Interferon gamma induces cellular protein alteration and increases replication of porcine circovirus type 2 in PK-15 cells.

Porcine circovirus type 2 (PCV2) infections may lead to the development of subclinical signs or chronic systemic syndromes, collectively known as "porcine circovirus-associated disease" (PCVAD) in swine. Interferon gamma (IFN-γ) is known to enhance PCV2 replication in vitro, and immune mediators may act as pivotal factors in triggering PCV2 infection progression toward PCVAD. We determined the effects of IFN-γ on PCV2 replication in PK-15 cells. PCV2 was cultured in the presence or absence of exogenous swine IFN-γ (swIFNγ). Growth curve analysis in PK-15 cells revealed that PCV2 could replicate to a significantly higher titer in swIFNγ medium. To investigate the host cell response upon PVC2 infection, differential expression of proteins in PCV2-infected PK-15 cells with or without swIFNγ stimulation was analyzed by proteomics (LC-MS/MS) analysis. A large proportion of the differentially expressed proteins in swIFNγ-treated PCV2-infected cells were found to be involved in apoptosis, cellular stress responses, cell survival/proliferation pathways, and inflammatory responses. We further confirmed the expression of these differentially expressed proteins at the mRNA levels by qRT-PCR. PCV2 infection in PK-15 cells in the presence of IFN-γ resulted in upregulation of cellular proteins in responses to stress, cell survival, and cell proliferation (Hsp90, MAP3K7, RAS-GTPase, c-myc, and 14-3-3 epsilon) as well as in an increase in the levels of proteins (CASP9 and TRAF5) related to the apoptosis pathways. Thus, PCV2 exploits several cellular biological processes through IFN activation for enhancing viral replication. This is the first evidence of IFN-γ promoting PCV2 replication in vitro via a mechanism similar to that used by several human viruses.

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein.

Background and Aim: African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process. Materials and Methods: SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA). Results: The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 µg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001). Conclusion: This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well.

An amino acid substitution in HCV core antigen limits its use as a reliable measure of HCV infection compared with HCV RNA.

Hepatitis C virus (HCV) is a viral pathogen that causes chronic hepatitis, which can lead to cirrhosis and hepatocellular carcinoma. Detection of HCV RNA is the standard method used to diagnose the disease and monitor antiviral treatment. A quantification assay for the HCV core antigen (HCVcAg) has been proposed as a simplified alternative to the HCV RNA test for predicting active HCV infection, with the aim of achieving the global goal of eliminating hepatitis. The objective of this study was to determine the correlation between HCV RNA and HCVcAg, as well as the impact of amino acid sequence heterogeneity on HCVcAg quantification. Our findings demonstrated a strong positive correlation between HCV RNA and HCVcAg across all HCV genotypes (1a, 1b, 3a, and 6), with correlation coefficients ranging from 0.88 to 0.96 (p < 0.001). However, in some cases, samples with genotypes 3a and 6 exhibited lower HCVcAg levels than expected based on the corresponding HCV RNA values. Upon the core amino acid sequence alignment, it was observed that samples exhibiting low core antigen levels had an amino acid substitution at position 49, where threonine was replaced by either alanine or valine. Core mutation at this position may correlate with one of the epitope regions recognized by anti-HCV monoclonal antibodies. The present findings suggest that the utilization of HCVcAg as a standalone marker for HCV RNA might not provide adequate sensitivity for the detection of HCV infection, especially in cases where there are variations in the amino acid sequence of the core region and a low viral load of HCV RNA.

Qualitative hepatitis C virus RNA assay identifies active infection with sufficient viral load for treatment among Phetchabun residents in Thailand.

The World Health Organization envisions the elimination of viral hepatitis by 2030 through reducing prevalence and transmission, increasing diagnostic screening, and expanding treatment coverage. Efforts to micro-eliminate hepatitis in Phetchabun province in Thailand, a region where the prevalence of hepatitis C virus (HCV) infection and liver cancer is higher than elsewhere in the country, began with evaluating the province-wide burden of HCV. Here, we describe a feasibility study to assess active HCV infection by screening Phetchabun residents ages 35 to 69 years for anti-HCV antibodies by using a rapid diagnostic test (RDT) at the point of care. Positive anti-HCV results were further evaluated for active infection using qualitative HCV RNA assay, followed by quantitative HCV viral load determination in a subset of samples. Currently, we have identified 6.2% (10,621/170,163) anti-HCV positive individuals, of whom 74.9% (3,930/5,246) demonstrated detectable viral RNA. Quantitative test found that 97.5% (1,001/1,027) had HCV viral load ≥5,000 IU/mL. Thus, primary screening with anti-HCV RDT followed by qualitative HCV RNA evaluation could identify active and chronic HCV infection in almost all individuals with a viral load ≥5,000 IU/mL, which is the current threshold for treatment dictated by Thailand's National Health Security Office. Our data suggest that qualitative HCV RNA evaluation may obviate the need for the more expensive quantitative HCV viral load test and reduce a significant barrier toward HCV elimination in a middle-income country.

Encapsidated-CpG ODN enhances immunogenicity of porcine circovirus type 2 virus-like particles.

A DNA fragment containing CpG motifs (CpG ODN) is one of the potent immunopotentiators used to improve vaccine efficacy. It can enhance a protective immunity by stimulating both innate and adaptive immune responses. In this study, we designed and constructed a recombinant plasmid carrying the combined CpG ODN to generate an immunopotentiator for boosting the immunogenicity of porcine circovirus type 2 (PCV2) virus-like particles (VLPs). The capsid protein of PCV2b was expressed in insect cells and purified by affinity chromatography. The purified capsid protein was incubated with the CpG ODN in the reaction that allowed VLPs formation and encapsidation of the CpG ODN to occur simultaneously. Morphology of the reassembled VLPs was similar to the PCV2 virions as observed using an electron microscope. When the CpG ODN-encapcidated VLPs was treated with DNase I, the VLPs could protect the packaged CpG ODN from the enzyme digestion. Moreover, we immunized mice subcutaneously with VLPs, CpG ODN-loaded VLPs, or phosphate buffer saline for three times at two-week intervals. The results showed that the CpG ODN-loaded VLPs could elicit significantly higher levels of PCV2-specific neutralizing antibodies and interferon gamma (IFN-γ) expression in the immunized mice compared to those conferred by the VLPs alone. Conclusively, we have proved that the CpG ODN incorporated in VLPs can serve as a potent immunopotentiator for PCV2 vaccine development.

A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production.

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.

Data on production of mammalian stable cells expressing secretory BEFV transmembrane deleted G protein.

Generation of stable cell lines is a widely used technique for continuous recombinant protein production. Advantages of the constitutive stable over the transient protein expression are uniformity of the expression across cell populations as well as high quantity and consistency of the protein yields. This data describe step-by-step procedure for the production of glycoprotein without a transmembrane domain (GΔTM) of bovine ephemeral fever virus (BEFV) by mammalian stable cells. LentiX-293T cells were transfected with four plasmid constructs to generate a recombinant lentivirus. Subsequently, 293T cells were transduced by the recombinant virus and the polyclonal stable cell pools were then selected by puromycin. Next, limiting dilution was performed from each cell pool to isolate the monoclonal stable cells expressing GΔTM protein. Western blot analysis showed that all monoclonal cell clones could stably express GΔTM protein. The data confirms that the stable 293T cell line expressing the secretory GΔTM protein is an attractive platform for antigen production.

The immunogenicity of the secretory GΔTM protein of bovine ephemeral fever virus stably expressed by mammalian cells.

Bovine ephemeral fever virus (BEFV) causes an acute febrile disease in cattle and water buffalo. The disease has an impact on dairy and beef production in tropical and subtropical countries. Vaccination is used for disease prevention and control. In this study, we developed a recombinant lentivirus to produce mammalian stable cells expressing histidine-tagged BEFV G protein with a deleted transmembrane domain (GΔTM) as a secretory protein. In addition, guinea pigs were immunised with the purified GΔTM protein and booster immunised at a 3-week interval. The mammalian stable cells were able to continuously produce GΔTM protein for a minimum of 25 passages. All of the mammalian stable cells expressing GΔTM protein could react specifically with a BEFV convalescent bovine serum. Serum samples from the immunised guinea pigs could react strongly and specifically with the purified GΔTM protein. Moreover, post-immunised guinea pig sera contained antibodies that could neutralise BEFV. These results indicate that the G protein without a transmembrane domain can be used as a subunit vaccine for the prevention and control of BEFV. The availability of the mammalian stable cells, which constitutively express GΔTM protein, could facilitate the potential use of the secretory protein for BEFV diagnosis and vaccine development.

Genetic diversity of porcine circovirus type 2 (PCV2) in Thailand during 2009-2015.

Porcine circovirus type 2 (PCV2), the essential cause of porcine circovirus associated disease (PCVAD), has evolved rapidly and it has been reported worldwide. However, genetic information of PCV2 in Thailand has not been available since 2011. Herein, we studied occurrence and genetic diversity of PCV2 in Thailand and their relationships to the global PCV2 based on ORF2 sequences. The results showed that 306 samples (44.09%) from 56 farms (80%) were PCV2 positive by PCR. Phylogenetic trees constructed by both neighbor-joining and Bayesian Inference yielded similar topology of the ORF2 sequences. Thai PCV2 comprise four clusters: PCV2a (5.5%), PCV2b (29.41%), intermediate clade 1 (IM1) PCV2b (11.03%) and PCV2d (54.41%). Genetic shift of PCV2 in Thailand has occurred similarly to the global situation. The shift from PCV2b to PCV2d was clearly observed during 2013-2014. The viruses with genetically similar to the first reported PCV2 in 2004 have still circulated in Thailand. The first Thai PCV2b and PCV2d were closely related to the neighboring countries. The haplotype network analysis revealed the relationship of PCV2 in Thailand and other countries. These results indicate that genetic diversity of PCV2 in Thailand is caused by genetic drift of the local strains and intermittent introduction of new strains or genotypes from other countries. Genetic evolution of PCV2 in Thailand is similar to that occurs globally.