Cross-species transmission of an emerging porcine circovirus (PCV4): First molecular detection and retrospective investigation in dairy cows.

Porcine circovirus 4 (PCV4), a novel porcine circovirus identified in pigs, has recently been proved to be pathogenic to piglets. However, little is known about its cross-species transmission, and demonstration of PCV4 in dairy cows is lacking. To explore whether the PCV4 genome exists in dairy cows, 1170 fecal samples were collected from dairy farms in 7 cities in Henan Province of China during 2012-2021, and screened by qPCR for the presence of PCVs (PCV2-PCV4). The detection results showed that the positive rate of PCV4 in dairy cows was 2.22 % (26/1170), but all fecal samples were negative for PCV2 and PCV3. Three full-length and five partial genomes of PCV4 strains were acquired, of which two PCV4 strains (NY2012-DC and XC2013-DC) were achieved from 2012 and 2013, indicating that PCV4 has been circulating in dairy cows in Henan Province of China for at least 10 years. The three PCV4 strains sequenced in this study shared high identity (97.5-99.5 %) with reference strains at the genome level. In phylogenetic analysis, three genotypes (PCV4a, PCV4b and PCV4c) were temporarily confirmed by analyzing 44 strains, and one amino acid variation in Rep (V239L) and three amino acid variations in Cap (N27S, R28G and M212L) were considered as a conserved genotype specific molecular marker. Analyzed from three perspectives (cross-time, cross-species and transboundary), the high nucleotide homology of PCV4 strains indicated the PCV4 evolutionary rate might be slow. Overall, this study was the first to report the detection of PCV4 in dairy cows and conducted a long-term retrospective investigation of PCV4 in Henan Province of China, which has important implications for understanding the genetic diversity and cross-species transmission of the ongoing PCV4 cases.

Modulation of Gut Microbiota, Short-Chain Fatty Acid Production, and Inflammatory Cytokine Expression in the Cecum of Porcine Deltacoronavirus-Infected Chicks.

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea and induces proinflammatory cytokine responses in piglets. Our previous research showed that the specific-pathogen-free (SPF) chicks exhibited mild diarrhea and low fecal viral shedding, along with cecum lesions after PDCoV infection. Disturbances in the homeostasis of the gut microbiota have been associated with various diseases. We aimed to explore the effects of PDCoV infection on chick gut microbiota, short-chain fatty acid (SCFAs) production, and inflammatory cytokine expression in chicks, and also to investigate the relationship between gut microbiota and SCFAs or inflammatory cytokine expression of the PDCoV-infected chicks. Results obtained using 16S rRNA sequencing showed that infection with PDCoV strain HNZK-02 significantly altered the composition of chick gut microbiota, with the reduced abundance of Eisenbergiella and Anaerotruncus genera at 5 days post-inoculation (dpi) (P < 0.05), and an increased abundance of Alistipes genus at 17 dpi (P < 0.05). The production of SCFAs in the cecum of PDCoV HNZK-02-infected chicks, including acetic acid, propionic acid, and butyric acid, decreased in all cases. The expression of inflammatory cytokines (interferon-γ, tumor necrosis factor-α, and interleukin-10) was increased in the cecum tissue and serum of the PDCoV HNZK-02-infected chicks when detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Further analysis showed significant correlation between bacterial genera and SCFAs or inflammatory cytokines expression in cecum of the PDCoV infected chicks. These findings might provide new insight into the pathology and physiology of PDCoV in chicks.

Porcine deltacoronavirus infection alters bacterial communities in the colon and feces of neonatal piglets.

Porcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus that causes watery diarrhea in piglets. Little is known regarding the alteration of the gut microbiota in PDCoV-induced diarrhea piglets. In this study, 5-day-old piglets were experimentally infected with PDCoV strain CH-01, and all piglets developed typical clinical disease, characterized by acute and severe watery diarrhea. Histologic lesions were limited to the villous epithelium of the duodenum and ileum. Gut microbiota profiles in the colon and feces of piglets inoculated with PDCoV were investigated using 16S rRNA sequencing. The results showed that PDCoV infection reduced bacterial diversity and significantly altered the composition of the microbiota from the phylum to the genus level in the colon and feces of piglets. Firmicutes (phylum), Lactobacillaceae (family), and Lactobacillus (genus) were significantly increased (p < .01), while the abundance of Bacteroidetes (phylum) was markedly reduced in the colon and feces of the PDCoV-infected piglets (p < .01) when compared to those of the healthy piglets. Furthermore, microbial function prediction indicated that the changes in the intestinal flora also affected the nucleotide transport and metabolism, defense, translation, and transcription function of the intestinal microbiota. The current study provides new insight into the pathology and physiology of PDCoV.

A nano silicon adjuvant enhances inactivated transmissible gastroenteritis vaccine through activation the Toll-like receptors and promotes humoral and cellular immune responses.

Inactivated transmissible gastroenteritis virus (TGEV) vaccines are widely used in swine herds in China. These are limited, however, by the need to elicit both humoral and cellular immunity, as well as the efficiency of adjuvants. In this study, a 70-nm nano silicon particle was applied with inactivated TGEV vaccine in mice, and its immune-enhancing effects and mechanism of action investigated. We found that nano silicon applied with inactivated TGEV vaccine induced high antibody titers, increase IL-6, TNF-α and IFN-γ expression, and stimulate CD3+ T cell proliferation with a high CD4+/CD8+ T lymphocyte ratio. Nano silicon could quickly activate innate and adaptive immunity by stimulating Toll-like receptor signaling pathways, indicating that the nano silicon adjuvant enhanced long-term humoral and early cellular immune responses when combined with inactivated TGEV vaccine. Nano silicon could be considered for use as an antigen- carrier and adjuvant for veterinary vaccines.

Porcine parvovirus infection activates inflammatory cytokine production through Toll-like receptor 9 and NF-κB signaling pathways in porcine kidney cells.

Porcine parvovirus virus (PPV) is an animal virus that has caused high economic losses for the swine industry worldwide. Previous studies demonstrated that PPV infection induced significant production of interleukin 6 (IL-6) in vitro and in vivo. However, the inflammatory cytokines and specific signaling pathways induced during PPV infection remain largely unknown. In the present study, we analyzed the expression levels of IL-6 in PPV-infected porcine kidney 15 (PK-15) and the results showed that PPV infection induced the increase of IL-6 mRNA expression in a dose-dependent manner. We also detected the expression of toll-like receptor 9 (TLR9) signaling proteins in the mRNA expressing level, when compared with the control group, the TLR9 expression in mRNA level increased at 24h in PK-15 cells after PPV infection and reached the peak level at 48h. In addition, the transcript profile of nuclear factor kappa B (NF-κB) signal pathway relating genes (MyD88, IRAK1, TRAF6, TAK1α, IκBκB and NF-κB) expression levels increased at different times. Furthermore, to verify the IL-6 expression was specific with the TLR9 expression and then by activating the NF-κB signal pathway, TLR9 and NF-κB specific inhibitors were applied during PPV infection, separately, the result indicated that the expression of IL-6 was decreased after inhibitor treatment. Taken together, PPV infection significantly induced IL-6 expression and this induction depended on NF-κB activation and TLR9 signaling pathways in PK-15 cell.

Construction and immunogenicity of a recombinant pseudorabies virus co-expressing porcine circovirus type 2 capsid protein and interleukin 18.

A novel recombinant pseudorabies virus (PRV) expressing porcine circovirus type 2 (PCV2) capsid protein and IL-18 was constructed. The PCV2 open reading frame 2 (ORF2) and porcine IL-18 genes were amplified by PCR and then inserted into the PRV transfer vector (pG) to produce a recombinant plasmid (pGO18). Plasmid pGO18 was transfected into porcine kidney cell (PK15) pre-infected with PRV HB98 vaccine strain to generate a recombinant virus. The recombinant virus PRV-ORF2-IL18 was purified by green fluorescent plaque purification and the inserts were confirmed by PCR, enzyme digestion, sequencing, and Western blot. The humoral and cellular responses induced by the recombinant virus were assessed in mice. Mice (n=10) were immunized with PRV-ORF2-IL18, PRV-ORF2, PRV HB98, or inactivated PCV2. PRV-ORF2-IL18 elicited high titers of ELISA and serum neutralizing antibodies and strong cell-mediated immune responses in mice as indicated by anti-PCV2 ELISA, PRV-neutralizing assay, PCV2 specific lymphocyte proliferation assay, CD3(+), CD4(+) and CD8(+) T lymphocytes analysis, respectively. And PRV-ORF2-IL18 immunization protected mice against a lethal challenge of a virulent PRV Fa strain and significantly reduced the amount of PCV2 viremia. These results suggest an adjuvant effect of IL-18 on cellular immune responses. The recombinant virus might be an attractive candidate vaccine for preventing PCV2 and PRV infections in pigs.

Molecular detection and genomic characterization of Torque teno sus virus 1 and 2 from domestic pigs in central China.

In the present study, Torque teno sus viruses (TTSuVs) were detected in tissue and blood samples obtained from domestic pigs in central China, and complete genomes of TTSuVs were characterized. A total of three tissue samples (3/20, 15 %) from post-weaning multisystemic wasting syndrome-affected pigs and 30 blood samples (30/40, 75 %) from healthy pigs were positive for Torque teno sus virus 1 (TTSuV1) and/or 2 (TTSuV2). Two TTSuV strains (TTV1Hn54 and TTV2Hn93) comprising 2,794 and 2,875 nucleotides, respectively, each had four open reading frames (ORFs) and the untranslated region with TATA box and GC-rich region. Genomic sequence of TTV2Hn93 strain was unique in length compared with other TTSuV2 genomic sequences. Interestingly, three rolling-circle replication (RCR) motif-IIIs (YXXK) which were located at amino acid (aa) position 166-169, 328-331, and 379-382, respectively, were found in the ORF1 of TTV1Hn54. Two RCR motif-IIIs (YXXK) at the aa position 105-108 and 480-483 respectively, were also identified in the ORF1 of TTV2Hn93. Phylogenetic tree based on complete genomes showed that TTV1Hn54 strain was designated into type TTSuV1b and had a slight high sequence identity of 91 % with the Canada strain (JQ120664). TTV2Hn93 strain was classified into subtype TTSuV2d and shared the highest identity (97 %) with the Spain strain (GU570207).

Simultaneous detection of porcine parvovirus and porcine circovirus type 2 by duplex real-time PCR and amplicon melting curve analysis using SYBR Green.

The development of a SYBR Green-based duplex real-time PCR is described for simultaneous detection of porcine parvovirus (PPV) and porcine circovirus type 2 (PCV-2) genomes. Viral genomes were identified in the same sample by their distinctive melting temperature (T(m)) which is 77.5°C for PPV VP2 313bp amplicon and 82.3°C for PCV-2 ORF2 171bp amplicon, respectively. The detection limit of the method was 0.01TCID(50)/mL for PPV and PCV-2, about 10 times more sensitive than conventional PCR. In addition, PPV and PCV-2 viral load were measured in 126 field samples, confirming the sensitivity and specificity, and the result showed that 70/126 samples were positive for PPV and 92/126 samples were positive for PCV2 by the duplex real-time PCR. This method may be a useful alternative rapid and reliable method for the detection of PPV/PCV-2 co-infection.

Enhancing immune responses to inactivated porcine parvovirus oil emulsion vaccine by co-inoculating porcine transfer factor in mice.

Inactivated porcine parvovirus (PPV) vaccines are available commercially and widely used in the breeding herds. However, inactivated PPV vaccines have deficiencies in induction of specific cellular immune response. Transfer factor (TF) is a material that obtained from the leukocytes, and is a novel immune-stimulatory reagent that as a modulator of the immune system. In this study, the immunogenicity of PPV oil emulsion vaccine and the immuno-regulatory activities of TF were investigated. The inactivated PPV oil emulsion vaccines with or without TF were inoculated into BALB/c mice by subcutaneous injection. Then humoral and cellular immune responses were evaluated by indirect enzyme-linked immunosorbent assays (ELISA), fluorescence-activated cell sorter analyses (FACS). The results showed that the PPV specific immune responses could be evoked in mice by inoculating with PPV oil emulsion vaccine alone or by co-inoculation with TF. The cellular immune response levels in the co-inoculation groups were higher than those groups receiving the PPV oil emulsion vaccine alone, with the phenomena of higher level of IFN-γ, a little IL-6 and a trace of IL-4 in serum, and a vigorous T-cell response. However, there was no significant difference in antibody titers between TF synergy inactivated vaccine and the inactivated vaccine group (P>0.05). In conclusion, these results suggest that TF possess better cellular immune-enhancing capability and would be exploited into an effective immune-adjuvant for inactivated vaccines.

Inhibitory effects of indigowoad root polysaccharides on porcine reproductive and respiratory syndrome virus replication in vitro.

BACKGROUND: Indigowoad root polysaccharide (IRPS) is a natural polysaccharide isolated from the traditional Chinese medicinal herb Radix Isatidis, and has many kinds of biological activities. However, the IRPS antiviral activity, especially the anti-porcine reproductive and respiratory syndrome virus (PRRSV) effect, has not been evaluated. METHODS: PRRSV was propagated in the MARC-145 cell line, and viral titre was determined by cytopathic effect and expressed as the 50% tissue culture infection dose (TCID(50)) in the current study. The cell cytotoxic effect of IRPS toward MARC-145 was evaluated by MTT assay firstly, then the inhibitory effects of IRPS on PRRSV replication in vitro were investigated by determining the effect of IRPS upon a single replicative cycle of PRRSV in MARC-145 cells. The effects of IRPS on viral RNA and protein synthesis in PRRSV-infected cells were investigated using real-time PCR and double-antibody (sandwich) ELISA. RESULTS: IRPS was able to effectively suppress the infectivity of the PRRSV in a dose-dependent manner, especially by adding IRPS during the PRRSV infection. IRPS could affect the attachment of PRRSV to MARC-145 cells, and also inhibit the viral RNA and protein synthesis. CONCLUSIONS: IRPS has an antiviral effect on PRRSV replication in MARC-145 cells and might be useful in medical development for antiviral research. However, the precise mechanism of the host and viral targets of IRPS are unknown, so further studies should be conducted to investigate the precise mechanism of IRPS inhibitory effect on PRRSV infection.

Enhancement of the immunogenicity of an infectious laryngotracheitis virus DNA vaccine by a bicistronic plasmid encoding glycoprotein B and interleukin-18.

A DNA vaccine against infectious laryngotracheitis virus (ILTV) can induce specific humoral and cell-mediated immunity. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. To determine if co-expression of a cytokine can result in a more potent ILTV DNA vaccine, immunogenicity and protective efficacy of a monocistronic vector encoding the glycoprotein B (gB) of ILTV was compared to that of a bicistronic vector separately encoding the gB and chicken interleukin-18. Humoral and cellular responses induced by the DNA vaccines administered to the quadriceps muscle of chickens were evaluated. There were significant differences in antibody levels elicited by either monocistronic or bicistronic DNA vaccines as determined by ELISA. The percentages of CD3(+), CD3(+)CD8(+) and CD3(+)CD4(+) subgroups of peripheral blood T-lymphocytes in chickens immunized with the bicistronic DNA vaccine were higher than those in chickens immunized with monocistronic DNA vaccine. When chickens were challenged with a virulent CG strain of ILTV, the protective efficacy was enhanced significantly after immunization with the bicistronic DNA vaccine. These results demonstrated that co-expression of an adjuvant cytokine from a bicistronic DNA vaccine may be an effective approach to increasing ILTV DNA vaccine immunogenicity.

Use of a Multiplex RT-PCR Assay for Simultaneous Detection of the North American Genotype Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus and Japanese Encephalitis Virus.

A multiplex reverse transcriptase-polymerase chain reaction (multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously, in swine. Specific primers for each of the 3 RNA viruses, North American genotype porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus, and swine influenza virus, were used in the testing procedure. The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses. The assay was also effective in detecting one or more of the same viruses in various combinations in specimens, including lymph nodes, lungs, spleens, and tonsils, collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses. The results from the multiplex RT-PCR were confirmed by virus isolation. The relative efficiency (compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.

Cloning, in vitro expression, and bioactivity of interleukin-18 isolated from a domestic porcine breed found in Henan.

To evaluate the effects of recombinant porcine interleukin-18 (rpIL-18) on the replication of viruses in host cells and proliferation of lymphocytes, porcine IL-18 (pIL-18) isolated from a domestic big-white porcine breed found in the Henan province (HN) was cloned using a reverse transcriptase-PCR. The cloned HN pIL-18 contained an ORF of 579 base pairs encoding a 192-amino-acid precursor protein. The amino acid sequence of HN pIL-18 was compared with all the other pIL-18 amino acid sequences and varied by at least one amino acid to the consensus of all the others available. HN pIL-18 mature protein gene was inserted into a prokaryotic vector pGEX-4T-1 and expressed in Escherichia coli BL21. The expression of glutathione-S-transferase-pIL18 fusion protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The rpIL-18 induced in vitro proliferation of concanavalin-A-stimulated porcine splenocytes, as revealed by the MTT assay. We studied the antiviral activities of the rpIL-18 on the replication of porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine parvovirus (PPV) cultured in two homologous cell lines. The results suggested that rpIL-18 can stimulate the proliferation of lymphocytes and inhibit viral pathogens infecting the porcine population.

Nitric oxide inhibits the replication cycle of porcine parvovirus in vitro.

This study investigated the inhibitory effect and mechanism of nitric oxide (NO) on porcine parvovirus (PPV) replication in PK-15 cells. The results showed that two NO-generating compounds, S-nitroso-L-acetylpenicillamine (SNAP) and L-arginine (LA), at a noncytotoxic concentration could reduce PPV replication in a dose-dependent manner and that this anti-PPV effect could be reversed by the NO synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME). By assaying the steps of the PPV life cycle, we also show that NO inhibits viral DNA and protein synthesis. This experiment provides a frame of reference for the study of the anti-viral mechanism of NO.

A TaqMan-based real-time polymerase chain reaction for the detection of porcine parvovirus.

A real-time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect porcine parvovirus (PPV). Real-time PCR was optimized to quantify PPV using a detection system (Rotor Gene 2000 detector) and a dual-labeled fluorogenic probe. The gene-specific labeled fluorogenic probe for the VP2 gene of PPV was used to detect PPV. Quantitation of PPV was accomplished by a standard curve plotting cycle threshold values (Ct) against each dilution of standard plasmids. When the specificity of the assay using specific PPV primers was evaluated by testing the PPV standard strain and other viruses, no cross-reactions were detected with non-PPV reference viruses. The detection limit of real-time PCR for PPV was 2.08log10 genome copy equivalent (gce). In this study, a real-time PCR assay was performed on 80 clinical samples and compared with a conventional PCR assay. In 48 of 80 samples, PPV DNA was detected by the conventional PCR assay. All samples positive for PPV DNA by the conventional PCR assay were also positive by the real-time PCR assay, and 12 of 32 samples that tested negative for PPV DNA by the conventional method tested positive by the real-time PCR assay. Using the real-time PCR assay, the number of samples in which PPV was detected increased by 15%. Therefore, it is considered to be a useful tool for the detection of PPV.

Phenotypic and molecular characterization of TEM-116 extended-spectrum beta-lactamase produced by a Shigella flexneri clinical isolate from chickens.

Extended-spectrum beta-lactamases (ESBLs) produced by a clinical isolate of Shigella flexneri from chickens were detected with confirmatory phenotypic tests of the Clinical and Laboratory Standards Institute, and minimum inhibitory concentrations of several antibacterial drugs against the isolate were determined by the twofold dilution method. The genotype and subtype of the ESBL-producing S. flexneri isolate were identified by PCR amplifying of ESBL genes and DNA sequencing analysis. The results revealed that the isolate was able to produce ESBLs. They were resistant to third-generation cephalosporins such as ceftiofur and ceftriaxone and showed characteristics of multidrug resistance. The ESBL gene from the S. flexneri isolate was of the TEM type. Sequence analysis indicated that the TEM-type gene had 99.1% and 99.2% identity to TEM-1D ESBL and TEM-1 beta-lactamase, respectively, at the nucleotide level. The amino acid sequence inferred from the TEM-type gene revealed three substitutions compared with the TEM-1 and TEM-1D enzymes: Ser51Gly, Val82Ila and Ala182Val. When it was compared with TEM-116 (99.8% identity), there were only two mutations (A(151)G and T(403)C) in the TEM-type gene, resulting in the substitution of Ser to Gly at position 51 in the amino acid sequence. The TEM type was a TEM-116 derivative.

[Prokaryotice expression of the NS1 gene of PPV and renaturation of the recombinant protein].

The antigen of NS1 gene of PPV was amplified by PCR, and the amplified fragments were cloned into the prokaryotic expression vector pGEX-4T-1. The insert position, the size and the frame were identified by PCR, restriction enzyme digestion and the sequence analysis of the recombinant plasmids. The sequence analysis results of pGEX-NS1-HN1 showed that the prokaryotic expression vector was successfully constructed. The target gene was successfully expressed in the host cell BL21 when induced with IPTG. The expression was optimized with proper inducing conditions of 1.0mmol/L IPTG, 10 hours and 37 degree C induction. The expression of the target protein added up to 29.8% of the total bacterial protein. The results of SDS-PAGE indicated that molecular weight of the expressed protein was about 52kDa and the expressed protein mainly existed in the inclusion body. Western blot analysis proved the recombinant protein has good reactive ability against PPV positive serum. The pGEX-NS1-HN1 inclusion body was dissolved with 8mol/L urea. Then the expressed protein was renatured by dilution method and the systems of GSH and GSSG. ELISA detection proved the renaturation protein has good biological activity.

[Construction of the eukaryotic expression vector with IL-2 gene and VP2 gene of PPV and research on immunogenicity].

To construct gene vaccine of PPV and to investigate the effects of interleukin 2 (IL-2) as an adjuvant on immune responses in mouse, the recombinant expression plasmid of pCIneo-IL2-VP2 was constructed and transfected into PK-15 cells by lipofectamine, the expressed product was detected by immunofluore assay. To study the immune effects of DNA vaccine in vitro and in vivo, mice were used as the animal model. The recombinant plasmid pCIneo-IL2-VP2, the control plasmid pCI-neo and the PPV live vaccine were immunized by intramuscular injection. Anti-PPV antibodies were measured by ELISA, lymphocyte proliferation activity was detected using MTT method, and the specific killing activities of CTL were assayed too. The results show that the immunized mice produced PPV antibody after one week, and reached to highest after four weeks. Compared with the control group, the pCIneo-IL2-VP2 immunized group produced significant differences in the antibody titers, the lymphocyte proliferation activity and the specific killing activities of CTL. The pCIneo-IL2-VP2 induced humoral and cellular immunity responses similarly to that the live vaccine induced. These results manifested that the PPV DNA vaccine successfully induced humoral and cellular immunity response in mice with the IL-2 gene as an adjuvant.