Whole-genome sequencing and genetic characteristics of representative porcine reproductive and respiratory syndrome virus (PRRSV) isolates in Korea.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a macrophage-tropic arterivirus with extremely high genetic and pathogenic heterogeneity that causes significant economic losses in the swine industry worldwide. PRRSV can be divided into two species [PRRSV1 (European) and PRRSV2 (North American)] and is usually diagnosed and genetically differentiated into several lineages based on the ORF5 gene, which constitutes only 5% of the whole genome. This study was conducted to achieve nonselective amplification and whole-genome sequencing (WGS) based on a simplified sequence-independent, single-primer amplification (SISPA) technique with next-generation sequencing (NGS), and to genetically characterize Korean PRRSV field isolates at the whole genome level. METHODS: The SISPA-NGS method coupled with a bioinformatics pipeline was utilized to retrieve full length PRRSV genomes of 19 representative Korean PRRSV strains by de novo assembly. Phylogenetic analysis, analysis of the insertion and deletion (INDEL) pattern of nonstructural protein 2 (NSP2), and recombination analysis were conducted. RESULTS: Nineteen complete PRRSV genomes were obtained with a high depth of coverage by the SISPA-NGS method. Korean PRRSV1 belonged to the Korean-specific subtype 1A and vaccine-related subtype 1C lineages, showing no evidence of recombination and divergent genetic heterogeneity with conserved NSP2 deletion patterns. Among Korean PRRSV2 isolates, modified live vaccine (MLV)-related lineage 5 viruses, lineage 1 viruses, and nation-specific Korean lineages (KOR A, B and C) could be identified. The NSP2 deletion pattern of the Korean lineages was consistent with that of the MN-184 strain (lineage 1), which indicates the common ancestor and independent evolution of Korean lineages. Multiple recombination signals were detected from Korean-lineage strains isolated in the 2010s, suggesting natural interlineage recombination between circulating KOR C and MLV strains. Interestingly, the Korean strain GGYC45 was identified as a recombinant KOR C and MLV strain harboring the KOR B ORF5 gene and might be the ancestor of currently circulating KOR B strains. Additionally, two novel lineage 1 recombinants of NADC30-like and NADC34-like viruses were detected. CONCLUSION: Genome-wide analysis of Korean PRRSV isolates retrieved by the SISPA-NGS method and de novo assembly, revealed complex evolution and recombination in the field. Therefore, continuous surveillance of PRRSV at the whole genome level should be conducted, and new vaccine strategies for more efficient control of the virus are needed.

Comparison of the pathogenicity of porcine reproductive and respiratory syndrome virus (PRRSV)-1 and PRRSV-2 in pregnant sows.

To date, few studies related to the evaluation of the pathogenicity of different PRRSV isolates using a reproductive model have been undertaken, and the main focus has remained on respiratory models using young pigs. This study aimed to evaluate the pathogenicity of two PRRSV-1 isolates (D40 and CBNU0495) and two PRRSV-2 isolates (K07-2273 and K08-1054) in a reproductive model. Pregnant sows were experimentally infected with PRRSV at gestational day 93 or used as an uninfected negative control. Sera were collected at 0, 3, 7, 14, and 19 days post-challenge (dpc) for virological and serological assays. At 19 dpc, all sows were euthanized, and their fetuses were recovered by performing cesarean section and immediately euthanized for sample collection. Here, compared to the other isolates, the CBNU0495 isolate replicated most efficiently in the pregnant sows, and K07-2273 produced the highest rate of reproductive failure even though it did not replicate as efficiently as the other isolates in sows and fetuses, indicating that vertical transmission and reproductive failure due to PRRSV infection do not have any significant correlation with the viral loads in samples from sows and fetuses. Similarly, the viral loads and the histopathological lesions did not show any correlation with each other, as the PRRSV-2-infected groups displayed more prominent and frequent histopathological lesions with lower viral loads than the PRRSV-1-infected groups. However, viral loads in the myometrium/endometrium might be related to the spreading of PRRSV in the fetuses, which affected the birth weight of live fetuses. This study contributes to a better understanding of the pathogenicity of the most prevalent Korean PRRSVs in a reproductive model.

Prevalence of porcine parvovirus 1 through 7 (PPV1-PPV7) and co-factor association with PCV2 and PRRSV in Korea.

BACKGROUND: Classical porcine parvovirus (PPV1) and novel porcine parvoviruses designated porcine parvovirus 2 through 7 (PPV2-PPV7) are widespread in pig populations. The objective of this study was to investigate the prevalence rates of PPV1-PPV7 in Korea by detecting PPVs in serum, lung and fecal samples and to elucidate the association of PPVs with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory virus (PRRSV), major pathogens involved in porcine respiratory disease complex (PRDC). A total of 286 serum, 481 lung, and 281 fecal samples collected from 2018 to 2020 were analyzed. RESULTS: The results showed that PPVs are widespread in Korea; the highest detection rates were found in lung samples and ranged from 7.9% (PPV1) to 32.6% (PPV2). Regarding age groups, fattening pigs had the highest detection rates of PPVs, ranging from 6.4% (PPV1) to 36.5% (PPV6); this finding suggests the chronic nature of PPV infections and the continual circulation of these viruses. When compared with PCV2- and PRRSV-negative lung samples, PCV2-positive samples with or without PRRSV positivity had significantly higher detection levels of PPV1 and PPV6. In contrast, the prevalence of PPV2 and PPV7 was significantly higher in PRRSV-infected lung samples regardless of PCV2 detection. PPV5 was detected significantly more frequently in samples  with both PCV2 and PRRSV positivity. CONCLUSIONS: This study could offer a better understanding of the role of PPVs in PCV2 and/or PRRSV infection though further studies are needed to experimentally assess the impact of PPVs in coinfections.

Temporal lineage dynamics of the ORF5 gene of porcine reproductive and respiratory syndrome virus in Korea in 2014-2019.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the Korean swine industry. Despite efforts including improved biosecurity and vaccination protocols, the virus continues to circulate and evolve. Based on phylogenetic analysis of open reading frame 5 (ORF5), Korean PRRSVs are known to form not only globally circulating lineages but also country-specific lineages (Lin Kor A, B, and C). To understand the recent epidemiological status of PRRSV in Korea, a total of 1349 ORF5 sequences of Korean PRRSV isolates from 2014 to 2019 were analyzed. Phylogenetic analysis was conducted using the maximum-likelihood method, and temporal changes in the relative prevalence of lineages were investigated. The analysis showed that PRRSV1 and PRRSV2 were both highly prevalent throughout the years examined. Among the PRRSV1 isolates, subgroup A (90.1%) and vaccine-like subgroup C (9.0%) composed most of the population. For PRRSV2 isolates, vaccine-like lineage 5 (36.3%) was dominant, followed by Lin Kor B (25.9%), Kor C (16.6%), lineage 1 (11.6%), and Kor A (9.1%). The PRRSV2 lineage 1 population increased from 2014 (1.8%) to 2019 (29.6%) in Korea due to the continual spread of sublineage 1.8 (NADC30-like) and introduction of sublineage 1.6 into the country. Additional genetic analysis, including analysis of non synonymous and synonymous mutations, revealed evidence of diversification and positive selection in immunologically important regions of the genome, suggesting that current vaccination is failing and promoting immune-mediated selection. Overall, these findings provide insights into the epidemiological and evolutionary dynamics of cocirculating viral lineages, and constant surveillance of PRRSV occurrence is needed.

Integrated time-serial transcriptome networks reveal common innate and tissue-specific adaptive immune responses to PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is the most important viral disease causing severe economic losses in the swine industry. However, mechanisms underlying gene expression control in immunity-responsible tissues at different time points during PRRSV infection are poorly understood. We constructed an integrated gene co-expression network and identified tissue- and time-dependent biological mechanisms of PRRSV infection through bioinformatics analysis using three tissues (lungs, bronchial lymph nodes [BLNs], and tonsils) via RNA-Seq. Three groups with specific expression patterns (i.e., the 3-dpi, lung, and BLN groups) were discovered. The 3 dpi-specific group showed antiviral and innate-immune signalling similar to the case for influenza A infection. Moreover, we observed adaptive immune responses in the lung-specific group based on various cytokines, while the BLN-specific group showed down-regulated AMPK signalling related to viral replication. Our study may provide comprehensive insights into PRRSV infection, as well as useful information for vaccine development.

Evaluation of local and systemic immune responses in pigs experimentally challenged with porcine reproductive and respiratory syndrome virus.

The host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) from infected pigs is currently poorly understood. To better understand the dynamics of host-pathogen interactions, seventy-five of 100 pigs infected with PRRSV-JA142 and 25 control pigs were euthanized at 3, 10, 21, 28 and 35 days post-challenge (dpc). Blood, lung, bronchoalveolar lavage (BAL) and bronchial lymph node (BLN) samples were collected to evaluate the cellular immune responses. The humoral responses were evaluated by measuring the levels of anti-PRRSV IgG and serum virus-neutralizing (SVN) antibodies. Consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between 3 and 10 dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by 28 dpc. At peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived DC/macrophage and conventional DC frequencies were increased, and these effects coincided with the early induction of local T-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, BAL, and BLN as early as 10 dpc. Conversely, the systemic T-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between 3 and 10 dpc. Taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining PRRSV through the early induction of T-cell responses at the sites of virus replication.

Effect of polymorphisms in porcine guanylate-binding proteins on host resistance to PRRSV infection in experimentally challenged pigs.

Guanylate-binding proteins (GBP1 and GBP5) are known to be important for host resistance against porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, the effects of polymorphisms in GBP1 (GBP1E2 and WUR) and GBP5 on host immune responses against PRRSV were investigated to elucidate the mechanisms governing increased resistance to this disease. Seventy-one pigs [pre-genotyped based on three SNP markers (GBP1E2, WUR, and GBP5)] were assigned to homozygous (n = 36) and heterozygous (n = 35) groups and challenged with the JA142 PRRSV strain. Another group of nineteen pigs was kept separately as a negative control group. Serum and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 7, 14, 21 and 28 days post-challenge (dpc). Viremia and weight gain were measured in all pigs at each time point, and a flow cytometry analysis of PBMCs was performed to evaluate T cell activation. In addition, 15 pigs (5 pigs per homozygous, heterozygous and negative groups) were sacrificed at 3, 14 and 28 dpc, and the local T cell responses were evaluated in the lungs, bronchoalveolar lavage cells (BALc), lymph nodes and tonsils. The heterozygous pigs showed lower viral loads in the serum and lungs and higher weight gains than the homozygous pigs based on the area under the curve calculation. Consistently, compared with the homozygous pigs, the heterozygous pigs exhibited significantly higher levels of IFN-α in the serum, proliferation of various T cells (γδT, Th1, and Th17) in PBMCs and tissues, and cytotoxic T cells in the lungs and BALc. These results indicate that the higher resistance in the pigs heterozygous for the GBP1E2, WUR and GBP5 markers could be mediated by increased antiviral cytokine (IFN-α) production and T cell activation.

Characterization of Clostridium novyi isolated from a sow in a sudden death case in Korea.

BACKGROUND: Multifocal spherical nonstaining cavities and gram-positive, rod-shaped, and endospore-forming bacteria were found in the liver of a sow that died suddenly. Clostridium novyi type B was identified and isolated from the sudden death case, and the isolate was characterized by molecular analyses and bioassays in the current study. RESULTS: C. novyi was isolated from the liver of a sow that died suddenly and was confirmed as C. novyi type B by differential PCR. The C. novyi isolate fermented glucose and maltose and demonstrated lecithinase activity, and the cell-free culture supernatant of the C. novyi isolate exhibited cytotoxicity toward Vero cells, demonstrating that the isolate produces toxins. In addition, whole-genome sequencing of the C. novyi isolate was performed, and the complete sequences of the chromosome (2.29 Mbp) and two plasmids (134 and 68 kbp) were identified for the first time. Based on genome annotation, 7 genes were identified as glycosyltransferases, which are known as alpha toxins; 23 genes were found to be related to sporulation; 12 genes were found to be related to germination; and 20 genes were found to be related to chemotaxis. CONCLUSION: C. novyi type B was isolated from a sow in a sudden death case and confirmed by biochemical and molecular characterization. Various virulence-associated genes were identified for the first time based on whole-genome sequencing.

Combined Enrichment and Quantitative Polymerase Chain Reaction to Improve Sensitivity and Reduce Time of Detection of Listeria monocytogenes in Mushrooms.

This study evaluated a combined method for the detection of Listeria monocytogenes in mushrooms, involving enrichment and quantitative real-time polymerase chain reaction (qPCR), to improve sensitivity and reduce detection time. The growth of L. monocytogenes was evaluated in Listeria enrichment broth (LEB) with modified carbon and nitrogen sources, increasing sodium concentrations, and added micronutrients. Primers targeting the L. monocytogenes iap (iap1 and iap2), hlyA (hlyA1-hlyA6), and prfA (prfA1-prfA4) genes were developed and their sensitivity and specificity were evaluated. The greatest increase in L. monocytogenes cell count was observed after 6-h incubation at 30°C in LEB+2 × FAC (LEB plus 20 mL/L ferric ammonium citrate), where cell count increased by 1.4 log CFU (colony-forming unit)/mL, compared with 0.9 log CFU/mL in LEB (p < 0.05). iap2 primers targeting the iap gene showed high specificity and were the most sensitive among those tested, with a detection limit of 2 log CFU/mL in LEB medium, 3.1 log CFU/g in golden needle mushroom, and 3.5 log CFU/g in large oyster mushroom. When applied to detection in golden needle mushrooms, a combination of 3-h incubation in LEB+2 × FAC medium and qPCR analysis with iap2 primers permitted detection of L. monocytogenes, even at an inoculum of 1 log CFU/g. Similarly, in large oyster mushrooms, 10-h enrichment in LEB+2 × FAC medium resulted in a cell count of 3.7 log CFU/g. These results indicate that a combined detection method, using LEB+2 × FAC medium for enrichment followed by qPCR with iap2 primer pair, can reduce enrichment time and improve the sensitivity and specificity of L. monocytogenes detection in mushrooms.

Characterisation of Pasteurella multocida isolates from pigs with pneumonia in Korea.

BACKGROUND: Pasteurella multocida is responsible for significant economic losses in pigs worldwide. In clinically diseased pigs, most P. multocida isolates are characterised as subspecies multocida, biovar 2 or 3 and capsular type A or D; however, there is little information regarding subspecies, biovars, and other capsular types of P. multocida isolates in Korea. Here, we provided information covering an extended time period regarding P. multocida in pigs with pneumonia in Korea using phenotypic and genotypic characterisations and data associated with the minimum inhibitory concentrations. RESULTS: The overall prevalence of P. multocida between 2008 and 2016 was 16.8% (240/1430), with 85% of the P. multocida isolates (204/240) coinfected with other respiratory pathogens. Of the 240 isolates, 166 were included in this study; all of these P. multocida isolates were characterised as subspecies multocida and the most prevalent phenotypes were represented by biovar 3 (68.7%; n = 114) and capsular type A (69.9%; n = 116). Additionally, three capsular type F isolates were identified, with this representing the first report of such isolates in Korea. All biovar 1 and 2 isolates were capsular types F and A, respectively. The virulence-associated gene distribution was variable; all capsular type A and D isolates harboured pmHAS and hsf-1, respectively (P < 0.001), with type F (biovar 1) significantly correlated with hsf-1 (P < 0.05) and pfhA (P < 0.01), biovar 2 highly associated with pfhA and pmHAS, and biovar 3 significantly correlated with hsf-1, pmHAS, and hgbB (P < 0.001), whereas biovar 13 was related only to hgbB (P < 0.05). The highest resistance rate was found to be to oxytetracycline (63.3%), followed by florfenicol (16.3%). CONCLUSIONS: P. multocida subspecies multocida, biovar 3, and capsular type A was the most prevalent isolate in this study, and our findings indicated the emergence of capsular type F in Korea. Moreover, prudent use of oxytetracycline and florfenicol is required because of the identified high resistance rates. Further studies are required for continuous monitoring of the antimicrobial resistance, prevalence, and epidemiological characterisation of P. multocida, and experimental infection models are needed to define the pathogenicity of capsular type F.

Evaluation of the Inhibitory Effects of (E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one (DiNap), a Natural Product Analog, on the Replication of Type 2 PRRSV In Vitro and In Vivo.

DiNap [(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one], an analog of a natural product (the chalcone flavokawain), was synthesized and characterized in this study. Porcine reproductive and respiratory syndrome virus (PRRSV) is the most challenging threat to the swine industry worldwide. Currently, commercially available vaccines are ineffective for controlling porcine reproductive and respiratory syndrome (PRRS) in pigs. Therefore, a pharmacological intervention may represent an alternative control measure for PRRSV infection. Hence, the present study evaluated the effects of DiNap on the replication of VR2332 (a prototype strain of type 2 PRRSV). Initially, in vitro antiviral assays against VR2332 were performed in MARC-145 cells and porcine alveolar macrophages (PAMs). Following this, a pilot study was conducted in a pig model to demonstrate the effects of DiNap following VR2332 infection. DiNap inhibited VR2332 replication in both cell lines in a dose-dependent manner, and viral growth was completely suppressed at concentrations ≥0.06 mM, without significant cytotoxicity. Consistent with these findings, in the pig study, DiNap also reduced viral loads in the serum and lungs and enhanced the weight gain of pigs following VR2332 infection, as indicated by comparison of the DiNap-treated groups to the untreated control (NC) group. In addition, DiNap-treated pigs had fewer gross and microscopic lesions in their lungs than NC pigs. Notably, virus transmission was also delayed by approximately 1 week in uninfected contact pigs within the same group after treatment with DiNap. Taken together, these results suggest that DiNap has potential anti-PRRSV activity and could be useful as a prophylactic or post-exposure treatment drug to control PRRSV infection in pigs.

Plant synthetic GP4 and GP5 proteins from porcine reproductive and respiratory syndrome virus elicit immune responses in pigs.

MAIN CONCLUSION: We demonstrated successful overexpression of porcine reproductive and respiratory syndrome virus (PRRSV)-derived GP4D and GP5D antigenic proteins in Arabidopsis. Pigs immunized with transgenic plants expressing GP4D and GP5D proteins generated both humoral and cellular immune responses to PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, the most economically significant disease affecting the swine industry worldwide. However, current commercial PRRSV vaccines (killed virus or modified live vaccines) show poor efficacy and safety due to concerns such as reversion of virus to wild type and lack of cross protection. To overcome these problems, plants are considered a promising alternative to conventional platforms and as a vehicle for large-scale production of recombinant proteins. Here, we demonstrate successful production of recombinant protein vaccine by expressing codon-optimized and transmembrane-deleted recombinant glycoproteins (GP4D and GP5D) from PRRSV in planta. We generated transgenic Arabidopsis plants expressing GP4D and GP5D proteins as candidate antigens. To examine immunogenicity, pigs were fed transgenic Arabidopsis leaves expressing the GP4D and GP5D antigens (three times at 2-week intervals) and then challenged with PRRSV at 6-week post-initial treatment. Immunized pigs showed significantly lower lung lesion scores and reduced viremia and viral loads in the lung than pigs fed Arabidopsis leaves expressing mYFP (control). Immunized pigs also had higher titers of PRRSV-specific antibodies and significantly higher levels of pro-inflammatory cytokines (TNF-α and IL-12). Furthermore, the numbers of IFN-γ+-producing cells were higher, and those of regulatory T cells were lower, in GP4D and GP5D immunized pigs than in control pigs. Thus, plant-derived GP4D and GP5D proteins provide an alternative platform for producing an effective subunit vaccine against PRRSV.

Salt-Induced, Continuous Deposition of Supramolecular Iron(III)-Tannic Acid Complex.

One-step assembly of iron(III)-tannic acid (Fe3+-TA) complex forms nanothin (∼10 nm) films on various substrates within minutes. In this deposition scheme, however, the film does not grow continuously over time even though Fe3+-TA complex is still abundant in the coating solution. In this paper, we report that the salt addition dramatically changes the one-off coating characteristic to continuous one, and each salt has its optimum concentration ( CMFT) that produces maximum film thickness. For detailed investigation of the salt effects, we employed various salts, including LiCl, NaCl, KCl, CaCl2, SrCl2, BaCl2, NaBr, and NaNO3, and found that only cations played an important role in the continuous deposition of the Fe3+-TA complex, with smaller CMFT values for the cations of higher valency and larger size. On the basis of the results, we suggested that the positively charged cations screened the negative surface charges of Fe3+-TA complex particles, leading to coagulation and continuous deposition, further supported by the ζ-potential measurement and time-resolved dynamic light-scattering analysis.

Analysis of peptide-SLA binding by establishing immortalized porcine alveolar macrophage cells with different SLA class II haplotypes.

Primary porcine alveolar macrophages (PAM) are useful for studying viral infections and immune response in pigs; however, long-term use of these cells is limited by the cells' short lifespan. We immortalized primary PAMs by transfecting them with both hTERT and SV40LT and established two immortalized cell lines (iPAMs) actively proliferating even after 35 passages. These cells possessed the characteristics of primary PAMs, including strong expression of swine leukocyte antigen (SLA) class II genes and the inability to grow anchorage-independently. We characterized their SLA genes and subsequently performed peptide-SLA binding assays using a peptide from porcine circovirus type 2 open reading frame 2 to experimentally measure the binding affinity of the peptide to SLA class II. The number of peptides bound to cells measured by fluorescence was very low for PK15 cells (7.0% ± 1.5), which are not antigen-presenting cells, unlike iPAM61 (33.7% ± 3.4; SLA-DQA*0201/0303, DQB1*0201/0901, DRB1*0201/1301) and iPAM303 (73.3% ± 5.4; SLA DQA*0106/0201, DQB1*0202/0701, DRB1*0402/0602). The difference in peptide binding between the two iPAMs was likely due to the allelic differences between the SLA class II molecules that were expressed. The development of an immortal PAM cell panel harboring diverse SLA haplotypes and the use of an established method in this study can become a valuable tool for evaluating the interaction between antigenic peptides and SLA molecules and is important for many applications in veterinary medicine including vaccine development.

The prevalence and genetic characteristics of porcine circovirus type 2 and 3 in Korea.

BACKGROUND: Porcine circovirus-associated diseases (PCVAD), caused by porcine circovirus type 2 (PCV2), threaten the pig industry worldwide. Five genotypes of PCV2 were recently identified: PCV2a, PCV2b, PCV2c, PCV2d and PCV2e. In addition, a novel porcine circovirus from a case of a sow with dermatitis, nephropathy syndrome and reproductive failure has been identified based on metagenomic analysis and classified as porcine circovirus type 3 (PCV3). Therefore, the current study was conducted to determine the prevalence and genetic characteristics of PCV2 and PCV3 in clinical samples. RESULTS: A total of 471 samples (161 tissue samples of lungs and lymph nodes from 34 farms and 310 serum samples from 47 farms) were tested for PCV2. Among them, 171 samples from 59 farms that had been positive for PCV2 were genotyped. Another 690 samples (296 tissue samples of lungs and lymph nodes from 91 farms, 108 samples of aborted foetuses from 26 farms, and 286 serum samples from 47 farms) were tested for PCV3. Based on PCV2 genotyping results, PCV2d was the most prevalent genotype (107 of 171 samples), and co-infections with combinations of PCV2a, 2b and 2d were identified in 48 samples from 17 farms. A total of 14 samples from 11 farms were also positive for both PCV2 and PCV3. For PCV3, 57 samples (9.8%) from 32 farms (23.2%) were positive. Among the 108 aborted foetuses from 26 farms, only 2 samples were positive for PCV3. Based on sequence comparisons, PCV2d shares 89.6-91.0% and 93.2-94.3% homology with PCV2a and PCV2b, respectively; 98.6-100% homology is shared among PCV2d strains. The PCV3 strains identified in this study share 98.0-99.5% homology. CONCLUSIONS: Our study concludes that PCV2d has become the most predominant genotype in Korea. PCV3 was also identified in clinical samples, though no significant association with clinical symptoms was observed in PCV3-positive cases.

In vitro immune responses of porcine alveolar macrophages reflect host immune responses against porcine reproductive and respiratory syndrome viruses.

BACKGROUND: Currently, an in vitro immunogenicity screening system for the immunological assessment of potential porcine reproductive and respiratory syndrome virus (PRRSV) vaccine candidates is highly desired. Thus, in the present study, two genetically divergent PRRSVs were characterized in vitro and in vivo to identify an in vitro system and immunological markers that predict the host immune response. Porcine alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs) collected from PRRSV-negative pigs were used for in vitro immunological evaluation, and the response of these cells to VR2332c or JA142c were compared with those elicited in pigs challenged with the same viruses. RESULTS: Compared with VR2332c or mock infection, JA142c induced increased levels of type I interferons and pro-inflammatory cytokines (TNF-α, IL-1α/ß, IL-6, IL-8, and IL-12) in PAMs, and these elevated levels were comparable to the cytokine induction observed in PRRSV-challenged pigs. Furthermore, significantly greater numbers of activated CD4+ T cells, type I helper T cells, cytotoxic T cells and total IFN-γ+ cells were observed in JA142c-challenged pigs than in VR2332c- or mock-challenged pigs. CONCLUSIONS: Based on these results, the innate immune response patterns (particularly IFN-α, TNF-α and IL-12) to specific PRRSV strains in PAMs might reflect those elicited by the same viruses in pigs.

Geographic distribution and molecular analysis of porcine reproductive and respiratory syndrome viruses circulating in swine farms in the Republic of Korea between 2013 and 2016.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes devastating disease characterized by reproductive failure and respiratory problems in the swine industry. To understand the recent prevalence and genetic diversity of field PRRSVs in the Republic of Korea, open reading frames (ORFs) 5 and 7 of PRRSV field isolates from 631 PRRS-affected swine farms nationwide in 2013-2016 were analyzed along with 200 Korean field viruses isolated in 2003-2010, and 113 foreign field and vaccine strains. RESULTS: Korean swine farms were widely infected with PRRSVs of a single type (38.4 and 37.4% for Type 1 and Type 2 PRRSV, respectively) or both types (24.2%) with up to approximately 83% nucleotide sequence similarity to prototype PRRSVs (Lelystad or VR2332). Phylogenetic analysis based on the ORF5 nucleotide sequence revealed that Korean Type 1 field isolates were classified as subgroups A, B, and C under subtype 1, while Korean Type 2 field isolates were classified as lineages 1 and 5 as well as three Korean lineages (kor A, B, and C) with the highest infection prevalence in subgroup A (50.5%) and lineage 5 (15.3%) for Type 1 and Type 2 PRRSV, respectively, among ORF5-positive farms. In particular, the lineages kor B and C were identified as novel lineages in this study, and lineage kor B comprised only the field viruses isolated from Gyeongnam Province in 2014-2015, establishing regionally unique genetic characteristics. It has also recently been confirmed that commercialized vaccine-like viruses (subgroup C) of Type 1 PRRSV and NADC30-like viruses of Type 2 PRRSV (lineage 1) are spreading rapidly in Korean swine farms. The Korean field viruses were also expected to be antigenically variable as shown in the high diversity of neutralizing epitopes and N-glycosylation sites. CONCLUSIONS: This up-to-date information regarding recent field PRRSVs should be taken into consideration when creating strategies for the application of PRRS control measures, including vaccination in the field.

The Attenuation Phenotype of a Ribavirin-Resistant Porcine Reproductive and Respiratory Syndrome Virus Is Maintained during Sequential Passages in Pigs.

UNLABELLED: In a previous study, ribavirin-resistant porcine reproductive and respiratory syndrome virus (PRRSV) mutants (RVRp13 and RVRp22) were selected, and their resistance against random mutation was shown in cultured cells. In the present study, these ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability during three pig-to-pig passages in comparison with modified live virus (MLV) (Ingelvac PRRS MLV). Pigs challenged with RVRp22 had significantly lower (P< 0.05) viral loads in sera and tissues than pigs challenged with MLV or RVRp13 at the first passage, and the attenuated replication of RVRp22 was maintained until the third passage. Viral loads in sera and tissues dramatically increased in pigs challenged with MLV or RVRp13 during the second passage. Consistently, all five sequences associated with the attenuation of virulent PRRSV in RVRp13 and MLV quickly reverted to wild-type sequences during the passages, but two attenuation sequences were maintained in RVRp22 even after the third passage. In addition, RVRp22 showed a significantly lower (P< 0.001) mutation frequency in nsp2, which is one of the most variable regions in the PRRSV genome, than MLV. Nine unique mutations were found in open reading frames (ORFs) 1a, 2, and 6 in the RVRp22 genome based on full-length sequence comparisons with RVRp13, VR2332 (the parental virus of RVRp13 and RVRp22), and MLV. Based on these results, it was concluded that RVRp22 showed attenuated replication in pigs; further, because of the high genetic stability of RVRp22, its attenuated phenotype was stable even after three sequential passages in pigs. IMPORTANCE: PRRSV is a rapidly evolving RNA virus. MLV vaccines are widely used to control PRRS; however, there have been serious concerns regarding the use of MLV as a vaccine virus due to the rapid reversion to virulence during replication in pigs. As previously reported, ribavirin is an effective antiviral drug against many RNA viruses. Ribavirin-resistant mutants reemerged by escaping lethal mutagenesis when the treatment concentration was sublethal, and those mutants were genetically more stable than parental viruses. In a previous study, two ribavirin-resistant PRRSV mutants (RVRp13 and RVRp22) were selected, and their higher genetic stability was shown in vitro Consequently, in the present study, both of the ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability in vivo RVRp22 was found to exhibit higher genetic and phenotypic stability than MLV, and nine unique mutations were identified in the RVRp22 genome based on a full-length sequence comparison with the RVRp13, VR2332, and MLV genomes.

Influence of Road Proximity on the Concentrations of Heavy Metals in Korean Urban Agricultural Soils and Crops.

The urban agricultural (UA) environment near active roadways can be degraded by traffic-related particles (i.e., exhaust gases and road dust), which may contain heavy metals. The current study investigated changes in heavy-metal [cadmium (Cd), copper (Cu), chromium (Cr) nickel (Ni), lead (Pb) and zinc (Zn)] concentrations in soils located near highly trafficked roads in Korea and the subsequent uptake of these metals by Chinese cabbage. Heavy-metal plant concentrations were determined in both washed and unwashed plant leaves to determine whether foliar deposition played any role in plant metal uptake. Soil concentrations of Cd, Cu, Pb, and Zn were all lower than the Korean standard soil limits and showed no significant influence from road traffic. In contrast, both Ni and Cr concentrations in soils collected within 10 m of the road were 4 and 5 times greater, respectively, than those in soils collected 70 m from the road. Heavy-metal concentrations in unwashed Chinese cabbage leaf collected at 5 m from the road were consistently greater than those of washed leaf samples, thus indicating the deposition of traffic-related particles on the plant surface. With the exception of Cu, all heavy-metal concentration in washed plant samples collected at 5 m also showed greater accumulation compared with samples collected further away. This was mainly attributed to increased total soil heavy-metal concentrations and increased metal phytoavailability induced by decreases in soil pH near the road. However, overall heavy-metal soil concentrations were well lower than the allowable concentrations, and the levels observed in plants collected in this study were considered not to currently pose a significant risk to human health. However, some traffic-related heavy metals, in particular Cr and Ni, were being accumulated in the roadside UA environment, which may warrant some caution regarding the environment and/or health issues in the future.

Impact of wastewater derived dissolved organic carbon on reduction, mobility, and bioavailability of As(V) and Cr(VI) in contaminated soils.

In this work, the effects of various wastewater sources (storm water, sewage effluent, piggery effluent, and dairy effluent) on the reduction, and subsequent mobility and bioavailability of arsenate [As(V)] and chromate [Cr(VI)] were compared using both spiked and field contaminated soils. Wastewater addition to soil can increase the supply of carbon, nutrients, and stimulation of microorganisms which are considered to be important factors enhancing the reduction of metal(loid)s including As and Cr. The wastewater-induced mobility and bioavailability of As(V) and Cr(VI) were examined using leaching, earthworm, and soil microbial activity tests. The rate of reduction of As(V) was much less than that of Cr(VI) both in the presence and absence of wastewater addition. Wastewater addition increased the reduction of both As(V) and Cr(VI) compared to the control (Milli-Q water) and the effect was more pronounced in the case of Cr(VI). The leaching experiment indicated that Cr(VI) was more mobile than As(V). Wastewater addition increased the mobility and bioavailability of As(V), but had an opposite effect on Cr(VI). The difference in the mobility and bioavailability of Cr(VI) and As(V) between wastewater sources can be attributed to the difference in their dissolved organic carbon (DOC) content. The DOC provides carbon as an electron donor for the reduction of As(V) and Cr(VI) and also serves as a complexing agent thereby impacting their mobility and bioavailability. The DOC-induced reduction increased both the mobility and bioavailability of As, but it caused an opposite effect in the case of Cr.