Japanese encephalitis virus NS1 and NS1' proteins induce vimentin rearrangement via the CDK1-PLK1 axis to promote viral replication.

The host cytoskeleton plays crucial roles in various stages of virus infection, including viral entry, transport, replication, and release. However, the specific mechanisms by which intermediate filaments are involved in orthoflavivirus infection have not been well understood. In this study, we demonstrate that the Japanese encephalitis virus (JEV) remodels the vimentin network, resulting in the formation of cage-like structures that support viral replication. Mechanistically, JEV NS1 and NS1' proteins induce the translocation of CDK1 from the nucleus to the cytoplasm and interact with it, leading to the phosphorylation of vimentin at Ser56. This phosphorylation event recruits PLK1, which further phosphorylates vimentin at Ser83. Consequently, these phosphorylation modifications convert the typically filamentous vimentin into non-filamentous "particles" or "squiggles." These vimentin "particles" or "squiggles" are then transported retrogradely along microtubules to the endoplasmic reticulum, where they form cage-like structures. Notably, NS1' is more effective than NS1 in triggering the CDK1-PLK1 cascade response. Overall, our study provides new insights into how JEV NS1 and NS1' proteins manipulate the vimentin network to facilitate efficient viral replication. IMPORTANCE: Japanese encephalitis virus (JEV) is a mosquito-borne orthoflavivirus that causes severe encephalitis in humans, particularly in Asia. Despite the availability of a safe and effective vaccine, JEV infection remains a significant public health threat due to limited vaccination coverage. Understanding the interactions between JEV and host proteins is essential for developing more effective antiviral strategies. In this study, we investigated the role of vimentin, an intermediate filament protein, in JEV replication. Our findings reveal that JEV NS1 and NS1' proteins induce vimentin rearrangement, resulting in the formation of cage-like structures that envelop the viral replication factories (RFs), thus facilitating efficient viral replication. Our research highlights the importance of the interplay between the cytoskeleton and orthoflavivirus, suggesting that targeting vimentin could be a promising approach for the development of antiviral strategies to inhibit JEV propagation.

mosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7-JEV complex to facilitate virus infection in vivo and in vitro Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

Porcine 2', 5'-oligoadenylate synthetases inhibit Japanese encephalitis virus replication in vitro.

The 2', 5'-oligoadenylate synthetases (OAS) are antiviral proteins and several isoforms have been identified as flavivirus-resistance biomarkers in human and mouse. The expression kinetics and antiviral functions of porcine OAS family (OAS1, OAS2, and OASL) in PK-15 cells following infection by Japanese encephalitis virus (JEV) were evaluated in the present study. The endogenous expression of the three OAS genes was efficiently induced by IFN-α treatment in PK-15 cells. However, expression of pOAS1 and pOAS2 responded more quickly than pOASL. Infection by JEV also induced the expression of the pOAS isoforms, but at a significantly lower level than that observed following IFN-α stimulation. Transient overexpression of pOASL and pOAS1 inhibited JEV replication more efficiently than OAS2 overexpression. Interestingly, knockdown of pOAS2 expression by siRNA treatment led to the highest increase in JEV multiplication. Co-silencing of RNase L and each pOAS revealed that the anti-JEV function of pOAS1 and pOAS2 were RNase L dependent, while the antiviral activity of pOASL was not. In conclusion, all pOAS isoforms play a significant role in the response to JEV infection, and are differentially induced by different stimuli. The alternative pathways of antiviral activity stimulated by OASL require further study.

Isolation and genome characterization of a novel duck Tembusu virus with a 74 nucleotide insertion in the 3' non-translated region.

During investigations into the outbreak of duck Tembusu virus (DTMUV) infection in 2011 in China, a DTMUV strain (DTMUV-AH2011) was isolated from the affected ducks. The length of the genome of the DTMUV-AH2011 strain was found to be 11,064 nucleotides and to possess 10,278 nucleotides of one open reading frame (ORF), flanked by 94 nucleotides of the 5' non-translated region (NTR) and 692 nucleotides of the 3' NTR. In comparison with five fully sequenced TMUV genomes, the genome of DTMUV-AH2011 had a 74 nucleotide insertion in the 3' NTR. Comparison of the DTMUV-AH2011 fully deduced amino acid sequences with those of other Tembusu virus strains reported recently in China showed they had a highly conserved polyprotein precursor, sharing 98.9% amino acid identities, at least. The overall divergences of amino acid substitutions were randomly distributed among viral proteins except for the protein NS4B, the protein NS4B was unchanged. Knowledge of the biological characters of DTMUV and the potential role of the insertion in the 3' NTR in RNA replication will be useful for further studies of the mechanisms of virus replication and pathogenesis.

BP8, a novel peptide from avian immune system, modulates B cell developments.

The bursa of Fabricius (BF) is the key humoral immune organ unique to birds, and is critical for early B-lymphocyte proliferation and differentiation. However, the molecular basis and mechanisms through which the BF regulates B cell development are not fully understood. In this study, we isolated and identified a new bursal peptide (BP8, AGHTKKAP) by RP-HPLC and MALDI-TOF-MS. BP8 promoted colony-forming pre-B formation, bound B cell precursor, regulated B cell development in vitro as well as in vivo, upstream of the EBF-E2A-Pax5 regulatory complex and increased immunoglobulin secretion. These data revealed a bursal-derived multifunctional factor BP8 as a novel biomaterial which is essential for the development of the immune system. This study elucidates further the mechanisms involved in humoral immune system and has implications in treating human diseases.

BP5 regulated B cell development promoting anti-oxidant defence.

Bursa of Fabricius is the humoral immune system for B cell differentiation and antibody production. Bursopentine (BP5) is a novel immunomodulatory peptide and significantly stimulated an antigen-specific immune response in mice. BP5 was also found to protect LPS-activated murine peritoneal macrophages from oxidative stress. In this study, the effects of BP5 on B cell development were examined. The results suggested that BP5 markedly promoted B cell development by increasing CFU-pre B, and affected the redox homeostasis regulation of B cells. To study the molecular mechanism of effect of bursal-derived BP5, this research utilized 2D-E and MALDI-TOF/TOF to analyze the differentially expressed proteins of BP5-treated WEHI-231 cells. The results showed that BP5 affected the redox homeostasis regulation of WEHI-231 cells and induced alterations in the protein expression profiles related to the oxidoreduction coenzyme metabolic process, precursor metabolites and energy, proteolysis, RNA splicing and translation and cellular process, respectively. BP5 also induced glucose-6-phosphate dehydrogenase (G6PD) activity, an essential anti-oxidant cofactor. We found that the redox homeostasis regulation effect of BP5 was reduced in G6PD-deficient cells. These data suggested that BP5 affected the redox balance toward reducing conditions by promoting the expression of G6PD, which in turn regulated the glutathione redox cycle and other processes.

Effects of Goose Astrovirus Type 2 Infection on Peripheral Blood Lymphocyte and Macrophage Activity In Vitro.

Goose astrovirus type 2 (GAstV-2) is a novel pathogen causing visceral gout in goslings; it not only causes necrosis of renal epithelial cells but also causes spleen damage, indicating that GAstV-2 induces immunosuppression in goslings. However, to date, the interaction between GAstV-2 and immune cells remains unclear. In this study, peripheral blood lymphocytes and macrophages were isolated from goslings without GAstV-2 infection and then inoculated in vitro with GAstV-2, and the virus localization in the lymphocytes and macrophages, proliferation and apoptosis of lymphocytes, and phagocytic activity, reactive oxygen species (ROS) and nitric oxide (NO) production, and cell polarity in macrophages were determined. The results showed that GAstV-2 was observed in the cytoplasm of CD4 and CD8 T cells and macrophages, indicating that GAstV-2 can infect both lymphocytes and macrophages. GAstV-2 infection reduced the lymphocyte proliferation induced by Concanavalin A and lipopolysaccharide stimulation and increased the lymphocyte apoptosis rate and mRNA expression of Fas, demonstrating that GAstV-2 causes damage to lymphocytes. Moreover, GAstV-2 infection enhanced phagocytic activity and production of ROS and NO and induced a proinflammatory phenotype in macrophages (M1 macrophages), indicating that macrophages play an antiviral role during GAstV-2 infection. In conclusion, these results demonstrate that GAstV-2 infection causes damages to lymphocytes, and host macrophages inhibit GAstV-2 invasion during infection.

JEV infection leads to dysfunction of lysosome by downregulating the expression of LAMP1 and LAMP2.

Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What's more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4+ cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131-204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function.

Aminoguanidine alleviates gout in goslings experimentally infected with goose astrovirus-2 by reducing kidney lesions.

Goose astrovirus (GAstV)-2, a novel pathogen identified in 2018, mainly causes visceral gout in goslings, leading to approximately 50% mortality. At present, no commercial veterinary products are available to prevent and treat the disease. Our previous studies showed that nitric oxide (NO) and inducible NO synthase (iNOS) were markedly higher in the kidney and spleen of goslings infected with GAstV-2, but their effects during GAstV-2 infection remain unclear. In the present study, goslings were intraperitoneally injected with aminoguanidine (AG)-an iNOS inhibitor-to examine the role of NO during GAstV-2 infection. AG significantly decreased the serum NO concentration and iNOS mRNA expression in the kidney. Moreover, AG reduced the mortality, serum uric acid and creatinine content, and urate deposition in visceral organs and joints. Histopathological analysis demonstrated that AG reduced renal tubular cell necrosis, inflammatory cell infiltration, glycogen deposition in glomerular mesangium, and interstitial fibrosis, suggesting alleviation of kidney lesions. Furthermore, AG decreased the expression of renal injury markers such as KIM-1 and desmin; inflammatory cytokine-related genes such as IL-1ß, IL-8, and MMP-9; and autophagy-related genes and proteins such as LC3II, ATG5, and Beclin1. However, quantitative real-time PCR and immunohistochemistry showed that treatment with AG did not affect the kidney and liver viral load. These findings suggest that AG decreases the mortality rate and kidney lesions in goslings infected with GAstV-2 through mechanisms associated with autophagy and inhibition of inflammatory cytokine production in the kidney but not with GAstV-2 replication.

Developmental Dynamics of the Gut Virome in Tibetan Pigs at High Altitude: A Metagenomic Perspective across Age Groups.

Tibetan pig is a geographically isolated pig breed that inhabits high-altitude areas of the Qinghai-Tibetan plateau. At present, there is limited research on viral diseases in Tibetan pigs. This study provides a novel metagenomic exploration of the gut virome in Tibetan pigs (altitude ≈ 3000 m) across three critical developmental stages, including lactation, nursery, and fattening. The composition of viral communities in the Tibetan pig intestine, with a dominant presence of Microviridae phages observed across all stages of development, in combination with the previous literature, suggest that it may be associated with geographical locations with high altitude. Functional annotation of viral operational taxonomic units (vOTUs) highlights that, among the constantly increasing vOTUs groups, the adaptability of viruses to environmental stressors such as salt and heat indicates an evolutionary response to high-altitude conditions. It shows that the lactation group has more abundant viral auxiliary metabolic genes (vAMGs) than the nursery and fattening groups. During the nursery and fattening stages, this leaves only DNMT1 at a high level. which may be a contributing factor in promoting gut health. The study found that viruses preferentially adopt lytic lifestyles at all three developmental stages. These findings not only elucidate the dynamic interplay between the gut virome and host development, offering novel insights into the virome ecology of Tibetan pigs and their adaptation to high-altitude environments, but also provide a theoretical basis for further studies on pig production and epidemic prevention under extreme environmental conditions.

Identification and characterization of novel immunomodulatory bursal-derived pentapeptide-II (BPP-II).

The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.

Interaction between hTIM-1 and Envelope Protein Is Important for JEV Infection.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic virus, is one of the most important causes of human viral encephalitis. JEV relies on various attachment or entry co-factors to enter host cells. Among these co-factors, hTIM-1 has been identified as an attachment factor to promote JEV infection through interacting with phosphatidylserine (PS) on the viral envelope. However, the reasons why JEV prefers to use hTIM-1 over other PS binding receptors are unknown. Here, we demonstrated that hTIM-1 can directly interact with JEV E protein. The interaction between hTIM-1 and JEV relies on specific binding sites, respectively, ND114115 in the hTIM-1 IgV domain and K38 of the E protein. Furthermore, during the early stage of infection, hTIM-1 and JEV are co-internalized into cells and transported into early and late endosomes. Additionally, we found that the hTIM-1 soluble ectodomain protein effectively inhibits JEV infection in vitro. Moreover, hTIM-1-specific antibodies have been shown to downregulate JEV infectivity in cells. Taken together, these findings suggested that hTIM-1 protein directly interacts with JEV E protein and mediates JEV infection, in addition to the PS-TIM-1 interaction.

MDA5 with Complete CARD2 Region Inhibits the Early Replication of H9N2 AIV and Enhances the Immune Response during Vaccination.

Chicken melanoma differentiation-associated gene 5 (MDA5) is a member of the RLRs family that recognizes the viral RNAs invading cells and activates downstream interferon regulatory pathways, thereby inhibiting viral replication. The caspase activation and recruitment domain (CARD) is the most important region in MDA5 protein. However, the antiviral and immune enhancement of MDA5 with the CARD region remains unclear. In this study, two truncated MDA5 genes with different CARD regions, namely MDA5-1 with CARD1 plus partial CARD2 domain and MDA5-2 with CARD1 plus complete CARD2 domain, were cloned via reverse transcription PCR and ligated into plasmid Flag-N vector to be Flag-MDA5-1 and Flag-MDA5-2 plasmids. DF-1 cells were transfected with two plasmids for 24 h and then inoculated with H9N2 virus (0.1 MOI) for 6 h to detect the levels of IFN-ß, PKR, MAVS, and viral HA, NA, and NS proteins expression. The results showed that MDA5-1 and MDA5-2 increased the expression of IFN-ß and PKR, activated the downstream molecule MAVS production, and inhibited the expression of HA, NA, and NS proteins. The knockdown of MDA5 genes confirmed that MDA5-2 had a stronger antiviral effect than that of MDA5-1. Furthermore, the recombinant proteins MDA5-1 and MDA5-2 were combined with H9N2 inactivated vaccine to immunize SPF chickens subcutaneously injected in the neck three times. The immune response of the immunized chicken was investigated. It was observed that the antibody titers and expressions of immune-related molecules from the chicken immunized with MDA5-1 and MDA5-2 group were increased, in which the inducing function of MDA5-2 groups was the highest among all immunization groups. These results suggested that the truncated MDA5 recombinant proteins with complete CARD2 region could play vital roles in antiviral and immune enhancement. This study provides important material for the further study of the immunoregulatory function and clinical applications of MDA5 protein.

The design and recombinant protein expression of a consensus porcine interferon: CoPoIFN-α.

CoPoIFN-α is a recombinant non-naturally occurring porcine interferon-α (IFN-α). It was designed by scanning 17 porcine IFN-α nonallelic subtypes and assigning the most frequently occurring amino acid in each position. We used a porcine IFN-α (PoIFN-α) derived from domestic pig as a control. Both porcine IFN-α genes were introduced into yeast expression vector PpICZα-A and expressed in Pichia pastoris. The antiviral unit of these two IFN-αs were assayed in MDBK, PK-15 and MARC-145 cells against vesicular stomatitis virus (VSV), and their inhibitory abilities on pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) replication were also examined, respectively. We found the antiviral activity (units/mg) of CoPoIFN-α was 46.4, 63.6 and 53.5-fold higher than that of PoIFN-α for VSV inhibition in MDBK, PK-15 and MARC-145 cells, 4.8-fold higher for PRV inhibition in PK-15 cells, and 5-fold higher for PRRSV inhibition in MARC-145 cells. Our results also showed that the PRV and PRRSV-specific cytopathic effect (CPE) could be inhibited in the cells pretreated with CoPoIFN-α and PoIFN-α, and the virus titers in the cells pretreated with CoPoIFN-α were lower than those cells pretreated with PoIFN-α by 10-20-fold. The antiproliferative activity of CoPoIFN-α was significantly higher than that of PoIFN-α on a molar basis. The mRNA level of Mx1 and OAS1 genes in PK-15 cells induced by CoPoIFN-α were enhanced about 4.6-fold and 3.2-fold compared to that induced by PoIFN-α. Based on a homology model of CoPoIFN-α and IFNAR2, all of the different residues between native PoIFN-α and CoPoIFN-α were not involved in IFNAR1 binding site, and there is no direct interaction between these residues and IFNAR2, either. We speculate that the higher activity of CoPoIFN-α was likely due to the electrostatic potential introduced by residue Arg156 around the binding site or a structural perturbation caused by these different residues. This may enhance the overall binding affinity of CoPoIIFN-α and the receptors. Thus, CoPoIFN-α may have the potential to be used in therapy of porcine diseases.

Functional analysis of the interferon-stimulated response element of porcine circovirus type 2 and its role during viral replication in vitro and in vivo.

BACKGROUND: Porcine circovirus type 2 (PCV2) is associated with post-weaning multi-systemic wasting syndrome (PMWS) in young weaned pigs. Immune stimulation was found to activate the replication of PCV2 and exacerbate the clinical outcome of the infection. Proper amount of interferon-α (IFN-α) is able to enhance PCV2 infection and production in Porcine kidney-15 (PK-15) cells when administered after inoculation. METHODS: In the present study, luciferase reporter assays, construction of mutant viruses, Analysis the replication efficiency and the response to IFN-α treatment in PK-15 cells and animal experiments were carried out to analyze the function of interferon-stimulated response element (ISRE) of PCV2 and its role during viral replication in vitro and in vivo. RESULTS: A functional viral ISRE sequence, 5'-CTGAAAACGAAAGA-3', was identified in Rep gene promoter (Prep) of PCV2. PCV2 Prep is composed of two mini promoters, the proximal one span the sequence +1 to -106, containing an ISRE while the distal mini promoter is composed of three tandem GC box like sites locate at -85 to -194. It was demonstrated that viral ISRE is necessary for porcine IFN-α initiated luciferase expression enhancement and it plays an important role in affecting the replication efficiency of PCV2 in vivo and in vitro. CONCLUSIONS: These findings provide a theoretical basis for the Phenomenon of immunostimulation is able to enhance PCV2 infection, and improve the understanding of the complicated mechanisms involved in the host and pathogen interactions of PCV2.

Multiple linear epitopes (B-cell, CTL and Th) of JEV expressed in recombinant MVA as multiple epitope vaccine induces a protective immune response.

Epitope-based vaccination might play an important role in the protective immunity against Japanese encephalitis virus (JEV) infection. The purpose of the study is to evaluate the immune characteristics of recombinant MVA carrying multi-epitope gene of JEV (rMVA-mep). The synthetic gene containing critical epitopes (B-cell, CTL and Th) of JEV was cloned into the eukaryotic expression vector pGEM-K1L, and the rMVA-mep was prepared. BALB/c mice were immunized with different dosages of purified rMVA-mep and the immune responses were determined in the form of protective response against JEV, antibodies titers (IgG1 and IgG2a), spleen cell lymphocyte proliferation, and the levels of interferon-γ and interleukin-4 cytokines. The results showed that live rMVA-mep elicited strongly immune responses in dose-dependent manner, and the highest level of immune responses was observed from the groups immunized with 107 TCID50 rMVA-mep among the experimental three concentrations. There were almost no difference of cytokines and neutralizing antibody titers among 107 TCID50 rMVA-mep, recombinant ED3 and inactivated JEV vaccine. It was noteworthy that rMVA-mep vaccination potentiates the Th1 and Th2-type immune responses in dose-dependent manner, and was sufficient to protect the mice survival against lethal JEV challenge. These findings demonstrated that rMVA-mep can produce adequate humoral and cellular immune responses, and protection in mice, which suggested that rMVA-mep might be an attractive candidate vaccine for preventing JEV infection.

A noval strategy of deletion in PK gene for construction of a vaccine candidate with exellent safety and complete protection efficiency against high virulent Chinese pseudorabies virus variant.

A variant of pseudorabies virus (PRV) with enhanced pathogenicity have emerged in many vaccinated swine herds in China since 2011. PRVΔTK&gE-AH02, a previously described TK/gE deletion PRV strain arising from the PRV variant AH02LA, has been shown to be safe for PRV antibody positive piglets, and could provide protection against emerging PRV variants. However, inoculation of PRVΔTK&gE-AH02 into PRV antibody negative neonatal piglets caused lethal infection. In the study, in order to attenuate the virulence of PRVΔTK&gE-AH02, an additional deletion of 1∼x223C13 bp of US3 (the serine/threonine kinase, PK) gene was performed to generate a TK/PK/gE deletion PRV variant (PRVΔTK&PK&gE-AH02). We found that the growth kinetics of PRVΔTK&PK&gE-AH02 was similar to that of PRVΔTK&gE-AH02. Mice inoculated with PRVΔTK&PK&gE-AH02 in different dose (104.0∼x223C107.0 TCID50) survived and showed no observable clinical symptoms. No virus was detected in the brains or lungs of the mice inoculated with PRVΔTK&PK&gE-AH02. Moreover, mice inoculated with PRVΔTK&PK&gE-AH02 and PRVΔTK&gE-AH02 showed similar survival against virulent PRV AH02LA strain. Importantly, safety test showed no clinical symptoms in PRV antibody negative neonatal piglets that were intranasally inoculated with PRVΔTK&PK&gE-AH02 at a dose of 106.5 TCID50, indicating that the virulence of PRVΔTK&PK&gE-AH02 was significantly mitigated. Piglets immunized with PRVΔTK&PK&gE-AH02 exhibited a high serum neutralization index. All piglets inoculated intramuscularly (I.M.) with 1 mL (105.0 TCID50) PRVΔTK&PK&gE-AH02 were completely protected against challenge intranasally (I.N.) with 2LD50 (106.5TCID50) PRV AH02LA strain. In summary, our results indicate that deletion of 1∼x223C13 bp of US3 (PK) can provide a useful way for further attenuation of PRV and the PRVΔTK&PK&gE-AH02 might be a promising vaccine candidate for controlling of the virulent PRV variants in China.

Japanese Encephalitis Virus (JEV) NS1' Enhances the Viral Infection of Dendritic Cells (DCs) and Macrophages in Pig Tonsils.

Pigs are the amplifying hosts of Japanese encephalitis virus (JEV). Currently, the safe and effective live attenuated vaccine made of JEV strain SA14-14-2, which does not express NS1', is widely used in humans and domestic animals to prevent JEV infection. In this study, we constructed the NS1' expression recombinant virus (rA66G) through a single nucleotide mutation in NS2A of JEV strain SA14-14-2. Animal experiments showed that NS1' significantly enhanced JEV infection in pig central nervous system (CNS) and tonsil tissues. Pigs shed virus in oronasal secretions in the JEV rA66G virus inoculation group, indicating that NS1' may facilitate the horizontal transmission of JEV. Additionally, dendritic cells (DCs) and macrophages are the main target cells of JEV infection in pig tonsils, which are an important site of persistent JEV infection. The reduction of major histocompatibility complex class II (MHC II) and activation of inducible nitric oxide synthase (iNOS) in pig tonsils caused by viral infection may create a beneficial environment for persistent JEV infection. These results are of significance for JEV infection in pigs and lay the foundation for future studies of JEV persistent infection in pig tonsils. IMPORTANCE Pigs are amplification hosts for Japanese encephalitis virus (JEV). JEV can persist in the tonsils for months despite the presence of neutralizing antibodies. The present study shows that NS1' increases JEV infection in pig tonsils. In addition, DCs and macrophages in the tonsils are the target cells for JEV infection, and JEV NS1' promotes virus infection in DCs and macrophages. This study reveals a novel function of JEV NS1' protein and lays the foundation for future studies of JEV persistent infection in pig tonsils.

P34L Mutation of swine TIM-1 enhances its ability to mediate Japanese encephalitis virus infection.

Japanese encephalitis virus (JEV) is a major causative agent of neurological infection affecting humans and pigs. Human T Cell Immunoglobulin and Mucin Domain 1 (hTIM-1) enhances the infection of JEV through virion-associated phosphatidylserine (PS) binding. Here, five swine TIM-1 (sTIM-1) gene variants were cloned from pig lung tissues by reverse-transcriptase polymerase chain reaction (RT-PCR). Sequence alignment analysis revealed that the gene homology between the sTIM-1 and hTIM-1 was 42.3-43.8%. Furthermore, ectopic expression of all five sTIM-1 variants in 293 T cells can promote JEV entry and infection. However, sTIM-1 V3 exhibited significantly less potent at promoting virus entry compared to the other four variants. Further studies revealed that the 34th amino acid of sTIM-1is critical for the entry of JEV, which is Pro34 in sTIM-1V3 while Leu34 in other four sTIM-1 variants. Mechanically, leucine at locus 34 was associated with the membrane distribution of sTIM-1, thereby affecting viral entry and infection. In total, our findings provide evidence that the PS receptor sTIM-1 promotes the infection of JEV and that the 34th amino acid position is critical for sTIM-1 to mediate viral infection.

Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of goose astrovirus genotypes 1 and 2.

Goose astrovirus (GAstV) is a novel pathogen that was discovered in 2018. It has two genotypes, GAstV-1 and GAstV-2, and both can cause visceral gout of goslings and result in significant economic losses. The present work aimed to develop a duplex TaqMan real-time quantitative reverse transcription PCR (RT-qPCR) assay to distinguish the two genotypes. MegAlign software was used to design two pairs of primers and a pair of matched probes based on the open reading frame 2 (ORF2) sequence with the greatest difference between GAstV-1 and GAstV-2, and primer and probe concentrations and annealing temperatures were optimised. Fluorescence signals were obtained for GAstV-1 and GAstV-2 in the FAM and VIC channels, respectively, but no fluorescent signal was observed for other pathogens. The detection limit for GAstV-1 and GAstV-2 was 33.3 and 33.7 DNA copies/µL, respectively. Intra- and inter-assay variability tests revealed excellent reproducibility. Furthermore, the assay detected GAstV-1 and GAstV-2 in allantoic fluids (100% positive) spiked with viruses, and 70 clinical gout gosling samples were examined, of which 11.4% were positive for GAstV-1, 74.3% were positive for GAstV-2%, and 5.7% were positive for mixed infection. In summary, the developed duplex RT-qPCR assay has high specificity, sensitivity, and reproducibility, and can be used in the clinic for detection of GAstV-1 and GAstV-2.