Isolation and Characterization of a Novel Recombinant Classical Pseudorabies Virus in the Context of the Variant Strains Pandemic in China.

Pseudorabies virus (PRV) variants were discovered in immunized pigs in Northern China and have become the dominant strains since 2011, which caused huge economic losses. In this study, a classical PRV strain was successfully isolated in a PRV gE positive swine farm. The complete genome sequence was obtained using a high-throughput sequencing method and the virus was named JS-2020. The nucleotide homology analysis and phylogenetic tree based on complete genome sequences or gC gene showed that the JS-2020 strain was relatively close to the classical Ea strain in genotype II clade. However, a large number of amino acid variations occurred in the JS-2020 strain compared with the Ea strain, including multiple immunogenic and virulence-related genes. In particular, the gE protein of JS-2020 was similar to earlier Chinese PRV strains without Aspartate insertion. However, the amino acid variations analysis based on major immunogenic and virulence-related genes showed that the JS-2020 strain was not only homologous with earlier PRV strains, but also with strains isolated in recent years. Moreover, the JS-2020 strain was identified as a recombinant between the GXGG-2016 and HLJ-2013 strains. The pathogenicity analysis proved that the PRV JS-2020 strain has typical neurogenic infections and a strong pathogenicity in mice. Together, a novel recombinant classical strain was isolated and characterized in the context of the PRV variant pandemic in China. This study provided some valuable information for the study of the evolution of PRV in China.

Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

LGP2 Promotes Type I Interferon Production To Inhibit PRRSV Infection via Enhancing MDA5-Mediated Signaling.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the global pig industry, which modulates the host's innate antiviral immunity to achieve immune evasion. RIG-I-like receptors (RLRs) sense viral RNA and activate the interferon signaling pathway. LGP2, a member of the RLR family, plays an important role in regulating innate immunity. However, the role of LGP2 in virus infection is controversial. Whether LGP2 has a role during infection with PRRSV remains unclear. Here, we found that LGP2 overexpression restrained the replication of PRRSV, while LGP2 silencing facilitated PRRSV replication. LGP2 was prone to interact with MDA5 and enhanced viral RNA enrichment and recognition by MDA5, thus promoting the activation of RIG-I/IRF3 and NF-κB signaling pathways and reinforcing the expression of proinflammatory cytokines and type I interferon during PRRSV infection. Meanwhile, there was a decreased protein expression of LGP2 upon PRRSV infection in vitro. PRRSV Nsp1 and Nsp2 interacted with LGP2 and promoted K63-linked ubiquitination of LGP2, ultimately leading to the degradation of LGP2. These novel findings indicate that LGP2 plays a role in regulating PRRSV replication through synergistic interaction with MDA5. Moreover, targeting LGP2 is responsible for PRRSV immune evasion. Our work describes a novel mechanism of virus-host interaction and provides the basis for preventing and controlling PRRSV. IMPORTANCE LGP2, a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), shows higher-affinity binding to RNA and work synergism with RIG-I or MDA5. However, LGP2 has divergent responses to different viruses, which remains controversial in antiviral immune responses. Here, we present the detailed process of LGP2 in positively regulating the anti-PRRSV response. Upon PRRSV infection, LGP2 was prone to bind to MDA5 and enhanced MDA5 signaling, manifesting the enrichment of viral RNA on MDA5 and the activation of downstream IRF3 and NF-κB, which results in increased proinflammatory cytokines and type I interferon expression, ultimately inhibiting PRRSV at the early stage of infection. Moreover, PRRSV Nsp1 and Nsp2 interacted with LGP2 via ubiquitin-proteasome pathways, thus blocking LGP2-mediated immune response. This research helps us understand the host recognition and innate antiviral response to PRRSV infection by neglected pattern recognition receptors, which sheds light on the detailed mechanism of virus-host interaction.

SLC1A3 facilitates Newcastle disease virus replication by regulating glutamine catabolism.

As obligate intracellular parasites, viruses rely completely on host metabolic machinery and hijack host nutrients for viral replication. Newcastle disease virus (NDV) causes acute, highly contagious avian disease and functions as an oncolytic agent. NDV efficiently replicates in both chicken and tumour cells. However, how NDV reprograms host cellular metabolism for its efficient replication is still ill-defined. We previously identified a significantly upregulated glutamate transporter gene, solute carrier family 1 member 3 (SLC1A3), during NDV infection via transcriptome analysis. To investigate the potential role of SLC1A3 during NDV infection, we first confirmed the marked upregulation of SLC1A3 in NDV-infected DF-1 or A549 cells through p53 and NF-κB pathways. Knockdown of SLC1A3 inhibited NDV infection. Western blot analysis further confirmed that glutamine, but not glutamate, asparagine, or aspartate, was required for NDV replication. Metabolic flux data showed that NDV promotes the decomposition of glutamine into the tricarboxylic acid cycle. Importantly, the level of glutamate and glutaminolysis were reduced by SLC1A3 knockdown, indicating that SLC1A3 propelled glutaminolysis for glutamate utilization and NDV replication in host cells. Taken together, our data identify that SLC1A3 serves as an important regulator for glutamine metabolism and is hijacked by NDV for its efficient replication during NDV infection. These results improve our understanding of the interaction between NDV and host cellular metabolism and lay the foundation for further investigation of efficient vaccines.

Metabolomics Analysis of PK-15 Cells with Pseudorabies Virus Infection Based on UHPLC-QE-MS.

Viruses depend on the metabolic mechanisms of the host to support viral replication. We utilize an approach based on ultra-high-performance liquid chromatography/Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass (UHPLC-QE-MS) to analyze the metabolic changes in PK-15 cells induced by the infections of the pseudorabies virus (PRV) variant strain and Bartha K61 strain. Infections with PRV markedly changed lots of metabolites, when compared to the uninfected cell group. Additionally, most of the differentially expressed metabolites belonged to glycerophospholipid metabolism, sphingolipid metabolism, purine metabolism, and pyrimidine metabolism. Lipid metabolites account for the highest proportion (around 35%). The results suggest that those alterations may be in favor of virion formation and genome amplification to promote PRV replication. Different PRV strains showed similar results. An understanding of PRV-induced metabolic reprogramming will provide valuable information for further studies on PRV pathogenesis and the development of antiviral therapy strategies.

High Pathogenicity of a Chinese NADC34-like PRRSV on Pigs.

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to be prevalent in China since 2018 and became one of the main epidemic strains in some areas of China. Yet, the pathogenicity of NADC34-like PRRSV tested by experimental infection has seldomly been investigated. In this study, we infected pigs with JS2021NADC34 PRRSV, a Chinese NADC34-like PRRSV isolated in Jiangsu province in 2021, to study the pathogenicity of this virus strain. Pigs infected with this virus had lasting fever and reduced body weight with high morbidity and mortality. Histopathological changes, including interstitial pneumonia, lymphocyte depletion, acute hemorrhage, and infiltration of neutrophils in the lymphoid tissues, were observed with the viral proteins detected by immunohistochemistry staining using PRRSV-specific antibody. These results suggested that JS2021NADC34 PRRSV is highly pathogenic to pigs. As it is the latest emerging PRRSV strain in China, the prevalence and pathogenicity of NADC34-like PRRSV need to be further investigated. IMPORTANCE NADC34 PRRSV was initially reported in the United States in 2018. Subsequently, this virus strain spread to other countries, including Peru, South Korea, and China. The virus was first found circulating in Northeast China and then spread to more than 10 provinces in China. NADC34 PRRSV causes severe abortion of sows and high mortality of piglets, which lead to huge economic losses to the Chinese pig industry. However, the pathogenicity of NADC34 PRRSV was rarely experimentally evaluated on pigs. In this study, pigs were infected with JS2021NADC34 PRRSV, a Chinese NADC34-like PRRSV isolated in Jiangsu province in 2021. The infected pigs had lasting fever and reduced body weight with high morbidity and mortality. Interstitial pneumonia, lymphocyte depletion, acute hemorrhage, and infiltration of neutrophils were observed in the lymphoid tissues, and high virus load was proved by immunohistochemistry staining. The above results indicated that NADC34 PRRSV has high pathogenicity on pigs.

Newcastle disease virus degrades SIRT3 via PINK1-PRKN-dependent mitophagy to reprogram energy metabolism in infected cells.

Lacking a self-contained metabolism network, viruses have evolved multiple mechanisms for rewiring the metabolic system of their host to hijack the host's metabolic resources for replication. Newcastle disease virus (NDV) is a paramyxovirus, as an oncolytic virus currently being developed for cancer treatment. However, how NDV alters cellular metabolism is still far from fully understood. In this study, we show that NDV infection reprograms cell metabolism by increasing glucose utilization in the glycolytic pathway. Mechanistically, NDV induces mitochondrial damage, elevated mitochondrial reactive oxygen species (mROS) and ETC dysfunction. Infection of cells depletes nucleotide triphosphate levels, resulting in elevated AMP:ATP ratios, AMP-activated protein kinase (AMPK) phosphorylation, and MTOR crosstalk mediated autophagy. In a time-dependent manner, NDV shifts the balance of mitochondrial dynamics from fusion to fission. Subsequently, PINK1-PRKN-dependent mitophagy was activated, forming a ubiquitin chain with MFN2 (mitofusin 2), and molecular receptor SQSTM1/p62 recognized damaged mitochondria. We also found that NDV infection induces NAD+-dependent deacetylase SIRT3 loss via mitophagy to engender HIF1A stabilization, leading to the switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis. Overall, these studies support a model that NDV modulates host cell metabolism through PINK1-PRKN-dependent mitophagy for degrading SIRT3.Abbreviations: AMPK: AMP-activated protein kinase; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECAR: extracellular acidification rate; hpi: hours post infection LC-MS: liquid chromatography-mass spectrometry; mito-QC: mCherry-GFP-FIS1[mt101-152]; MFN2: mitofusin 2; MMP: mitochondrial membrane potential; mROS: mitochondrial reactive oxygen species; MOI: multiplicity of infection; 2-NBDG: 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose; NDV: newcastle disease virus; OCR: oxygen consumption rate; siRNA: small interfering RNA; SIRT3: sirtuin 3; TCA: tricarboxylic acid; TCID50: tissue culture infective doses.

Isolation and genomic characterization of a Chinese NADC34-like PRRSV isolated from Jiangsu province.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important causative agents to swine industry, which has been epidemic more than 30 years. The emergence and recombination of new virus strains bring great challenges to the prevention and control of PRRSV. In the present study, we reported and characterized a novel PRRSV strain, designated as JS2021NADC34, which was for the first time isolated from clinical samples in Jiangsu province, China. Phylogenetic analysis demonstrated that JS2021NADC34 belonging to sublineage 1.5 of PRRSV-2 and was highly related to NADC34-like strains. Genetically, JS2021NADC34 strain had a continuous 100 aa depletion in NSP2, as compared to VR-2332 strain, which was consistent with most reported NADC34-like strains. Moreover, there were several amino acid substitutions occurred in the antigenic regions of GP2-GP5. Similar to other reported NADC34-like PRRSV in China, JS2021NADC34 had no recombination with other domestic strains, which indicates this sublineage of PRRSV may be directly transported from the United States and have not undergone extensive mutation and recombination with local strains yet.

Induction of UPR Promotes Interferon Response to Inhibit PRRSV Replication via PKR and NF-κB Pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) was previously shown to induce a certain level of cellular stress during viral replication. Unfolded protein response (UPR) is a cellular stress response responsible for coping with stress and cellular survival. However, the pathway leading to the induction of UPR that may influence PRRSV replication is still unknown. Here, we found that PRRSV infection induced UPR prior to interferon response. Induction of UPR significantly enhanced the expression of interferon and interferon-related genes, thus leading to the suppression of PRRSV infection. Next, we explored the underlying mechanisms of UPR-induced antiviral response. We found that induction of UPR promoted the expression of protein kinase R (PKR), and PKR was highly correlated with the reduction of PRRSV replication. Furthermore, tunicamycin stimulation and PKR overexpression activated NF-κB and interferon response at the early stage of PRRSV infection, thus reinforcing the expression of type I interferons and proinflammatory cytokines and leading to inhibition of PRRSV. In addition, PRRSV nsp4 was shown to reduce the expression of PKR. These findings might have implications for our understandings of the host's immune mechanism against PRRSV and a new strategy of PRRSV to evade the host antiviral immunity.

Development and application of an indirect ELISA for the detection of antibody to porcine circovirus 4 in pigs.

Porcine circovirus type 4 (PCV4) was first reported in 2019 in China. So far, the viral DNA was detected from both healthy and diseased pigs in China and South Korea by using molecular techniques including PCR and real-time PCR. In contrast, a serological survey regarding the presence of PCV4 antibodies in the pig population was seldomly reported. In the present study, we describe the development of an indirect enzyme-linked immunosorbent assay (ELISA) based on capsid protein for the detection of PCV4 antibodies in swine sera. After validating the specificity and sensitivity, the ELISA was used in a retrospective serological survey for PCV4 antibodies in pig sera from Jiangsu Province of China. Note that 3.44% of analyzed samples collected between 2018 and 2021 were tested positive for PCV4 antibodies. However, PCV4 genome was absent in all ELISA-positive serum samples. Therefore, the dynamic of viremia and antibody response to PCV4 infection in pigs warrant further investigation.

Meclizine Inhibits Pseudorabies Virus Replication by Interfering With Virus Entry and Release.

Pseudorabies virus (PRV) is a pathogen that causes substantial economic losses to the swine industry. With the emergence and widespread of PRV variants since 2011 in China, current commercial vaccines cannot provide complete protection against PRV infection. Therefore, antiviral drugs may work as an alternative way to control and prevent PRV. In this study, the inhibitory effects and underlying molecular mechanisms of meclizine against PRV were studied. Meclizine displayed a significant inhibitory effect against PRV when it was added before, simultaneously with, or after virus infection. The inhibitory effect of meclizine occurred during viral entry and cell-to-cell spreading but not at viral attachment into PK-15 cells. Meclizine also inhibited viral particle release at the late stage of infection. The antiviral effect of meclizine was tested in mice, and the results showed that meclizine reduced the severity of clinical symptoms and the viral loads in tissues, and delayed the death, after PRV challenge. The above results indicated that meclizine had an inhibitory effect on PRV. Our findings will contribute to the development of potential therapeutic drugs against PRV infection.

In Vitro and In Vivo Metabolomic Profiling after Infection with Virulent Newcastle Disease Virus.

Newcastle disease (ND) is an acute, febrile, highly contagious disease caused by the virulent Newcastle disease virus (vNDV). The disease causes serious economic losses to the poultry industry. However, the metabolic changes caused by vNDV infection remain unclear. The objective of this study was to determine the metabolomic profiling after infection with vNDV. DF-1 cells infected with the vNDV strain Herts/33 and the lungs from Herts/33-infected specific pathogen-free (SPF) chickens were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) in combination with multivariate statistical analysis. A total of 305 metabolites were found to have changed significantly after Herts/33 infection, and most of them belong to the amino acid and nucleotide metabolic pathway. It is suggested that the increased pools of amino acids and nucleotides may benefit viral protein synthesis and genome amplification to promote NDV infection. Similar results were also confirmed in vivo. Identification of these metabolites will provide information to further understand the mechanism of vNDV replication and pathogenesis.

Hemagglutinin-neuraminidase and fusion proteins of virulent Newcastle disease virus cooperatively disturb fusion-fission homeostasis to enhance mitochondrial function by activating the unfolded protein response of endoplasmic reticulum and mitochondrial stress.

The fusogenically activated F and HN proteins of virulent NDV induce complete autophagic flux in DF-1 and A549 cells. However, the effect of both glycoproteins on mitochondria remains elusive. Here, we found that F and HN cooperation increases mitochondrial biogenesis but does not cause the mitochondria damage. We observed that both glycoproteins change the morphological characteristics and spatial distribution of intracellular mitochondria. F and HN cooperate cooperatively to induce ER stress and UPRmt. Our preliminary data suggested that F and HN cooperatively disturb mitochondrial fusion-fission homeostasis to enhance mitochondrial biogenesis, and eventually meet the energy demand of syncytium formation.

Suppressor of cytokine signaling 3 plays an important role in porcine circovirus type 2 subclinical infection by downregulating proinflammatory responses.

Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated diseases and usually evokes a subclinical infection, without any obvious symptoms, in pigs. It remains unclear how PCV2 leads to a subclinical infection. In this study, we found that peripheral blood mononuclear cells (PBMCs) from PCV2-challenged piglets with no significant clinical symptoms exhibited increased expression of suppressor of cytokine signaling (SOCS) 3, but no significant changes in the expression of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α; this differed from piglets that displayed significant clinical symptoms. IL-6- and TNF-α-mediated signalings were inhibited in PBMCs from subclinical piglets. Elevated SOCS3 levels inhibited IL-6- and TNF-α-mediated NF-kappa-B inhibitor alpha degradation in PBMCs and PK-15 cells. SOCS3 production was also increased in PCV2-infected PK-15 porcine kidney cells, and IL-6 and TNF-α production that was induced by PCV2 in PK-15 cells was significantly increased when SOCS3 was silenced by a small interfering RNA. SOCS3 interacted with signal transducer and activator of transcription 3 and TNF-associated receptor-associated factor 2, suggesting mechanisms by which SOCS3 inhibits IL-6 and TNF-α signaling. We conclude that SOCS3 plays an important role in PCV2 subclinical infection by suppressing inflammatory responses in primary immune cells.

Baculovirus expression of the N-terminus of porcine heat shock protein Gp96 improves the immunogenicity of recombinant PCV2 capsid protein.

Porcine circovirus type 2 (PCV2) causes significant economic losses to the swine industry worldwide. Heat shock proteins (Hsps) can be used as modulators to enhance both innate and adaptive immune responses. In the present study, recombinant baculoviruses expressing the PCV2Cap protein and the N-terminal 22-370 amino acids of porcine Gp96 (Gp96N), Hsp90, and Hsp70 (rBac-cap/Gp96N, rBac-cap/Hsp90 and rBac-cap/Hsp70, respectively) were constructed and the immune responses were examined in mice and piglets. The mouse experiments showed that rBac-cap/Gp96N increased the titers of specific anti-PCV2 neutralizing antibodies, proliferative responses of peripheral blood mononuclear cells (PBMCs) and IFN-γ levels compared to rBac-cap/Hsp90, rBac-cap/Hsp70, or rBac-cap. The pig experiments showed that the levels of anti-PCV2 antibody, proliferative responses of PBMCs, and IFN-γ in the rBac-cap/Gp96N groups were increased compared to those in rBac-cap group. There were no clear clinical signs of infection following PCV2 challenge in pigs inoculated with recombinant rBac-cap/Gp96N and rBac-cap, and the relative daily weight gains were higher than those in the challenge control (CC) group. The pathological lesions, extent of viremia, and viral loads of the vaccinated groups were milder than those in the CC group. Meanwhile, the extent of viremia and viral load present in the rBac-cap/Gp96N group were significantly lower than those in the rBac-cap group. These results indicated that porcine Gp96N effectively increased the humoral and cell-mediated immune responses of PCV2Cap. Gp96N presents an attractive adjuvant or immunotargeting strategy to enhance the protective efficacy of PCV2 subunit vaccines in swine.