Swine Testicle Cells Research Articles

Construction and Immunogenicity of a Recombinant Porcine Pseudorabies Virus (PRV) Expressing the Major Neutralizing Epitope Regions of S1 Protein of Variant PEDV.

Porcine epidemic diarrhea virus (PEDV) infection causes severe diarrhea and high mortality in neonatal piglets. Pseudorabies causes acute and often fatal infections in young piglets, respiratory disorders in growing pigs, and reproductive failure in sows. In late 2011, pseudorabies virus (PRV) variants occurred in Bartha-K61-vaccine-immunized swine herds, resulting in economic losses to the global pig industry. Therefore, it is essential to develop a safe and effective vaccine against both PEDV and PRV infections. In this study, we constructed a recombinant virus rPRV-PEDV S1 expressing the major neutralizing epitope region (COE, SS2, and SS6) of the PEDV S1 protein by homologous recombination technology and CRISPR/Cas9 gene editing technology, and then evaluated its biological characteristics in vitro and immunogenicity in pigs. The recombinant virus rPRV-PEDV S1 had similar growth kinetics in vitro to the parental rPRV NY-gE-/gI-/TK- strain, and was proven genetically stable in swine testicle (ST) cells and safe for piglets. PEDV S1-specific antibodies were detected in piglets immunized with rPRV-PEDV S1 on the 7th day post-immunization (dpi), and the antibody level increased rapidly at 14-21 dpi. Moreover, the immunized piglets receiving the recombinant virus exhibited alleviated clinical signs and reduced viral load compared to the unvaccinated group following a virulent PEDV HN2021 strain challenge. Also, piglets immunized with rPRV-PEDV S1 developed a PRV-specific humoral immune response and elicited complete protection against a lethal PRV NY challenge. These data indicate that the recombinant rPRV-PEDV S1 is a promising vaccine candidate strain for the prevention and control of PEDV and PRV infections.

Porcine ex-vivo intestinal mucus has age-dependent blocking activity against transmissible gastroenteritis virus

Transmissible gastroenteritis virus (TGEV) causes high mortality in young piglets (< 3 days of age). With aging, the susceptibility/morbidity/mortality rates drop. We previously hypothesized that the age-related changes in the intestinal mucus could be responsible for this resistance. Hence, this study investigated the effect of porcine intestinal mucus from 3-day and 3-week-old pigs on the free mobility of the virulent TGEV Miller strain, and on the infection in swine testicle (ST) cells. Single particle tracking (SPT) revealed that TGEV had significantly higher diffusion coefficients in 3-day mucus compared to 3-week mucus. TGEV and charged and uncharged control nanoparticles diffused freely in 3-day mucus but were hindered by 3-week mucus in the diffusion model; TGEV mimicked the diffusion behavior of negatively charged carboxylated particles. Inoculation of ST cells with TGEV in the presence of 3-week mucus resulted in a significantly lower average number of infected cells (30.9 ± 11.9/5 fields) compared with 3-day mucus (84.6 ± 16.4/5 fields). These results show that 3-week mucus has a significant TGEV-blocking activity compared to 3-day mucus in free diffusion and infection of the underlying susceptible cells. Additionally, a label-free proteomics analysis revealed an increased expression of mucin 13, known for negatively regulating the tight junctions in intestinal epithelium, in 3-day-old pigs. In 3-week-old pigs, a higher expression of mucin 2, a type of secreted mucin which is known for inhibiting coronavirus infection, was observed. Concludingly, this study demonstrated a protective effect of 3-week mucus against viral infections.

Emergence of a novel GIII Getah virus variant in pigs in Guangdong, China, 2023.

From May to July of 2023, one pig farm in Heyuan city, Guangdong Province of China, suffered severe piglet death and sow reproductive disorders. The common pig viruses and bacteria tested negative. To uncover the possible cause of the disease, a metagenomic analysis was performed in the pooled small intestine samples from three 8-day-old diseased piglets. The results showed that Getah virus (GETV), an RNA virus, might be the potential pathogen that affects pig health. Subsequently, GETV nucleotide was detected in all of the 15 samples collected from three diseased piglets using quantitative reverse transcription PCR, suggesting GETV as the main pathogen of the disease. A GETV strain, designated as GDHYLC23, was successfully isolated using the swine testicle cell line. Sequence analysis showed that the epidemic strain had a unique 32-nucleotide repeat insertion in the 3' noncoding region. Phylogenetic analysis showed that GDHYLC23 belonged to the pandemic group III. The identification of GETV with new variations implies the continuous evolution of the virus, which poses potential threats to the swine industry.IMPORTANCEPig farms are faced with emerging and re-emerging viruses that may cause substantial economic loss. The identification of potentially pathogenic viruses helps to prevent and control the spread of diseases. In this study, by using metagenomic analysis, we found that a neglected virus, GETV with a unique insertion in the genome, was the main pathogen in one pig farm that suffered severe piglet death and sow reproductive disorders. Although the potential impact of such an insertion on viral pathogenicity is unknown, the surveillance of the continuing evolution of GETV in pig farms cannot be ignored.

Antiviral effect of baicalein onPorcine Deltacoronavirusinfectionbyregulating the inflammatory responses through PI3K-Akt-NF-κB signaling pathwayin cultured cells.

Porcine deltacoronavirus (PDCoV) is an newly emerged enteropathogenic coronavirus, mainly causing diarrhea in suckling piglets, and also has the potential for cross-species transmission.However, there are no effective vaccines or specific therapeutic agents for PDCoV. This study investigates the antiviral properties of baicalein against PDCoV infection in swine testicle cells (ST). It reveals that baicalein exerts a dose-dependent inhibitory effect on PDCoV replication, primarily targeting the replication stage of the viral infection by impeding viral RNA and protein synthesis. Furthermore, treatment with baicalein leads to reduced phosphorylation of PI3K, AKT, and NF-κB p65 proteins, along with decreased mRNA levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α). These results signify that PDCoV replication is inhibited through the inhibition of the PI3K-Akt-NF-κB protein signaling pathway, thereby suppressing the inflammatory response. In conclusion, it underscores the potential of baicalein as a therapeutic candidate for treating PDCoV infection.

Porcine deltacoronavirus enters ST cells by clathrin-mediated endocytosis and does not require Rab5, Rab7, or Rab11.

Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus threatening pig industries worldwide. Our previous work showed that PDCoV enters porcine kidney (PK-15) cells through a caveolae-dependent pathway, but the entry mechanism for PDCoV into swine testicle (ST) cells remains unclear. Mechanisms of virus entry can be different with different virus isolates and cell types. Here, we determined that PDCoV enters ST cells via clathrin-mediated endocytosis. Additionally, we found that PDCoV entry does not require Rab5, Rab7, or Rab11. These findings provide additional understanding of the entry mechanisms of PDCoV and possible antiviral targets.

Procyanidins inhibit zearalenone-induced apoptosis and oxidative stress of porcine testis cells through activation of Nrf2 signaling pathway

The mycotoxin zearalenone (ZEA) in food and feed seriously harms human and animal health. How to reduce its toxicity is an important direction of current research on food safety. This study aim to assess the effects of procyanidins (PC) on cell apoptosis caused by ZEA and to clarify the role of Nrf2 in the process. Swine testicle (ST) cells were treated with ZEA (57.5 μmol/L) and/or PC (10 mg/L) for 24 h. Cell viability was detected by CCK-8 assay. Cell apoptosis and the level of ROS were detected by flow cytometry. The expression levels of mRNA and protein was detected by qRT-PCR and western blotting. Our results showed that ZEA reduced the antioxidant capacity of the ST cells, induced the cell apoptosis and inhibited the gene and protein expression of Nrf2 and its downstream genes (ho-1,nqo1), while PC improved the cell antioxidant capacity, reduced the degree of ZEA-induced cell apoptosis and promoted the gene and protein expression of Nrf2 and its downstream genes. However, when the Nrf2 small molecule inhibitor ML385 was added, the ability of PC to inhibit ZEA-induced cell apoptosis and promote the expression of Nrf2 and its downstream genes were decreased. Our results demonstrated that ZEA induced oxidative stress and apoptosis of ST cells, which were alleviated by PC intervention via activating Nrf2 signaling pathway. This finding of this study provided a molecular basis for the clinical application of PC to prevent ZEN-caused reproductive toxicity.

Environmental stability of porcine respiratory coronavirus in aquatic environments.

Coronaviruses (CoVs) are a family of viruses that are best known as the causative agents of human diseases like the common cold, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) and COVID-19. CoVs spread by human-to-human transmission via droplets or direct contact. There is, however, concern about potential waterborne transmission of SARS-CoV-2, the virus responsible for COVID-19, as it has been found in wastewater facilities and rivers. To date, little is known about the stability of SARS-CoV-2 or any other free coronavirus in aquatic environments. The inactivation of terrestrial CoVs in seawater is rarely studied. Here, we use a porcine respiratory coronavirus (PRCV) that is commonly found in animal husbandry as a surrogate to study the stability of CoVs in natural water. A series of experiments were conducted in which PRCV (strain 91V44) was added to filtered and unfiltered fresh- and saltwater taken from the river Scheldt and the North Sea. Virus titres were then measured by TCID50-assays using swine testicle cell cultures after various incubation times. The results show that viral inactivation of PRCV in filtered seawater can be rapid, with an observed 99% decline in the viral load after just two days, which may depend on temperature and the total suspended matter concentration. PRCV degraded much slower in filtered water from the river Scheldt, taking over 15 days to decline by 99%, which was somewhat faster than the PBS control treatment (T99 = 19.2 days). Overall, the results suggest that terrestrial CoVs are not likely to accumulate in marine environments. Studies into potential interactions with exudates (proteases, nucleases) from the microbial food web are, however, recommended.

Identification of Cleavage Sites Proteolytically Processed by NS2B-NS3 Protease in Polyprotein of Japanese Encephalitis Virus.

Understanding the proteolytic processing of polyprotein mediated by NS2B-NS3 protease contributes to the exploration of the mechanisms underlying infection of Japanese encephalitis virus (JEV), a zoonotic flavivirus. In this study, eukaryotic and prokaryotic cell models were employed to identify the cleavage sites mediated by viral NS2B-NS3 protease in JEV polyprotein. Artificial green fluorescent protein (GFP) substrates that contained the predicted cleavage site sequences of JEV polyprotein were expressed in swine testicle (ST) cells in the presence and absence of JEV infection, or co-expressed in E. coli with the recombinant NS2B-NS3 protease that was generated by fusing the N-terminal protease domain of NS3 to the central hydrophilic domain of NS2B. The cleavage of GFP substrates was examined by western blot. Among twelve artificial GFP substrates containing the cleavage site sequences predictively processed by host cell and/or NS2B-NS3 proteases, all sites were found to be cleaved by host cell proteases with different efficiencies. The sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions, but not the sites at internal NS3, internal NS4A and NS4B/NS5 junctions were identified to be cleaved by JEV NS2B-NS3 protease. These data provide insight into the proteolytic processing of polyprotein, which is useful for understanding JEV replication and pathogenesis.

Antiviral activities of Radix isatidis polysaccharide against pseudorabies virus in swine testicle cells

BackgroundRadix isatidis has been used in China and other Asian countries for its antiviral and anti-inflammatory effects for thousands of years. However, the antiviral effect of Radix isatidis polysaccharide against pseudorabies virus (PRV) is still unknown.MethodsThe polysaccharide were isolated from extract of the roots of Radix isatidis. MTT assays were used to determine the preventive effect, inhibitory effect and antiviral effect of Radix isatidis polysaccharide on PRV in vitro.ResultsThis study found that different concentrations of polysaccharides from this plant can inhibit PRV replication by 14.674–30.840%, prevent infection at rates of 6.668–14.923%, and kill this virus at rates of 32.214–67.422%.ConclusionThese results broaden the understanding of this traditional Chinese herb and provide a theoretical basis for further research. Moreover, Radix isatidis polysaccharide could be used for antiviral therapy.

Characterization of a recombinant pseudorabies virus expressing porcine parvovirus VP2 protein and porcine IL-6

BackgroundPorcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. Interleukin-6 (IL-6) has the roles to support host immune response to infections as a pleiotropic cytokine. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV.MethodsThe recombinant plasmid, pGVP2-IL6, was constructed by porcine IL-6 gene substituting for EGFP gene of the PRV transfer plasmid pGVP2-EGFP containing VP2 gene of PPV. Plasmid pGVP2-IL6 was transfected into swine testicle cells pre-infected with the virus rPRV-VP2-EGFP strain through homologous recombination and plaque purification to generate a recombinant virus rPRV-VP2-IL6. The recombinant PRV was further identified by PCR and DNA sequencing, and the expression of the VP2 protein and porcine IL-6 was analyzed by reverse transcription-PCR (RT-PCR) and Western blot. The virus titer was calculated according to Reed and Muench method. The immunogenicity of the recombinant virus was preliminarily evaluated in mice by intramuscular administration twice with the rPRV-VP2-IL6 at 4-week intervals.ResultsA recombinant virus rPRV-VP2-IL6 was successfully constructed and confirmed in this study. The properties of rPRV-VP2-IL6 were similar to the parental virus HB98 in terms of growth curve, morphogenesis and virus plaque sizes, and rPRV-VP2-IL6 was proliferated in different cell types. It induced specific antibodies against PPV as well as a strong increase of PPV-specific lymphocyte proliferation responses in mice immunized with rPRV-VP2-IL6, and provided partial protection against the virulent PPV challenge. rPRV-VP2-IL6 also induced a high level of neutralizing antibodies against PRV, and significantly reduced the mortality rate of (1 of 10) following virulent PRV challenge compared with the control (10 of 10).ConclusionsThe recombinant rPRV-VP2-IL6 might be a potential candidate vaccine against PRV and PPV infections in pigs.

In vitro and in silico studies reveal capsid-mutant Porcine circovirus 2b with novel cytopathogenic and structural characteristics

Porcine circovirus 2 (PCV2) is an icosahedral, non-enveloped, and single-stranded circular DNA virus that belongs to the family Circoviridae, genus Circovirus, and is responsible for a complex of different diseases defined as porcine circovirus diseases (PCVDs). These diseases – including postweaning multisystemic wasting syndrome (PMWS), enteric disease, respiratory disease, porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure – are responsible for large economic losses in the pig industry. After serial passages in swine testicle (ST) cells of a wild-type virus isolated from an animal with PMWS, we identified three PCV2b viruses with capsid protein (known as Cap protein) cumulative mutations, including two novel mutants. The mutant viruses were introduced into new ST cell cultures for reisolation and showed, in comparison to the wild-type PCV2b, remarkable viral replication efficiency (> 1011 DNA copies/ml) and cell death via necrosis, which were clearly related to the accretion of capsid protein mutations. The analysis of a Cap protein/capsid model showed that the mutated residues were located in solvent-accessible positions on the external PCV2b surface. Additionally, the mutated residues were found in linear epitopes and participated in pockets on the capsid surface, indicating that these residues could also be involved in antibody recognition. Taking into account the likely natural emergence of PCV2b variants, it is possible to consider that the results of this work increase knowledge of Circovirus biology and could help to prevent future serious cases of vaccine failure that could lead to heavy losses to the swine industry.

Continuous Passaging of a Recombinant C-Strain Virus in PK-15 Cells Selects Culture-Adapted Variants that Showed Enhanced Replication but Failed to Induce Fever in Rabbits.

Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that RecCpp80+10 failed to induce fever in rabbits, whereas RecCpp40+10 caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.

Effect of RNA binding motif protein 3 overexpression on Caspase 3 expression in swine testiclar cell under cold exposure

To investigate the change of apoptosis protein (Caspase 3) expression when RNA binding motif protein 3 (RBM3) overexpression in swine testicle (ST) cell line under cold stress (32℃) condition. In present study, RBM3 overexpression lentiviral vector (pLenti6/V5-GW/EmGFP-RBM3) and empty viral vector (pLenti6/V5-GW/EmGFP-DEST) with green fluorescent protein (GFP) that were successfully constructed in our laboratory were transfected into ST cells as overexpression virus group (OEV), empty vector virus (EVV) group and establishment of wildtype cell (WTC) group as the control group. The real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect expression of RBM3 mRNA and protein in each group, then cells in each group were cultured at 37℃ or 32℃ (2 h, 4 h, 8 h), the changes of Caspase 3 expression in each group were detected by enzyme linked immunosorbent assay (ELISA). Relative RBM3 gene and protein expression in OEV group were significantly higher than those in EVV group and WTC group. At the temperature of 37℃ and at 32℃ (2 h, 4 h, 8 h) in cold stress experiment, Caspase 3 expression in OEV group was significantly lower than those in EVV group and WTC group. RBM3 overexpression in ST cell line exposured to cold can significantly decreased the level of Caspase 3 expression, this study provides an experimental basis of RBM3 resistance against apoptosis of ST cells induced by low temperature.

A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.

Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.

Establishment and preliminary application of an assay for the detection of porcine parvovirus in cells used for production

Objective To establish an assay for the detection of porcine parvovirus (PPV) and to verify its application for monitoring cells used for production. Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1). Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated. A stock of PPV strains was prepared by infecting swine testicle (ST) cells with PPV strains. An assay for the detection of PPV infection was developed by using ST cells as sensitive cells. A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay. The sensitivity of ST cell infection-PCR test was analyzed. The cell samples used for production of biological products were detected by using the established assay. Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses. The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98. The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl. Both of the intra- and inter-coefficient of variation (CV) were less than 5% in Ct values. The intra- and inter-CV in copies of detection were 5%-15% and 30%-40% respectively. The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml. The PPV strains were detected in cell samples with no interference. The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml. All of the 22 cell samples were negative for PPV by using the real-time fluorescent quantitative PCR assay. Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully. The application of the two assays was conducive to further enhance the safety of using cells for production and therapy. Key words: Porcine parvovirus (PPV); Real-time fluorescent quantitative PCR assay; Swine testicle (ST) cells; Infection assay; Infection-PCR assay

Inhibitory effect of silver nanomaterials on transmissible virus-induced host cell infections

Coronaviruses belong to the family Coronaviridae, which primarily cause infection of the upper respiratory and gastrointestinal tract of hosts. Transmissible gastroenteritis virus (TGEV) is an economically significant coronavirus that can cause severe diarrhea in pigs. Silver nanomaterials (Ag NMs) have attracted great interests in recent years due to their excellent anti-microorganism properties. Herein, four representative Ag NMs including spherical Ag nanoparticles (Ag NPs, NM-300), two kinds of silver nanowires (XFJ011) and silver colloids (XFJ04) were selected to study their inhibitory effect on TGEV-induced host cell infection in vitro. Ag NPs were uniformly distributed, with particle sizes less than 20 nm by characterization of environmental scanning electron microscope and transmission electron microscope. Two types of silver nanowires were 60 nm and 400 nm in diameter, respectively. The average diameter of the silver colloids was approximately 10 nm. TGEV infection induced the occurring of apoptosis in swine testicle (ST) cells, down-regulated the expression of Bcl-2, up-regulated the expression of Bax, altered mitochondrial membrane potential, activated p38 MAPK signal pathway, and increased expression of p53 as evidenced by immunofluorescence assays, real-time PCR, flow cytometry and Western blot. Under non-toxic concentrations, Ag NPs and silver nanowires significantly diminished the infectivity of TGEV in ST cells. Moreover, further results showed that Ag NPs and silver nanowires decreased the number of apoptotic cells induced by TGEV through regulating p38/mitochondria-caspase-3 signaling pathway. Our data indicate that Ag NMs are effective in prevention of TGEV-mediated cell infection as a virucidal agent or as an inhibitor of viral entry and the present findings may provide new insights into antiviral therapy of coronaviruses.

Cultivation of PCV2 in swine testicle cells using the shell vial technique and monitoring of viral replication by qPCR and RT-qPCR

Porcine circovirus type 2 (PCV2) is difficult to isolate. Currently, no published articles have used the shell vial technique to isolate PCV2. In addition, the action of d-glucosamine on swine testicle cells (ST) has not been evaluated properly. Thus, the aim of this study was to determine an optimal concentration of d-glucosamine and to test the shell vial technique for PCV2 propagation in ST cells. The optimal concentration of d-glucosamine was determined to be 100mM. Because PCV2 is noncytopathic, the traditional adsorption was compared to the shell vial technique for 15 passages by qPCR, and RT-qPCR for passages 12 through 15. The quantities of viral DNA (P=0.013) and ORF1-mRNA detected with the shell vial technique were two-fold higher than the obtained with traditional adsorption. The levels of ORF2-mRNA were similar for both methods; however, by passage 15, a six-fold increase in levels was observed with the shell vial technique. Therefore, the shell vial technique was more efficient for the cultivation of PCV2, and qPCR/RT-qPCR can be used to monitor viral replication. In addition, a high viral load (>2.7×1010 DNA copies/ml) and high levels of viral mRNA expression indicated that the ST cells were persistently infected.

Characterization in vitro and in vivo of a novel porcine parainfluenza virus 5 isolate in Korea

A novel porcine parainfluenza 5 (pPIV5), KNU-11, in the genus Rubulavirus of the subfamily Paramyxovirinae, was isolated from the lung of a piglet in Korea in 2011. To understand the importance of this virus as an infectious agent, in vitro and in vivo characteristics of KNU-11 virus was investigated. KNU-11 was remarkably cytopathogenic, showing distinct cell rounding and clumping evident in porcine alveolar macrophage (PAM), porcine kidney (PK-15), and swine testicle (ST) cells within 12h postinfection and capable of hemagglutinating guinea pig red blood cells. Interestingly, this cytopathology was found to be absent in cell lines from other mammalian species. To evaluate the in vitro immunity of the pPIV5 isolate, we sought to explore alteration of inflammatory cytokine and chemokine expression in PAM cells infected with KNU-11 by using quantitative real-time RT-PCR. Most cytokine and chemokine genes including type 1 interferons (IFN-α/β) and IFN-related antiviral genes were found to be significantly elevated in KNU-11 virus-infected PAM cells. A serum neutralization test-based serosurvey demonstrated that neutralizing antibodies against KNU-11 are readily detected in domestic swine populations, suggesting high prevalence of pPIV5 in Korean pig farms. Animal studies showed that KNU-11 fails to establish an acute respiratory illness, indicating that pPIV5 is non- or very mildly pathogenic to pigs.

Establishment of real-time TaqMan-fluorescence quantitative RT-PCR assay for detection and quantitation of ten kinds of porcine inflammation markers mRNA

Real-time PCR assays based on TaqMan probes for detection of porcine inflammation markers including interferon-α (IFN-α), interferon-β (IFN-β), retinoic acid-inducible gene 1 (RIG-1), toll-like receptor 9 (TLR-9), interferon regulatory factors (IRF-3, IRF-7), Janus kinase (JAK-1, JAK-2), signal transducers and activators of transcription (STAT-1, STAT-2) were established in this study. The results indicated that the established assays were highly specific and sensitive with a detection limit of 1.0×101copies/μl, and coefficient of variations was less than three percent for both inter- and intra-assay. The established assays were used to detect mRNA levels of these inflammation markers and β-actin in swine testicle (ST) cells transfected with polyinosinic: polycytidylic acid (poly (I:C)). The results showed that the transcription levels (mRNA) of IFN-α, IFN-β, RIG-1 and IRF-7 were up-regulated in ST cells transfected with poly (I:C), and the transcription levels (mRNA) of TLR-9, IRF-3, JAK-1, JAK-2, STAT-1 and STAT-2 showed minimal change. The real-time PCR assays established in this study with high specificity, sensitivity and reproducibility could be used to quantify mRNA levels of porcine inflammation markers.

Interferon induction and suppression in swine testicle cells by porcine parvovirus and its proteins

Porcine parvovirus (PPV) is a major causative agent of reproductive failure in swine, which currently affects the swine industry worldwide. Although PPV was identified several years ago, little is known about how it overcomes host innate immunity. In this study, we used quantitative real-time PCR and a luciferase reporter assay to determine whether PPV infection induces type I interferon (IFN-α and IFN-β) and whether PPV infection blocks dsRNA-induced IFN-β promoter activation in cell cultures. The results indicate that PPV does not induce type I interferon and that the NS2 protein of PPV could blocks dsRNA-induced IFN-β promoter activation.