Whole genome sequencing analysis of seven unknown resistance mechanisms of carbapenem-resistant Klebsiella pneumoniae strains resistance to ceftazidime-avibactam.

AIMS: To investigate alternative resistance mechanisms among seven ceftazidime-avibactam (CZA)-resistant carbapenem-resistant Klebsiella pneumoniae (CRKP) strains lacking common antimicrobial resistance genes (ARGs) using whole genome sequencing. METHODS AND RESULTS: ARG and virulence factors (VFs) were screened using the ARG database CARD and the VF database, respectively, and identified using genomic annotation data with BLAST+. Six strains were ST11 sequence types (STs), and one was ST2123. ST11 strains harbored more ARGs than the ST2123 strains. All seven strains carried multiple ARGs with efflux-mediated antibiotic resistance, including oqxA, oqxB, tet (A), qacEdltal, CRP, H-NS, Kpn-E, F, G, H, acrA, LptD, acrB, acrD, cpxA, mdtB, and mdtC. These efflux-mediated ARGs were identified in most strains and even all strains. Whole genome sequencing revealed that the ST11 strain carried multiple potential prophages, genomic islands, and integrative and conjugative elements, while the ST2123 strain carried an independent potential prophages and a genomic island. CONCLUSIONS: Whole genome sequencing analysis revealed that these seven CZA-resistant CRKP strains lacking common ARGs exhibited efflux-mediated antibiotic resistance-associated ARGs. The main mechanism by which CRKP resists CZA is antibiotic inactivation. Except for tet (A), no ARGs and validation experiments related to efflux were found. This study's results provide a new possibility for the resistance mechanism of CRKP to CZA, and we will verify this conclusion through experiments in the future.

Comparative pharmacokinetic analysis of sporoderm-broken and sporoderm-removed Ganoderma lucidum spore in rat by using a sensitive plasma UPLC-QqQ-MS method.

Previous studies have found that removing the sporoderm significantly enhanced antitumor and immunoregulatory activities of Ganoderma lucidum spore (GLS) compared with breaking the sporoderm. However, the pharmacokinetics of sporoderm-removed GLS (RGLS) and sporoderm-broken GLS (BGLS) remain elusive. To compare the pharmacokinetic differences between the two products, we developed a UPLC-QqQ MS method for determining nine representative triterpenoid concentrations. Chloramphenicol was used as an internal standard. The samples were separated on a reversed-phase column using acetonitrile-0.1% formic acid and water-0.1% formic acid as mobile phases. Nine triterpenoids were analyzed using multiple reaction monitoring mode. The results showed that the area under the concentration-time curve from dosing to time t of all nine components was increased in RGLS compared with BGLS. And the time to the maximum concentration in BGLS was delayed compared with that of RGLS. These indicated that the absorption of RGLS was better than that of BGLS, and the sporoderm might hinder the absorption of the active components. These results increase our understanding of the bioavailability of BGLS and RGLS and indicate that increased bioavailability is one of the main reasons for the enhanced efficacy of RGLS.

Clinical outcomes of endodontic microsurgery in complicated cases with large or through-and-through lesions: a retrospective longitudinal study.

OBJECTIVES: To investigate the clinical outcomes of endodontic microsurgery in complicated cases presenting with large or through-and-through lesions. MATERIALS AND METHODS: We retrospectively collected and analyzed preoperative, intraoperative, and follow-up data from 143 complicated cases that underwent endodontic microsurgery. Clinical outcomes were assessed in terms of tooth survival and surgery success. Cox regression analysis was used to evaluate the survival rate and identify associated risk factors. Additionally, the success rate was compared across different postoperative periods, and potential factors contributing to surgical failure were identified through binary logistic regression. RESULTS: The overall survival and success rates were 93.0% and 91.7%, respectively. The Cox regression model identified four risk factors affecting tooth survival, including apicoectomy of four teeth (HR = 35.488; P = 0.0002), an open apex observed on preoperative radiographs (HR = 6.300; P = 0.025), the performance of guided tissue regeneration technique (HR = 8.846; P = 0.028), and a palatal surgical approach (HR = 8.685; P = 0.030). The success rate demonstrated an initial increase in the early postoperative period (from 0.5 to 2 years; P = 5.8124e-30), followed by stabilization (from 2 to 9 years; P = 0.298). Surgery success rate significantly declined when apicoectomy involved four teeth (OR = 109.412; P = 0.002). CONCLUSIONS: Endodontic microsurgery demonstrates satisfactory outcomes in complicated cases, maintaining a stable success rate after two years. However, tooth survival and surgery success are significantly compromised when apicoectomy involves four teeth. Factors such as guided tissue regeneration, an open apex, and the palatal surgical approach are associated with an increased risk of tooth extraction. CLINICAL RELEVANCE: Despite achieving acceptable outcomes in complicated cases, endodontic microsurgery is adversely affected by the apicoectomy of four teeth.

Effects of ceramsite derived from sewage sludge combined with the O3-FeCl3/PAM process on the dewatering of waste-activated sludge and investigation of dewatering mechanisms.

The high water content of waste-activated sludge restricts the subsequent disposal of sludge. The dehydration properties of O3, ferric chloride (FeCl3)/polyacrylamide, and sludge ceramsite sand (SCS) were studied. Simultaneously, the effect of combining the three was investigated to support the deep dehydration of waste-activated sludge. Experimental results showed that with13.42 mg/(g dry solids (DS)) of O3, 109.89 mg/(g DS) of FeCl3, and 100 mesh dosage of 70% DS of sludge ceramsite on weight, the highest sludge net yield was 7.13 kg/(m2·h) and the minimum specific resistance to filtration of sludge cake was 1.02 × 1012 (m/kg). Compared with the compressibility of the raw sludge, the compressibility of the sludge cake decreased by 37.48%. Moreover, the YN (net yield) increased by 73.55%. The results demonstrate that the structure of cracking, flocculation, and hydrophobic framework is the mechanism of sludge dewatering in this combined process. This combined treatment process provides a new perspective for the realization of deep dewatering of sludge and is anticipated to be a successful sludge dehydration method.

Analysis of hypervirulent Klebsiella pneumoniae and classic Klebsiella pneumoniae infections in a Chinese hospital.

AIMS: To evaluate the clinical and genetic virulence characteristics of critically ill patients with hypervirulent Klebsiella pneumoniae (hvKP) and classic KP (cKP) infection. METHODS AND RESULTS: The patients included in this retrospective study (n = 225) were grouped according to their hvKP (n = 114) or cKP (n = 111) status, and their clinical characteristics were analysed and compared. Cox multivariate analysis was conducted to determine the risk factors for hvKP infection. Length of hospital stay, length of intensive care unit stay, duration of mechanical ventilation and 28-day survival rate were similar between the groups. However, the incidence of septic shock was higher in the hvKP group (16.7%) than in the cKP group (8.1%). CONCLUSIONS: There was a high rate of hvKP infection in this population. Compared to patients with cKP infection, those with hvKP infection showed a higher probability of having septic shock; nevertheless, survival and length of hospital stay were similar between the groups. Risk factors for hvKP infection included hospital-acquired infection and renal insufficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents relevant information on the characteristics of hvKP infection in a Chinese population, and this promotes early diagnosis and supports the view that the prevalence of hvKP is high in China.

Hepatoprotective Activity of Ethanol Extract of Rice Solid-State Fermentation of Ganoderma tsugae against CCl4-Induced Acute Liver Injury in Mice.

Ganoderma tsugae is well known as a medicinal mushroom in China and many Asian countries, while its fermentation technique and corresponding pharmacological activity are rarely reported. In this study, a wild G. tsugae strain (G42) with high triterpenoid content was screened from nine strains by rice solid-state fermentation, and 53.86 mg/g triterpenoids could be produced under optimized conditions; that is, inoculation amount 20%, fermentation temperature 27 °C, and culture time 45 days. The hepatoprotective activity of G42 ethanol extract was evaluated by CCl4-induced liver injury in mice, in which changes in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), oxidation-related factors, and inflammatory cytokines in serum or liver samples demonstrated the therapeutic effect. In addition, the ethanol extract of G42 reduced the incidence of necrosis and inflammatory infiltration, and decreased protein expression levels of phosphor-nuclear factor-κB (NF-κB), interleukin-Iß (IL-1ß), and nuclear factor erythroid-2-related factor 2 (NRF2). The chemical composition of the ethanol extract was analyzed by high-resolution mass spectrometry and molecular networking. Three main triterpenoids, namely platycodigenin, cucurbitacin IIb, and ganolecidic acid B were identified. This work provided an optimized fermentation method for G. tsugae, and demonstrated that its fermentation extract might be developed as a functional food with a hepatoprotective effect.

Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum.

Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and "sandwiched" by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78-100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. KEY POINTS: • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.

Characterization and reverse genetic establishment of cattle derived Akabane virus in China.

BACKGROUND: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. RESULTS: An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. CONCLUSION: We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Prognostic Value of Prealbumin in Liver Cancer: A Systematic Review and Meta-Analysis.

Accumulated studies have reported the prognostic significance of prealbumin in liver cancer, but the results were not conclusive. The aim of this study was to evaluate the association between pretreatment serum prealbumin and clinical outcome of liver cancer patients through a meta-analysis. We comprehensively searched EMBASE, PubMed, Web of Science and the Cochrane library to identify eligible studies. The pooled hazard ratios (HRs) and their 95% confidence intervals (CIs) were utilized to evaluate the prognostic value of pretreatment serum prealbumin in overall survival (OS) and recurrence-free survival (RFS) of liver cancer patients. A total of 3470 patients from 10 eligible studies were finally included for analysis. The combined effects of prealbumin on liver cancer patients' OS and RFS were HR = 1.83, 95% CI: 1.46-2.30, P < 0.001 and HR = 1.47, 95% CI: 1.01-2.14, P = 0.045, respectively. Sensitivity and subgroup analysis showed that the pooled HR of prealbumin on liver cancer patients' OS was stable. Since potential publication bias was identified in the OS studies, the trim-and-fill method therefore was performed to explore publication bias, and the results showed reliability. This meta-analysis shows that low pretreatment serum prealbumin is significantly associated with poor prognosis of liver cancer patients.

The Laplace approach in microrheology.

When coupled with the generalized Stokes-Einstein (GSE) equation, it is often reported that micro-rheology probes the dynamic properties differently than do macroscopic rheological measurements, especially in relatively condensed systems. In the present work, we empirically examine the GSE in its widely used form: following an analytical continuation, the Fourier transformed particle mean-square displacement (MSD) is used to determine the dynamic moduli [G'(ω) and G''(ω)] and we compare the results with those obtained by direct inverse Laplace transform calculation of the relevant viscoelastic functions (either relaxation modulus or creep compliance) from the MSD. The results show that the inverse Laplace approaches can differ from the Fourier approach and give better agreement with macroscopic rheological measurements when this is the case. Some instances of agreement between the Fourier approach and the direct Laplace transform approaches are also shown. It is recommended that micro-rheology MSD data be interpreted using one of the direct Laplace transform based approaches.

The deubiquitinating enzyme MoUbp8 is required for infection-related development, pathogenicity, and carbon catabolite repression in Magnaporthe oryzae.

Deubiquitination is an essential regulatory step in the Ub-dependent pathway. Deubiquitinating enzymes (DUBs) mediate the removal of ubiquitin moieties from substrate proteins, which are involved in many regulatory mechanisms. As a component of the DUB module (Ubp8/Sgf11/Sus1/Sgf73) in the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex, Ubp8 plays a crucial role in both Saccharomyces cerevisiae and humans. In S. cerevisiae, Ubp8-mediated deubiquitination regulates transcriptional activation processes. To investigate the contributions of Ubp8 to physiological and pathological development of filamentous fungi, we generated the deletion mutant of ortholog MoUBP8 (MGG-03527) in Magnaporthe oryzae (syn. Pyricularia oryzae). The ΔMoubp8 strain showed reduced sporulation, pathogenicity, and resistance to distinct stresses. Even though the conidia of the ΔMoubp8 mutant were delayed in appressorium formation, the normal and abnormal (none-septum or one-septum) conidia could finally form appressoria. Reduced melanin in the ΔMoubp8 mutant is highly responsible for the attenuated pathogenicity since the appressoria of the ΔMoubp8 mutant was much more fragile than those of the wild type, due to the defective turgidity. The weakened ability to detoxify or scavenge host-derived reactive oxygen species (ROS) further restricted the invasion of the pathogen. We also showed that carbon derepression, on the one hand, rendered the ΔMoubp8 strain highly sensitive to allyl alcohol, on the other hand, it enhances the resistance of the MoUBP8 defective strain to deoxyglucose. Overall, we suggest that MoUbp8 is not only required for sporulation, melanin formation, appressoria development, and pathogenicity but also involved in carbon catabolite repression of M. oryzae.

The S Gene Is Necessary but Not Sufficient for the Virulence of Porcine Epidemic Diarrhea Virus Novel Variant Strain BJ2011C.

The recently emerged highly virulent variants of porcine epidemic diarrhea virus (PEDV) have caused colossal economic losses to the worldwide swine industry. In this study, we investigated the viral virulence determinants by constructing a series of chimeric mutants between the highly virulent strain BJ2011C and the avirulent strain CHM2013. When tested in the 2-day-old piglet model, wild-type (WT) BJ2011C caused severe diarrhea and death of the piglets within 72 h. In contrast, its chimeric derivative carrying the S gene from CHM2013 (BJ2011C-SCHM) was avirulent to the piglets. Moreover, reciprocal substitution of the BJ2011C S gene (CHM2013-SBJ) did not enable CHM2013 to gain any virulence. However, when the whole structural protein-coding region of BJ2011C (CHM2013-SPBJ) was swapped, CHM2013 started to gain the ability to efficiently colonize the intestinal tract and caused diarrhea in piglets. A further gain of virulence required additional acquisition of the 3' untranslated region (UTR) of BJ2011C, and the resultant virus (CHM2013-SP + 3UTRBJ) caused more severe diarrhea and death of piglets. Together, our findings suggest that the virulence of PEDV epidemic strains is a multigenic event and that the S gene is only one of the necessary determinants.IMPORTANCE The recently emerged highly virulent PEDV variants are the major cause of the global porcine epidemic diarrhea (PED) pandemic. The S gene of the variants undergoes remarkable variations and has been thought to be the virulence determinant for the enhanced pathogenesis. Our studies here showed that the S gene is only part of the story and that full virulence requires cooperation from other genes. Our findings provide insight into the pathogenic mechanism of the highly virulent PEDV variants and have implications for future vaccine development.

LncRNA HOTTIP-Mediated HOXA11 Expression Promotes Cell Growth, Migration and Inhibits Cell Apoptosis in Breast Cancer.

As the most common cause of cancer death in women, the pathogenesis of breast cancer still remains unclear. Here, we reported a long non-coding RNA (lncRNA), HOTTIP (HOXA transcript at the distal tip), that may play an important role in the pathogenesis of breast cancer. Using gain-and-loss-of experiments in vitro and in vivo, we observed the marked upregulation of HOTTIP/HOXA11 in the breast cancer cell line, MCF-7, and the downregulation of HOTTIP or HOXA11, which might inhibit cell proliferation and migration but promote cell apoptosis in breast cancer MCF-7 cells. In addition, by further rescue experiments with HOXA11 overexpression, we uncovered a novel potential regulatory mechanism between HOTTIP and one of its physical HOXA clusters, HOXA11. Hence, HOTTIP may mediate, at least partly, HOXA11 expression involved in cell growth, migration, and apoptosis of breast cancer MCF-7 cells.

WD40-repeat protein MoCreC is essential for carbon repression and is involved in conidiation, growth and pathogenicity of Magnaporthe oryzae.

Carbon catabolite repression (CCR) is a common regulatory mechanism used by microorganisms to prioritize use of a preferred carbon source (usually glucose). The CreC WD40-repeat protein is a major component of the CCR pathway in Aspergillus nidulans. To clarify the function of the CreC ortholog from Magnaporthe oryzae in regulating gene expression important for pathogenesis, MoCreC was identified and genetically characterized. The vegetative growth rate of the MoCreC deletion mutant on various carbon sources was reduced. The MoCreC mutant produced fewer conidia and with about 60% of conidia having septation defects. Appressorium formation was impaired in the MoCreC mutant. Although some appressoria of the mutant could penetrate the leaf surface successfully, the efficiency of penetration and invasive growth of infection hyphae was reduced, resulting in attenuated virulence toward host plants. The CCR was defective as the mutant was more sensitive to allyl alcohol in the presence of glucose, and 2-deoxyglucose was unable to fully repress utilization of secondary carbon sources. qRT-PCR results indicated that the genes encoding cell wall degradation enzymes, such as ß-glucosidase, feruloyl esterase and exoglucanase, were upregulated in MoCreC mutant. Taken together, we conclude that MoCreC is a major regulator of CCR and plays significant roles in regulating growth, conidiation, and pathogenicity of M. oryzae.

Functional characterization of a new promoter isolated from torque teno sus virus 1.

Torque teno sus virus 1 (TTSuV1) has a non-enveloped, single-stranded, negative-sense circular DNA genome, and it is widely distributed in pigs. Open reading frame 1 (ORF1) of TTSuV1 can be transcribed into mRNA and then translated into protein; however, its promoter has not yet been identified. We used a dual-luciferase reporter system, involving pGL3-Basic and pRL-TK, to identify the promoter of TTSuV1 ORF1. Our results revealed that the sequence between nucleotides 196 and 525 promoted the transcription of the firefly luciferase gene. The core sequence of the promoter was between nucleotides 250 and 400. A comparison of the identified TTSuV1 ORF1 promoter with that from cytomegalovirus (CMV) suggested that the two promoters were similar in strength. Our findings provide new information regarding the molecular biology of TTSuV1 and have revealed a new promoter that can be used in plasmids for numerous applications.

Characterization of monoclonal antibodies that recognize the amino- and carboxy-terminal epitopes of the pseudorabies virus UL42 protein.

The pseudorabies virus (PRV) UL42 protein, known as the DNA polymerase processivity factor, is an essential protein required for viral replication. The in vitro function of UL42 has been characterized; however, there is little information concerning the linear B cell epitopes of UL42 that are recognized during humoral immune responses. We generated and characterized six UL42-reactive monoclonal antibodies (mAbs) from mice that had been immunized with a recombinant form of UL42. Through western blotting analysis, we identified two regions of UL42 (amino acids 39-148 and 302-384) that reacted with these mAbs. We then synthesized a panel of UL42-derived peptides spanning the two regions and screened the six mAbs. We were able to identify three linear epitopes ((116)SGGVLDALK(124), (354)KRPAAPR(360), and (360)RMYTPIAK(367)) by enzyme-linked immunosorbent assays. The (116)SGGVLDALK(124) epitope was located at the amino-terminus, while the other two epitopes were at the carboxy-terminus. Using these mAbs, we found that UL42 localized to the nucleus during viral replication and could be immunoprecipitated from PRV-infected PK-15 cells. We also established a UL42 mAb-based immunoperoxidase monolayer assay for the determination of PRV titers. Sequence analysis showed that the linear epitopes of UL42 were highly conserved among PRV strains. Taken together, our results indicate that the six generated mAbs could be useful tools for investigating the structure and function of UL42 during viral replication. In addition, these mAbs could be applied to diagnostic and therapeutic approaches for the effective control of PRV infections.

Characterization and application of monoclonal antibodies against Mycoplasma hyorhinis pyruvate dehydrogenase E1 complex subunit alpha.

Mycoplasma hyorhinis is commonly found in the respiratory tract of pigs and is the etiological agent of polyserositis. The metabolic enzymes of M. hyorhinis may play important roles in host-pathogen interactions. We immunized BALB/c mice with sodium deoxycholate-extracted antigens (DOC-Ags) and screened 10 hybridomas that secreted antibodies against various M. hyorhinis proteins. Pyruvate dehydrogenase E1 complex subunit alpha (PDHA) was identified as the protein that reacted with five of the 10 monoclonal antibodies (mAbs). Sequence analysis indicated that PDHA was highly conserved among M. hyorhinis strains, but not among other mycoplasmas. We predicted the three-dimensional structure of PDHA and identified three epitopes ((277)RTEEEEK(283), (299)KDKKYITDE(307), and (350)LKEQKQHAKDY(360)). The mAb 1H12 we generated was used to detect M. hyorhinis PDHA in vitro and in piglets infected with M. hyorhinis. We observed that PDHA was mainly located in the epithelial cells of the lungs. Our results indicate that the mAbs we generated could be used to further investigate the structure and function of M. hyorhinis PDHA. In addition, they could be used in the differential diagnosis of M. hyorhinis and other mycoplasmas.

Capsid proteins from PCV2a genotype confer greater protection against a PCV2b strain than those from PCV2b genotype in pigs: evidence for PCV2b strains becoming more predominant than PCV2a strains from 2000 to 2010s.

Two major porcine circovirus type 2 (PCV2) genotypes, PCV2a and PCV2b, are recognized. PCV2a was predominant in the global pig population until 2000 while PCV2b became predominant from 2003 onward. The aim of this study was to analyze the immune protection conferred by two PCV2a and two PCV2b capsid proteins (Caps) in pigs challenged with a mutant PCV2b/YJ (mPCV2b/YJ) strain. Pigs vaccinated with PCV2a/LG-Cap and PCV2a/CL-Cap elicited significantly higher levels of PCV2-specific antibodies and neutralizing antibodies compared with PCV2b/JF-Cap and mPCV2b/YJ-Cap. Following a mPCV2b/YJ challenge, no viremia was detected in the PCV2a/LG-Cap and PCV2a/CL-Cap groups, while viremias were found in 20 and 40 % of the pigs in the PCV2b/JF-Cap and mPCV2b/YJ-Cap groups, respectively. Viral loads in the inguinal lymph nodes of pigs from the PCV2b/JF-Cap and mPCV2b/YJ-Cap groups were significantly higher than those in the PCV2a/LG-Cap and PCV2a/CL-Cap groups, but significantly lower than those of the challenge control group. Furthermore, PCV2 antigens were not detected in the inguinal lymph nodes of pigs from commercial vaccine groups, as well as the PCV2a/LG-Cap and PCV2a/CL-Cap groups, but were found in the challenge control (100 %, 5/5), PCV2b/JF-Cap (20 %, 1/5), and mPCV2b/YJ-Cap (20 %, 1/5) groups. These findings suggest that mPCV2b/YJ-Cap and PCV2b/JF-Cap were less immunogenic than PCV2a/LG-Cap and PCV2a/CL-Cap. We speculate that a genotypic shift from PCV2a to PCV2b might be the result of the majority of PCV2a strains being more immunogenic than the majority of PCV2b strains. These results provide a possible explanation for why PCV2b strains are more likely to cause epidemics than PCV2a strains. It tells us that PCV2 pathogenesis may be associated with its immunogenicity to some extent.

Prevalence of emerging porcine parvoviruses and their co-infections with porcine circovirus type 2 in China.

A total of 450 samples from domestic pigs in China were tested for porcine parvoviruses (PPVs) and co-infections with porcine circovirus type 2 (PCV2), and their complete capsid genes were sequenced. The prevalence of PPV1, PPV2, PPV3, PPV4, and PCV2 was 5.56 %, 39.56 %, 45.11 %, 21.56 %, and 47.33 %, respectively, and co-infection with PCV2 occurred in 4 % (PPV1), 22.44 % (PPV2), 24 % (PPV3), and 12 % (PPV4) of the samples. Phylogenetic analysis revealed two main lineages for each virus, and residues that differentiated these viruses were identified. The co-infections of emerging PPVs and PCV2 were prevalent, indicating their cooperative roles in porcine circovirus-associated diseases.

Identification of three PPV1 VP2 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed recombinant VP2 protein.

Porcine parvovirus type 1 (PPV1) is a major causative agent of embryonic and fetal death in swine. The PPV1 VP2 protein is closely associated with viral immunogenicity for eliciting neutralizing antibodies, but its antigenic structures have been largely unknown. We generated three monoclonal antibodies (MAbs) against baculovirus-expressed recombinant PPV1 VP2 protein. A PEPSCAN analysis identified the minimal B cell linear epitopes of PPV1 VP2 based on these MAbs. Three core epitopes, (228)QQITDA(233), (284)RSLGLPPK(291), and (344)FEYSNGGPFLTPI(356), were defined and mapped onto three-dimensional models of the PPV1 virion and VP2 monomer. The epitope (228)QQITDA(233) is exposed on the virion surface, and the other two are located inside the protein. An alignment of the PPV1 VP2 amino acid sequences showed that (284)RSLGLPPK(291) and (344)FEYSNGGPFLTPI(356) are absolutely conserved, whereas (228)QQITDA(233) has a single substitution at residue 233 in some (S → A or T). We developed a VP2 epitope-based indirect enzyme-linked immunosorbent assay (iELISA) to test for anti-PPV1 antibodies. In a comparative analysis with an immunoperoxidase monolayer assay using 135 guinea pig sera, the VP2-epitope-based iELISA had a concordance rate of 85.19 %, sensitivity of 83.33 %, and specificity of 85.47 %. MAb 8H6 was used to monitor VP2 during the PPV1 replication cycle in vitro with an indirect immunofluorescence assay, which indicated that newly encapsulated virions are released from the nucleus at 24 h postinfection and the PPV1 replication cycle takes less than 24 h. This study provides valuable information clarifying the antigenic structure of PPV1 VP2 and lays the foundations for PPV1 serodiagnosis and antigen detection.