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In order to investigate the apoptosis triggered by porcine circovirus type 2 (PCV2) in lymphocytes and the underlying mechanism, the levels of apoptosis and the expression levels of miRNA were examined by flow cytometry, Western blotting and real-time PCR (qPCR). The mimics or inhibitors of miR-125a-5p, an apoptosis-related miRNA, were transfected into PK-15 cells, and the apoptosis rate was examined upon overexpression or inhibition of mir-125a-5p. The target gene of mir-125a-5p was predicted by bioinformatics method, and the regulation of mir-125a-5p on the target gene was analyzed by luciferase reporter assay. The expressions of Bcl-2, Bax, cytochrome C and caspase-3 were detected by Western blotting. The results showed that exosomes secreted by PK-15 cells infected with PCV2 significantly increased the lymphocyte apoptosis rate, which was dose-dependent in certain concentration range. The expression of miR-125a-5p was dramatically increased. The apoptosis rate was increased significantly in the cells transfected with miR-125a-5p. It was predicted that there were binding sites of miR-125a-5p at

To obtain more information of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) transmission via faeces in/between farms, 360 swine faecal samples were randomly collected from different farms in China from 2017 to 2019. Sixty-two ORF5 genes were amplified by PCR from 120 positive samples identified by real-time RT-PCR and further characterized by sequencing. Phylogenetic analysis based on the ORF5 gene revealed that these strains can be divided into four lineages: lineage 1 (NADC30-like), lineage 3 (QYYZ-like), lineage 5.1 (VR2332-like) and lineage 8.7 (JXA1-like), with 62.9% (39/62) NADC30-like virus, 21% (13/62) QYYZ-like virus, 1.6% (1/62) VR2332-like virus and 14.5% (9/62) for JAX1-like virus. In particular, 14 PRRSVs including lineage 1, 5.1 and 8.7 can be isolated from 120 positive faecal samples, which further suggests that faecal transmission may be an important factor in the spread of PRRSV in farms. Full-length genome sequencing analysis showed that 14 isolates share 83.1%-97.7% homology with each other and 82.3%-96.1% identity with NADC30, 83.2%-99.7% with VR2332, 79.6%-87.2% with QYYZ and 82.6%-98.9% with JXA1 and CH-1a, and only 60.1%-60.7% with LV. Recombination events were observed in the six out of 14 strains. Collectively, the data of this study are useful for understanding the spread of PRRSV via faeces. Additionally, the virus was isolated from positive faecal samples, suggesting that faecal transmission may be an important factor in the spread of PRRSV in farms.

The objective of this study was to assess the genetic diversity of porcine reproductive and respiratory syndrome virus circulating in Fujian province (southeastern China). Based on 53 ORF5 nucleotide sequences collected from nine sites, both highly pathogenic (sublineage 8.7) and lineage 1 strains were circulating in Fujian in 2009-2014 along with lineages 3 and 5.1. Notably, the lineage 1 strains were closely related to the NADC30 strain circulating in North America and were the predominant strains in 2014. In addition, we found that nonstructural protein 2 (NSP2) was the most variable nonstructural protein in Fujian isolates, with a 36-amino-acid (aa) insertion and seven different deletions detected in the 53 sequences examined. Similarly, analysis of GP5 amino acid sequences showed that the isolates were highly variable in primary neutralizing epitopes. Interesting, FJ3.2 and FJ7-2 strains have the mutation N44K, but they exhibited high replication and high titers in MARC-145 and PAM cells. The complete genome sequences determined for 12 type 2 isolates were 82.1-99.3% identical and were 15,016-15,407 nucleotides (nt), in length excluding the poly(A) tail. The strains also shared 88.2-99.4% identity with strain VR2332 (the prototype North American strain), 83.4-99.2% identity with strain JXA1 (the prototype high-pathogenicity Chinese strain), 88.2-97.1% identity with strain CH-1a (the prototype classical Chinese strain), and 82.9-97.1% identity with strain NADC30 (the prototype NADC30-like strain). Strikingly, phylogenetic and molecular evolutionary analyses indicated that strain FJW05 is a spontaneous recombinant between a circulating lineage 1 virus and the vaccine strain JXA1-R, which is derived from the highly pathogenic strain JXA-1. Collectively, the data highlight the epidemiology of porcine reproductive and respiratory syndrome in Fujian and may aid in selecting a suitable vaccine for use on pig farms.

From 2014 to 2015, four novel highly pathogenic PRRS virus (HP-PRRSV) strains named 14LY01-FJ, 14LY02-FJ 15LY01-FJ, and 15LY02-FJ were isolated from high morbidity (100%) and mortality (40%-80%) in piglets and sows in Fujian Province. To further our knowledge about these novel virus strains, we characterized their complete genomes and determined their pathogenicity in piglets. Full-length genome sequencing analysis showed that these four isolates were closely related to type 2 (North American type, NA-type) isolates, with 88.1%-96.3% nucleotide similarity, but only 60.6%-60.8% homology to the Lelystad virus (LV) (European type, EU-type). The full length of the four isolates was determined to be 15017 or 15018 nucleotides (nt), excluding the poly(A) tail. Furthermore, the four isolates had three discontinuous deletions (aa 322-432, aa 483, and aa 504-522) within hypervariable region II (HV-II) of Nsp2, as compared to the reference strain VR-2332. This deletion pattern in the four isolates is consistent with strain MN184 and strain NADC30 isolated from America. Phylogenetic and molecular evolutionary analyses indicated that these virulent strains originated from a natural recombination event between the JXA1-like HP-PRRSV (JXA-1 is one of the earliest Chinese HP-PRRSV strains; sublineage 8.7) and the NADC30-like (lineage 1) PRRSV. Animal experiments demonstrated that these four strains caused significant weight loss and severe histopathological lung lesions as compared to the negative control group. High mortality rate (40% or 80%) was found in piglets infected with any one of the four strains, similar to that found with other Chinese HP-PRRSV strains. This study showed that the novel variant PRRSV was HP-PRRSV, and it is therefore critical to monitor PRRSV evolution in China and develop a method for controlling PRRS.

Parainfluenza virus 5 (PIV5) is widespread in mammals and humans. Up to now, there is little information about PIV5 infection in lesser pandas. In this study, a PIV5 variant (named ZJQ-221) was isolated from a lesser panda with respiratory disease in Guangzhou zoo in Guangdong province, southern China. The full-length genome of ZJQ-221 was found to be 15,246 nucleotides and consisted of seven non-overlapping genes encoding eight proteins (i.e., NP, V, P, M, F, SH, HN and L). Sequence alignment and genetic analysis revealed that ZJQ-221 shared a close relationship with a PIV5 strain of canine-origin (1168-1) from South Korea. The findings of this study confirm the presence of PIV5 in lesser panda and indicate this mammal as a possible natural reservoir. Furthermore they highlight the urgent need to strengthen viral surveillance and control of PIV5 in zoo animals. Electronic supplementary material The online version of this article (doi:10.1007/s00705-017-3245-0) contains supplementary material, which is available to authorized users.

Porcine circovirus type 2 (PCV2) has been causing huge economic losses in Chinese swine herds since it was first identified in China in 1999. Genotypes of PCV2 except for PCV2c coexist in swine herds in China, which may facilitate virus recombination. In the current study, six novel PCV2 strains were detected in China, and these strains shared high nucleotide similarity of the Rep gene with the PCV2c strain DK1987PMWSfree and high homology of the Cap gene with PCV2d. Genome sequence analysis revealed that the complete genomes of these strains were 1767 nucleotides (nt) in length and shared 99.8%-99.9% nucleotide identity with each other and 91.7%-98.7% with representative strains. Phylogenetic analysis, sequencing analysis, base-by-base comparisons and comprehensive recombination analysis demonstrated that these six strains originated from recombination within the Rep gene between PCV2c and PCV2d strains. Surprisingly, further investigation through theoretical recombination analysis of Chinese PCV2 GenBank sequences showed that these novel patterns of recombinant PCV2 strains have been generated since 2010. Collectively, our findings provide additional evidence of inter-genotypic recombination of PCV2.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most devastating swine diseases worldwide, resulting in immense economic losses. PRRS virus (PRRSV) is divided into two major genotypes, European (type 1) and the North American (type 2). Type 1 PRRSV have recently emerged in Fujian province (South China), and this might have a significant impact on the Chinese pig industry. From 2013 to 2014, two type 1 PRRSV strains, named FJEU13 and FJQEU14, were isolated from piglets and sows with respiratory problems and reproductive disorders in Fujian province. The full genome length of the two isolates was 14,869-15,062 nucleotides (nt), excluding the poly(A) tail. These isolates shared 86.0-89.9% sequence identity with the prototypic strains Lelystad virus (LV) and 82.8-92% with Chinese type 1 PRRSV strains, but only 59.9-60.1% with the North American reference strain VR-2332. However, they were 82.9% identical to each other. Nonstructural protein 2 (Nsp2) and ORF3-ORF5 were the most variable regions when compared to other type 1 PRRSV strains. Nsp2 and ORF3 contained multiple discontinuous deletions and a 204-bp deletion in NSP2 in isolate FJQEU14, which has never been described in other Chinese type 1 PRRSV strains. All of these results might be useful for understanding the epidemic status of type 1 PRRSV in China.

The objective of the present study was to determine the cross-protection of Ingelvac PRRS MLV against challenge with the new lineage 1 PRRSV emerged in China in pigs. Two lineage 1 PRRSV strains (FJZ03 and FJWQ16 originated from recombination event between NADC30 and JXA1-like strain). We found that pigs vaccinated with the vaccine were protected against challenge with the FJZ03 as shown by fewer days of clinical fever, reduced lung pathology scores, lower PRRS virus load in the blood and developed broadly neutralizing antibodies with high titers to FJZ03. In contrast, vaccine provided limited protection against challenge with FJWQ16 with higher fever, lower antibody titers, lower neutralizing antibodies and higher viral loads in blood. These results demonstrate PRRSV-MLV provides incomplete protection against new lineage 1 PRRSVs.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important viral swine diseases, resulting in immense economic losses in Chinese pig industry. Currently, four major lineages: lineage 1 (NADC30-like), 3 (QYYZ-like), 5.1 (VR2332-like) and 8.7 (JXA1-like) of type 2 PRRSV (North American type) have been circulating in China based on classification system, which have caused concern about the potential of virus recombination. In the present study, a novel variant of PRRSV strain named FJLIUY-2017 was isolated from abortion rate (25%) in pregnant gilts in Fujian Province in China in 2017. To further our knowledge about the novel virus strain, we characterized the complete genome of FJLIUY-2017. Comparison to PRRS sequences in GenBank confirmed the absence of close relatives (

PRRS virus (PRRSV) has undergone rapid evolution and resulted in immense economic losses worldwide. In the present study, a PRRSV strain named FJ0908 causing high abortion rate (25%) and mortality (40%) was detected in a swine herd in China. To determine if a new PRRSV genotype had emerged, we characterized the genetic characteristics of FJ0908. Phylogenetic analysis indicated that FJ0908 was related to 1-7-4-like strains circulating in the United States since 2014. Furthermore, the ORF5 sequence restriction fragment length polymorphism (RFLP) pattern of FJ0908 was 1-7-4. Additionally, FJ0908 had a 100 aa deletion (aa329–428) within nsp2, as compared to VR-2332, and the deletion pattern was consistent with most of 1-7-4 PRRSVs. Collectively, the data of this study contribute to the understanding of 1-7-4-like PRRSV molecular epidemiology in China.

To more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the Nsp2 and ORF5 gene sequences of 35 representative PRRSV isolates from 2008 to 2012. Sequence analysis revealed that the Nsp2 and ORF5 genes have undergone genetic variation. Furthermore, the isolate FJLYDX04 contains five insertions at positions 599 to 603 and is the first isolate from China reported to have an insertion in Nsp2. Our results suggest that the highly pathogenic PRRSV has become the dominant strain in China and that Chinese PRRSV has undergone rapid evolution and can circumvent immune responses induced by currently used vaccines.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most devastating swine diseases worldwide, resulting in immense economic losses. PRRS virus (PRRSV) has undergone rapid evolution since its first recognition in 1990s. In the present study, a PRRSV strain named FJXS15 causing high morbidity and mortality was isolated from piglets and sows from a farm participating in vaccination in China. Phylogenetic and molecular evolutionary analyses revealed that FJXS15 was highly similar to the JXA1-R vaccine strain (a live attenuated virus vaccine strain derived from the highly pathogenic PRRSV JXA1) in the ORF1a (nt 901-)−ORF4 (-nt 419) coding regions, as well as to FJZ03 (lineage 1, NADC30-like) in the 5′-UTR, ORF5a−ORF7 coding regions, and 3′-UTR, suggestive of a natural recombination event. Recombination analyses showed that recombination events occurred in two inter-lineage recombination events between Lineages 1 and 8 based on based on classification system (Shi et al., 2010), and two recombination breakpoints at positions 1–1092 and 13771–15537 of the sequence alignment (with reference to the VR-2332 strain). Animal experiments demonstrated that FJXS15-infected animals had more severe histopathological lung lesions than did JXA1-R-infected and control groups. A 25% mortality rate was found in FJXS15-infected piglets, which was similar to that found with other Chinese HP-PRRSV strains. Thus, the recombinant virus is a highly virulent PRRSV. Moreover, this report provides evidence for inter-subgenotypic recombination between the JXA1-R vaccine virus and a circulating Lineage 1 virus.

Porcine circovirus 2 (PCV2) has been prevalent in swine herds in China since 2002, causing severe economic loss to the pig industry. The number of live pigs in southeast China is > 20 million. Since information on the genetic variation of PCV2 in the Fujian province is limited, the objective of the present work was to investigate the epidemiological and evolutionary characteristics of PCV2 in southeast China from 2013 to 2017. Of the 685 samples collected from 90 different swine herds from 2013 to 2017, 356 samples from 84 different swine herds were positive for PCV2. PCV2a, PCV2b, PCV2d, and PCV2e co-existed in the Fujian province, with PCV2d being the predominant circulating strain in swineherds and PCV2e being reported for the first time in China. Strikingly, PCV2-FJ-water DNA comes from contaminated river water and not infected animals. Sequence comparison among all isolates indicated that 95 isolates shared approximately 78.7%–100% nucleotide identity and 74.5%–100% amino acid identity for open reading frame 2 (ORF2). Amino acid alignment showed that the Cap protein of PCV2e differed markedly from those of PCV2a, PCV2b, PCV2c, and PCV2d. These results indicated that various PCV2 genotypes exist in China, and that PCV2 is continuously evolving, leading to rapid emergence of new variant stains.

In an effort to develop a safe and effective vaccine for the prevention of enterotoxigenicEscherichia coli(ETEC) F41 infections, we have developed a surface antigen display system using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. The recombinant fusion proteins comprised of PgsA and fimbrial protein of F41 were stably expressed inLactobacillus casei525. Surface localization of the fusion protein was verified by immunoblotting, immunofluorescence microscopy, and flow cytometry. Oral inoculation of recombinantL. casei525 into specific-pathogen-free BALB/c mice resulted in significant mucosal immunoglobulin A (IgA) titers that remained elevated for >16 weeks. High levels of IgG responses in sera specific for F41 fimbriae were also induced, with prominent IgG1 titers as well as IgG2a and IgG2b titers. The helper T-cell (Th) response was Th2-cell dominant, as evidenced by increased mucosal and systemic interleukin-4-producing T cells and a concomitant elevation of serum IgG1 antibody responses. More than 80% of the mice were protected against challenge with a 2 × 104-fold 50% lethal dose of standard-type F41 (C83919). The induced antibodies were important for eliciting a protective immune response against F41 infection. These results indicated that the use of recombinantL. casei525 could be a valuable strategy for future vaccine development for ETEC.

Mucosal immunity plays an important role in protecting pigs against transmissible gastroenteritis virus (TGEV) infection. To elicit mucosal immune response against TGEV, we developed a surface antigen display system using the poly-[.gamma]-glutamate synthetase A (pgsA) protein of Bacillus subtilis as an anchoring matrix to express recombinant fusion proteins of pgsA and nucleocapsid protein of TGEV in Lactobacillus casei. Surface location of fusion protein was verified by ELISA and indirect immunofluorescence test. Oral and intranasal inoculations of pregnant sow and mice with recombinant L. casei resulted in high levels of serum immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA) against recombinant N protein as demonstrated by ELISA. More importantly, the level of specific sIgA in colostrum significantly increased compared with that of IgG. The serum IgG levels of the piglets increased after suckling colostrum produced by sows was previously inoculated with recombinant L. casei. These results indicate that immunization with recombinant L. casei expressing TGEV N protein on its surface elicited high levels of specific sIgA and circulating IgG against TGEV N protein.

Mucosal immunization is advantageous over other routes of antigen delivery because it can induce both mucosal and systemic immune responses. In this study, we have developed fimbriae protein of enterotoxigenic Escherichia coli (ETEC) F41 was stably expressed on the surface Lactobacillus casei 525. The method of expressing vaccine antigens in L. casei induces both systemic and mucosal immunity after oral or intranasal administration. We demonstrate that an oral or intranasal vaccine based on live recombinant L. casei 525 protects infant mice from ETEC F41 infection. This platform technology can be applied to design oral or intranasal vaccine delivery vehicles against several microbial pathogens.

To exploit a safe and effective vaccine for the prevention against K99 or K88 infections of enterotoxigenic Escherichia coli (ETEC), we have developed a mucosal delivery vehicle based on Lactobacillus casei CICC 6105 using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. To evaluate the immunization effect of the recombinant strains (harboring plasmids pLA-K99-K88-LTB, pLA-K99, and pLA-K88), anti-ETEC K99 or K88 antibody responses, T-cell proliferation, and cytokines by intracellular staining (ICS) were investigated after specific pathogen-free (SPF) C57BL/6 mice orally inoculated with these recombinant strains. After oral vaccination into C57BL/6 mice, all recombinant strains were proved to be immunogenic and able to elicit high levels of mucosal immunoglobulin A (IgA) titers in bronchoalveolar lavage fluids, intestinal fluids and prominent systemic immunoglobulin G and IgG subclasses (IgG1, IgG2b, and IgG2a) responses in sera. Using the T-cell proliferation assay, the stimulation index (SI) of groups immunized with pLA-K99/L. casei and pLA-K88/L. casei reached to 2.73 and 2.64, respectively, versus 2.56 in a group immunized with pLA-K99-K88-LTB/L. casei. A detailed analysis of the cell-mediated immune responses by ICS showed the number of specific CD8(+) T cells expressing cytokines (IFN-γ, TNF-α, and IL-2) and granule-associated proteins (CD107a) was higher than that of specific CD4(+) T cells secreted by immune spleen cells upon restimulation in vitro with peptides. Next, the results showed that DCs activated in vitro with recombinant L. casei enhance specific T-cell proliferation and promote T cells to produce both Th1 and Th2 cytokines. More than 80% of the vaccinated mice were protected after challenge with a lethal dose of standard strains C83912 and C83902. These results demonstrate that recombinant L. casei can induce specific humoral and mucosal antibodies and cellular immune response against protective antigens upon oral administration.

In order to represent a promising strategy for mucosal vaccination, oral or intranasal immunization of Specific Pathogen Free (SPF) BALB/c mice were performed. The mucosal immunity, systemic immune and protective immune responses were compared after immunization with the recombinant Lactobacillus casei (L. casei) harboring enterotoxigenic Escherichia coli (ETEC) F41.The recombinant fusion proteins were detected by Western blot. Surface localization of the fusion protein was verified by immunofluorescence microscopy and flow cytometry. Six-week-old female SPF BALB/c mice (160 heads) were divided into 4 groups for immunization and control. Oral and intranasal immunization of mice was performed with the recombinant strain L. casei harboring pLA-F41 or pLA. For oral immunization, the mice were inoculated daily on days 0 to 4, 7 to 11, 21 to 25, and 49 to 53. A lighter schedule was used for nasal immunization (days 0 to 2, 7 to 9, 21 and 49). Specific anti-F41 IgG antibody in the serum and specific anti-F41 secret immunoglobulin A (sIgA) antibody in the lung, intestines, vagina fluid and feces of mice were detected by indirect ELISA. The mice orally or intranasally immunized with pLA-F41/L. casei and pLA/IL. casei were challenged with standard-type ETEC F41 (C83919) (2 x 10(3) LD50).Mice immunized with pLA-F41/L. casei could produce remarkable anti-F41 antibody level. More than 90% survived in oral immunization group whereas more than 85% survived in intranasal immunization group after challenged with C83919, all dead in the control group. Ninety percent of the pups survived in oral immunization group whereas 80% survived in intranasal immunization group after challenged with C83919, but only a 5% survival rate for pups that were either immunized with a control pLA vector or unimmunized.Oral or intranasal immunization with recombinant L. casei displaying ETEC F41 antigens on the surface induced effective and similar systemic and mucosal immune responses against the ETEC F41.

K88ac-LT(B) gene derived from pQE30-K88ac-LT(B) was cloned into the expression vector pLA and then the recombinant vector was transformed into the competent cells Lactobacillus casei 525. The recombinant bacteria were grown at 37 degrees C, in MRS broth. Western blotting analysis with rabbit-anti-K88ac-LT(B) polyclonal serum indicated that the recombinant protein reacted with the specific antibodies. The results showed that the molecular weight of the recombinant protein was about 71.2 kD. The K88ac-LT(B) fusion protein on the cell surface was confirmed by immunofluorescence mciroscopy and flow cytometric analysis. In addition, the survival of recombinant Lactobacillus casei 525 was studied in imitative gastrointestinal environments such as artificial gastro fluid (pH 1.5-5.5), artificial intestinal fluid, bile(0.3-3.0 g/L). The results indicated that the recombinant strain survived well in artificial gastric fluids at pH 2.5-4.5 in 5 h. The recombinant Lactobacillus casei 525 could slowly grow in the artificial intestinal fluid for different time, and could survive in 0.3% bile.

In an effort to develop a safe and effective vaccine for the prevention of enterotoxigenic Escherichia coli (ETEC) K99 infections, we have developed a surface antigen display system using pgsA (poly-gamma-glutamate synthetase A) as an anchoring matrix. The recombinant fusion proteins comprised of pgsA and fimbriae protein of ETEC K99 were stably expressed on Lactobacillus casei. Surface localization of the fusion protein was verified by immunoblotting, immunofluorescence microscopy and flow cytometry. Specific Pathogen Free (SPF) BALB/c mice orally or intranasally vaccinated with recombinant L. casei resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA against ETEC K99, as demonstrated by enzyme-linked immunosorbent assays using purified fimbriae peptides. The serum antibody isotypes elicited were predominantly IgG1 and IgG2a. Vaccinated SPF BALB/c mice were evaluated by oral challenge with standard-type ETEC C83912 after the last booster immunization. More than 80% of immunized mice survived regardless of the immune route. The antibody titers elicited following oral immunization were lower than those following intranasal immunization but the protective efficacy was in the same order of magnitude. These results indicate that mucosal immunization with recombinant L. casei expressing ETEC K99 fimbriae protein on its surface provides an effective means for eliciting a protective immune response against the ETEC K99.

Irradiation with ultrasound is found to facilitate the synthesis of 5,5-disubstituted hydantoins. A higher yield and shorter reaction time give this method obvious advantages over the conventional ones.