Molecular epidemiology and population immunity of SARS-CoV-2 in Guangdong (2022-2023) following a pivotal shift in the pandemic.

The SARS-CoV-2 Omicron variant sparked the largest wave of infections worldwide. Mainland China eased its strict COVID-19 measures in late 2022 and experienced two nationwide Omicron waves in 2023. Here, we investigated lineage distribution and virus evolution in Guangdong, China, 2022-2023 by comparing 5813 local viral genomes with the datasets from other regions of China and worldwide. Additionally, we conducted three large-scale serological surveys involving 1696 participants to measure their immune response to the BA.5 and XBB.1.9 before and after the corresponding waves. Our findings revealed the Omicron variants, mainly the BA.5.2.48 lineage, causing infections in over 90% of individuals across different age groups within a month. This rapid spread led to the establishment of widespread immunity, limiting the virus's ability to further adaptive mutation and dissemination. While similar immune responses to BA.5 were observed across all age groups after the initial wave, children aged 3 to 11 developed a stronger cross immune response to the XBB.1.9 strain, possibly explaining their lower infection rates in the following XBB.1 wave. Reinfection with Omicron XBB.1 variant triggered a more potent neutralizing immune response among older adults. These findings highlight the impact of age-specific immune responses on viral spread in potential future waves.

Intermediate open state of CD4-bound HIV-1 env heterotrimers in asia CRFs.

The HIV-1 envelope glycoprotein (Env) plays crucial role in viral infection by facilitating viral attachment to host cells and inducing fusion of the virus with the host cell membrane. This fusion allows the HIV-1 viral genome to enter the target cell then triggering various stages of the viral life cycle. The native Env directly interacts with the main receptor CD4 and the co-receptor (CCR5 or CXCR4) in human cell membrane then induces membrane fusion. The elucidation of the structure of Env with CD4 and co-receptors in different HIV-1 subtypes is essential for the understanding of the mechanism of virus entry. Here we report the Cryo-EM structure of the CD4-bound HIV-1 heterotrimeric Env from Asia prevalent CRF07_BC CH119 strain. In this structure, the binding of three CD4 molecules with Env induced extensively conformational changes in gp120, resulting in the transformation of the Env from close state to intermediate open state. Additionally, the conformational shift of V1/V2 loops of the heterotrimeric Env allosterically expose the V3 loop and promoting the further interactions with co-receptor CCR5 or CXCR4. These findings not only illustrate the structural complexity and plasticity of HIV-1 Env but also give new insights how the biological trimeric Env initialize the immune recognition and membrane fusion.

Genomic epidemiology of CVA10 in Guangdong, China, 2013-2021.

Hand, Foot and Mouth Disease (HFMD) is a highly contagious viral illness primarily affecting children globally. A significant epidemiological transition has been noted in mainland China, characterized by a substantial increase in HFMD cases caused by non-Enterovirus A71 (EV-A71) and non-Coxsackievirus A16 (CVA16) enteroviruses (EVs). Our study conducts a retrospective examination of 36,461 EV-positive specimens collected from Guangdong, China, from 2013 to 2021. Epidemiological trends suggest that, following 2013, Coxsackievirus A6 (CVA6) and Coxsackievirus A10 (CVA10) have emerged as the primary etiological agents for HFMD. In stark contrast, the incidence of EV-A71 has sharply declined, nearing extinction after 2018. Notably, cases of CVA10 infection were considerably younger, with a median age of 1.8 years, compared to 2.3 years for those with EV-A71 infections, possibly indicating accumulated EV-A71-specific herd immunity among young children. Through extensive genomic sequencing and analysis, we identified the N136D mutation in the 2 A protein, contributing to a predominant subcluster within genogroup C of CVA10 circulating in Guangdong since 2017. Additionally, a high frequency of recombination events was observed in genogroup F of CVA10, suggesting that the prevalence of this lineage might be underrecognized. The dynamic landscape of EV genotypes, along with their potential to cause outbreaks, underscores the need to broaden surveillance efforts to include a more diverse spectrum of EV genotypes. Moreover, given the shifting dominance of EV genotypes, it may be prudent to re-evaluate and optimize existing vaccination strategies, which are currently focused primarily target EV-A71.

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Quantifying rigidity for thermally stable Cr3+ phosphors.

Near-infrared (NIR) phosphors with high thermal stability are significant for NIR light-emitting diodes (LEDs). For a decade, Debye temperature has been a successful indicator of structural rigidity and thermal stability for phosphors, but some exceptions exist due to its dependence on atomic mass. Inspired by the Debye temperature model that relates the elastic properties of solids, our density functional theory calculations revealed that the Vickers hardness of Cr3+-doped NIR phosphors was negatively correlated with Stokes shifts (Pearson's R = -0.81) and positively correlated with thermal stabilities (Pearson's R = 0.85) within a set of 13 distinct material types. Highlighting the predictive power of Vickers hardness, two new NIR phosphors were investigated: KMg(PO3)3:Cr3+ showed low thermal stability, correlating with its lower Vickers hardness, in contrast to the high thermal stability and correspondingly higher Vickers hardness of La2MgSnO6:Cr3+. Vickers hardness can be used to screen potential hosts for Cr3+-doped NIR phosphors with high thermal stabilities, due to the advantages of the predictable feature by density functional theory calculation and low independence on atomic mass.

Influence of framework disordering on the luminescence performance of Cr3+-doped near-infrared phosphors: a case study of A3B6O14-type hosts.

The luminescence efficiency and thermal stability are enduring topics in the realm of phosphors. It is acknowledged that the structural transformation from disorder to order results in increased lattice rigidity, consequently inducing heightened efficiency and enhanced thermal stability. In this case study of the structural evolution of Ca3Ga2Ge4O14:Cr3+, NaCa2GaGe5O14:Cr3+ and Na2CaGe6O14:Cr3+ near-infrared (NIR) phosphors, a significant paradox is revealed: the incongruent relationship between the fluctuating degrees of disorder and the simultaneous improvements in efficiency and thermal stability. By drawing on insights gained from structural analysis, optical investigations, and theoretical calculations, a notable revelation surfaces: the primary factor affecting rigidity and optical performance is not the disordering of the entire lattice, but rather the disordering of the framework itself. The findings elucidate the principle of framework-order engineering for crafting high-performance NIR phosphors.

METTL14 and FTO mediated m6A modification regulate PCV2 replication by affecting miR-30a-5p maturity.

The epigenetic modification of the N6-methyladenosine (m6A) methylation plays an important role in virus infection and replication. However, its role in Porcine circovirus type 2 (PCV2) replication has not been well studied. Here, we demonstrated that m6A modifications are increased in PK-15 cells after PCV2 infection. In particular, PCV2 infection could increase the expression of methyltransferase METTL14 and demethylase FTO. Moreover, interfering with METTL14 accumulation reduced the m6A methylation level and virus reproduction, whereas depleting the FTO demethylase enhanced the m6A methylation level and stimulated virus reproduction. Besides, we showed that METTL14 and FTO regulate PCV2 replication by affecting the process of miRNA maturity, especially the miRNA-30a-5p. Taken together, our results demonstrated that the m6A modification positively affects PCV2 replication and the role of m6A modification in the replication mechanism of the PCV2 virus provides a new idea for the prevention and control of the PCV2.

A chimeric PCV rescued virus with the immunogenic cap gene of PCV3 cloned into the genomic backbone of the nonpathogenic PCV1 induces specific antibodies but with no pathogenic in pigs.

A chimeric PCV called PCV1-3 with the immunogenic Cap gene of pathogenic PCV type 3(PCV3) cloned into the genomic skeleton of the nonpathogenic PCV1 was rescued and inoculated into PCV3 negative piglets. The results of fluorescence quantitative PCR showed that the PCV1-3 DNA detected in serum and tissues was negative. The pathogenicity of piglets showed that PCV 1-3 did not cause the clinical characteristics and pathological changes. The viral neutralization assay revealed that infected pigs could produce antibodies and neutralize the viral activity. All results showed that chimeric virus induced specific antibodies but with no pathogenic in pigs, which provided new candidate strains for the development of PCV3 vaccine.

CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy.

RNA interference (RNAi) is a major form of antiviral defense in host cells, and Ago2 and Dicer are the major proteins of RNAi. The Senecavirus A (SVA) is a reemerging virus, resulting in vesicular lesions in sows and a sharp decline in neonatal piglet production. In this study, CRISPR/Cas9 technology was used to knock out Ago2 and Dicer genes in BHK-21 cell lines used for SVA vaccine production. Cell clones with homozygous frameshift mutations of Ago2 and Dicer genes were successfully identified. The two knockout cell lines were named BHK-DicerΔ- and BHK-Ago2Δ-. Results showed that the two genes' knockout cell lines were capable of stable passage and the cell growth rate did not change significantly. The replication rate and virus titers of SVA were significantly increased in knockout cell lines, indicating that RNAi could inhibit SVA replication. In addition, compared with normal cells, autophagy was significantly enhanced after SVA-infected knockout cell lines, while there was no significant difference in autophagy between the knockout and normal cell lines without SVA. The results confirmed that SVA could enhance the autophagy in knockout cells and promote viral replication. The two knockout cell lines can obtain viruses with high viral titers and have good application prospects in the production of SVA vaccine. At the same time, the RNAi knockout cell lines provide convenience for further studies on RNAi and SVA resistance to RNAi, and it lays a foundation for further study of SVA infection characteristics and screening of new therapeutic drugs and drug targets.

Complete genome and pathogenesis of a novel recombinant Senecavirus A isolate in P.R. China.

Senecavirus A (SVA), formerly called Seneca Valley virus (SVV) was first isolated from the USA in 2002. This study isolated an SVA strain from a pig herd in Shandong Province, PR China and designated it SVA-CH-SDGT-2017. The full-length genome, excluding the poly(A) tails of the SVA isolates, was 7280 nucleotides long, with the genomic organization resembling and sharing high nucleotide identities of 90.7-96.9 % with other previously reported SVA isolates. To investigate the pathogenicity of the SVA isolates, experimental infections of pigs were performed. The SVA strains successfully infected the pigs, as evidenced by the presence of virus shedding and robust serum neutralizing antibody responses. In addition, the contact-exposed experiment showed that the virus shedding of the contact-exposed pigs was approximately a 100-fold reduced compared to that of the inoculated group, indicating that the virus is capable of transmission to pigs. Our findings provide useful data for studying the pathogenesis and transmission of SVA in pigs.

Mutation of the sixth amino acid of the Rep protein has no effect on porcine circovirus 2b but enhances porcine circovirus 2d replication in vitro.

Porcine circovirus type 2 (PCV2) is the etiological agent of post-weaning multisystemic wasting syndrome (PMWS). The original prevalent genotype, PCV2a, has been replaced by genotypes 2b and 2d in the swine population worldwide. The Rep protein is critical for viral replication. Comparison of a large number of Rep protein amino acid (aa) sequences showed that three sites distinguish genotype 2b from genotype 2d. In order to analyze the effect of exchanging the amino acids (asparagine and serine) at position 6 in the Rep proteins of PCV2b and PCV2d, two wild-type and two mutant viruses were rescued. Real-time quantitative PCR and a one-step growth curve were used to determine the viral load to assess the replication ability of the rescued viruses. The results showed that there was no significant difference in in vitro performance between the wild-type PCV2b and the mutated virus, while the mutation of PCV2d enhanced viral replication.

PCV2 Triggers PK-15 Cell Apoptosis Through the PLC-IP3R-Ca2+ Signaling Pathway.

The endoplasmic reticulum (ER) plays an essential role in Ca2+ concentration balance and protein biosynthesis. During infection, the virus needs to complete its life process with the help of ER. At the same time, ER also produces ER stress (ERS), which induces apoptosis to resist virus infection. Our study explored the Ca2+ concentration, ERS, and the apoptosis mechanism after porcine circovirus 2 (PCV2) infection. We show here that PCV2 infection induces the increased cytoplasmic Ca2+ level and PK-15 cell ER swelling. The colocalization of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptor (IP3R) in the cytoplasm was observed by laser confocal microscopy. Western blot and quantitative polymerase chain reaction experiments confirmed that PLC and IP3R expression levels increased after PCV2 infection, and Ca2+ concentration in the cytoplasm increased after virus infection. These results suggest that PCV2 infection triggers ERS of PK-15 cells via the PLC-IP3R-Ca2+ signaling pathway to promote the release of intracellular Ca2+ and led to cell apoptosis.

Efficient influence of ssDNA virus PCV2 replication by CRISPR/Cas9 targeting of the viral genome.

Porcine circovirus type 2 (PCV2), a ubiquitous pathogen that primary cause of postweaning multisystemic wasting syndrome (PMWS), had caused significant morbidity and mortality in swine populations with huge economic losses in the worldwide swine industry. Currently, looking for effective antiviral drugs for PCV2 infection remains an important works. In our study, CRISPR/Cas9 system was used to further detected the key sites of PCV2 replication. We designed 8 single guide RNAs (sgRNA) by targeting essential genes across the genome of PCV2. Western-blot(WB), Cell counting kit-8 for high-throughput sgRNA screening were applied to detect PCV2 replication levels. The results showed that Oc8, O13, O134, NQT and NPS sgRNAs can edit the PCV2 genome efficiently and inhibit PCV2 replication in PK-15 cell; H3 sgRNA cannot edit the PCV2 genome successfully; NAT sgRNA can edit the PCV2 genome efficiently to improve the PCV2 replication in PK-15 cell; O26 sgRNA can edit the PCV2 genome successfully but it is not known yet of its effect on PCV2 replication, besides the Cas9 expression had no effect on cell viability. These data suggest that CRISPR/Cas9 system targeting PCV2 essential genes may serve as a novel therapeutic agent against PCV2 infection in the future.

Molecular epidemiology of enteroviruses associated with severe hand, foot and mouth disease in Shenzhen, China, 2014-2018.

In this study, we investigated the epidemiology and molecular characteristics of enteroviruses associated with severe hand, foot and mouth disease (HFMD) in Shenzhen, China, during 2014-2018. A total of 137 fecal specimens from patients with severe HFMD were collected. Enterovirus (EV) types were determined using real-time reverse transcription polymerase chain reaction (RT-PCR), RT nested PCR, and sequencing. Sequences were analyzed using bioinformatics programs. Of 137 specimens tested, 97 (70.8%), 12 (8.8%), and 10 (7.3%) were positive for EV-A71, coxsackievirus A6 (CVA6), and CVA16, respectively. Other pathogens detected included CVA2 (2.9%, 4/137), CVA10 (2.9%, 4/137), CVA5 (0.7%, 1/137), echovirus 6 (E6) (0.7%, 1/137) and E18 (0.7%, 1/137). The most frequent complication in patients with proven EV infections was myoclonic jerk, followed by aseptic encephalitis, tachypnea, and vomiting. The frequencies of vomiting and abnormal eye movements were higher in EV-A71-infected patients than that in CVA6-infected or CVA16-infected patients. Molecular phylogeny based on the complete VP1 gene revealed no association between the subgenotype of the virus and disease severity. Nevertheless, 12 significant mutations that were likely to be associated with virulence or the clinical phenotype were observed in the 5'UTR, 2Apro, 2C, 3A, 3Dpol and 3'UTR of CVA6. Eight significant mutations were observed in the 5'UTR, 2B, 3A, 3Dpol and 3'UTR of CVA16, and 10 significant mutations were observed in the 5'UTR, VP1, 3A and 3Cpro of CVA10. In conclusion, EV-A71 is still the main pathogen causing severe HFMD, although other EV types can also cause severe complications. Potential virulence or phenotype-associated sites were identified in the genomes of CVA6, CVA16, and CVA10.

Synthesis of ortho-Phenolic Sulfilimines via an Intermolecular Sulfur Atom Transfer Cascade Reaction.

To expand the toolbox for the synthesis of ortho-phenolic sulfilimines, sigmatropic rearrangements were introduced to the field of sulfilimine chemistry. Herein we report a N-H sulfenylation/[2,3]-sigmatropic rearrangement cascade reaction. This mild reaction enables commercially available thiols to serve as the sulfenylation reagent and generates water as the sole byproduct. Moreover, the reaction has a wide substrate scope and can be conducted on a gram scale with excellent reaction efficiency.

Genetic characterization of Porcine circovirus type 2 from 2013 to 2018 in Shandong Province, China.

PCV2 belongs to the genus Circovirus, family Circoviridae, who is recognized as the causative agents of postweaning multisystemic wasting syndrome. Since being found to China in 2000, it has caused serious damage to the pig industry. In this study, we downloaded 40 PCV2 genome-wide sequences uploaded to GenBank from 2013 to 2018 in Shandong Province, including 23 uploaded by our laboratory. Construction of a genome-wide evolution tree using MEGA V5.0 software. Phylogenetic tree analysis indicated that the genotype of PCV2 in Shandong Province was: three genotypes coexisted (2a, 2b, 2d); among them, PCV2d has become the main genotype in the province due to its number and spread range. Amino acid sequence analysis of different genotypes of ORF2 showed that specific amino acid sites exist in different genotypes, with the most significant range of 81-160; different genotypes of PCV2 can be distinguished at the molecular level. This study found that due to the increase in infections of the PCV2d genotype in recent years, it may replace PCV2b as the dominant base in Shandong.

Crystal structure of PppA from Pseudomonas aeruginosa, a key regulatory component of type VI secretion systems.

The Type VI secretion system (T6SS) is a membrane protein complex related to inter-bacterial competitions and host-pathogen interactions in Pseudomonas aeruginosa. The T6SS is regulated by a great variety of regulatory mechanisms at multiple levels, including post-translational modification with threonine phosphorylation mediated by Ser/Thr protein kinase PpkA and phosphatase PppA. The T6SS is activated by PpkA via Thr phosphorylation of Fha, and PppA can antagonize PpkA. PppA is a PP2C-family protein phosphatase and plays a key role in the disassembly and reassembly of T6SS organelles. Herein, we report the first crystal structure of PppA from Pseudomonas aeruginosa, which was determined at a resolution of 2.10 Å. The overall structure consists of a bacteria PPM structural core and a flexible flap subdomain. PppA harbors a catalytic pocket containing two manganese ions which correspond to the canonical dinuclear metal center of Ser/Thr protein phosphatases including the bacterial PPM phosphatases and human PP2C. The flexibility and the diversity of the sequence of flap subdomain across the homologues might provide clues for substrates specific recognition of phosphatases.

Near-Complete Genome Sequences of 12 Coxsackievirus Group A Strains from Hand, Foot, and Mouth Disease and Herpangina Cases with Different Clinical Symptoms.

Coxsackievirus group A (CV-A) strains are important pathogens of hand, foot, and mouth disease and herpangina. We report here the near-complete genome sequences of 12 CV-A strains isolated from infants and children with different clinical diseases. The presented data will be very useful for future genome-based epidemiological studies.

Molecular surveillance of coxsackievirus A16 reveals the emergence of a new clade in mainland China.

Coxsackievirus A16 (CV-A16) of the genotypes B1a and B1b have co-circulated in mainland China in the past decades. From 2013 to 2017, a total of 3,008 specimens from 3,008 patients with mild hand, foot, and mouth disease were collected in the present study. Viral RNA was tested for CV-A16 by a real-time RT-PCR method, and complete VP1 sequences and full-length genome sequences of CV-A16 strains from this study were determined by RT-PCR and sequencing. Sequences were analyzed using a series of bioinformatics programs. The detection rate for CV-A16 was 4.1%, 25.9%, 10.6%, 28.1% and 12.9% in 2013, 2014, 2015, 2016 and 2017, respectively. Overall, the detection rate for CV-A16 was 16.5% (497/3008) in this 5-year period in Shenzhen, China. One hundred forty-two (142/155, 91.6%) of the 155 genotype B1 strains in the study belonged to subgenotype B1b, and 13 (13/155, 8.4%) strains belonged to subgenotype B1a. Two strains (CVA16/Shenzhen174/CHN/2017 and CVA16/Shenzhen189/CHN/2017) could not be assigned to a known genotype. Phylogenetic analysis of these two strains and other Chinese CV-A16 strains indicated that these two CV-A16 strains clustered independently in a novel clade whose members differed by 8.4%-11.8%, 8.4%-12.1%, and 14.6%-14.8% in their nucleotide sequences from those of Chinese B1a, B1b, and genotype D strains, respectively. Phylogenetic analysis of global CV-A16 strains further indicated that the two novel CV-A16 strains from this study grouped in a previously uncharacterized clade, which was designated as the subgenogroup B3 in present study. Meanwhile, phylogenetic reconstruction revealed two other new genotypes, B1d and B4, which included a Malaysian strain and two American strains, respectively. The complete genome sequences of the two novel CV-A16 strains showed the highest nucleotide sequence identity of 92.3% to the Malaysian strain PM-15765-00 from 2000. Comparative analysis of amino acid sequences of the two novel CV-A16 strains and their relatives suggested that variations in the nonstructural proteins may play an important role in the evolution of modern CV-A16.

The correlation between the mutual deletions of amino acids within porcine circovirus rep protein and the discrepancy of replication.

Porcine circovirus (PCV) has two potential open reading frames, ORF1 and ORF2. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA. In some studies, PCV1 replicated more efficiently in PK-15 cells than PCV2 was elucidated. PCV1 compared with PCV2; there is some amino acids' deficiency on Rep protein. To identify whether the above amino acids deletion affects the replication of PCV1 and PCV2, we constructed three double copy clones by overlap extension PCR. The 2PCV2(vV) clone deleted the valine of Rep protein in the backbone of PCV2 genome. The 2PCV2(dSGR) clone inserted serine, glycine and arginine of Rep protein successively in the backbone of PCV2 genome. The 2PCV2(dSGR&vV) clone inserted serine, glycine and arginine as well as deleted the valine of Rep protein in the backbone of PCV2 genome. These clones we constructed with amino acid mutations and parental DNA clones were all transfected in PK-15 cells that free of PCV contamination, and their growth characteristics in vitro were determined and compared, to evaluating the replication of the mutant infectious DNA clones. Our results showed that the double copy infectious clones with amino acid mutations could be rescued in vitro. The 2PCV2(vV) replicated more efficiently than parental viruses 2PCV2 and 2PCV1 but the replicated ability of 2PCV2(dSGR) and 2PCV2(dSGR&vV) is attenuated than parental viruses 2PCV2 and 2PCV1. We can determine the valine is the important amino acid that cause PCV1 replicated more efficiently in PK-15 cells than PCV2 primarily. These findings are benefit for exploring the mechanisms of viral replication in pigs and important implications for PCV2 vaccine development.