Porcine Reproductive And Respiratory Syndrome Virus Isolates Research Articles

Evolution Characterization and Pathogenicity of an NADC34-like PRRSV Isolated from Inner Mongolia, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen that causes severe abortions in sows and high piglet mortality, resulting in huge economic losses to the pig industry worldwide. The emerging and novel PRRSV isolates are clinically and biologically important, as there are likely recombination and pathogenic differences among PRRSV genomes. Furthermore, the NADC34-like strain has become a major epidemic strain in some parts of China, but the characterization and pathogenicity of the latest strain in Inner Mongolia have not been reported in detail. In this study, an NADC34-like strain (CHNMGKL1-2304) from Tongliao City, Inner Mongolia was successfully isolated and characterized, and confirmed the pathogenicity in pigs. The phylogenetic tree showed that this strain belonged to sublineage 1.5 and had high homology with the strain JS2021NADC34. There is no recombination between CHNMGKL1-2304 and any other domestic strains. Animal experiments show that the CHNMGKL1-2304 strain is moderately virulent to piglets, which show persistent fever, weight loss and high morbidity but no mortality. The presence of PRRSV nucleic acids was detected in both blood, tissues, nasal and fecal swabs. In addition, obvious pathological changes and positive signals were observed in lung, lymph node, liver and spleen tissues when subjected to hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). This report can provide a basis for epidemiological investigations and subsequent studies of PRRSV.

Computational antigenic insights into the novel NADC-34-like Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) isolate YC-2020

In this computational study, we advanced the understanding of the antigenic properties of the NADC-34-like isolate of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), named YC-2020, relevant in veterinary pathology. We utilized sequence comparison analyses of the M and N proteins, comparing them with those of NADC34, identifying substantial amino acid homology that allowed us to highlight conserved epitopes and crucial variants. Through the application of Clustal Omega for multiple sequence alignment and platforms like Vaxijen and AllerTOP for predicting antigenic and allergenic potential, our analyses revealed important insights into the conservation and variation of epitopes essential for the development of effective diagnostic tools and vaccines. Our findings, aligned with initial experimental studies, underscore the importance of these epitopes in the development of targeted immunodiagnostic platforms and significantly contribute to the management and control of PRRSV. However, further studies are required to validate the computational predictions of antigenicity for this new viral isolate. This approach underscores the potential of computational models to enable ongoing monitoring and control of PRRSV evolution in swine. While this study provides valuable insights into the antigenic properties of the novel PRRSV isolate YC-2020 through computational analysis, it is important to acknowledge the limitations inherent to in silico predictions, specifically, the absence of laboratory validation.

Isolation and pathogenicity comparison of two novel natural recombinant porcine reproductive and respiratory syndrome viruses with different recombination patterns in Southwest China.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the swine industry. Frequent mutations and recombinations account for PRRSV immune evasion and the emergence of novel strains. In this study, we isolated and characterized two novel PRRSV-2 strains from Southwest China exhibiting distinct recombination patterns. They were designated SCABTC-202305 and SCABTC-202309. Phylogenetic results indicated that SCABTC-202305 was classified as lineage 8, and SCABTC-202309 was classified as lineage 1.8. Amino acid mutation analysis identified unique amino acid substitutions and deletions in ORF5 and Nsp2 genes. The results of the recombination analysis revealed that SCABTC-202305 is a recombinant with JXA1 as the major parental strain and NADC30 as the minor parental strain. At the same time, SCABTC-202309 is identified as a recombinant with NADC30 as the major parental strain and JXA1 as the minor parental strain. In this study, we infected piglets with SCABTC-202305, SCABTC-202309, or mock inoculum (control) to study the pathogenicity of these isolates. Although both isolated strains were pathogenic, SCABTC-202305-infected piglets exhibited more severe clinical signs and higher mortality, viral load, and antibody response than SCABTC-202309-infected piglets. SCABTC-202305 also caused more extensive lung lesions based on histopathology. Our findings suggest that the divergent pathogenicity observed between the two novel PRRSV isolates may be attributed to variations in the genetic information encoded by specific genomic regions. Elucidating the genetic determinants governing PRRSV virulence and transmissibility will inform efforts to control this devastating swine pathogen.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the most critical pathogens impacting the global swine industry. Frequent mutations and recombinations have made the control of PRRSV increasingly difficult. Following the NADC30-like PRRSV pandemic, recombination events involving PRRSV strains have further increased. We isolated two novel field PRRSV recombinant strains, SCABTC-202305 and SCABTC-202309, exhibiting different recombination patterns and compared their pathogenicity in animal experiments. The isolates caused higher viral loads, persistent fever, marked weight loss, moderate respiratory clinical signs, and severe histopathologic lung lesions in piglets. Elucidating correlations between recombinant regions and pathogenicity in these isolates can inform epidemiologic tracking of emerging strains and investigations into viral adaptive mechanisms underlying PRRSV immunity evasion. Our findings underscore the importance of continued genomic surveillance to curb this economically damaging pathogen.

Assessment of temperature and time on the survivability of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) on experimentally contaminated surfaces.

Fomites might be responsible for virus introduction in swine farms, highlighting the importance of implementing practices to minimize the probability of virus introduction. The study's objective was to assess the efficacy of different combinations of temperatures and holding-times on detecting live PRRSV and PEDV on surfaces commonly found in supply entry rooms in swine farms. Two PRRSV isolates (MN 184 and 1-4-4 L1C variant) and one PEDV isolate (NC 49469/2013) were inoculated on cardboard and aluminum. An experimental study tested combinations of four temperatures (20°C, 30°C, 40°C, and 50°C) and six holding-times (15 minutes, 60 minutes, 6 hours, 12 hours, 24 hours, and 36 hours) for the presence of the viruses on each surface type. After virus titration, virus presence was assessed by assessing the cytopathic effects and immunofluorescence staining. The titers were expressed as log10 TCID50/ml, and regression models; half-lives equations were calculated to assess differences between treatments and time to not detect the live virus. The results suggest that the minimum time that surfaces should be held to not detect the virus at 30°C was 24 hours, 40°C required 12 hours, and 50°C required 6 hours; aluminum surfaces took longer to reach the desired temperature compared to cardboard. The results suggest that PRRSV 1-4-4 L1C variant had higher half-lives at higher temperatures than PRRSV MN 184. In conclusion, time and temperature combinations effectively decrease the concentration of PRRSV and PEDV on different surfaces found in supply entry rooms in swine farms.

Introduction of a PRRSV-1 strain of increased virulence in a pig production structure in Spain: virus evolution and impact on production

BackgroundA strain of Porcine reproductive and respiratory syndrome virus (PRRSV), showing characteristics of enhanced virulence, affected a pyramidal production system from Spain with 7600 sows in 4 genetic nuclei and 13,000 sows in multipliers. Different PRRSV strains circulating in this production system from December 2020 to October 2021 were detected and sequenced. The spread of each isolate was examined and their impact on health and production in three of the affected farms was evaluated.ResultsThe newly emerged PRRSV isolate with enhanced virulence entered the system before the onset of the study (January 2020) and afterwards four significantly different clades were detected during the study period in different farms, probably because of independent introduction events. The diversification of the enhanced virulence strain was higher for those clades (substitution rates up to 1.1% nucleotides/year) compared to other PRRSV strains present in the production system (up to 0.17%), suggesting a faster spread and adaptation. The impact of the infection in the first affected farm was dramatic, with an average abortion rate above 27% during 17 weeks before returning to the baseline production. Fertile sow mortality reached 6.5% for 39 weeks. In two farms infected later by other clades of this enhanced virulence strain, the impact was less acute; despite the fact that for parameters such as the proportion of stillbirths or mummies, more than 10 months were needed to recover pre-outbreak values. In the examined nurseries, mortalities reached peaks between 28 and 50% and several months were needed to return to normality.ConclusionIntroduction of a PRRSV strain of enhanced virulence in a production system where several farms were previously positive for other PRRSV strains, resulted in a fast spread such as would be observed in naïve farms. The productive and health impact was very high taking several months to return to normality.

Genome-Wide Characterization of QYYZ-Like PRRSV During 2018-2021.

In the last decade, the emergence of QYYZ-like porcine reproductive and respiratory syndrome virus (PRRSV) has attracted increasing attention due to the high incidence of PRRSV mutation and recombination. However, the endemic status and genomic characteristics of the QYYZ-like strains are unclear. From 2018 to October 2021, 24 QYYZ-like PRRSV isolates were obtained from 787 PRRSV-positive clinical samples. Only one QYYZ-like positive sample was from a northern province, and the rest were from central and southern provinces. We selected 9 samples for whole-genome sequencing, revealing genome lengths of 15,008–15,316 nt. We retrieved all the available whole-genome sequences of QYYZ-like PRRSVs isolated in China from 2010 to 2021 (n = 28) from GenBank and analyzed them together with the new whole-genome sequences (n = 9). Phylogenetic tree analysis based on the ORF5 gene showed that all QYYZ-like PRRSV strains belonged to sublineage 3.5 but were clustered into three lineages (sublineage 1.8, sublineage 3.5, and sublineage 8.7) based on whole-genome sequences. Genomic sequence alignment showed that QYYZ-like strains, have characteristic amino acids insertions or deletions in the Nsp2 region (same as NADC30, JXA1 and QYYZ) and that thirteen strains also had additional amino acid deletions, mostly between 468 and 518 aa. Moreover, QYYZ-like strains (sublineage 3.5) have seven identical characteristic amino acid mutations in ORF5. Recombination analysis revealed that almost all QYYZ-like complete genome sequences (36/37) were products of recombination and mainly provided structural protein fragments (GP2-N) for the recombinant strains. Overall, QYYZ-like strains were mainly prevalent in central and southern China from 2018 to 2021, and these strains provided recombinant fragments in the PRRSV epidemic in China.

Evolutionary Analysis of Four Recombinant Viruses of the Porcine Reproductive and Respiratory Syndrome Virus From a Pig Farm in China.

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens causing substantial economic losses to the Chinese swine industry. In this study, we analyzed the complete genome sequences of four PRRSV isolates (PRRSV2/CN/SS0/2020, PRRSV2/CN/SS1/2021, PRRSV2/CN/L3/2021, and PRRSV2/CN/L4/2020) isolated from a single pig farm from 2020 to 2021. The genomes of the four isolates were 14,962–15,023 nt long, excluding the poly (A) tails. Comparative analysis of the genome sequences showed that the four isolates shared 93.2–98.1% homology and they had no close PRRSV relatives registered in the GenBank (<92%). Furthermore, PRRSV2/CN/SS0/2020 and PRRSV2/CN/SS1/2021 had characteristic 150-aa deletions (aa481+aa537-566 +aa628–747) that were identical to the live attenuated virus vaccine strain TJM-F92 (derived from the HP-PRRSV TJ). Further analysis of the full-length sequences suggests that the four isolates were natural recombinant strains between lineages 1 (NADC30-like), 3 (QYYZ-like), and 8.7 (JXA1-like). Animal experiments revealed discrepancies in virulence between PRRSV2/CN/SS0/2020 and PRRSV2/CN/L3/2021. The strain with high homology to HP-PRRSV demonstrates higher pathogenicity for pigs than the other isolate with low homology to HP-PRRSV. Taken together, our findings suggest that PRRSVs have undergone genome evolution by recombination among field strains/MLV-like strains of different lineages.

Evolution Characterization and Pathogenicity of a Porcine Reproductive and Respiratory Syndrome Virus Isolate from a Pig Farm in Shandong Province, China.

In recent years, porcine reproductive and respiratory syndrome virus (PRRSV) strains have been experiencing extensive recombination in Chinese swine farms. This recombination usually happens in NADC30/34 strains and highly pathogenic (HP) PRRSV strains. This study identified a new PRRSV isolate that shared 99% and 99.1% nucleotide identity with CH-1a and CH-1R at the genomic level, respectively. After purification by viral plaque assay, this isolate was named PRRSV CSR1801. The isolate did not experience any recombination with other PRRSV strains common in swine herd epidemics in China, which means it still maintains the stable features of the classical PRRSV strain and did not easily recombine with other PRRSV strains. Further analysis of the pathogenicity of the PRRSV isolate CSR1801 was performed in piglets. The results indicated that none of the inoculated piglets showed the typical clinical manifestations of PRRS, which presented with runny noses, rough back hair, rectal temperatures always below 40.5 °C, and no deaths. Additionally, no obvious histopathological lesions such as severe interstitial pneumonia could be observed in the lungs of the piglets. Hence, the PRRSV isolate CSR1801 should be classified as a classical-like PRRSV strain. This classical PRRSV strain showed genetic stability and maintained low pathogenicity. This study may provide new clues for further understanding the genetic evolution and pathogenicity of PRRSV and may also be an important reference for the prevention and control of PRRS in swine farms.

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

BackgroundPorcine 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.MethodsThe 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.ResultsNineteen 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.ConclusionGenome-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.

Epidemiological and Genetic Characteristics of Porcine Reproductive and Respiratory Syndrome Virus in South China Between 2017 and 2021.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to the swine industry in China and has caused enormous losses every year. To monitor the epidemiological and genetic characteristics of PRRSV in South China, 6,795 clinical samples from diseased pigs were collected between 2017 and 2021, and 1,279 (18.82%) of them were positive for PRRSV by RT-PCR detecting the ORF5 gene. Phylogenetic analysis based on 479 ORF5 sequences revealed that a large proportion of them were highly-pathogenic PRRSVs (409, 85.39%) and PRRSV NADC30-like strains (66, 13.78%). Furthermore, 93.15% of these highly-pathogenic strains were found to be MLV-derived. We next recovered 11 PRRSV isolates from the positive samples and generated the whole genome sequences of them. Bioinformatic analysis showed that seven isolates were MLV-derived. Besides, six isolates were found to be recombinant strains. These eleven isolates contained different types of amino acid mutations in their GP5 and Nsp2 proteins compared to those of the PRRSVs with genome sequences publicly available in GenBank. Taken together, our findings contribute to understanding the prevalent status of PRRSV in South China and provide useful information for PRRS control especially the use of PRRSV MLV vaccines.

A NADC30-like PRRSV causes serious intestinal infections and tropism in piglets

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to China's swine industry. Currently, a novel type 2 PRRSV, called the NADC30-like strain, is epidemic in numerous provinces of China. In this study, a NADC30-Like PRRSV strain was isolated in primary alveolar macrophage (PAM) cells from fecal samples collected from a local pig farm, which suffered severe diarrhea. A pathogenicity comparison study was conducted in 6–week‐old piglets by inoculating highly pathogenic HP-PRRSV and NADC30-Like PRRSV isolates. RT-qPCR revealed detection of NADC30-Like PRRSV but not the HP-PRRSV in the intestine. PRRSV infection-related lesions were observed in the intestine were further confirmed by histopathological and immunohistochemical examination (IHC). In addition, severe virus infections were also detected by RT-qPCR. Based on clinical observation and pathogenicity experiments, we confirmed that NADC30-Like PRRSV gained more tissue tropism, especially in the small intestine. This may be the one reason explaining why NADC-Like 30 PRRSV become a major epidemic strain in China since the first outbreak in 2013.

Novel characteristics of Chinese NADC34-like PRRSV during 2020-2021.

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) strains were first detected in China in 2017, with epidemic potential. In this study, the phylogenetic, epidemic, and recombinant properties of NADC34-like PRRSV in China were evaluated comprehensively. From 2020 to October 2021, 82 NADC34-like PRRSV isolates were obtained from 433 PRRSV-positive clinical samples. These strains accounted for 11.5% and 28.6% of positives in 2020 and 2021, respectively, and have spread to eight provinces. We selected 15 samples for whole-genome sequencing, revealing genome lengths of 15,009-15,113nt. Phylogenetic analysis revealed that Chinese NADC34-like strains cluster with American sublineage 1.5 strains and do not form an independent branch. Recombination analysis revealed that six of fifteen complete genome sequences were derived from recombination between NADC34-like and NADC30-like or HP-PRRSV; all of the strains recombined with local strains in China, exhibiting a complex recombination pattern. Partial Nsp2 sequence alignment showed that nine of fifteen isolates had a 100 aa continuous deletion (similar to that in IA/2014/NADC34); other isolates had a 131 aa discontinuous deletion (similar to that in NADC30). Five of them also had additional amino acid deletions, all of which are reported for the first time here. In the last 2 years, NADC34-like PRRSV has become one of the main epidemic strains in some areas of China; it has changed significantly, its homology has decreased significantly, and it has undergone complex recombination with local Chinese strains. These results are of great significance for understanding the current epidemic situation of PRRSV in China.

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.

Nanopore-Based Direct RNA-Sequencing Reveals a High-Resolution Transcriptional Landscape of Porcine Reproductive and Respiratory Syndrome Virus.

The TRS-mediated discontinuous transcription process is a hallmark of Arteriviruses. Precise assessment of the intricate subgenomic RNA (sg mRNA) populations is required to understand the kinetics of viral transcription. It is difficult to reconstruct and comprehensively quantify splicing events using short-read sequencing, making the identification of transcription-regulatory sequences (TRS) particularly problematic. Here, we applied long-read direct RNA sequencing to characterize the recombined RNA molecules produced in porcine alveolar macrophages during early passage infection of porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequencing two PRRSV isolates, namely XM-2020 and GD, we revealed a high-resolution and diverse transcriptional landscape in PRRSV. The data revealed intriguing differences in subgenomic recombination types between the two PRRSVs while also demonstrating TRS-independent heterogeneous subpopulation not previously observed in Arteriviruses. We find that TRS usage is a regulated process and share the common preferred TRS in both strains. This study also identified a substantial number of TRS-mediated transcript variants, including alternative-sg mRNAs encoding the same annotated ORF, as well as putative sg mRNAs encoded nested internal ORFs, implying that the genetic information encoded in PRRSV may be more intensively expressed. Epigenetic modifications have emerged as an essential regulatory layer in gene expression. Here, we gained a deeper understanding of m5C modification in poly(A) RNA, elucidating a potential link between methylation and transcriptional regulation. Collectively, our findings provided meaningful insights for redefining the transcriptome complexity of PRRSV. This will assist in filling the research gaps and developing strategies for better control of the PRRS.

Evaluation of the intranasal route for porcine reproductive and respiratory disease modified-live virus vaccination

BackgroundIn pigs, modified live virus (MLV) vaccines against porcine reproductive and respiratory syndrome virus (PRRSV) are commonly used and administered by intramuscular (IM) injection. In contrast, PRRSV, as a primary respiratory pathogen, is mainly transmitted via the intranasal (IN) route. The objective of this study was to evaluate the efficacy of a commonly used commercial PRRSV MLV delivered IN compared to the IM route. MethodsFifty-four pigs were divided into five treatment groups. All vaccinated groups received the same MLV vaccine but administered via different routes. Group IN-JET-VAC was vaccinated with an automated high pressure prototype nasal jet device (IN-JET-VAC, n = 12), group IN-MAD-VAC was vaccinated with a mucosal atomization device (IN-MAD-VAC, n = 12), group IM-VAC was vaccinated intramuscularly (IM-VAC; n = 12) according to label instructions, while the NEG-CONTROL (n = 6) and the POS-CONTROL (n = 12) groups were both unvaccinated. At 28 days post vaccination all vaccinated groups and the POS-CONTROL pigs were challenged with a pathogenic US PRRSV isolate. Blood and nasal swabs were collected at regular intervals, and all pigs were necropsied at day 10 post challenge (dpc) when gross and microscopic lung lesions were assessed. ResultsPrior to challenge most vaccinated pigs had seroconverted to PRRSV. Clinical signs (fever, inappetence) were most obvious in the POS-CONTROL group from dpc 7 onwards. The vaccinated groups were not different for PRRSV viremia, seroconversion, or average daily weight gain. However, IN-JET-VAC and IN-MAD-VAC had significantly higher neutralizing antibody levels against the vaccine virus at challenge. ConclusionsComparable vaccine responses were obtained in IN and IM vaccinated pigs, suggesting the intranasal administration route as an alternative option for PRRSV vaccination.

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.

The Ability of Porcine Reproductive and Respiratory Syndrome Virus Isolates to Induce Broadly Reactive Neutralizing Antibodies Correlates With In Vivo Protection.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most relevant diseases of swine. The condition is caused by PRRS virus (PRRSV), an extremely variable virus of the Arteriviridae family. Its heterogeneity can be responsible, at least partially, of the poor cross-protection observed between PRRSV isolates. Neutralizing antibodies (NAs), known to play a role in protection, usually poorly recognize heterologous PRRSV isolates, indicating that most NAs are strain-specific. However, some pigs develop broadly reactive NAs able to recognize a wide range of heterologous isolates. The aim of this study was to determine whether PRRSV isolates that induce broadly reactive NAs as determined in vitro are able to confer a better protection in vivo. For this purpose two in vivo experiments were performed. Initially, 40 pigs were immunized with a PRRSV-1 isolate known to induce broadly reactive NAs and 24 additional pigs were used as controls. On day 70 after immunization, the pigs were divided into eight groups composed by five immunized and three control pigs and exposed to one of the eight different heterologous PRRSV isolates used for the challenge. In the second experiment, the same experimental design was followed but the pigs were immunized with a PRRSV-1 isolate, which is known to generate mostly strain-specific NAs. Virological parameters, specifically viremia and the presence of challenge virus in tonsils, were used to determine protection. In the first experiment, sterilizing immunity was obtained in three groups, prevention of viremia was observed in two additional groups, although the challenge virus was detected occasionally in the tonsils of immunized pigs, and partial protection, understood as a reduction in the frequency of viremia compared with controls, was recorded in the remaining three groups. On the contrary, only partial protection was observed in all groups in the second experiment. The results obtained in this study confirm that PRRSV-1 isolates differ in their ability to induce cross-reactive NAs and, although other components of the immune response might have contributed to protection, pigs with cross-reactive NAs at the time of challenge exhibited better protection, indicating that broadly reactive NAs might play a role in protection against heterologous reinfections.

Transcriptomic profiling reveals different innate immune responses in primary alveolar macrophages infected by two highly homologous porcine reproductive and respiratory syndrome viruses with distinct virulence

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates show high genetic and pathogenic diversity. The mechanisms underlying different virulence of PRRSV isolates are still not fully clarified. Two highly homologous PRRSV isolates (XJ17-5 and JSTZ1712-12) with distinct virulence were identified in our previous study. To evaluate the association between host responses and different virulence, here we investigated the transcriptomic profiles of porcine alveolar macrophages (PAMs) infected with these two isolates. RNA-Seq results showed that there are 1932 differential expression genes (DEGs) between two PRRSV infected groups containing 1067 upregulation and 865 downregulation genes. Compared with the avirulent JSTZ1712-12 infected group, GO analysis identified significant enrichment gene sets not only associated with virus infection but also innate immune response in the virulent XJ17-5 infected group. In addition, KEGG analysis indicated significantly enriched genes associated with NOD-like and RIG-I-like receptor signaling pathways in XJ17-5 vs JSTZ1712-12 group. Furthermore, XJ17-5 isolate induced significantly higher levels of innate immune response associated genes (IL-1β, CXCL2, S100A8, OAS2, MX1, IFITM3, ISG15 and IFI6) than JSTZ1712-12 isolate, which were further confirmed by real-time PCR. Given that these two isolates share similar replication efficiency in vivo and in vitro, our results indicated that distinct virulence of PRRSV isolates is associated with different host innate immune responses.

Chimeric HP-PRRSV2 containing an ORF2-6 consensus sequence induces antibodies with broadly neutralizing activity and confers cross protection against virulent NADC30-like isolate

Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection.

Newly-designed primer pairs for the detection of type 2 porcine reproductive and respiratory syndrome virus genes

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, caused by PRRS virus (PRRSV), that critically affects the swine industry. While the detection of PRRSV genes plays a key role in PRRS control, the PRRSV genome is known to undergo frequent mutation. Nevertheless, primer pairs widely used for the detection of PRRSV genes were designed between 1995 and 2010. The reliability of these primer pairs for the detection of currently circulating PRRSVs is therefore questionable. Here, we investigated the sensitivity of the previously reported primer pairs to detect PRRSV genes that have been recently isolated or detected in Japan. In addition, based on nucleotide sequences from the recent Japanese PRRSVs, we designed four new primer pairs for the detection of PRRSV genes. The sensitivity and specificity of the new primer pairs were evaluated by quantitative reverse transcription PCR using RNA extracted from PRRSV isolates, swine serum, and oral fluid specimens collected from PRRS-affected pigs, and swine sera collected from a PRRSV-free pig farm in Japan. One of novel primer pairs used in our study exhibited greater sensitivity than the previously reported primer pairs, and is thus more reliable for the detection of PRRSV genes.