The Development of a Sensitive Droplet Digital Polymerase Chain Reaction Test for Quantitative Detection of Goose Astrovirus.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

Phylogenetically evolutionary analysis provides insights into the genetic diversity and adaptive evolution of porcine deltacoronavirus.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is one of the emerging swine enteric coronaviruses (SECoVs), which has been widely prevalent in the North America and Asia. In addition to causing severe diarrhea in piglets, PDCoV also shows the potential to infect diverse host species, including calves, chickens, turkey poults, and humans. However, the clinical pathogenicity and genetic evolution of PDCoV is still not fully understood. RESULTS: Here, we recorded an outbreak of a novel recombinant PDCoV strain (CHN-HeN06-2022) in a large nursery fattening pig farm. Genomic analysis showed that the CHN-HeN06-2022 strain shared 98.3-98.7% sequence identities with the Chinese and American reference strains. To clarify the evolutionary relationships, phylogenetic analysis was performed using the PDCoV genome sequences available in the GenBank database. Based on genetic distance and geographical distribution, the phylogenetic tree clearly showed that all the PDCoV sequences could be divided into lineage 1 and lineage 2, which were further classified into sublineage 1.1 (Chinese strains), 1.2 (the North American strains), 2.1 (the Southeast Asian strains), and 2.2 (Chinese strains). Corresponding to the evolutionary tree, we found that, compared to lineage 1, lineage 2 strains usually contain a continuous 6-nt deletion in Nsp2 and a 9-nt deletion in Nsp3, respectively. Furthermore, recombination analysis suggested that the CHN-HeN06-2022 occurred segments exchange crossed Nsp2 and Nsp3 region between sublineage 1.1 and sublineage 2.1. Combined with previously reported recombinant strains, the highest recombination frequency occurred in Nsp2, Nsp3, and S gene. Additionally, we identified a total of 14 amino acid sites under positive selection in spike protein, most of which are located in the regions related with the viral attachment, receptor binding, and membrane fusion. CONCLUSIONS: Taken together, our studies provide novel insights into the genetic diversity and adaptive evolution of PDCoV. It would be helpful to the development of vaccine and potential antiviral agent.

Development of a unique sandwich enzyme-linked immunosorbent assay based on monoclonal antibodies for the specific detection of the egg drop syndrome virus.

RESEARCH HIGHLIGHTS: A sandwich ELISA was developed to detect EDSV using the mAbs 5G4 and HRP-6G6.The sandwich ELISA maintained high specificity and sensitivity.The sandwich ELISA had equivalent consistency with real-time PCR assay.

Design and Identification of a Novel Antiviral Affinity Peptide against Fowl Adenovirus Serotype 4 (FAdV-4) by Targeting Fiber2 Protein.

Outbreaks of hydropericardium hepatitis syndrome caused by fowl adenovirus serotype 4 (FAdV-4) with a novel genotype have been reported in China since 2015, with significant economic losses to the poultry industry. Fiber2 is one of the important structural proteins on FAdV-4 virions. In this study, the C-terminal knob domain of the FAdV-4 Fiber2 protein was expressed and purified, and its trimer structure (PDB ID: 7W83) was determined for the first time. A series of affinity peptides targeting the knob domain of the Fiber2 protein were designed and synthesized on the basis of the crystal structure using computer virtual screening technology. A total of eight peptides were screened using an immunoperoxidase monolayer assay and RT-qPCR, and they exhibited strong binding affinities to the knob domain of the FAdV-4 Fiber2 protein in a surface plasmon resonance assay. Treatment with peptide number 15 (P15; WWHEKE) at different concentrations (10, 25, and 50 µM) significantly reduced the expression level of the Fiber2 protein and the viral titer during FAdV-4 infection. P15 was found to be an optimal peptide with antiviral activity against FAdV-4 in vitro with no cytotoxic effect on LMH cells up to 200 µM. This study led to the identification of a class of affinity peptides designed using computer virtual screening technology that targeted the knob domain of the FAdV-4 Fiber2 protein and may be developed as a novel potential and effective antiviral strategy in the prevention and control of FAdV-4.

Pseudorabies Virus Regulates the Extracellular Translocation of Annexin A2 To Promote Its Proliferation.

Pseudorabies virus (PRV) infection causes enormous economic losses to the pork industry and severe health consequences in many hosts. Annexin A2 (ANXA2) is a membrane-associated protein with various intracellular functions associated with many viral infections. However, the role of ANXA2 in alphaherpesvirus replication is still not explored. In the present study, we identified the interaction between ANXA2 and PRV US3. The deficiency of ANXA2 significantly restricted PRV proliferation. PRV infection or US3 overexpression led to ANXA2 extracellular translocation. Furthermore, we confirmed that PRV or US3 could lead to the phosphorylation of the Tyr23 ANXA2 and Tyr419 Src kinase, which was associated with the ANXA2 cell surface transposition. US3 can also bind to Src in an ANXA2-independent manner and enhance the interaction between Src and ANXA2. Additionally, inhibitors targeting ANXA2 (A2ti-1) or Src (PP2) could remarkably inhibit PRV propagation in vitro and protect mice from PRV infection in vivo. Collectively, our findings broaden our understanding of the molecular mechanisms of ANXA2 in alphaherpesvirus pathogenicity and suggest that ANXA2 is a potential therapeutic target for treating alphaherpesvirus-induced infectious diseases. IMPORTANCE PRV belongs to the alphaherpesvirus and has recently re-emerged in China, causing severe economic losses. Recent studies also indicate that PRV may pose a potential public health challenge. ANXA2 is a multifunctional calcium- and lipid-binding protein implicated in immune function, multiple human diseases, and viral infection. Herein, we found that ANXA2 was essential to PRV efficient proliferation. PRV infection resulted in the extracellular translocation of ANXA2 through phosphorylation of ANXA2 and Src. ANXA2 and Src formed a complex with PRV US3. Importantly, inhibitors targeting ANXA2 or Src prevented PRV infection in vitro and in vivo. Therefore, our studies reveal a novel strategy by which alphaherpesvirus modifies ANXA2 to promote its replication and highlight ANXA2 as a target in developing novel promising antivirus agents in viral therapy.

Development and application of a recombinase-aided amplification and lateral flow assay for rapid detection of pseudorabies virus from clinical crude samples.

Pseudorabies virus (PRV) is an economically important viral agent affecting the swine industry in China. Accurate, rapid and simple detection is critical to PRV control and eradication. In the present study, a visible and low equipment-dependent recombinase-aid amplification assay integrated with lateral flow assay (RAA-LFA) was successfully developed to detect the PRV against the gE gene. The RAA-LFA did not react with the other swine pathogens, indicating the method has a good specificity. The limit of detection (LOD) for this RAA-LFA method was 21 copies per reaction against standard plasmids containing gE gene. Notably, the RAA-LFA can detect as low as 6.0 × 100 50 % tissue culture infective dose (TCID50) viral titer per reaction under nucleic-acid-extraction free condition. Clinical detection showed that the results detected by RAA-LFA were completely consistent with that of the qPCR assay. Taken together, the developed PRV RAA-LFA method provides approachability, comparable accuracy and sensitivity tool for PRV point-of-care testing (POCT), which is valuable to PRV control in areas where equipment and personnel resources are scarce.

Precise Assembly of Multiple Antigens on Nanoparticles with Specially Designed Affinity Peptides.

Antigen proteins, assembled on nanoparticles, can be recognized by antigen-presenting cells effectively to enhance antigen immunogenicity. The ability to simultaneously display multiantigens on the same nanoparticle could have numerous applications but remained technical challenges. Here, we described a method for precise assembly of multiple antigens on nanoparticles with specially designed affinity peptides. First, we designed and screened affinity peptides with high affinity and specificity, which could respectively target the key amino acid residues of classical swine fever virus (CSFV) E2 protein or porcine circovirus type 2 capsid protein (PCV2 Cap) accurately. Then, we conjugated the antigen proteins to poly(lactic acid-glycolic acid) copolymer (PLGA) and Gram-positive enhancer matrix (GEM) nanoparticles through the peptides and perfectly assembled two kinds of multiantigen display nanoparticles with different particle sizes. Subsequently, the immunological properties of the assembled nanoparticles were tested. The results showed that the antigen display nanoparticles could promote the maturation, phagocytosis, and proinflammatory effects of antigen-presenting cells (APCs). Besides, compared with the antigen proteins, multiantigen display nanoparticles could induce much higher levels of antibodies and neutralizing antibodies in mice. This strategy may provide a technical support for the study of protein structure and the research and development of polyvalent vaccines.

Core antigenic advantage domain-based ELISA to detect antibody against novel goose astrovirus in breeding geese.

Goose astrovirus (GAstV), the major causative agent of visceral and joint gout in goslings, is a novel pathogen greatly threatening waterfowl industry. Importantly, the horizontal and vertical transmissibility of GAstV posed a great challenge for disease prevention and control. Given the absence of commercial vaccine, restricting vertical transmission and protecting susceptible goslings must be a priority. Although many detection methods have been established, there is no serological method to detect GAstV-specific antibody, greatly limiting inspection and elimination of infected breeding bird. In this study, the B-cell epitopes of GAstV capsid protein were predicted, and its core antigenic advantage domain (shCAP) was expressed and purified. After authenticating the antigenicity, the recombinant shCAP protein was taken as the coating antigen, and an easily accessible indirect enzyme-linked immunosorbent assay (ELISA) was established to detect GAstV-specific antibody. The working conditions, including antigen concentration, serum dilution and incubation time, blocking buffer concentration, and color developing time, were gradually optimized by checkerboard titration. The cut-off OD450 value of the indirect ELISA for positive sample was 0.379, and the analytical sensitivity was 1:800. There was no cross-reaction with sera against goose parvovirus (GPV), Tembusu virus (TUMV), H5 and H7 subtype avian influenza virus (AIV H5 + H7), and Newcastle disease virus (NDV). The assay was further applied to examine 73 breeding goose serum samples and shared excellent agreement of 93.5% (68/73) with western blot, which also suggested that GAstV is circulating in the goose population in China. In conclusion, the developed indirect ELISA is simple, specific, and sensitive, which will be greatly useful to screen GAstV infection and block vertical transmission. KEY POINTS: • B-cell epitopes of GAstV capsid protein were predicted and expressed as immunogen • A core antigenic advantage domain-based ELISA was established to detect GAstV-specific antibody • The established ELISA will contribute to inspection and elimination of infected breeding geese and provide a useful tool for large scale serological testing of GAstV in geese.

Comprehensive Analysis of Codon Usage Patterns in Chinese Porcine Circoviruses Based on Their Major Protein-Coding Sequences.

Porcine circoviruses (PCVs) are distributed in swine herds worldwide and represent a threat to the health of domestic pigs and the profits of the swine industry. Currently, four PCV species, including PCV-1, PCV-2, PCV-3 and PCV-4, have been identified in China. Considering the ubiquitous characteristic of PCVs, the new emerged PCV-4 and the large scale of swine breeding in China, an overall analysis on codon usage bias for Chinese PCV sequences was performed by using the major proteins coding sequences (ORF1 and ORF2) to better understand the relationship of these viruses with their host. The data from genome nucleotide frequency composition and relative synonymous codon usage (RSCU) analysis revealed an overrepresentation of AT pair and the existence of a certain codon usage bias in all PCVs. However, the values of an effective number of codons (ENC) revealed that the bias was of low magnitude. Principal component analysis, ENC-plot, parity rule two analysis and correlation analysis suggested that natural selection and mutation pressure were both involved in the shaping of the codon usage patterns of PCVs. However, a neutrality plot revealed a stronger effect of natural selection than mutation pressure on codon usage patterns. Good host adaptation was also shown by the codon adaptation index analysis for all these viruses. Interestingly, obtained data suggest that PCV-4 might be more adapted to its host compared to other PCVs. The present study obtained insights into the codon usage pattern of PCVs based on ORF1 and ORF2, which further helps the understanding the molecular evolution of these swine viruses.

Efficacy of a live attenuated highly pathogenic PRRSV vaccine against a NADC30-like strain challenge: implications for ADE of PRRSV.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) infection can cause severe reproductive failure in sows and respiratory distress in pigs of all ages, leading to major economic losses. To date, there are still no effective strategies to prevent and control PRRSV. Antibody-dependent enhancement (ADE), a phenomenon in which preexisting non-neutralizing antibodies or sub-neutralizing antibodies facilitate virus entry and replication, may be a significant obstacle in the development of effective vaccines for many viruses, including PRRSV. However, the contribution of ADE to PRRSV infection remains controversial, especially in vivo. Whether attenuated PRRSV vaccines prevent or worsen subsequent disease in pigs infected by novel PRRSV strains requires more research. In the present study, in vivo experiments were conducted to evaluate ADE under different immune statuses, which were produced by waiting different lengths of time after vaccination with a commercially available attenuated highly pathogenic PRRSV (HP-PRRSV) vaccine (JXA1-R) before challenging the pigs with a novel heterologous NADC30-like strain. RESULTS: Piglets that were vaccinated before being challenged with PRRSV exhibited lower mortality rates, lower body temperatures, higher bodyweight gain, and lower viremia. These results demonstrate that vaccination with JXA1-R alleviated the clinical signs of PRRSV infection in all vaccinated groups. CONCLUSIONS: The obtained data indicate that the attenuated vaccine test here provided partial protection against the NADC30-like strain HNhx. No signs of enhanced PRRSV infection were observed under the applied experimental conditions. Our results provide some insight into the molecular mechanisms underlying vaccine-induced protection or enhancement in PRRSV.

Fine mapping of linear B cell epitopes on capsid protein of porcine circovirus 3.

Porcine circovirus type 3 (PCV3) is an emerging swine pathogen associated with acute porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, and multisystemic inflammation. Current evidence shows that PCV3 is spread worldwide, and its high incidence may pose a threat to the global pig industry. Capsid (Cap) protein is the sole structural protein which plays an important role in inducing protective immunity against PCV3 infection. In this study, monoclonal antibodies (mAbs) against Cap protein of PCV3 were produced by the hybridoma technique. Subsequently, 12 serial overlapping peptides (P1 to P12) spanning the entire region of Cap were synthesized to determine the B cell epitope regions using the mAbs. Results from dot-blot and peptide ELISA identified that P3, P9, and P10 were the major B cell antigenic regions. Fine mapping by shorter N- and C-terminal truncated peptides confirmed that the motifs 57NKPWH61, 140KHSRYFT146, and 161QSLFFF166 were linear B cell epitopes, which were highly conserved among different PCV3 strains. Interestingly, we found that the motif 140KHSRYFT146 was highly conserved in all reported types of PCVs (i.e., PCV1, PCV2, PCV3, and PCV4), except for the substitution (Y → K → R) of the first residue. This is the first research to identify B cell epitopes of PCV3 Cap, and these findings may lead to a better understanding of the antibody-antigen interaction and provide some guidance for PCV3 vaccine design.Key points• The recombinant Cap protein of PCV3 was expressed and purified in soluble form. • PCV3 Cap-specific mAbs prepared in this study had no cross-reactivity with PCV1/PCV2 Cap. • This is the first report of three conserved linear B cell epitopes on PCV3 Cap. • The minimal residues of the epitopes were 57-61 aa, 140-146 aa, and 161-166 aa.

Gold nanoparticles enhance immune responses in mice against recombinant classical swine fever virus E2 protein.

OBJECTIVE: To create a novel subunit vaccine that used AuNPs as carriers to enhance immune responses in mice against recombinant classical swine fever virus E2 protein (CSFV E2). RESULTS: Gold nanoparticles (AuNPs) were successfully coupled to the E2 protein and formed stable particle complexes called E2 conjugated AuNPs (E2-AuNPs). In vitro studies have shown that the E2-AuNPs complex has the same immunogenicity as the E2 protein, and AuNPs can promote the phagocytosis of the E2 protein by antigen-presenting cells (APCs). In vivo results of BALB/c mice showed that the antibody levels, lymphocyte proliferation index, IFN-γ and IL-10 cytokines induced by E2-AuNPs were relatively higher than those of E2 or AuNPs group. CONCLUSIONS: This finding demonstrated the potential of using AuNPs as a carrier to enhance the body's immune response for developing CSFV subunit vaccines. This model also contributes to the development of other flavivirus subunit vaccines, such as hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV).

Phylogenetic analysis of porcine circovirus type 2 (PCV2) between 2015 and 2018 in Henan Province, China.

BACKGROUND: Porcine circovirus type 2 (PCV2) is the pathogen of porcine circovirus associated diseases (PCVAD) and one of the main pathogens in the global pig industry, which has brought huge economic losses to the pig industry. In recent years, there has been limited research on the prevalence of PCV2 in Henan Province. This study investigated the genotype and evolution of PCV2 in this area. RESULTS: We collected 117 clinical samples from different regions of Henan Province from 2015 to 2018. Here, we found that the PCV2 infection rate of PCV2 was 62.4%. Thirty-seven positive clinical samples were selected to amplify the complete genome of PCV2 and were sequenced. Based on the phylogenetic analysis of PCV2 ORF2 and complete genome, it was found that the 37 newly detected strains belonged to PCV2a (3 of 37), PCV2b (21 of 37) and PCV2d (13 of 37), indicating the predominant prevalence of PCV2b and PCV2d strains. In addition, we compared the amino acid sequences and found several amino acid mutation sites among different genotypes. Furthermore, the results of selective pressure analysis showed that there were 5 positive selection sites. CONCLUSIONS: This study indicated the genetic diversity, molecular epidemiology and evolution of PCV2 genotypes in Henan Province during 2015-2018.

Nanovaccine Confers Dual Protection Against Influenza A Virus And Porcine Circovirus Type 2.

BACKGROUND: The influenza A virus (IAV) is known for its high variability and poses a huge threat to the health of humans and animals. Pigs play a central role in the cross-species reassortment of IAV. Ectodomain of matrix protein 2 (M2e) is the most conserved protective antigen in IAV and can be used to develop nanovaccines through nanoparticles displaying to increase its immunogenicity. However, the high immunogenicity of nanoparticles can cause the risk of off-target immune response, and excess unwanted antibodies may interfere with the protective efficacy of M2e-specific antibodies. Therefore, it is necessary to select reasonable nanoparticles to make full use of antibodies against nanoparticles while increasing the level of M2e-specific antibodies. Porcine circovirus type 2 (PCV2) is the most susceptible virus in pigs and can promote IAV infection. It is meaningful to develop a vaccine that can simultaneously control swine influenza virus (SIV) and PCV2. METHODS: In the present study, M2e of different copy numbers were inserted into the capsid (Cap) protein of PCV2 and expressed in Escherichia coli to form self-assembled chimeric virus-like particles (VLPs) nanovaccine. BALB/c mice and pigs were immunized with these nanovaccines to explore optimal anti-IAV and anti-PCV2 immunity. RESULTS: Cap is capable of carrying at least 81 amino acid residues (three copies of M2e) at its C-terminal without impairing VLPs formation. Cap-3M2e VLPs induced the highest levels of M2e-specific immune responses, conferring protection against lethal challenge of IAVs from different species and induced specific immune responses consistent with PCV2 commercial vaccines in mice. In addition, Cap-3M2e VLPs induced high levels of M2e-specific antibodies and PCV2-specific neutralizing antibodies in pigs. CONCLUSION: Cap-3M2e VLP is an economical and promising bivalent nanovaccine, which provides dual protection against IAV and PCV2.

A Universal Influenza Nanovaccine for "Mixing Vessel" Hosts Confers Potential Ability to Block Cross-Species Transmission.

Influenza A virus (IAV), a deadly zoonotic pathogen, poses a tremendous threat and burden to global health systems. Pigs act as "mixing vessel" hosts to support and generate new pandemic viruses. Preventing the spread of IAV in pigs effectively can delay or even block cross-species transmission. Universal vaccines based on the highly conserved ectodomain of influenza matrix protein 2 (M2e) have been widely reported, but have not been applied due to inadequate protection. Porcine circovirus type 2 (PCV2) causes immunosuppression and promotes swine influenza virus (SIV) infection. Here, M2e is inserted into capsid protein of PCV2 without burying the neutralizing epitopes and self-assembles to form a bivalent nanovaccine. Inoculation with the nanovaccine induces robust M2e- and PCV2-specific immune responses. The nanovaccine confers protection against lethal challenges of IAV from different species in mice, and significantly reduces SIV titers in pigs' respiratory tract and blocks SIV transmission. These results indicate that the nanovaccine is an economical and promising PCV2 and universal IAV bivalent vaccine, and it will synergistically and powerfully offer potential ability to block IAV cross-species reassortment and transmission.

Porcine reproductive and respiratory syndrome virus induces interleukin-1ß through MyD88/ERK/AP-1 and NLRP3 inflammasome in microglia.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection which caused severe reproductive failure and respiratory disorders in swine is accompanied with severe nervous symptoms. Our previous studies demonstrated that microglia, the resident innate immune cells in central nervous system (CNS), could support PRRSV infection and replication in vitro. And PRRSV infection led to the increased expressions of large amounts of proinflammatory cytokines and chemokines which contributed to neuropathogenesis of PRRSV. Interleukin-1ß (IL-1ß) is one of the increased proinflammatory cytokines, which possesses diverse functions in immune response upon virus infection, including activation of innate immune and modulation of adaptive immune responses. Importantly, considerable evidences indicated that 1L-1ß is involved in neuronal injury. Here, we demonstrated that PRRSV infection up-regulated IL-1ß expression at both the mRNA and protein levels in microglia in a dose-dependent manner. Myeloid differentiation primary response gene 88 (MyD88), extracellular signal-regulated kinase1/2 (ERK) and activator protein 1 (AP-1) were involved in PRRSV induced IL-1ß production in microglia. Moreover, NOD-like receptor protein 3 (NLRP3) inflammasome is activated by PRRSV in microglia, which is required for IL-1ß secretion. Taken together, our data indicated that PRRSV infection could induce IL-1ß up-regulation, which was likely mediated by MyD88/ERK/AP-1 and NLRP3 inflammasome. These findings will provide new insights into the molecular mechanisms of IL-1ß production and some implications for neuropathogenesis of PRRSV.

Effects of Isatis root polysaccharide in mice infected with H3N2 swine influenza virus.

Isatis root polysaccharide (IRPS) has gained attention in the field of virology. However, very few studies have evaluated the effects of IRPS on H3N2 swine influenza virus (SIV). The antiviral activities of IRPS against SIV were investigated in vitro through three different modes and in vivo in an experimental mouse model of SIV infection. Mice were treated by oral gavage with various doses of IRPS before being experimentally infected with SIV A/swine/Henan/2010(H3N2). The antiviral effects of IRPS were evaluated by clinical signs, weight, histopathology, cytokine levels in lung homogenates and serum nitric oxide (NO) and IgG levels at 2, 5 and 9 d post-infection. IRPS demonstrated an inhibitory effect on SIV in Madin-Darby canine kidney cells. Additionally, IRPS significantly improved symptoms, reduced pathological changes and enhanced serum levels of NO and IgG in SIV-infected mice. Furthermore, detection of cytokines in lung homogenates showed IRPS could alter cytokine production to improve immune responses and systemic ability to repair inflammation. Moreover, IRPS extenuated the pulmonary inflammatory response. The results show that various concentrations of IRPS exert antiviral effects in vitro and in vivo. In an experimental mouse model of SIV infection, IRPS at a dose of 75 mg/kg was effective.

Nanoparticle orientationally displayed antigen epitopes improve neutralizing antibody level in a model of porcine circovirus type 2.

Recent advancements in biotechnology have enabled the rapid identification and subsequent expression of pathogenic microbial major antigens that induce protective immune responses. However, subunit vaccines have not been successfully commercialized mainly due to the lack of sufficient levels of neutralizing antibodies (NAs). High levels of NA rely on the efficient recognition and cross-linking of multiple neutralizing epitopes with B-cell receptors (BCRs). Nanoparticles are able to display coupled antigenic arrays at high density and provide multiple binding molecular scenarios with BCRs. The high-resolution antigenic structure makes it possible to accurately display stable neutralizing epitopes. Therefore, the development of a nanovaccine that orientationally displays neutralizing epitopes is a feasible strategy. To address this hypothesis, the capsid (Cap) protein of porcine circovirus type 2 as model antigen was conjugated to gold nanoparticles (AuNPs) through direct reaction of the mercapto group of the unique cysteines with AuNPs, rendering Cap-AuNPs to have neutralizing epitopes on outer surface and an immunodominant epitope buried within the inner surface. In vitro studies showed that AuNPs promoted the phagocytosis of Cap protein and NA levels were significantly improved, meanwhile antibody levels against the immunodominant epitope was significantly reduced. In mouse studies, Cap-AuNP-immunized mice displayed a high production of interleukin (IL)-4, IL-10, and interferon-γ, suggesting that Cap-AuNPs can effectively activate CD4+ and CD8+ T cells and balance Th1 and Th2 cellular responses. This study presents a new vaccine design strategy based on antigen structure, where nanoparticles are coupled to antigens in well-ordered arrays and orientationally display neutralizing epitopes to enhance NA levels.

Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China.

In several parts of China, there have been a large number of hydropericardium syndrome (HPS) outbreaks caused by serotype 4 fowl adenovirus (FAdV-4) in broiler chickens since 2015. These outbreak-associated FAdV-4 strains were distinct from previous circulating strains which did not lead to severe HPS outbreaks. To better understand the molecular epidemiology of the currently circulating FAdV strains for effective diagnosis and treatment of HPS, we isolated 12 HPS outbreak-associated FAdV-4 strains from different regions in central China and investigated their molecular characteristics by performing phylogenetic analyses based on the hexon genes. Our results indicated the FAdV-4 strains in this study all belonged to serotype FAdV-4, species FAdV-C. And in comparison with ON1, KR5, MX-SHP95, PK-01, PJ-06 strains within the cluster where outbreak-associated FAdV-4 strains were located, the nucleotide sequence divergence were 1.31, 1.10, 1.42, 2.77 and 2.84%, respectively. Phylogenetic analyses revealed the hexon genes of the 12 outbreak-associated strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor and we suggested that these outbreak-associated FAdV-4 strains originate from earlier strains in India.