A natural recombinant PRRSV between HP‐PRRSV JXA1‐like and NADC30‐like strains

Porcine reproductive and respiratory syndrome virus (PRRSV ) is a major economically significant pathogen that has adversely affected China's swine industry. Currently, a novel type 2 PRRSV , called the NADC 30‐like strain, is epidemic in numerous provinces of China, and commercial vaccines provide limited protection for infected animals. The extensive recombination phenomenon among NADC 30‐like PRRSV s is identified as a unique molecular characteristic of the virus. However, our understanding of how recombination influences NADC 30‐like PRRSV s is largely inadequate. In this study, we analysed the genetic characteristics of a recombinant NADC 30‐like PRRSV (SC ‐d) and examined its pathogenicity compared with a non‐recombinant NADC 30‐like PRRSV (SD ‐A19) and a highly pathogenic PRRSV (HuN4). SC ‐d has three discontinuous deletions in nsp2, consistent with NADC 30 isolated from the United States in 2008. Furthermore, we identified four recombination breakpoints in the SC ‐d genome, which separated the SC ‐d genome into four regions (regions A, B, C and D). Regions A and C are closely related to the JXA 1‐like strain, one of the earliest Chinese HP ‐PRRSV strains, and regions B and D are closely related to the NADC 30 strain. Moreover, SC ‐d inoculated piglets exhibited a persistent fever, moderate weight loss, mild thymus atrophy and obvious microscopic lung lesions. In summary, the recombinant NADC 30‐like PRRSV SC ‐d strain displayed a higher pathogenicity than the non‐recombinant NADC 30‐like PRRSV SD ‐A19 strain; however, the pathogenicity of the NADC 30‐like PRRSV SC ‐d was lower compared with the HP ‐PRRSV HuN4 strain in piglets. Our findings demonstrate that recombination is responsible for the enormous genetic diversity and pathogenicity variance of the NADC 30‐like PRRSV in China. This study provides a theoretical basis for developing a more reasonable PRRSV control and prevention strategy.     

Outbreak Investigation of NADC30‐Like PRRSV in South‐East China

Epidemiological outbreak investigations were conducted on NADC30‐like porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the prevalence of the disease in south‐east China in 2015. Two more provinces were found to have NADC30‐like PRRSV circulating besides previously reported six provinces. Phylogenetic analysis showed that these virus isolates were clustered in an independent branch and shared high nucleotide similarity to NADC30, a type 2 PRRSV that has been isolated in Unite States in 2008. One NADC30‐like PRRSV strain from Henan province was successfully isolated on porcine alveolar macrophages and was tested on 6‐week‐old specific pathogen‐free pigs for pathogenic study. The virus‐inoculated pigs showed typical PRRSV clinical symptoms, but all pigs survived throughout the study with a period of 14 days. At necropsy, the lungs of infected pigs developed PRRSV‐specific interstitial pneumonia, and virus antigen was detected in lung samples. Therefore, our results indicated NADC30‐like PRRSV has widely spread in China and could cause clinical disease on pigs.     

Genetic characterization of a novel recombinant PRRSV2 from lineage 8, 1 and 3 in China with significant variation in replication efficiency and cytopathic effects

There are four major porcine reproductive and respiratory syndrome virus 2 (PRRSV2) lineages circulating in China based on classification system, including lineages 1 (NADC30‐like), 3 (QYYZ‐like), 5.1 (VR2332‐like) and 8 (JXA1‐like/CH‐1a‐Like), which leads to the potential recombination. In the present study, a novel variant of PRRSV2 strain named JS18‐3 was isolated from piglets suffering severe breathing difficulties in Jiangsu Province of China in 2018. Full‐length genome analysis indicated that JS18‐3 shared 86.5%, 87.9%, 84.2%, 82.2% and 86.4% nucleotide similarity with PRRSVs CH‐1a, JXA1, VR2332, QYYZ and NADC30, respectively. 4871–6635 of JS18‐3 shared the highest identity of 99.3% in nucleotide sequence with HP‐PRRSV representative strain JXA1 indicating ongoing evolution to HP‐PRRSV. JS18‐3 was classified into classical lineage 8 of PRRSV2 based on phylogenetic analysis of complete genome and ORF5. Genomic break points in structural (ORF3) and non‐structural (NSP2, NSP3) regions of genomes were detected in recombination analysis. JS18‐3 is a recombinant isolate from lineages 8, 1 and 3. Replication enhancement and severe cytopathic effects caused by JS18‐3 were observed in Marc‐145 cells and porcine alveolar macrophages (PAMs) as compared to JX07, a typical strain of lineage 8. Pathogenicity results indicated that piglets inoculated with JS18‐3 presented persistent fever, dyspnoea, serious microscopic lung lesions and lymph node congestion. The study suggests that lineage 8 of PRRSV2 is involved in continuing evolution by genetic recombination and mutation leading to outbreaks in vaccinated pigs in China.     

Emergence of a novel highly pathogenic porcine reproductive and respiratory syndrome virus in China

From 2014 to 2015, four novel highly pathogenic PRRS virus (HP‐PRRSV) strains named 14LY01‐FJ, 14LY02‐FJ 15LY01‐FJ, and 15LY02‐FJ were isolated from high morbidity (100%) and mortality (40%–80%) in piglets and sows in Fujian Province. To further our knowledge about these novel virus strains, we characterized their complete genomes and determined their pathogenicity in piglets. Full‐length genome sequencing analysis showed that these four isolates were closely related to type 2 (North American type, NA‐type) isolates, with 88.1%–96.3% nucleotide similarity, but only 60.6%–60.8% homology to the Lelystad virus (LV) (European type, EU‐type). The full length of the four isolates was determined to be 15017 or 15018 nucleotides (nt), excluding the poly(A) tail. Furthermore, the four isolates had three discontinuous deletions (aa 322–432, aa 483, and aa 504–522) within hypervariable region II (HV‐II) of Nsp2, as compared to the reference strain VR‐2332. This deletion pattern in the four isolates is consistent with strain MN184 and strain NADC30 isolated from America. Phylogenetic and molecular evolutionary analyses indicated that these virulent strains originated from a natural recombination event between the JXA1‐like HP‐PRRSV (JXA‐1 is one of the earliest Chinese HP‐PRRSV strains; sublineage 8.7) and the NADC30‐like (lineage 1) PRRSV. Animal experiments demonstrated that these four strains caused significant weight loss and severe histopathological lung lesions as compared to the negative control group. High mortality rate (40% or 80%) was found in piglets infected with any one of the four strains, similar to that found with other Chinese HP‐PRRSV strains. This study showed that the novel variant PRRSV was HP‐PRRSV, and it is therefore critical to monitor PRRSV evolution in China and develop a method for controlling PRRS.     

A novel NADC30‐like porcine reproductive and respiratory syndrome virus (PRRSV) plays a limited role in the pathogenicity of porcine circoviruses (PCV2 and PCV3) and PRRSV co‐infection

Co‐infection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCVs) is commonly observed under field conditions and elicits more severe diseases than any singular infection. In this study, the co‐infection of PRRSV, PCV2 and PCV3 was analyzed in tissue samples collected from 150 pigs from April 2016 to April 2018. PRRSV, PCV2 and PCV3 was detected in 55 (36.67%), 43 (28.67%) and 3 (2%) of 150 pigs respectively. Remarkably, one lung sample (SD17‐36) collected from a diseased pig was co‐infected with PRRSV, PCV2 and PCV3. The complete genomes of SD17‐36 viruses of PRRSV, PCV2 and PCV3 were determined, which belong to the subgroups of NADC30‐like PRRSV, PCV2d and PCV3a respectively. Sequence comparison showed that PRRSV SD17‐36 isolate contains a N33 deletion in GP5. Animal challenge study showed that the novel NADC30‐like PRRSV SD17‐36 isolate is low pathogenic. Our results indicate that the co‐infection of PRRSV and PCVs might cause diseases even when PRRSV plays a limited role in the pathogenicity of the co‐infection.     

Pathogenicity and Distribution of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in Pigs

The pathogenesis of highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) strain (HuN4) is poorly understood. Therefore, highly pathogenic PRRSV strain (HuN4) and its derivative strain (HuN4‐F112) (obtained by propagation in MARC145 cells for 112 passages) were inoculated into a total of 48 PRRSV‐sero‐negative pigs (age: 4–5 weeks) by the intranasal route. Virological, pathological and in situ hybridization analyses were performed. The results exhibited that pigs infected with HuN4 showed a loss of appetite, decrease in body weight, raised body temperature and respiratory symptoms, along with interstitial pneumonia lesions. In the HuN4 group, multifocal interstitial pneumonia with macrophage infiltration was found in the lung. The lesions in the lymph node were characterized by collapsed follicles, depletion of germinal centres and reduction in lymphocytes. Perivascular cuffing and glial nodules were observed in the brains of some pigs. By comparison, the HuN4‐F112 group had milder lesions. PRRSV was detected in macrophages, alveolar epithelial cells and vascular endothelial cells in the tonsil and lymph nodes. The PRRSV amounts in the pigs infected with HuN4 were 10⁵–10⁹ copies/ml in the blood and 10¹⁰–10¹¹ copies/g in the lung tissues, whereas the virus amounts with HuN4‐F112 were 10^2.15–10^3.13 copies/ml in the blood and 10^3.0–10^3.6 copies/g in the lung. Our results demonstrate that the PRRS HuN4 virus infects alveolar epithelial cells, macrophages and vascular endothelial cells causing diffuse alveolar damage and lymph node necrosis. Its higher pathogenicity compared with HuN4‐F112 virus may be explained in part by higher replication rate in the previously mentioned organs.     

Porcine reproductive and respiratory syndrome virus NADC30‐like strain accelerates Streptococcus suis serotype 2 infection in vivo and in vitro

Porcine reproductive and respiratory syndrome (PRRS), an economically significant pandemic disease, commonly results in increased impact of bacterial infections, including those by Streptococcus suis (S. suis). In recent years, PRRS virus (PRRSV) NADC30‐like strain has emerged in different regions of China, and coinfected with S. suis and PRRSV has also gradually increased in clinical performance. However, the mechanisms involved in host innate responses towards S. suis and their implications of coinfection with NADC30‐like strain remain unknown. Therefore, the pathogenicity of NADC30‐like strain and S. suis serotype 2 (SS2) coinfection in vivo and in vitro was investigated in this study. The results showed that NADC30‐like increased the invasion and proliferation of SS2 in blood and tissues, resulting in more severe pneumonia, myocarditis, and peritonitisas well as higher mortality rate in pigs. In vitro, NADC30‐like strain increased the invasion and survival of SS2 in porcine alveolar macrophages (PAM) cells, causing more drastic expression of inflammatory cytokines and activation of NF‐ĸB signalling. These results pave the way for understanding the interaction of S. suis with the swine immune system and their modulation in a viral coinfection.     

Pathology and Virus Distribution in the Lung and Lymphoid Tissues of Pigs Experimentally Inoculated with Three Distinct Type 1 PRRS Virus Isolates of Varying Pathogenicity

Porcine reproductive and respiratory syndrome (PRRS) continues to be the most economically important disease of swine worldwide. The appearance of highly pathogenic PRRS virus (PRRSV) strains in Europe and Asia has raised concerns about this disease and initiated increased efforts to understand the pathogenesis. In this study, we have compared the pathology and the virus distribution in tissues of pigs experimentally inoculated with three different genotype 1 PRRSV isolates. Sixty 5‐week‐old pigs were inoculated intranasally with a) the Lelystad virus (LV), b) a field strain from the UK causing respiratory clinical signs (UK) or c) a highly pathogenic strain from Belarus (BE). Sixteen animals were mock‐infected and used as controls. The animals were euthanized at 3, 7 and 35 days post‐infection (dpi), and lung and lymphoid tissues collected for histopathological examination and PRRSV detection by immunohistochemistry (IHC). Histopathological lesions consisted of interstitial pneumonia with mononuclear cell infiltrates in the lungs, lymphoid depletion, apoptosis and follicular hyperplasia in the spleen, lymph nodes and tonsil and lymphoid depletion in the thymus. Porcine reproductive and respiratory syndrome virus was detected mainly in monocytes–macrophages. BE‐infected animals showed the highest pathological scores and the highest presence of virus at 3 and 7 dpi, followed by the UK field strain and then LV. Moderate lesions were observed at 35 dpi with lesser detection of PRRSV by IHC in each infected group. The highly pathogenic BE strain induced more severe pathology in both lungs and lymphoid organs of pigs compared with the classic field isolate and the prototype LV. The increased severity of pathology was in correlation with the presence of a higher number of PRRSV‐infected cells in the tissues.     

A modified‐live porcine reproductive and respiratory syndrome virus (PRRSV)‐1 vaccine protects late‐term pregnancy gilts against heterologous PRRSV‐1 but not PRRSV‐2 challenge

The objective of this study was to determine the efficacy of a commercially available porcine reproductive and respiratory syndrome virus (PRRSV )‐1 modified‐live virus (MLV ) vaccine against PRRSV ‐1 and PRRSV ‐2 challenge in late‐term pregnancy gilts. Gilts were vaccinated with the PRRSV ‐1 MLV vaccine at 4 weeks prior to breeding and then challenged intranasally with PRRSV ‐1 or PRRSV ‐2 at 93 days of gestation. After PRRSV ‐1 challenge, vaccinated pregnant gilts had a significantly longer gestation period, significantly higher numbers of live‐born and weaned piglets and a significantly lower number of stillborn piglets at birth compared to unvaccinated pregnant gilts. No significant improvement in reproductive performance was observed between vaccinated and unvaccinated pregnant gilts following PRRSV ‐2 challenge. Vaccinated pregnant gilts also exhibited a significantly improved reproductive performance after challenge with PRRSV ‐1 compared to vaccinated pregnant gilts following PRRSV ‐2 challenge. The PRRSV ‐1 MLV vaccine was able to reduce PRRSV ‐1 but not PRRSV ‐2 viremia in pregnant gilts. Vaccinated gilts also showed a significantly higher number of PRRSV ‐1‐specific IFN ‐γ‐secreting cells (IFN ‐γ‐SC ) compared to PRRSV ‐2‐specific IFN ‐γ‐SC . The data presented here suggest that the vaccination of pregnant gilts with a PRRSV ‐1 MLV vaccine provides good protection against PRRSV ‐1 but only limited protection against PRRSV ‐2 challenge in late‐term pregnancy gilts based on improvement of reproductive performance, reduction in viremia and induction of IFN ‐γ‐SC .     

A potential endemic strain in China: NADC34‑like porcine reproductive and respiratory syndrome virus

Porcine respiratory and reproductive syndrome virus (PRRSV) causes an economically important disease affecting commercial pork production worldwide. NADC34‐like PRRSV has had a strong impact on the U.S. and Peruvian pig industries in recent years and also emerged in northeastern China in 2017. However, the endemic status of NADC34‐like PRRSV in China is unclear. In this study, we examined 650 tissue samples collected from 16 Provinces in China from 2018 to 2019. Six NADC34‐like PRRSV strains were detected in samples from three Provinces, and the complete genomes of four of these strains were sequenced. Phylogenetic analysis showed that these novel PRRSV strains belong to sublineage 1.5 (or NADC34‐like PRRSV), forming two groups in China. Sequence alignment suggested that these novel strains share the same 100‐aa deletion in the Nsp2 protein that was identified in IA/2014/NADC34 isolated from the United States in 2014. Recombination analysis revealed that five of eight complete genome sequences are derived from recombination between IA/2014/NADC34 and ISU30 or NADC30. The number and distribution of NADC34‐like PRRSVs is increasing in China. Importantly, compared with the currently endemic strain NADC30‐like PRRSV, NADC34‐like PRRSV has the potential to be an endemic strain in China. This study will help us understand the epidemic status of NADC34‐like PRRSV in China and provide data for further monitoring this type of PRRSV in China.     

Downregulation of Antigen‐Presenting Cells in Tonsil and Lymph Nodes of Porcine Reproductive and Respiratory Syndrome Virus‐Infected Pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) can persist in different organs of infected pigs, which suggests a failure in the immune response. Antigen‐presenting cells (APCs) play a pivotal role in the induction of effective T‐ and B‐cell responses. In this study, we investigated the changes in the different APC subpopulations and T‐ and B‐cell counts in the tonsil, retropharyngeal and mediastinal lymph nodes of pigs experimentally infected with a European PRRSV field isolate. Our results demonstrated that the expression of S100, SWC3, HLA‐DR molecule and CD3 was diminished in the studied organs throughout the study, observing a significant negative correlation between viral antigen and HLA‐DR expression in both retropharyngeal and mediastinal lymph nodes. In contrast, λ‐light chains showed an increase during the study. Taking all into account, after PRRSV infection, no enhancement in the number of APCs and T cells was observed, suggesting an impairment of the immune function which may allow the persistence of PRRSV into the organism.     

The infectious cDNA clone of commercial HP‐PRRS JXA1‐R‐attenuated vaccine can be a potential effective live vaccine vector

Multiple commercial porcine reproductive and respiratory syndrome (PRRS) modified live vaccines are currently utilized in Chinese swine herds due to the limited cross‐protection of vaccines and coexistence of different PRRS viruses. In this study, an infectious cDNA clone of the highly pathogenic PRRS (HP‐PRRS) vaccine JXA1‐R strain was generated. We successfully rescued the virus from direct in vitro DNA transfection of rJXA1‐R clone, which has similar growth kinetics to the parental JXA1‐R virus in Marc‐145 cells. To further evaluate the potential use of the cloned rJXA1‐R virus as a live vector for foreign gene expression, the enhanced green fluorescent protein (EGFP) was inserted between non‐structural and structural genes. Our results showed that the dynamic expression of EGFP can be visualized by live cell imaging system during the infection in Marc‐145 cells. The availability of our cloned JXA1‐R viruses provides a crucial platform to study the fundamental biology of HP‐PRRS virus vaccine and also serves as a potential effective vector for developing live vector vaccines against swine pathogens.     

Double knockout pigs deficient in N‐glycolylneuraminic acid and Galactose α‐1,3‐Galactose reduce the humoral barrier to xenotransplantation

Background

Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα‐1,3‐Gal (Gal) and N‐glycolylneuraminic acid (Neu5Gc) are two known xenoantigens.

Methods

We report the homozygous disruption of the α1, 3‐galactosyltransferase (GGTA1) and the cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase (CMAH) genes in liver‐derived female pig cells using zinc‐finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody‐binding and antibody‐mediated complement‐dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal.

Results

This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1.

Conclusions

This technology will accelerate the development of pigs for xenotransplantation research.     

Comparative Analysis of Immune Responses in Pigs to High and Low Pathogenic Porcine Reproductive and Respiratory Syndrome Viruses Isolated in China

The CH‐1a and HuN4 strains of porcine reproductive and respiratory syndrome virus (PRRSV) show different pathogenicities in pigs. To understand host immune responses against these viruses, we investigated the dynamic changes in cytokine levels produced in peripheral blood of piglets infected with the highly pathogenic PRRSV HuN4 strain or the CH‐1a strain. Clinical signs, virus loads and serum cytokine levels [interferon(IFN)‐α, Interleukin (IL)‐1, TNF‐α, IL‐6, IL‐12, IFN‐γ, IL‐10 and TGF‐β] were tested. The results showed that while piglets developed effective cellular immune responses against CH‐1a infection, those infected with HuN4 displayed ineffective cellular immunity, organ lesions and persistent elevated levels of immunoregulatory cytokines (IL‐10 and TGF‐β), which delayed the development of PRRSV‐specific immune responses. These results demonstrated that HuN4 infection induced higher cytokine levels than that of CH‐1a infection induced. The changes in inflammatory cytokines intensified the inflammatory reaction and damaged the tissues and organs.     

Emergence of novel recombination lineage 3 of porcine reproductive and respiratory syndrome viruses in Southern China

Lineage 3 of porcine reproductive and respiratory syndrome viruses, which belong to North America type 2, has a long epidemic history in China. The novel lineage 3 viruses constantly emerging in recent years are characterized by a high detection rate and significant pathogenicity. In this study, we investigated the prevalence of lineage 3 in southern China and selected two isolated strains for genome and virulence analyses. A cross‐sectional epidemiology investigation indicated that the prevalence of lineage 3 antigens was 35.68% (95% CI: 27.6–44.3%) among 227 samples collected from over 100 infected farms from January 2016 to July 2017 in southern China. Two novel isolates of lineage 3 were selected. After 20 passages, Marc‐145 cells were not susceptible to those viruses. Full‐length genome analysis indicated that the two strains share 95.2% homology with each other and 95.7%–96.2% with highly pathogenic porcine reproductive and respiratory syndrome viruses (HP‐PRRSVs; JXA1‐like strain, lineage 8.7). Phylogenetic and molecular evolutionary results showed that for the two isolates, HP‐PRRSV provides most of the ORF1 gene. Animal experiment revealed discrepancies in virulence between the strains. Although challenge resulted in 100% morbidity, the isolate carrying most of the HP‐PRRSV ORF1 caused severe clinical symptoms and 40% mortality, whereas the other isolate containing part of the ORF1 gene caused no mortality. Overall, these findings suggest that lineage 3 viruses might be commonly circulating in most of southern China. Frequent recombination events within HP‐PRRSVs of this lineage with changing virulence could represent potential threats to the pig industry.     

Pathogenicity and Molecular Characterization of Emerging Porcine Reproductive and Respiratory Syndrome Virus in Vietnam in 2007

In 2007, Vietnam experienced swine disease outbreaks causing clinical signs similar to the ‘porcine high fever disease’ that occurred in China during 2006. Analysis of diagnostic samples from the disease outbreaks in Vietnam identified porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV‐2). Additionally, Escherichia coli and Streptococcus equi subspecies zooepidemicus were cultured from lung and spleen, and Streptococcus suis from one spleen sample. Genetic characterization of the Vietnamese PRRSV isolates revealed that this virus belongs to the North American genotype (type 2) with a high nucleotide identity to the recently reported Chinese strains. Amino acid sequence in the nsp2 region revealed 95.7–99.4% identity to Chinese strain HUN4, 68–69% identity to strain VR‐2332 and 58–59% identity to strain MN184. A partial deletion in the nsp2 gene was detected; however, this deletion did not appear to enhance the virus pathogenicity in the inoculated pigs. Animal inoculation studies were conducted to determine the pathogenicity of PRRSV and to identify other possible agents present in the original specimens. Pigs inoculated with PRRSV alone and their contacts showed persistent fever, and two of five pigs developed cough, neurological signs and swollen joints. Necropsy examination showed mild to moderate bronchopneumonia, enlarged lymph nodes, fibrinous pericarditis and polyarthritis. PRRSV was re‐isolated from blood and tissues of the inoculated and contact pigs. Pigs inoculated with lung and spleen tissue homogenates from sick pigs from Vietnam developed high fever, septicaemia, and died acutely within 72 h, while their contact pigs showed no clinical signs throughout the experiment. Streptococcus equi subspecies zooepidemicus was cultured, and PRRSV was re‐isolated only from the inoculated pigs. Results suggest that the cause of the swine deaths in Vietnam is a multifactorial syndrome with PRRSV as a major factor.     

Development of universal and quadruplex real‐time RT‐PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses

Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA‐PRRSV2), highly pathogenic (HP‐PRRSV2) and NADC30‐like (NL‐PRRSV2)) are currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real‐time RT‐PCR assays using TaqMan‐MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real‐time RT‐PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real‐time RT‐PCR assays were further validated by comparing with a universal PRRSV conventional RT‐PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real‐time RT‐PCR assays and the conventional RT‐PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV‐positive, including 2 samples that were determined to be co‐infected with NL‐PRRSV2 and HP‐PRRSV2 isolates by the quadruplex real‐time RT‐PCR assay. ORF5 sequencing confirmed the real‐time RT‐PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA‐PRRSV2, NL‐PRRSV2 and HP‐PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.     

Evolutionary Dynamics of a Highly Pathogenic Type 2 Porcine Reproductive and Respiratory Syndrome Virus: Analyses of Envelope Protein‐Coding Genes

Porcine reproductive and respiratory syndrome virus (PRRSV) has long been an economically devastating swine viral disease. The recent emergence of a highly pathogenic type 2 PRRSV with high mobility and mortality in China, spreading in Vietnam, Laos, and Thailand has placed neighbouring countries at risk. This study applied a codon‐based extension of the Bayesian relaxed clock model and the fixed effects maximum‐likelihood method to investigate and compare the evolutionary dynamics of type 2 PRRSV for all of known structural envelope protein‐coding genes. By comparing the highly pathogenic type 2 PRRSV clade against the typical type 2 PRRSV clade, this study demonstrated that the highly pathogenic clade evolved at high rates in all of the known structural genes but did not display rapid evolutionary dynamics compared with typical type 2 PRRSV. In contrast, the ORF3, ORF5 and ORF6 genes of the highly pathogenic clade evolved in a qualitatively different manner from the genes of the typical clade. At the population level, several codons of the sequence elements that were involved in viral neutralization, as well as codons that were associated with in vitro attenuation/over‐attenuation, were predicted to be selected differentially between the typical clade and the highly pathogenic clade. The results of this study suggest that the multigenic factors of the envelope protein‐coding genes contribute to diversifying the biological properties (virulence, antigenicity, etc.) of the highly pathogenic clade compared with the typical clade of type 2 PRRSV.     

Similar frequency of Porcine circovirus 3 (PCV‐3) detection in serum samples of pigs affected by digestive or respiratory disorders and age‐matched clinically healthy pigs

Porcine circovirus 3 (PCV‐3) has been identified in pigs affected by different disease conditions, although its pathogenicity remains unclear. The objective of the present study was to assess the frequency of PCV‐3 infection in serum samples from animals suffering from post‐weaning respiratory or digestive disorders as well as in healthy animals. A total of 315 swine serum samples were analysed for PCV‐3 DNA detection by conventional PCR; positive samples were further assayed with a quantitative PCR and partially sequenced. Sera were obtained from 4 week‐ to 4 month‐old pigs clinically diagnosed with respiratory (n = 129) or digestive (n = 126) disorders. Serum samples of age‐matched healthy animals (n = 60) served as negative control. Pigs with clinical respiratory signs had a wide variety of pulmonary lesions including suppurative bronchopneumonia, interstitial pneumonia, fibrinous‐necrotizing pneumonia and/or pleuritis. Animals with enteric signs displayed histopathological findings like villus atrophy and fusion, catarrhal enteritis and/or catarrhal colitis. Overall, PCV‐3 DNA was detected in 19 out of 315 analysed samples (6.0%). Among the diseased animals, PCV‐3 was found in 6.2% (8 out of 129) and 5.6% (7 out of 126) of pigs with respiratory and digestive disorders, respectively. The frequency of PCV‐3 PCR positive samples among healthy pigs was 6.7% (4 out of 60). No apparent association was observed between PCR positive cases and any type of histopathological lesion. The phylogenetic analysis of the partial genome sequences obtained showed high identity among viruses from the three groups of animals studied. In conclusion, PCV‐3 was present in the serum of diseased and healthy pigs to similar percentages, suggesting that this virus does not seem to be causally associated with respiratory or enteric disorders.