Development of porcine induced pluripotent stem cells with a CD163 reporter system / 生物工程学报

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

Eukaryotic expression of GP5 and M protein of porcine reproductive and respiratory syndrome virus and immunogenicity evaluation / 生物工程学报

In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.

Age-specific Prevalence of Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus Type 2, and Mycoplasma hyopneumoniae in Korea Pig Farms

Porcine respiratory disease complex (PRDC) continues to be a significant economic problem to the swine industry. Porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and Mycoplasma hyopneumoniae (MH) are considered to be the most important pathogens that cause PRDC. In this study, we investigated the prevalence of antibodies against PRRSV and MH in the serum of sows and piglets from 89 domestic commercial pig farms by ELISA, and the presence of viral nucleic acids of PRRSV, including North American and European PRRS, and PCV2 was also investigated in the serum of sows and piglets from 89 domestic commercial pig farms by real-time PCR. In case of PRRSV, 78.7% (70/89) of sows were positive for PRRSV antibody, and 96.6% (86/89) of piglets were positive for PRRSV antibody. For MH, 76.4% (68/89) of sows showed positive for MH antibody. In the PRRSV viral nucleic acid detection experiment, 36.0% (32/89) of sows were positive for PRRSV nucleic acids, and virus nucleic acid was detected in 83.1% (74/89) of piglets. In case of virus type, both North American and European types were detected. In case of PCV2, 15.7% (14/89) of sows were positive for PCV2 nucleic acids. Conclusively, PCV2, PRRSV, and MH were widely distributed in pig farms in Korea. These prevalence data related with PRDC provides clinical information for vaccination strategy and development for the control of PRDC.

Evaluation of the efficacy of an attenuated live vaccine based on virulent porcine reproductive and respiratory syndrome virus 2 in young pigs / 대한수의학회지

The efficacy of the CA-2-MP120 vaccine, a cell culture-attenuated strain of virulent porcine reproductive and respiratory syndrome virus (PRRSV), was assessed in pigs. Despite the persistence of viremia in all vaccinated animals during the immunization period, the virus was not detected in vaccinated pigs following challenge. Furthermore, no pigs in the vaccinated group shed PRRSV nasally, orally or rectally throughout the experiment. Moreover, histopathological lung and lymph node lesions in the immunized group were much milder than those in the unimmunized and challenged group. These results indicated that CA-2-MP120 can provide effective protection against virulent wildtype PRRSV-2.

Acute porcine reproductive and respiratory syndrome outbreaksin immunized sow herds: from occurrence to stabilization under whole herd vaccination strategy / 대한수의학회지

Outbreaks of porcine reproductive and respiratory syndrome virus (PRRSV) in vaccinated sow herds from occurrence to stabilization were monitored and analyzed in terms of serology and reproductive performance. Three different conventional pig farms experienced severe reproductive failures with the introduction of a type 1 PRRSV. These farms had adopted mass vaccination of sows using a type 2 PRRSV modified live vaccine (MLV). Therefore, to control the type 1 PRRSV, an alternative vaccination program utilizing both type 1 and type 2 MLV was undertaken. Following whole herd vaccinations with both types of MLV, successful stabilization of PRRS outbreaks was identified based on serological data (no viremia and downward trends in ELISA antibody titers in both sows and suckling piglets) and recovery of reproductive performance. Additionally, through comparison of the reproductive parameters between outbreak and non-outbreak periods, it was identified that PRRSV significantly affected the farrowing rate and the number of suckling piglets per litter at all three pig farms. Comparison of reproductive parameters between periods when the different vaccination strategies were applied revealed that the number of piglets born in total and born dead per litter were significantly increased after the introduction of the type 1 PRRS MLV.

Preliminary assessment of correlation between T-lymphocyte responses and control of porcine reproductive and respiratory syndrome virus (PRRSV) in piglets born after in-utero infection of a type 2 PRRSV / 대한수의학회지

A preliminary study into the protective mechanisms of adaptive immunity against porcine reproductive and respiratory syndrome virus (PRRSV) in piglets (n = 9) born to a gilt challenged intranasally with a type-2 PRRSV. Immune parameters (neutralizing antibodies, CD3⁺CD4⁺, CD3⁺CD8⁺, CD3⁺CD4⁺CD8⁺ T-lymphocytes, and PRRSV-specific interferon (IFN)-γ secreting T-lymphocytes) were compared with infection parameters (macro- and microscopic lung lesion, and PRRSV-infected porcine alveolar macrophages (CD172α⁺PRRSV-N⁺ PAM) as well as with plasma and lymphoid tissue viral loads. Percentages of three T-lymphocyte phenotypes in 14-days post-birth (dpb) peripheral blood mononuclear cell (PBMC) had significant negative correlations with percentages of CD172α⁺PRRSV-N⁺ PAM (p 0.1) with infection parameters. The results indicate that T-lymphocytes contribute to controlling PRRSV replication in young piglets born after in-utero infection.

An enhanced immunochromatographic strip test using colloidal gold nanoparticle-labeled dual-type N proteins for detection of antibodies to PRRS virus

Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most important infectious diseases causing serious economic loss in the swine industry worldwide. Due to its increasing genetic diversity, a rapid and accurate diagnosis is critical for PRRS control. The immunochromatographic strip test (ICST) is a rapid and convenient type of immunoassay. In this study, an on-site immunochromatographic assay-based diagnostic method was developed for detection of PRRS virus (PRRSV)-specific antibodies. The method utilized colloidal gold nanoparticle-labeled dual-type nucleocapsid proteins encoded by open reading frame 7. We evaluated 991 field samples from pig farms and 66 serum samples from experimentally PRRSV-inoculated pigs. Based on true PRRSV-specific antibody-positive or

Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages

The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 in vivo. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2-MP120 had decreased virulence, eliciting weight gains, body temperatures, and histopathologic lesions similar to those in the negative control group. However, compared to CA-2-P100 infection, CA-2-MP120 yielded consistently higher viremia kinetics and enhanced antibody responses in pigs. All pigs inoculated with CA-2-MP120 developed viremia and seroconverted to PRRSV. During 20 passages in PAM cells, CA-2-MP120 acquired 15 amino acid changes that were mostly distributed in nsp2 and minor structural protein-coding regions. Among these changes, 6 mutations represented reversions to the sequences of the reference CA-2 and parental CA-2-P20 strains. These genetic drifts may be hypothetical molecular markers associated with PRRSV macrophage tropism and virulence. Our results indicate that the PAM-passaged CA-2-MP120 strain is a potential candidate for developing a live, attenuated PRRSV vaccine.

A new recombined porcine reproductive and respiratory syndrome virus virulent strain in China

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases worldwide. In the present study, a new virulent strain of PRRS virus (PRRSV), GDsg, was isolated in Guangdong province, China, and caused high fever, high morbidity, and high mortality in sows and piglets. The genome of this new strain was 15,413 nucleotides (nt) long, and comparative analysis revealed that GDsg shared 82.4% to 94% identity with type 2 PRRSV strains, but only 61.5% identity with type 1 PRRSV Lelystad virus strain. Phylogenetic analysis indicated that type 2 PRRSV isolates include five subgenotypes (I, II, III, IV, and V), which are represented by NADC30, VR-2332, GM2, CH-1a, and HuN4, respectively. Moreover, GDsg belongs to a newly emerging type 2 PRRSV subgenotype III. More interestingly, the newly isolated GDsg strain has multiple discontinuous nt deletions, 131 (19 + 18 + 94) at position 1404–1540 and a 107 nt insertion in the NSP2 region. Most importantly, the GDsg strain was identified as a virus recombined between low pathogenic field strain QYYZ and vaccine strain JXA1-P80. In conclusion, a new independent subgenotype and recombinant PRRSV strain has emerged in China and could be a new threat to the swine industry of China.

Genetic diversity and phylogenetic analysis of porcine reproductive and respiratory syndrome virus in southern China from 2007 to 2014

Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic variation. In this study, we characterized the genetic variation and evolutionary relationships among circulating PRRSV strains in southern China. We analyzed 29 NSP2 strains and 150 ORF5 strains from clinical samples collected in southern China during 2007–2014. The alignment results showed that the nucleotide identity similarities of the two genes among these strains were 80.5%–99.7% and 80.9%–100%, respectively. Phylogenetic analysis based on the NSP2 gene showed that highly pathogenic (HP)-PRRSV was still the dominant virus in southern China from 2013 to 2014. Compared with reference strains CH-1a and VR-2332, the field strain 131101-GD-SHC, which shared high homology with JXA1-P170, had a novel 12 amino acid deletion at position 499–510. Phylogenetic analysis based on the ORF5 gene showed that HP-PRRSV, VR2332-like strains, and QYYZ-like strains were simultaneously circulating in southern China from 2007 to 2014, suggesting that, in recent years, the type 2 PRRSV was more diverse in southern China. In conclusion, mutations in the decoy epitope and primary neutralizing epitope could be markers of viral evolution and used to study evolutionary relationships among PRRSV strains in China.

Duplex nested reverse transcriptase polymerase chain reaction for simultaneous detection of type 2 porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 from tissue samples

There are high levels of co-incidence of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in porcine tissue. This study established a duplex nested reverse transcriptase polymerase chain reaction (RT-PCR) method that targets the genomic RNA of type 2 PRRSV and the mRNA of PCV2 in infected tissues. The method amplified discriminative bands of 347 bp and 265 bp specific for type 2 PRRSV and PCV2, respectively. The limits of detection of the duplex nested RT-PCR were 10(1.5) TCID₅₀/mL for type 2 PRRSV and 10² infected cells/mL for PCV2. The kappa statistic, which measures agreement between methods, was 0.867, indicating a good level of agreement. This RNA-based duplex RT-PCR approach can be another way to detect type 2 PRRSV and PCV2 simultaneously and with improved convenience.

Preparation and identification of polyclonal antibodies specific for nsp4 protein of porcine reproductive and respiratory syndrome virus / 生物工程学报

To obtain specific antibodies against nsp4 protein of porcine reproductive and respiratory syndrome virus (PRRSV), nsp4 gene was amplified by RT-PCR and cloned into pET-28a(+) vector, designated pET28a-nsp4. pET28a-nsp4 was transformed into Escherichia coli Trasseta (DE3) cells and expressed after induction of IPTG. SDS-PAGE analysis showed that the recombinant protein was expressed in soluble form with the molecular weight of 26 kDa. The soluble fusion protein in the supernatant was purified using Ni+-NTA affinity chromatography. New Zealand rabbits were immunized by the purified nsp4 and anti-sera against nsp4 were obtained. The titer of polyclonal antibodies was about 106 and showed good specificity and sensitivity in the immunofluorescence assay and Western blotting analysis. The polyclonal antibodies also recognized native nsp4 form PRRSV infected Marc-145 cells, providing a useful tool in PRRSV replication mechanism study.

Genetic variation analysis of Nsp2, ORF5 and ORF3 of PRRSV SC-GY strain from Sichuan province / 中国兽医学报

To monitor genetic variation of porcine reproductive and respiratory syndrome virus (PRRSV),RT-PCR was used to identify a sample suspected of PRRSV infection.A PRRSV named SC-GY strain was obtained,and its Nsp2,ORF5 and ORF3 genes were used for sequence alignment and phylogenetic tree construction.The results showed that SC-GY strain is highly pathogenic PRRSV American variant strains with Nsp2 gene discontinuous deletion of 30 amino acids,ORF3 gene aa17 a serine (S) insert.Comparing to VR2332,CH-1a,JXA1,HUN4,NADC30,HENAN-XINX and SC2012,the Nsp2,ORF5 and ORF3 of SC-GY shared 70.3％-97.9％,82.4％-97.6％ and 83.1％-98.2％ of nucleotide similarity,and 62.3％-96.3％,78.0％-95.7％ and 81.6％-96.5％ of deduced amino acid similarity;and compared to LV they shared only 18.9％,60.8％ and 63.7％ of nucleotide similarity,and 14.0％,54.9％ and 57.2％ of deduced amino acid similarity.The phylogenetic tree revealed that the SC-GY formed independent small branches although it belonged to the same subgroup as highly pathogenic PRRSV strains.The results showed that in high frequency live vaccine immunization of currently PRRSV,the gene of PRRSV epidemic strain is still in constant variation.Vaccination of live PRRSV vaccines should be reduced and surveillance of PRRSV strains should be enhanced.

Augmented immune responses in pigs immunized with an inactivated porcine reproductive and respiratory syndrome virus containing the deglycosylated glycoprotein 5 under field conditions

PURPOSE: Porcine reproductive and respiratory syndrome virus (PRRSV) leads to major economic losses in the swine industry. Vaccination is the most effective method to control the disease by PRRSV. MATERIALS AND METHODS: In this study, the efficacy of a glycoprotein (GP) 5-modified inactivated vaccine was investigated in pigs. The study was performed in three farms: farm A, which was porcine reproductive and respiratory syndrome (PRRS)-negative, farm B (PRRS-active), which showed clinical signs of PRRS but had not used vaccines, and farm C (PRRS-stable), which had a history of endemic PRRS over the past years, but showed no more clinical signs after periodic administration of modified live virus vaccine. RESULTS: The inactivated vaccine induced great enhancement in serum neutralizing antibody titer, which was sufficient to protect pigs from further infections of PRRSV in a farm where pre-existing virus was circulating. CONCLUSION: These results indicated that vaccination with the inactivated vaccine composed of viruses possessing deglycosylated GP5 would provide enhanced protection to pigs from farms suffering from endemic PRRSV.

Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine

The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA-ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNAIL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4+ and CD8+ T lymphocytes, proliferation indices, and interferon-gamma expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4+ and CD8+ T lymphocytes, and significantly higher IFN-gamma production than the other inoculated pigs (p < 0.05).

Analysis of molecular variation in porcine reproductive and respiratory syndrome virus in China between 2007 and 2012 / 中国病毒学·英文版

In the present study, 89 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-struc-tural protein 2 (Nsp2) and glycoprotein 5 (GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discon-tinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007-2012 in China. Owing to the extensive use of representative vaccine strains, natu-ral recombination events occurred between strains. Three isolates-HH08, DY, and YN-2011-were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolu-tionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.

Detection of porcine reproductive and respiratory syndrome virus in oral fluid from naturally infected pigs in a breeding herd

The objectives of the present study were to evaluate the anatomic localization of porcine reproductive and respiratory syndrome virus (PRRSV) in naturally infected pigs and to determine whether oral fluid could be used to detect the virus in infected animals. Two sows, seven 2-month-old grower pigs, and 70 6-month-old gilts were included in this study. PRRSV in sera and oral fluid were identified by nested reverse transcription PCR (nRT-PCR) while lung, tonsil, and tissue associated with oral cavity were subjected to nRT-PCR, immunohistochemistry, and in situ hybridization. In sows, PRRSV was identified in oral fluid and tonsils. PRRSV was also detected in oral fluid, tonsils, salivary glands, oral mucosa, and lungs of all seven grower pigs. However, viremia was observed in only two grower pigs. Double staining revealed that PRRSV was distributed in macrophages within and adjacent to the tonsillar crypt epithelium. In gilts, the North American type PRRSV field strain was detected 3 to 8 weeks after introducing these animals onto the farm. These results confirm previous findings that PRRSV primarily replicates in tonsils and is then shed into oral fluid. Therefore, oral fluid sampling may be effective for the surveillance of PRRSV in breeding herds.

The signal sequence of type II porcine reproductive and respiratory syndrome virus glycoprotein 3 is sufficient for endoplasmic reticulum retention

The glycoprotein 3 (GP3) of type II porcine reproductive and respiratory syndrome virus has the characteristic domains of a membrane protein. However, this protein has been reported to be retained in the endoplasmic reticulum (ER) rather than transported to the plasma membrane of the cell. In this study, we performed confocal laser scanning microscopy analysis of variants of GP3 and foundthat the signal sequence of the GP3 led to confinement of GP3 in the ER, while the functional ortransmembrane domain did not affect its localization. Based on these results, we concludedthat the signal sequence of GP3 contains the ER retention signal, which might play an important role in assembly of viral proteins.

Pathologic studies in lymph nodes of pigs infected with porcine circovirus type 2, porcine reproductive and respiratory syndrome virus / 대한수의학회지

Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) have been suspected to have immunosuppressive effects on pigs. To investigate the correlation between these virus infection and the lesions of lymph nodes including sub-mandibular and inguinal lymph node, 44 pigs (PCV2 single, n = 14; PRRSV single, n = 10; PCV2/PRRSV, n = 14; negative control, n = 6) were examined by histopathology and immunohistochemistry. Histopathologically, granulomatous lymphadenitis characterized by lymphoid depletion with histiocytic cells infiltration was observed in PCV-2 single and PCV-2/PRRSV group. Immunohistochemically, there were significant reduction of B and T lymphocytes in lymph nodes of these groups, while the number of macrophages was increased. In only PRRSV infected group, germinal center hypertrophy and lymphoid necrosis were observed. Immunohistochemically, the number of CD3+ T lymphocytes was slightly increased. Severe lymphocytic depletion in PCV-2 infection-related lymph nodes might be associated with producing immunocompromised state in pig. Comparing with PCV-2 infected group, PRRSV produced minor effects on the changes in immune cell population in the lymph nodes of pigs. PRRSV may increase susceptibility of the disease in pigs by disruption of the first defense lines in target organs, such as the alveolar macrophages in lungs.

Genetic diversity of porcine reproductive and respiratory syndrome virus in Korea

The high genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been an obstacle to developing an effective vaccine for porcine reproductive and respiratory syndrome (PRRS). This study was performed to assess the degree of genetic diversity among PRRSVs from Korean pig farms where wasting and respiratory syndrome was observed from 2005 to 2009. Samples from 786 farms were tested for the presence of PRRSV using reverse transcription PCR protocol. A total of 117 farms were positive for type 1 PRRSV while 198 farms were positive for type 2. Nucleotide sequences encoding the open reading frame (ORF) 5 were analyzed and compared to those of various published PRRSV isolates obtained worldwide. Sequence identity of the ORF 5 in the isolates was 81.6~100% for type 1 viruses and 81.4~100% for type 2 viruses. Phylogenetic analysis of the ORF 5 sequences showed that types 1 and 2 PRRSVs from Korea were mainly classified into three and four clusters, respectively. The analyzed isolates were distributed throughout the clusters independent of the isolation year or geographical origin. In conclusion, our results indicated that the genetic diversity of PRRSVs from Korean pig farms is high and has been increasing over time.