline. To address these issues, we propose a shadow imaging bilinear model and design a novel three-branch residual (TBR) network for shadow removal. Our bilinear model reveals the single-image shadow removal process and can explain why simply increasing the brightness of shadow areas cannot remove shadows without artifacts. We considerably shorten the shadow removal pipeline by modeling illumination compensation and developing a single-stage shadow removal network without additional detection and refinement networks. Specifically, our network consists of three task branches, i.e., shadow image reconstruction, shadow matte estimation, and shadow removal. To merge these three branches and enhance the shadow removal branch, we design a model-based TBR module. Multiple TBR modules are cascaded to generate an intensive information flow and facilitate feature integration among the three branches. Thus, our network ensures the fidelity of nonshadow areas and restores the light intensity of shadow areas through three-branch collaboration. Extensive experiments demonstrate that our method outperforms the state-of-the-art methods. The model and code are available at https://github.com/nachifur/TBRNet.

Genome editing of pseudorabies virus in the CRISPR/Cas9 era: a mini-review.

Pseudorabies virus (PRV) is an important swine virus that has a significant impact on the global swine industry. PRV is a member of the herpesvirus family, specifically the alphaherpesvirus subfamily, and has been extensively utilized as a prototype herpesvirus. Notably, recent studies have reported that PRV sporadically spills over into humans. The PRV genome is approximately 150 kb in size and is difficult to manipulate at the genomic level. The development of clustered regularly interspaced short palindromic repeat-associated protein (CRISPR/Cas9) technology has revolutionized PRV genome editing. CRISPR/Cas9 has been widely used in the construction of reporter viruses, knock-out/knock-in of genes of interest, single virus tracking and antiviral strategies. Most importantly, for vaccine development, virulence gene knockout PRV vaccine candidates can be obtained within 2 weeks using CRISPR/Cas9. In this mini-review, we provide a concise overview of the application of CRISPR/Cas9 in PRV research and mainly share our experience with methods for efficiently editing the PRV genome. Through this review, we hope to give researchers better insight into the genome editing of pseudorabies virus.

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.