Genomic analysis of the differential response to experimental infection with porcine circovirus 2b

Porcine circovirus type 2 (PCV2) is the etiological agent of a group of associated diseases (PCVAD) that affect production efficiency and can lead to mortality. Using different crossbred lines of pigs, we analyzed host genetic variation of viral load, immune response and weight change following experimental infection with a PCV2b strain (n = 386). Pigs expressed variation in the magnitude and initiation of viremia and immune response recorded weekly until 28 days post‐infection. A higher viral load was correlated with weight gain (r = ?0.26, P < 0.0001) and presence of PCV2‐specific antibodies (IgM, r = 0.26–0.34, P < 0.0001; IgG, r = 0.17–0.20, P < 0.01). In genome‐wide association analyses of the responses at different time points, the proportions of phenotypic variation explained by combined effects of 56 433 SNPs were 34.8–59.4% for viremia, 10.1–59.5% for antibody response and 5.6–14.9% for weight change. Relationships between genomic prediction of overall viral load and weight gain during the first weeks of challenge were negative (?0.21 and ?0.24 respectively, P < 0.0001). Individuals that carried more favorable alleles across three SNPs on SSC9 (0.60 Mb) and SSC12 (6.8 and 18.2 Mb) partially explained this relationship, having lower viral load (P < 0.0001); lower viremia at day 14 (P < 0.0001), day 21 (P < 0.01) and day 28 (P < 0.05) and greater overall average daily gain during infection (ADG_i; P < 0.01), ADG_i at week 3 (P < 0.001) and week 4 (P < 0.01). These additive genetic relationships could lead to molecular solutions to improve animal health and reduce production costs.     

An experimental live chimeric porcine circovirus 1-2a vaccine decreases porcine circovirus 2b viremia when administered intramuscularly or orally in a porcine circovirus 2b and porcine reproductive and respiratory syndrome virus dual-challenge model

Commercially available inactivated vaccines against porcine circovirus type 2 (PCV2) have been shown to be effective in reducing PCV2 viremia. Live-attenuated, orally administered vaccines are widely used in the swine industry for several pathogens because of their ease of use yet they are not currently available for PCV2 and efficacy. The aims of this study were to determine the efficacy of a live-attenuated chimeric PCV2 vaccine in a dual-challenge model using PCV2b and porcine reproductive and respiratory syndrome virus (PRRSV) and to compare intramuscular (IM) and oral (PO) routes of vaccination. Eighty-three 2-week-old pigs were randomized into 12 treatment groups: four vaccinated IM, four vaccinated PO and four non-vaccinated (control) groups. Vaccination was performed at 3 weeks of age using a PCV1-2a live-attenuated vaccine followed by no challenge, or challenge with PCV2b, PRRSV or a combination of PCV2b and PRRSV at 7 weeks of age. IM administration of the vaccine elicited an anti-PCV2 antibody response between 14 and 28 days post vaccination, 21/28 of the pigs being seropositive prior to challenge. In contrast, the anti-PCV2 antibody response in PO vaccinated pigs was delayed, only 1/27 of the pigs being seropositive at challenge. At 21 days post challenge, PCV2 DNA loads were reduced by 80.4% in the IM vaccinated groups and by 29.6% in the PO vaccinated groups. PCV1-2a (vaccine) viremia was not identified in any of the pigs. Under the conditions of this study, the live attenuated PCV1-2a vaccine was safe and provided immune protection resulting in reduction of viremia. The IM route provided the most effective protection.     

Protective efficacy of a DNA vaccine encoding capsid protein of porcine circovirus‐like virus P1 against porcine circovirus 2 in mice

The capsid protein is the major immunogenic protein of porcine circovirus 2 (PCV2). The nucleotide sequence of porcine circovirus‐like virus P1 shares high homology with open reading frame (ORF) 2 of PCV2, and ORF1 of P1 encodes its structural protein. Mice were vaccinated twice intramuscularly with a plasmid expressing the P1 ORF1 protein (pcDNA3.1(+)‐ORF1) at 2‐week intervals. All animals vaccinated with pcDNA3.1(+)‐ORF1 developed higher specific anti‐P1 antibody levels, and had less PCV2 viremia and milder histopathological changes than PCV2‐challenged mice in the control group. Our results show that the P1 DNA vaccine elicited immune responses against PCV2 infection in a mouse model.

     

Protective immunity conferred by porcine circovirus 2 ORF2‐based DNA vaccine in mice

Post‐weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP‐N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP‐Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP‐Cap, LG‐strain vaccine, pEGFP‐N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP‐Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN‐γ and IL‐10), and a small PCV2 viral load. The mice treated with the pEGFP‐Cap and LG‐strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP‐Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS.     

2010–2015年华东地区猪圆环病毒2型的分子流行病学分析

【目的】猪圆环病毒2型(PCV2)是严重危害世界养猪业的重要传染病,即猪圆环病毒相关疾病(PCVAD)的重要病原,其致病机制及流行规律尚未阐明。本研究对2010–2015年间采集自中国华东地区的病料进行PCV2检测,以探讨中国华东地区PCV2的分子流行病学特征,分析PCV2的遗传演化规律。【方法】本研究利用PCR方法对384份断奶仔猪多系统衰竭综合征疑似发病猪样本进行检测,并对随机选取的42份阳性样品进行PCV2全基因组的测定和分析。【结果】华东地区普遍存在PCV2的感染,阳性率为41.15%。序列扩增获得42株PCV2全长基因组,同源性和系统进化树分析表明,基于PCV2 ORF2和全长核苷酸序列构建的系统进化树结构是基本一致的。从病料中测得的42株PCV2与11株参考毒株序列的ORF2基因的核苷酸和氨基酸序列同源性分别为87.0%–100.0%和82.5%–100.0%。遗传进化分析显示,53株PCV2毒株可分为4个基因型,即PCV2a、PCV2b、PCV2c和PCV2d。本文获得的42株序列ORF2基因的核苷酸和氨基酸序列同源性分别为89.6%–100.0%和86.8%–100%,...     

TNF‐α‐induced cardiomyocyte apoptosis contributes to cardiac dysfunction after coronary microembolization in mini‐pigs

This experimental study was designed to clarify the relationship between cardiomyocyte apoptosis and tumour necrosis factor‐alpha (TNF‐α) expression, and confirm the effect of TNF‐α on cardiac dysfunction after coronary microembolization (CME) in mini‐pigs. Nineteen mini‐pigs were divided into three groups: sham‐operation group (n = 5), CME group (n = 7) and adalimumab pre‐treatment group (n = 7; TNF‐α antibody, 2 mg/kg intracoronary injection before CME). Magnetic resonance imaging (3.0‐T) was performed at baseline, 6th hour and 1 week after procedure. Cardiomyocyte apoptosis was detected by cardiac‐TUNEL staining, and caspase‐3 and caspase‐8 were detected by RT‐PCR and immunohistochemistry. Furthermore, serum TNF‐α, IL‐6 and troponin T were analysed, while myocardial expressions of TNF‐α and IL‐6 were detected. Both TNF‐α expression (serum level and myocardial expression) and average number of apoptotic cardiomyocyte nuclei were significantly increased in CME group compared with the sham‐operation group. Six hours after CME, left ventricular end‐systolic volume (LVESV) was increased and the left ventricular ejection fraction (LVEF) was decreased in CME group. Pre‐treatment with adalimumab not only significantly improved LVEF after CME (6th hour: 54.9 ± 2.3% versus 50.4 ± 3.9%, P = 0.036; 1 week: 56.7 ± 4.2% versus 52.7 ± 2.9%, P = 0.041), but also suppressed cardiomyocyte apoptosis and the expression of caspase‐3 and caspase‐8. Meanwhile, the average number of apoptotic cardiomyocytes nuclei was inversely correlated with LVEF (r = ?0.535, P = 0.022). TNF‐α‐induced cardiomyocyte apoptosis is likely involved in cardiac dysfunction after CME. TNF‐α antibody therapy suppresses cardiomyocyte apoptosis and improves early cardiac function after CME.     

Enhancement of the immunogenicity of a porcine circovirus type 2 DNA vaccine by a recombinant plasmid coexpressing capsid protein and porcine interleukin‐6 in mice

The development of effective vaccines against porcine circovirus type 2 (PCV2) has been accepted as an important strategy in the prophylaxis of post‐weaning multisystemic wasting syndrome; a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, DNA vaccines usually induce weak immune responses. In this study, it was found that the efficacy of a DNA vaccine expressing Cap protein was improved by simultaneous expression of porcine IL‐6. A plasmid (pIRES‐ORF2/IL6) separately expressing both Cap protein and porcine IL‐6 was constructed and compared with another plasmid (pIRES‐ORF2) expressing Cap protein for its potential to induce PCV2‐specific immune responses. Mice were vaccinated i.m. twice at 3 week intervals and the induced humoral and cellular responses evaluated. All animals vaccinated with pIRES‐ORF2/IL6 and pIRES‐ORF2 developed specific anti‐PCV2 antibodies (according to enzyme‐linked immunosorbent assay) and a T lymphocyte proliferation response. The percentages of CD3⁺, CD3⁺CD8⁺, and CD3⁺CD4⁺ subgroups of peripheral blood T‐lymphocytes were significantly higher in mice immunized with pIRES‐ORF2/IL6 than in those that had received pIRES‐ORF2. After challenge with the virulent PCV2 Wuzhi isolate, mice vaccinated with pIRES‐ORF2/IL6 had significantly less viral replication than those vaccinated with pIRES‐ORF2, suggesting that the protective immunity induced by pIRES‐ORF2/IL6 is superior to that induced by pIRES‐ORF2.     

表达猪圆环病毒2型Cap蛋白的重组猪瘟兔化弱毒疫苗株的构建与鉴定

猪瘟(CSF)是由猪瘟病毒(CSFV)引起的一种毁灭性传染病,给养猪业造成重大经济损失。猪瘟兔化弱毒疫苗(C株)是一株非常安全、有效的优秀弱毒疫苗,对各年龄和品种的猪都极其安全,同时对不同基因亚型的CSFV均能提供有效的免疫保护。在现地,CSFV和猪圆环病毒2型(PCV2)混合感染的现象时常发生,有必要研制针对这两种病毒混合感染的二价疫苗。本研究首次构建了表达PCV2 Cap蛋白的重组C株,并评价了其在体内外的特性。结果表明,该重组病毒与C株具有相近的体外增殖特征,能够稳定表达Cap蛋白,在家兔体内具有与C株相似的生物学表型,在免疫家兔后10 d,抗CSFV E2抗体全部转阳,然而抗Cap抗体未能转阳。本研究为进一步优化表达PCV2Cap蛋白的重组C株奠定了基础。     

MicroRNA-98促进猪圆环病毒2型在3D4/21细胞中的复制

【目的】通过高通量测序的方法获得PCV2感染3D4/21细胞的miRNAs表达谱,并探讨miRNA-98在PCV2复制中的作用。【方法】本研究以猪肺泡巨噬细胞系3D4/21细胞为细胞模型,对PCV2感染过程中的3D4/21细胞进行miRNAs差异表达分析,筛选与病毒复制相关的特异性miRNAs,并探讨其在PCV2复制中的作用。【结果】经高通量测序,获得PCV2感染3D4/21细胞的miRNAs表达谱,结合实验室前期研究筛选获得miRNA-98。实验表明,miRNA-98的表达量随PCV2感染时间的延长而持续升高,其变化趋势与Cap蛋白表达变化基本一致,由此推测miRNA-98与PCV2复制正相关。过表达miRNA-98可显著上调Cap蛋白的表达量和PCV2的复制。进一步的研究表明,miRNA-98参与调节宿主免疫相关细胞因子的表达和PCV2的复制。【结论】miRNA-98可通过调节免疫相关细胞因子的表达调控宿主免疫功能,帮助PCV2逃逸宿主免疫,促进PCV2在3D4/21细胞中的复制。这些发现不仅为深入了解PCV2与宿主之间的关系提供了新视角,还有望为猪圆环病毒相关疾病的防控提供新的抗病毒策略。     

Genome‐wide association with delayed puberty in swine

An improvement in the proportion of gilts entering the herd that farrow a litter would increase overall herd performance and profitability. A significant proportion (10–30%) of gilts that enter the herd never farrow a litter; reproductive reasons account for approximately a third of gilt removals, with anestrous and failure to conceive the most common reasons for culling. Tools to select gilts for reproductive longevity through genomics or alternative phenotypes would be of great benefit to the producer. Ninety‐one gilts that failed to display behavioral estrus by 240 days (cases) and 127 pubertal littermates (controls) were genotyped with the Illumina Porcine SNP60 Beadchip. One hundred and seventy‐four SNPs with the most significant associations were genotyped in an additional 86 cases and 103 controls. Twelve of these associations were significant in the final analysis. The most significant (P < 1.5 × 10^?14) region associated with failure to attain puberty was on chromosome 4 surrounding the NHLH2 gene. Delayed pubertal development and age at first estrus have been associated with NHLH2 in mice. Because attainment of puberty is a complex trait, identifying genes that affect pubertal age would greatly contribute to our knowledge of reproductive development as well as overall fertility.     

猪圆环病毒2型感染对仔猪盲肠免疫功能与菌群的影响

【背景】肠炎是猪圆环病毒2型(porcine circovirus type 2,PCV2)感染猪的临床症状之一,其发生影响猪的生产性能。盲肠是单胃动物重要的消化器官,PCV2感染的影响值得探究。【目的】探究猪盲肠在PCV2感染后的免疫功能及菌群变化。【方法】将12头健康断奶仔猪随机分为对照组和感染组,每组6头,感染途径为口服和肌注,分别接种5mL,总接种量为10mL/头,对照组以同种方式接种PK15细胞培养物。分别检测在感染后56d(days post-infection,dpi)内血清中病毒载量、抗体动态及21dpi和56dpi盲肠的组织病理变化、病毒抗原含量、免疫功能及其内容物微生物菌群的变化。【结果】在21dpi,血清病毒核酸载量和抗体均达较高水平,PCV2抗原信号强,主要分布在盲肠黏膜上皮细胞和固有层中,盲肠分泌物SIgA含量显著降低,而T淋巴细胞增殖能力显著增高,感染猪盲肠上皮细胞严重脱落,肠腺萎缩,盲肠菌群多样性与丰度显著降低,有益菌属如弧菌属(Butyrivibrio)、瘤胃球菌属(Ruminococcaceae-NK4A214)显著降低,条件致病菌普雷沃氏菌属(Alloprevotella)显著增高;56dpi,病毒核酸载量降至7dpi水平,抗体持续较高水平,其他各项指标基本恢复到对照组水平。【结论】PCV2感染导致仔猪盲肠免疫功能紊乱,引起盲肠黏膜损伤,有益菌丰度降低、条件致病菌丰度增高,这些变化与病毒含量存在一定关系。     

载体表达的siRNA分子对猪圆环病毒2型复制的抑制作用

[目的]寻找一种基于RNA干扰技术的猪圆环病毒2型感染的防控方法.[方法]根据猪圆环病毒2型毒株基因组核苷酸序列,设计了3条特异性小干扰RNA(short interfering RNA,siRNA)分子,其中2条针对猪圆环病毒1型和2型复制酶基因(rep),1条针对猪圆环病毒2型核衣壳蛋白基因(cap),将合成的DNA片段退火形成双链,分别连接到RNAi-Ready pSIREN-RetroQ ZsGreen载体鼠源U6启动子下游,转化大肠杆菌得到阳性克隆,测序鉴定后分别命名为Retro-SH1,Retro-SH4,Retro-SH6.用上述质粒转染PCV2感染前、后的Dulac细胞及肌肉注射PCV2感染前、后的BALB/c小鼠,应用实时定量PCR试验评价其对病毒在细胞及小鼠体内复制的抑制作用,免疫组化法检测脾脏中病毒的存在.[结果]感染PCV2前或后转染500 ng Retro-SH1,Retro-SH4,Retro-SH6质粒能有效抑制PCV2在Dulac细胞上的复制,抑制率最高可达99%以上,对10株不同来源的临床分离株在细胞中复制的抑制作用同样明显,且不同毒株间差异不大.动物试验中,肌肉注射10μg上述不同siRNA分子对小鼠体内PCV2的复制有一定的抑制作用,其抑制率在26%至99%之间.[结论]载体表达的siRNA分子可能成为防控猪圆环病毒2型感染的一种新工具.     

猪圆环病毒2型双拷贝感染性DNA的构建及体外拯救

感染性分子克隆是研究病毒复制和致病机制的有力工具。本研究应用PCR诱变技术解决了外源片段易于自连的难题,成功将2个PCV2SD1株全基因组(DQ346683)头尾相接插入到真核生物表达载体pSK的多克隆位点中,构建重组质粒pSK-2PCV2;另外课题组成功构建含单个PCV2全基因组的pSK-PCV2和自身环化质粒ds-PCV2。将所得3种质粒分别转染无PCV污染的PK-15细胞系,经10次连续传代后,间接免疫荧光试验检测显示三者均在细胞核中聚集大量的病毒抗原;经RT-PCR检测都有PCV2特异性基因转录;透射电镜下可观察到直径约为17～20nm的典型PCV2病毒粒子;经测序鉴定所拯救出的病毒与亲本病毒核苷酸同源性为100%。拯救出的PCV2与亲本病毒具有相同的病毒学及分子生物学特性。本研究应用PCR诱变技术成功构建PCV2双拷贝感染性克隆,并经体外拯救证实其具有感染性,为进行PCV2分子特性及致病机理研究打下了基础。     

Genome‐wide association analysis of residual feed intake in Junmu No. 1 White pigs

Residual feed intake (RFI) is a measure of feed efficiency. Pigs with low RFI have reduced feed costs without compromising their growth. For marker‐assisted selection, it is helpful to identify genes or genetic markers associated with RFI in animals with improved feed efficiency at an early age. Using Illumina's PorcineSNP60 BeadChip, we performed a pilot genome‐wide association study of 217 Junmu No. 1 white male pigs phenotyped for RFI. Two‐step and one‐step methods were used separately to identify associated SNPs. Both methods obtained similar results. Twelve SNPs were identified as significantly associated with RFI at a Bonferroni adjusted P‐level < 9.7 × 10^?7, and 204 were found to have suggestive (moderately significant) association with RFI at P < 5 × 10^?5. NMBR, KCTD16, ASGR1, PRKCQ, PITRM1, TIAM1 and RND3 were identified as candidate genes for RFI.     

Genome‐wide association study identifies markers associated with meat ultimate pH in Duroc pigs

In this study we aimed to identify genomic regions associated with muscle pH, meat colour and water‐holding capacity in a population of 280 Italian Duroc pigs genotyped by the Illumina PorcineSNP60 v2 Genotyping BeadChip. After quality control, the remaining 32 597 SNPs and 278 subjects were used to perform a genome‐wide association study with the genabel package, using a kinship matrix in a model with the effects of sex, age and slaughter day. Bonferroni correction was applied, and the significant markers and regions were then further investigated to identify the nearest genes and the linkage disequilibrium (LD) between markers. Four markers (ASGA0082344, ASGA0095635, DBWU0000985 and CASI0005117) were significantly associated with ultimate pH (pH_u); no significant association was detected for the other traits. The four significant variants, located from 16.841 to 17.643 Mb on chromosome 3, were found within or close to the sequences of the sulfatase modifying factor 2 (SUMF2), lysine acetyltransferase 8 (KAT8), serine protease 8 (PRSS8) and phosphorylase kinase catalytic subunit gamma 2 (PHKG2) genes. The four associated markers lie in two LD blocks, suggesting that the observed effect is related to mutations located in two regions: the first one where SUMF2 is mapped and the second one where genes KAT8, PRSS8 and PHKG2 are located.