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.     

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.     

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.     

Suppression of osteoblast toll‐like receptor 2 signaling by endothelin‐1

Peripheral endothelin‐1 (ET‐1) levels are increased in chronic systemic disorders such as congestive cardiac failure, diabetes and chronic renal failure. Bone infections are also associated with poor prognoses in these conditions. In the present study, we examined the alterations in Toll‐like receptor 2 (TLR2) signaling induced by ET‐1 in an in vitro osteoblast cell model. The TLR2‐positive murine osteoblast cell line MC3T3‐E1 was treated with heat‐killed Listeria monocytogenes (HKLM), a TLR2 ligand, in the presence or absence of ET‐1. We examined TLR2 expression, intranuclear NF‐κB phosphorylation and interleukin 6 (IL‐6) production. ET‐1 suppressed cell surface expression of TLR2, NF‐κB phosphorylation and IL‐6 production. As TLR2 represents an important mechanism by which osteoblasts recognize bacterial pathogens, a continuously elevated ET‐1 status may impair pathogenic recognition by osteoblasts and consequently affect bone metabolism during infections. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:910–914, 2014.     

Pathogenicity of porcine reproductive and respiratory syndrome virus (ORF5 RFLP 1‐7‐4 viruses) in China

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and respiratory problems in piglets. PRRSV infection leads to substantial pig mortality and causing huge economic losses so that disease outbreaks caused by the new PRRSV strain from other regions have caused great concern in China. In this study, we analysed the pathogenicity of the novel ORF5 RFLP 1‐7‐4‐like PRRSV strain, named PRRSV‐ZDXYL‐China‐2018‐1 in pigs. The viral challenge test showed that PRRSV‐ZDXYL‐China‐2018‐1 infection can cause persistent fever, moderate dyspnoea, serum viraemia and interstitial pneumonia in piglets. The levels of viral loads in serum and PRRSV‐specific antigen were also detected in lung tissues were used one‐step Taq‐Man RT‐qPCR and Immunohistochemistry, respectively. At 28dpi, the level of specific antibodies was increased among infected piglets. Importantly, the new virus appeared be a moderately virulent isolate with pathogenicity compared to HP‐PRRSV strain LQ (JXA1‐like strain). Histological examination revealed severe monocyte haemorrhage and interstitial pneumonia associated with monocyte infiltration in the lung tissue of pigs infected with PRRSV‐ZDXYL‐China‐2018‐1 and LQ‐JXA1 strains. Immunohistochemistry (IHC) results showed positive brown‐red epithelial cells and macrophages in pig lungs. Therefore, it is critical to establish an effective strategy to control the spread of PRRSV in China.     

Osteoblast‐Derived TGF‐β1 Stimulates IL‐8 Release Through AP‐1 and NF‐κB in Human Cancer Cells

Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)‐8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone‐derived growth factors on the IL‐8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast‐derived TGF‐β1 is associated with osteolytic bone diseases. Materials and Methods: IL‐8 mRNA levels were measured using RT‐PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF‐β1, BMP‐2, and IGF‐1. DNA affinity protein‐binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c‐fos, c‐jun, p65, and p50 to the IL‐8 promoter. A transient transfection protocol was used to examine IL‐8, NF‐κB, and activator protein (AP)‐1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL‐8, AP‐1, and NF‐κB promoter in human cancer cells. Osteoblasts were transfected with TGF‐β1, BMP‐2, or IGF‐1 small interfering RNA, and the medium was collected after 48 h. TGF‐β1 but not BMP‐2 or IGF‐1 siRNA inhibited OBCM‐induced IL‐8 release in human cancer cells. In addition, TGF‐β1 also directly induced IL‐8 release in human cancer cells. Activation of AP‐1 and NF‐κB DNA‐protein binding and MAPKs after TGF‐β1 treatment was shown, and TGF‐β1–induced IL‐8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF‐β1, BMP‐2, and IGF‐1. TGF‐β1 is the major contributor to the activation of extracellular signal‐related kinase (ERK), p38, and c‐Jun N‐terminal kinase (JNK), leading to the activation of AP‐1 and NF‐κB on the IL‐8 promoter and initiation of IL‐8 mRNA and protein release, thereby promoting osteoclastogenesis.     

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 .     

Sequence and phylogenetic analyses of Nsp2‐HVII,ORF5, and ORF7 coding regions of highly pathogenic porcine reproductive and respiratory syndrome virus from Myanmar

In this study, PRRSVs that spread during the outbreaks of 2011 in Myanmar were investigated. Sequences and phylogenetic analyses of the Nsp2 middle hypervariable region (Nsp2‐HVII) encoding gene, ORF5, and ORF7 showed that they belonged to the North American (NA) genotype and were clustered with HP‐PRRSV strains from other Southeast Asian countries. The discontinuous 30‐amino acid deletions at positions 481 and 533–561 were found in the Nsp2‐HVII of all Myanmarese PRRSVs, implying their derivation from HP‐PRRSV. The phylogenetic trees also showed that Myanmarese strains were in the same group as other Southeast Asian strains from Cambodia, Thailand, and Laos suggesting their close relationships. Conversely, Vietnamese 07QN was in the same group as Chinese JXA1. The unique amino acid mutations found only in Myanmarese PRRSVs were L292F, P431S, and V621M in Nsp2‐HVII and E170G in GP5, which may be used as a marker for monitoring genetic diversity of newly emerging HP‐PRRSV strains.