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Abstract The prevalence of porcine reproductive and respiratory syndrome virus 1 (PRRSV1) isolates has continued to increase in Chinese swine herds in recent years. However, no effective control strategy is available for PRRSV1 infection in China. In this study, we generated the first infectious cDNA clone (rHLJB1) of a Chinese PRRSV1 isolate and subsequently used it as a backbone to construct an ORF2-6 chimeric virus (ORF2-6-CON). This virus contained a synthesized consensus sequence of the PRRSV1 ORF2-6 gene encoding all the envelope proteins. The ORF2-6 consensus sequence shared > 90% nucleotide similarity with four representative strains (Amervac, BJEU06-1, HKEU16 and NMEU09-1) of PRRSV1 in China. ORF2-6-CON had replication efficacy similar to that of the backbone rHLJB1 virus in primary alveolar macrophages (PAMs) and exhibited cell tropism in Marc-145 cells. Piglet inoculation and challenge studies indicated that ORF2-6-CON is not pathogenic to piglets and can induce enhanced cross-protection against a heterologous SD1291 isolate. Notably, ORF2-6-CON inoculation induced higher levels of heterologous neutralizing antibodies (nAbs) against SD1291 than rHLJB1 inoculation, which was concurrent with a higher percentage of T follicular helper (Tfh) cells in tracheobronchial lymph nodes (TBLNs), providing the first clue that porcine Tfh cells are correlated with heterologous PRRSV nAb responses. The number of SD1291-strain-specific IFNγ-secreting cells was similar in ORF2-6-CON-inoculated and rHLJB1-inoculated pigs. Overall, our findings support that the Marc-145-adapted ORF2-6-CON can trigger Tfh cell and heterologous nAb responses to confer improved cross-protection and may serve as a candidate strain for the development of a cross-protective PRRSV1 vaccine.

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus with constantly emerging recombinant and mutant strains. Because of the high genetic diversity of PRRSV, current vaccines only provide partial protection against the infection of heterologous strains, which makes it a challenge for PRRSV prevention and control. Tubercidin is a naturally extracted compound with potential antiviral properties. However, whether tubercidin has anti-PRRSV ability is unknown. Our study found that tubercidin showed effective antiviral effects on PRRSV replication. In terms of mechanism, tubercidin suppressed PRRSV at the entry, replication, and release steps of the viral life cycle. Additionally, we demonstrated that tubercidin treatment promoted the activation of retinoic acid-inducible gene I and nuclear factor kappa-light-chain-enhancer of activated B cell signaling pathway, thus increasing the type I interferon and inflammatory cytokine expression. Furthermore, tubercidin restrained the viral non-structural protein 2 expression and viral dsRNA synthesis and ultimately inhibited PRRSV replication. Hence, our data showed that tubercidin is promising and has potential antiviral ability against PRRSV replication in vitro.IMPORTANCEPorcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases, which causes huge economic loss worldwide. However, there is no effective therapeutic method for PRRS prevention and control. Here, we found that tubercidin, a naturally extracted adenosine analog, exhibited strong anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity. Mechanically, tubercidin inhibited viral binding, replication, and release. Tubercidin suppressed PRRSV non-structural protein 2 expression, which is important for the formation of replication and transcription complex, leading to the block of viral RNA synthesis and PRRSV replication. Moreover, tubercidin could activate retinoic acid-inducible gene I/nuclear factor kappa-light-chain-enhancer of activated B cell innate immune signaling pathway and increased the expression of interferons and proinflammatory cytokines, which was the other way to inhibit PRRSV replication. Our work evaluated the potential value of tubercidin as an antiviral agent on PRRSV replication and provided a new way to prevent PRRSV replication in vitro.

Infection of pigs with the pseudorabies virus (PRV) causes significant economic losses in the pig industry. Immunization with live vaccines is a crucial aspect in the prevention of pseudorabies in swine. The TK/gE/gI/11k/28k deleted pseudorabies vaccine is a promising alternative for the eradication of epidemic pseudorabies mutant strains. This study optimized the lyophilization of a heat-resistant PRV vaccine to enhance the quality of a live vaccine against the recombinant PRV rHN1201TK−/gE−/gI−/11k−/28k−. The A4 freeze-dried protective formulation against PRV was developed by comparing the reduction in virus titer after lyophilization and after seven days of storage at 37 °C. The formulation contains 1% gelatin, 5% trehalose, 0.5% poly-vinylpyrimidine (PVP), 0.5% thiourea, and 1% sorbitol. The A4 freeze-dried vaccine demonstrated superior protection and thermal stability. It experienced a freeze-dried loss of 0.31 Lg post-freeze-drying and a heat loss of 0.42 Lg after being stored at a temperature of 37 °C for 7 consecutive days. The A4 freeze-dried vaccine was characterized through XRD, FTIR, and SEM analyses, which showed that it possessed an amorphous structure with a consistent porous interior. The trehalose component of the vaccine formed stable hydrogen bonds with the virus. Long-term and accelerated stability studies were also conducted. The A4 vaccine maintained viral titer losses of less than 1.0 Lg when exposed to 25 °C for 90 days, 37 °C for 28 days, and 45 °C for 7 days. The A4 vaccine had a titer loss of 0.3 Lg after storage at 2–8 °C for 24 months, and a predicted shelf life of 6.61 years at 2–8 °C using the Arrhenius equation. The A4 freeze-dried vaccine elicited no side effects when used to immunize piglets and produced specific antibodies. This study provides theoretical references and technical support to improve the thermal stability of recombinant PRV rHN1201TK−/gE−/gI−/11k−/28k− vaccines.

Key points A canine-derived chimeric MAb 11D9 was produced by CHO cell lines. The MAb 11D9 exhibited high HI and neutralization titers against new CPV-2 variants. The MAb 11D9 had a high therapeutic effect in beagles infected with the new CPV-2a variant.

Abstract Feline herpesvirus type 1 (FHV-1) is a common causative agent of domestic cats’ rhinotracheitis in domestic cats, and it increasingly threatens wild felids worldwide. The endangered snow leopard (Panthera uncia) belongs to the family Felidae, and it is the top predator on the Tibetan Plateau. Here we report the identification and isolation of FHV-1 from three dead captive snow leopards that presented with sneezing and rhinorrhea. To explore the relationship between FHV-1 and their deaths, organs and nasal swabs were collected for histopathology, viral isolation and sequence analysis. The results revealed that all three snow leopards were infected with FHV-1. The first animal died primarily of cerebral infarction and secondary non-suppurative meningoencephalitis that was probably caused by FHV-1. The second animal died mainly of renal failure accompanied by interstitial pneumonia caused by FHV-1. The cause of death for the third animal was likely related to the concurrent reactivation of a latent FHV-1 infection. The gD and gE gene sequence alignment of the isolated FHV-1 isolate strain revealed that the virus likely originated from a domestic cat. It was found that FHV-1 infection can cause different lesions in snow leopards than in domestic cats and is associated with high risk of disease in wild felids. This suggests that there should be increased focus on protecting wild felids against FHV-1 infections originating from domestic cats.

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.

Immunoglobulin A (IgA) of sows is critically important for assessing piglets’ protective capacity against porcine epidemic diarrhea virus (PEDV). Here, we report a therapeutic chimeric anti-PEDV IgG/IgA expressed by Chinese hamster ovary (CHO) cells for oral treatment of PED. The chimeric anti-PEDV IgG/IgA was produced by the CHO cell lines, in which the heavy chain was constructed by combining the VH, Cγ1 and hinge regions of PEDV IgG mAb 8A3, and the Cα2 and Cα3 domains of a Mus musculus immunoglobulin alpha chain. The chimeric anti-PEDV IgG/IgA could neutralize the strains of CV777 (G1), P014 (G2) and HN1303 (G2) in vitro effectively, showing broad-spectrum neutralization activity. The in vivo challenge experiments demonstrated that chimeric anti-PEDV IgG/IgA (9C4) produced in the CHO cell supernatant could alleviate clinical diarrhea symptoms of the PEDV infection in piglets. In general, our study showed that chimeric anti-PEDV IgG/IgA produced from CHO cell line supernatants effectively alleviates PEDV infection in piglets, which also gives the foundation for the construction of fully functional secretory IgA by the J chain introduction to maximize the antibody therapeutic effect.

Pseudorabies caused by pseudorabies virus (PRV) infection is still a major disease affecting the pig industry; its eradication depends on effective vaccination and antibody (Ab) detection. For a more rapid and accurate PRV detection method that is suitable for clinical application, here, we established a poly(dimethylsiloxane)-based (efficient removal of non-specific binding) solid-phase protein chip platform (blocking ELISA) for dual detection of PRV gD and gE Abs. The purified gD and gE proteins expressed in baculovirus were coated into the highly hydrophobic nanomembrane by an automatic spotter, and the gray values measured by a scanner were used for the S/N (sample/negative) value calculation (gD and gE Abs standard, positive: S/N value ≤0.6; negative: S/N value >0.7; suspicious: 0.6 < S/N ≤ 0.7). The method showed an equal sensitivity in the gD Ab test of immunized pig serum samples compared to the neutralization test and higher sensitivity in the gE Ab test compared to the commercial gE Ab detection kit. In the clinical evaluation, we found an agreement of 100% (122/122) in the gD Ab detection compared to the neutralization test and an agreement of 97.5% (119/122) in the gE Ab detection compared to the commercial PRV gE Ab detection kit. In summary, the protein chip platform for dual detection of PRV gD and gE Abs showed high sensitivity and specificity, which is suitable for PRV immune efficacy evaluation and epidemic monitoring.

Abstract Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection.

Abstract Background Candida is the common conditionally pathogenic fungus that infected human and animal clinically. C. tropicalis had been isolated from the skin and hair of healthy pigs, but with no report of fatal infection in gastrointestinal diseases. Case presentation In a pig farm in Henan Province of China, about 20 % of pregnant and postpartum sows suffered from severe gastrointestinal diseases, with a mortality rate higher than 60 % in the diseased animals. The sows had gastrointestinal symptoms such as blood in stool and vomiting. Necropsy revealed obvious gastric ulcers, gastrointestinal perforation, and intestinal hemorrhage in the gastrointestinal tract, but no lesions in other organs. The microbial species in gastric samples collected from gastric ulcer of the diseased sows then was initially identified as Candida by using routine systems of microscopic examination, culture characteristics on the medium Sabouraud dextrose agar medium. The fungus was further identified as C. tropicalis by species-specific PCR and sequencing. This study revealed an infection of C. tropicalis in sows through gastrointestinal mucosa could cause fatal digestive system disease and septicemia. Conclusions For the first time, a strain of C. tropicalis was isolated and identified from the gastric tissue of sows with severe gastrointestinal diseases. PCR and sequencing of ITS-rDNA combined with morphology and histopathological assay were reliable for the identification of Candida clinically.

ABSTRACT G2 porcine epidemic diarrhea virus (G2 PEDV) and highly pathogenic porcine reproductive and respiratory syndrome virus 2 (HP-PRRSV2) are two of the most prevalent swine pathogens in China’s swine herds, and their coinfection occurs commonly. Several PED and PRRS vaccines have been utilized in China for decades, and systemic homologous neutralizing antibodies (shnAbs) in serum are frequently used to evaluate the protective efficacy of PED and PRRS vaccines. To develop a vaccine candidate against G2 PEDV and HP-PRRSV2 coinfection, in this study, we generated a chimeric virus (rJSTZ1712-12-S) expressing S protein of G2 PEDV using an avirulent HP-PRRSV2 rJSTZ1712-12 infectious clone as the viral vector. The rJSTZ1712-12-S strain has similar replication efficacies as the parental rJSTZ1712-12 virus. In addition, animal inoculation indicated that rJSTZ1712-12-S is not pathogenic to piglets and can induce shnAbs against both G2 PEDV and HP-PRRSV2 isolates after prime-boost immunization. However, passive transfer study in neonatal piglets deprived of sow colostrum showed that rJSTZ1712-12-S-induced shnAbs may only decrease PEDV and PRRSV viremia but cannot confer sufficient protection against dual challenge of high virulent G2 PEDV XJ1904-34 strain and HP-PRRSV2 XJ17-5 isolate. Overall, this study provides the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for the evaluation of protective efficacy of PED and PRRS bivalent vaccine (especially for the PED vaccine). IMPORTANCE Porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) coinfection occurs commonly and can synergistically reduce feed intake and pig growth. Vaccination is an effective strategy utilized for PED and PRRS control, and systemic homologous neutralizing antibodies (shnAbs) in serum are commonly used for protective efficacy evaluation of PED and PRRS vaccines. Currently, no commercial vaccine is available against PEDV and PRRSV coinfection. This study generated a chimeric vaccine candidate against the coinfection of prevalent PEDV and PRRSV in China. The chimeric strain can induce satisfied shnAbs against both PEDV and PRRSV after prime-boost inoculation in pigs. But the shnAbs cannot confer sufficient protection against PEDV and PRRSV coinfection in neonatal piglets. To the best of our knowledge, these findings provide the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for evaluating PED and PRRS bivalent vaccine protective efficacy.

One Gram-negative Bacillus was isolated from a brain sample of a pig with neurological symptoms. Pathological examination showed meningitis at necropsy. Ochrobactrum anthropi (O. anthropi) was successfully isolated from the brain sample and was confirmed by biochemical reaction results (API 20 NE) and gene sequencing. The strain was highly resistant to β-lactam antibiotics. Mice were experimentally infected with O. anthropi and showed typical meningitis. This is the first report on O. anthropi isolated from a pig, and indicates that O. anthropi may have a broader host spectrum of infection. Keywords: Ochrobactrum anthropi, Pig brain

The egg-drop syndrome ‘76 (EDS ‘76) caused by duck atadenovirus A (DAdV-1) infection in laying hens leads to the decrease in egg production, causing heavy economic losses in the poultry industry; thus, vaccines with high safety and immunogenicity are needed. In this study, the DAdV-1 fiber protein expressed in Escherichia coli with codon optimization showed the hemagglutination (HA) titer of 13 log2 after purification (0.6 mg/mL). Compared with inactivated EDS ‘76 vaccine, the specific pathogen-free chickens immunized with 0.4 mL fiber protein (HA titer of 11 log2) induced an equal level of HA inhibition (HI) titer and neutralizing antibodies. Meanwhile, after immunization with fiber protein, the lowest HI titer that could provide the effect to reduce egg production rate in laying hens after the challenge was 7 log2. Moreover, fiber protein with an HA titer of 7 log2 could induce an HI titer no

In China, the re-emerging pseudorabies virus (PRV) variant has caused large-scale outbreaks of pseudorabies in swine herds with classical PRV vaccine immunization since late 2011. Here, a recombinant PRV with TK/gI/gE/11k/28k deletion was constructed based on variant HN1201 strain isolated in 2012, by the bacterial artificial chromosome infectious clones. Compared with the parental virus, the recombinant PRV rHN1201TK−/gE−/gI−/11k−/28k− showed a similar virus grown curve and exhibited smaller plaques. The vaccination of rHN1201TK−/gE−/gI−/11k−/28k− could elicit an earlier and higher level of gB antibody, and the neutralizing antibodies elicited by rHN1201TK−/gE−/gI−/11k−/28k− were effective against both PRV classical and variant strains. Clinically, the body temperature of the pigs immunized with rHN1201TK−/gE−/gI−/11k−/28k− was significantly lower than that of the classical PRV vaccine immunized pigs, and the recombinant PRV could provide effective protection against the challenge with the PRV variant. These results imply that the rHN1201TK−/gE−/gI−/11k−/28k− could be a promising vaccine candidate for the prevention of the current epidemic of pseudorabies in China.

African swine fever (ASF) causes high morbidity and mortality of both domestic pigs and wild boars and severely impacts the swine industry worldwide. ASF virus (ASFV), the etiologic agent of ASF epidemics, mainly infects myeloid cells in swine mononuclear phagocyte system (MPS), including blood-circulating monocytes, tissue-resident macrophages, and dendritic cells (DCs). Since their significant roles in bridging host innate and adaptive immunity, these cells provide ASFV with favorable targets to manipulate and block their antiviral activities, leading to immune escape and immunosuppression. To date, vaccines are still being regarded as the most promising measure to prevent and control ASF outbreaks. However, ASF vaccine development is delayed and limited by existing knowledge gaps in viral immune evasion, pathogenesis, etc. Recent studies have revealed that ASFV can employ diverse strategies to interrupt the host defense mechanisms via abundant self-encoded proteins. Thus, this review mainly focuses on the antagonisms of ASFV-encoded proteins towards IFN-I production, IFN-induced antiviral response, NLRP3 inflammasome activation, and GSDMD-mediated pyroptosis. Additionally, we also make a brief discussion concerning the potential challenges in future development of ASF vaccine.

To analyze the evolutionary characteristics of the highly contagious porcine epidemic diarrhea virus (PEDV) at the molecular and structural levels, we analyzed the complete genomes of 647 strains retrieved from the GenBank database. The results showed that the spike (S) gene exhibited larger dS (synonymous substitutions per synonymous site) values than other PEDV genes. In the selective pressure analysis, eight amino acid (aa) sites of the S protein showed strong signals of positive selection, and seven of them were located on the surface of the S protein (S1 domain), suggesting a high selection pressure of S protein. Topologically, the S gene is more representative of the evolutionary relationship at the genome-wide level than are other genes. Structurally, the evolutionary pattern is highly S1 domain-related. The haplotype networks of the S gene showed that the strains are obviously clustered geographically in the lineages corresponding to genotypes GI and GII. The alignment analysis on representative strains of the main haplotypes revealed three distinguishable nucleic acid sites among those strains, suggesting a putative evolutionary mechanism in PEDV. These findings provide several new fundamental insights into the evolution of PEDV and guidance for developing effective prevention countermeasures against PEDV.

We isolated Japanese encephalitis virus (JEV) from brain samples of 2 seals with lethal encephalitis at Weihai Aquarium, Weihai, China, in 2017. We confirmed our findings by immunohistochemical staining and electron microscopy. Phylogenetic analysis showed this virus was genotype I. Our findings suggest that JEV might disseminate though infected zoo animals.

Foot-and-mouth disease (FMD), caused by FMD virus (FMDV), is a highly contagious and economically devastating viral disease of cloven-hoofed animals worldwide. In this study, the coexpression of small ubiquitin-like modifier (SUMO)–fused capsid proteins of FMDV serotype O by single plasmid in Escherichia coli was achieved with an optimal tandem permutation (VP0–VP3–VP1), showing a protein yield close to 1:1:1. After SUMO removal at a low level of protease activity (5 units), the assembled FMDV virus-like particles (VLPs) could expose multiple epitopes and have a size similar to the naive FMDV. Immunization of pigs with the FMDV VLPs could induce FMDV-specific humoral and cellular immune responses effectively, in a dose-dependent manner. These data suggested that the stable FMDV VLPs with multiple epitope exposure were effective for the induction of an immune response in pigs, which laid a foundation for the further development of the FMDV subunit vaccine.

Postweaning multisystemic wasting disease (PMWS) in piglets caused by porcine circovirus type 2 (PCV2) is one of the major threats to most pig farms worldwide. Among all the PCV types, PCV2 is the dominant genotype causing PMWS and associated diseases. Considerable efforts were made to study the virus-like-particle (VLP) assembly and the specific PCV2-associated epitope(s) in order to establish the solid foundation for engineered PCV2 vaccine development. Although the N-terminal fragment including Nuclear Localization Signal (NLS) sequence seems important for recombinant PCV2 capsid protein expression and VLP assembly, the detailed structural and functional information regarding this important fragment are largely unknown. In this study, we report crystal structure of PCV2 VLP assembled from N-terminal NLS truncated PCV2 capsid protein at 2.8 Å resolution and cryo-EM structure of PCV2 VLP assembled from full-length PCV2 capsid protein at 4.1Å resolution. Our in vitro PCV2 VLP assembly results show that NLS-truncated PCV2 capsid protein only forms instable VLPs which were easily disassembled in solution, whereas full-length PCV2 capsid protein forms stable VLPs due to interaction between 15PRSHLGQILRRRP27 (α-helix) and 33RHRYRWRRKN42 (NLS-B) in a repeated manner. In addition, our results also showed that N-terminal truncation of PCV2 capsid protein up to 27 residues still forms PCV2 particles in solution with similar size and immunogenicity, while N-terminal truncation of PCV2 capsid protein with more than 30 residues is not able to form stable PCV2 particles in solution, demonstrating the importance of interaction between the α-helix at N-terminal and NLS-B in PCV2 VLP formation. Moreover, we also report the cryo-EM structure of PCV2 VLP in complex with 3H11-Fab, a PCV2 type-specific neutralizing antibody, at 15 Å resolution. MAb-3H11 specifically recognizes one exposed epitope located on the VLP surface EF-loop (residues 128-143), which is further confirmed by PCV1-PCV2 epitope swapping assay. Hence, our results have revealed the structural roles of N-terminal fragment of PCV2 capsid protein in PCV2 particle assembly and pinpointed one PCV2 type-specific neutralizing epitope for the first time, which could provide clear clue for next generation PCV2 vaccine and diagnostic kits development.

Pseudorabies virus (PRV) belongs to the Herpesviridae family, and is an important veterinary pathogen. Highly pathogenic PRV variants have caused severe epidemics in China since 2011, causing huge economic losses. To tackle the epidemics, we identified a panel of mouse monoclonal antibodies (mAbs) against PRV glycoprotein B (gB) that effectively block PRV infection. Among these 15 mAbs, fourteen of them block PRV entry in a complement-dependent manner. The remaining one, 1H1 mAb, however can directly neutralize the virus independent of complement and displays broad-spectrum neutralizing activities. We further determined the crystal structure of PRV gB and mapped the epitopes of these antibodies on the structure. Interestingly, all the complement-dependent neutralizing antibodies bind gB at the crown region (domain IV). In contrast, the epitope of 1H1 mAb is located at the bottom of domain I, which includes the fusion loops, indicating 1H1 mAb might neutralize the virus by interfering with the membrane fusion process. Our studies demonstrate that gB contains multiple B-cell epitopes in its crown and base regions and that antibodies targeting different epitopes block virus infection through different mechanisms. These findings would provide important clues for antiviral drug design and vaccine development.

An early and yet indispensable step in the alphaherpesvirus infection is the engagement of host receptors by the viral envelope glycoprotein D (gD). Of the thus-far identified gD receptors, nectin-1 is likely the most effective in terms of its wide usage by multiple alphaherpesviruses for cell entry. The molecular basis of nectin-1 recognition by the gD protein is therefore an interesting scientific question in the alphaherpesvirus field. Previous studies focused on the herpes simplex virus (HSV) of the Simplexvirus genus, for which both the free gD structure and the gD/nectin-1 complex structure were reported at high resolutions. The structural and functional features of other alphaherpesviral gDs, however, remain poorly characterized. In the current study, we systematically studied the characteristics of nectin-1 binding by the gD of a Varicellovirus genus member, the pseudorabies virus (PRV). We first showed that PRV infects host cells via both human and swine nectin-1, and that its gD exhibits similar binding affinities for nectin-1 of the two species. Furthermore, we demonstrated that removal of the PRV gD membrane-proximal residues could significantly increase its affinity for the receptor binding. The structures of PRV gD in the free and the nectin-1-bound states were then solved, revealing a similar overall 3D fold as well as a homologous nectin-1 binding mode to its HSV counterpart. However, several unique features were observed at the binding interface of PRV gD, enabling the viral ligand to utilize different gD residues (from those of HSV) for nectin-1 engagement. These observed binding characteristics were further verified by the mutagenesis study using the key-residue mutants of nectin-1. The structural and functional data obtained in this study, therefore, provide the basis of receptor recognition by PRV gD.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically relevant viral pathogens in pigs and causes substantial losses in the pig industry worldwide each year. At present, PRRSV vaccines do not effectively prevent and control this disease. Consequently, it is necessary to develop new antiviral strategies to compensate for the inefficacy of the available vaccines. Histone deacetylase 6 (HDAC6) is an important member of the histone deacetylase family that is responsible for regulating many important biological processes. Studies have shown that HDAC6 has anti-viral activities during the viral life cycle. However, whether HDAC6 overexpression enhances resistance to PRRSV in pigs remains unknown. In this study, we used a somatic cell cloning method to produce transgenic (TG) pigs that constitutively overexpress porcine HDAC6. These TG pigs showed germ line transmission with continued overexpression of HDAC6. In vitro, virus-challenged porcine alveolar macrophages (PAMs) overexpressed HDAC6, which suppressed viral gene expression and PRRSV production. In vivo, resistance to PRRSV in TG pigs was evaluated by direct or cohabitation mediated infection with a highly pathogenic PRRSV (HP-PRRSV) strain. Compared with non-TG (NTG) siblings, TG pigs showed a significantly lower viral load in the lungs and an extended survival time after infection with HP-PRRSV via intramuscular injection. In the cohabitation study, NTG pigs housed with challenged NTG pigs exhibited significantly worse clinical symptoms than the other three in-contact groups. These results collectively suggest that HDAC6 overexpression enhances resistance to PRRSV infection both in vitro and in vivo. Our findings suggest the potential involvement of HDAC6 in the response to PRRSV, which will facilitate the development of novel therapies for PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a huge threat to the modern pig industry, and current vaccine prevention strategies could not provide full protection against it. Therefore, exploring new anti-PRRSV strategies is urgently needed. Ginsenoside Rg1, derived from ginseng and notoginseng, is shown to exert anti-inflammatory, neuronal apoptosis-suppressing and anti-oxidant effects. Here we demonstrate Rg1-inhibited PRRSV infection both in Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose-dependent manner. Rg1 treatment affected multiple steps of the PRRSV lifecycle, including virus attachment, replication and release at concentrations of 10 or 50 µM. Meanwhile, Rg1 exhibited broad inhibitory activities against Type 2 PRRSV, including highly pathogenic PRRSV (HP-PRRSV) XH-GD and JXA1, NADC-30-like strain HNLY and classical strain VR2332. Mechanistically, Rg1 reduced mRNA levels of the pro-inflammatory cytokines, including IL-1β, IL-8, IL-6 and TNF-α, and decreased NF-κB signaling activation triggered by PRRSV infection. Furthermore, 4-week old piglets intramuscularly treated with Rg1 after being challenged with the HP-PRRSV JXA1 strain display moderate lung injury, decreased viral load in serum and tissues, and an improved survival rate. Collectively, our study provides research basis and supportive clinical data for using Ginsenoside Rg1 in PRRSV therapies in swine.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to the global swine industry. Even though several control strategies have been applied, PRRS is still not effectively controlled due to the continuous emergence of new variants and limited cross-protection by current vaccines. During the routine epidemiological investigation in 2017, two PRRSV variants were identified from a severe abortion farm and a clinically healthy farm, respectively. The viruses were isolated and denominated as XJ17-5 and JSTZ1712-12. Genomic sequencing indicated that their genomes are both 14,960 bp in length sharing 99.45% nucleotide identity. Sequence alignments identified a discontinuous 30-amino-acid deletion and a continuous 120-amino-acid deletion in nsp2 of both isolates. Genome-based phylogenetic analysis confirmed that XJ17-5 and JSTZ1712-12 belong to the HP-PRRSV subtype but form a new branch with other isolates containing the same 150-amino-acid deletion in nsp2. Pathogenic analysis showed that XJ17-5 is highly virulent causing 60% mortality, while JSTZ1712-12 is avirulent for piglets. Furthermore, fragment comparisons identified 34-amino-acid differences between XJ17-5 and JSTZ1712-12 that might be associated with the distinct virulence. The identification of highly homologous HP-PRRSV variants with new genetic feature and distinct virulence contributes to further analyze the pathogenesis and evolution of PRRSV in the field.

In 2012, an unprecedented large-scale outbreak of disease in pigs in China caused great economic losses to the swine industry. Isolates from pseudorabies virus epidemics in swine herds were characterized. Evidence confirmed that the pathogenic pseudorabies virus was the etiologic agent of this epidemic.

We characterized isolates from porcine respiratory and reproductive syndrome virus epidemics in Vietnam and China in 2007. These isolates showed ≈99% identity at the genomic level. Genetic analysis indicated that they share a discontinuous deletion of 30 aa in nonstructural protein 2, which indicates that identical variants emerged in Vietnam and China.

Since April 2010, a severe outbreak of duck viral infection, with egg drop, feed uptake decline and ovary-oviduct disease, has spread around the major duck-producing regions in China. A new virus, named BYD virus, was isolated in different areas, and a similar disease was reproduced in healthy egg-producing ducks, infecting with the isolated virus. The virus was re-isolated from the affected ducks and replicated well in primary duck embryo fibroblasts and Vero cells, causing the cytopathic effect. The virus was identified as an enveloped positive-stranded RNA virus with a size of approximately 55 nm in diameter. Genomic sequencing of the isolated virus revealed that it is closely related to Tembusu virus (a mosquito-borne Ntaya group flavivirus), with 87-91% nucleotide identity of the partial E (envelope) proteins to that of Tembusu virus and 72% of the entire genome coding sequence with Bagaza virus, the most closely related flavivirus with an entirely sequenced genome. Collectively our systematic studies fulfill Koch's postulates, and therefore, the causative agent of the duck egg drop syndrome occurring in China is a new flavivirus. Flavivirus is an emerging and re-emerging zoonotic pathogen and BYD virus that causes severe egg-drop, could be disastrous for the duck industry. More importantly its public health concerns should also be evaluated, and its epidemiology should be closely watched due to the zoonotic nature of flaviviruses.

Porcine reproductive and respiratory syndrome (PRRS) is a severe viral disease in pigs, causing great economic losses worldwide each year. The causative agent of the disease, PRRS virus (PRRSV), is a member of the family Arteriviridae. Here we report our investigation of the unparalleled large-scale outbreaks of an originally unknown, but so-called "high fever" disease in China in 2006 with the essence of PRRS, which spread to more than 10 provinces (autonomous cities or regions) and affected over 2,000,000 pigs with about 400,000 fatal cases. Different from the typical PRRS, numerous adult sows were also infected by the "high fever" disease. This atypical PRRS pandemic was initially identified as a hog cholera-like disease manifesting neurological symptoms (e.g., shivering), high fever (40-42 degrees C), erythematous blanching rash, etc. Autopsies combined with immunological analyses clearly showed that multiple organs were infected by highly pathogenic PRRSVs with severe pathological changes observed. Whole-genome analysis of the isolated viruses revealed that these PRRSV isolates are grouped into Type II and are highly homologous to HB-1, a Chinese strain of PRRSV (96.5% nucleotide identity). More importantly, we observed a unique molecular hallmark in these viral isolates, namely a discontinuous deletion of 30 amino acids in nonstructural protein 2 (NSP2). Taken together, this is the first comprehensive report documenting the 2006 epidemic of atypical PRRS outbreak in China and identifying the 30 amino-acid deletion in NSP2, a novel determining factor for virulence which may be implicated in the high pathogenicity of PRRSV, and will stimulate further study by using the infectious cDNA clone technique.