Comparative pathogenicity of type 1 and type 2 isolates of porcine reproductive and respiratory syndrome virus (PRRSV) in a young pig infection model

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates are classified in two different genotypes, based on genomic heterogeneity: type 1, which comprises European type isolates, and type 2, which includes North American type isolates. It is believed that members of both genotypes differ in some biological properties including pathogenicity, however extensive studies comparing isolates of both genotypes have never been carried out. The objective of the present study was to compare the pathogenic properties of six different PRRSV isolates, three of type 1 and three of type 2, in a young pig infection model. For this purpose, a total of 105 3-week-old piglets were divided in 7 groups of 15 animals that were exposed on day 0 of the experiment to one of the six isolates tested or were mock infected (negative control group). Clinical signs and rectal temperatures were recorded daily and blood samples were taken on days 3, 6, 9, 12, 15, 18 and 21 of the experiment. On days 7, 14 and 21 post-inoculation five animals per group were sacrificed, macroscopic lung lesions were evaluated and different tissue samples were collected to determine viral organic distribution. The results obtained indicate that type 2 isolates are more pneumovirulent than type 1 isolates, as demonstrated by the recording of respiratory clinical signs only in pigs exposed to type 2 viruses and by the severity of macroscopic and microscopic lung lesions in those pigs. However, no clear differences could be established between genotypes in systemic clinical signs or viral load and viral distribution after challenge. These results support the general idea that type 2 isolates induce more severe respiratory disease than type 1 isolates.     

猪繁殖与呼吸综合征病毒河南分离株ORF5基因的克隆与变异分析

从河南郑州、新乡、周口不同地区猪场的急性病猪中分离到3株猪繁殖与呼吸综合征病毒(Porcine reproduc-tive and respiratory syndrome virus,PRRSV),分别命名为PRRSV Hn-1/06、PRRSV Hn-2/06和PRRSV Hn-3/06,细胞中和试验证实其血清型与美洲型一致。利用RT-PCR方法克隆了它们的ORF5基因,并对其基因序列和推导的氨基酸序列与7个不同来源的PRRSV毒株进行了同源性和亲缘关系比较分析,结果表明,3个分离株之间的ORF5基因及其推导的氨基酸均同源性均大于95.5%;与VR-2332标准北美洲株和疫苗株RespPRRS MLV间的同源性均低于与中国CH-1a毒株,而与流行的欧洲株间的同源性均低于54.5%。同时对推导氨基酸序列与6个北美洲型PRRSV株进行变异分析比较,证实其推导氨基酸序列发生了变异,特别是中和位点处第39位明显不同,表明河南省流行的PRRSV为北美洲型的变异株。     

猪繁殖与呼吸道综合征病毒(PRRSV)单克隆抗体的研制

利用猪繁殖与呼吸道综合征病毒（ＰＲＲＳＶ）国内分离株Ｊ１,采用反复差速离心法制备免疫抗原,长程免疫法免疫ＢＡＬＢ／ｃ小鼠,用间接ＥＬＩＳＡ方法检测抗体,通过细胞融合技术,并经３次亚克隆获得了１０株能稳定分泌抗ＰＲＲＳＶ单抗的杂交瘤细胞单克隆株（Ａ１Ｄ７Ｈ１０,Ａ１Ｄ７Ｈ１１,Ａ１Ｅ７Ｈ９,Ａ１Ｅ７Ｄ９,Ａ２Ｄ８Ｅ７,Ａ２Ｄ８Ｂ１１,Ｂ３Ｄ１１Ｄ６,Ｂ２Ｇ９Ａ９,Ｂ２Ｇ９Ｆ２）。这些细胞经体外连续传     

Effect of porcine circovirus type 2 (PCV2) vaccination on porcine reproductive and respiratory syndrome virus (PRRSV) and PCV2 coinfection

The objectives were to determine if PCV2 vaccination is effective in reducing disease and lesions associated with PRRSV and PCV2 coinfection and if there is a difference between intradermal (ID) and intramuscular (IM) route of PCV2 vaccination. Seventy-four, 21-day-old pigs were randomly allocated into one of six groups. On day 0, pigs were vaccinated with 2ml Suvaxyn((R)) PCV2 One Dose (Fort Dodge Animal Health, Inc.) by intramuscular (VAC-M-COINF) or intradermal (VAC-D-COINF) routes. On day 28, pigs were either singularly (PRRSV-only, PCV2-only) or coinfected (COINF) with PRRSV and PCV2. All pigs in all groups were necropsied on day 42. All vaccinated pigs seroconverted (IgM, IgG, and neutralizing antibodies) to PCV2 between 14 and 28 days post-vaccination. After challenge, all groups inoculated with PRRSV had reduced average daily gain compared to CONTROLS and PCV2-only (P<0.001). COINF pigs had significantly (P<0.05) reduced anti-PCV2-IgG antibody levels and neutralizing antibody levels compared to both vaccinated groups. COINF pigs had more severe lung lesions compared to VAC-M-COINF (P<0.05). COINF pigs had higher amounts of PCV2 DNA in serum samples and feces (P<0.05) and increased amounts of PCV2 in lymphoid tissues (P<0.05) compared to both vaccinated groups. In summary, PCV2 vaccination was effective at inducing a neutralizing antibody response and significantly reducing PCV2-associated lesions and PCV2 viremia in pigs coinfected with PCV2 and PRRSV. Differences between intradermal and intramuscular routes of vaccine administration were not observed.     

Synergism between porcine reproductive and respiratory syndrome virus (PRRSV) and Salmonella choleraesuis in swine

Porcine reproductive and respiratory syndrome virus (PRRSV) and Salmonella choleraesuis are two leading causes of economic loss in the swine industry. While respiratory disease is common in both S. choleraesuis and PRRSV infections, the factors that contribute to its development remain largely undefined. We investigated the interaction of PRRSV, S. choleraesuis, and stress in 5-week-old swine. All combinations of three factors (inoculation with S. choleraesuis on Day 0, PRRSV on Day 3, and treatment with dexamethasone on Days 3-7) were used to produce eight treatment groups in two independent trials. Fecal samples, tonsil and nasal swabs, serum samples and postmortem tissues were collected for bacteriologic and virologic examinations. No clinical signs were observed in pigs inoculated with only PRRSV or only S. choleraesuis. In contrast, pigs which were dually infected with S. choleraesuis and PRRSV exhibited unthriftiness, rough hair coats, dyspnea, and diarrhea. The pigs which received all three treatment factors were the most severely affected and 43% (three of seven) of the animals in this group died. Individuals in this group shed significantly higher quantities of S. choleraesuis in feces and had significantly higher serum PRRSV titers compared to other treatments (p < or = 0.05). In addition, S. choleraesuis and PRRSV were shed longer and by more pigs in this group than other groups and S. choleraesuis was recovered from more tissues in this group on Day 21 post inoculation. These results suggested that PRRSV, S. choleraesuis, and dexamethasone acted synergistically to produce a syndrome similar to that observed in the field.     

Genetics and geographical variation of porcine reproductive and respiratory syndrome virus (PRRSV) in Thailand

The Thai isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were obtained from the Chulalongkorn University-Veterinary Diagnostic Laboratory (CU-VDL). Virus isolation was confirmed by immunoperoxidase monolayer assay (IPMA) using SDOW-17. The virus genotype was determined using nested multiplex RT-PCR (nm RT-PCR) of ORF 1b. The nm RT-PCR was able to detect at least 10TCID50/ml of PRRSV. Of 137 Thai isolates, 66.42% belonged to the European (EU) genotype and 33.58% to the North American (US) genotype. ORF5 products of the eight US strains (00CS1, 01NP1, 01UD6, 02CB13, 02KK1, 02PB1, 02SP2 and 02SP3) and the six EU strains (01CB1, 01RB1, 02BR1, 02CB12, 02SB2 and 03RB1) were sequenced for genetic variation analysis. The US strains of the Thai isolates are clustered within the same group and are more closely related to the IAF-EXP91 from Canada (89-90% nucleotide identity), whereas the EU strains were very similar to the EU prototype, Lelystad virus (87-97.5% nucleotide identity). The ORF5 nucleotide identities within the US genotype tested in this study compared to the US prototype, VR-2332 varied from 83.7 to 85.2%, whereas 83.5-85.5% amino acid identities were found. Based on the phylogenetic tree, each pair of the Thai isolates (01NP1 and 02KK1, 00CS1 and 01UD6, and 01CB1 and 01RB1) was identical despite they were collected from different provinces. Therefore, there was no geographic influence on the spreading of PRRSV in Thailand. Interestingly, 02CB12 (EU genotype) shared over 99% similarity of the ORF5 nucleotide sequence and 98.6% of amino acid identity with the European vaccine, Porcillis (AF378819). However, modified live virus vaccines for PRRSV have not yet been used in the swine population in Thailand. The results suggested that both US and EU genotypes exist in Thailand, genetic variation does occur in both genotypes, and the sources of the viruses appear to be from Canada and Northern Europe, respectively. In addition, the spreading of PRRSV in Thailand might be due to introducing infected replacement pigs or infected semen into the farm.     

Replication characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) European subtype 1 (Lelystad) and subtype 3 (Lena) strains in nasal mucosa and cells of the monocytic lineage: indications for the use of new receptors of PRRSV (Lena)

Recently, it has been demonstrated that subtype 3 strains of European type porcine reproductive and respiratory syndrome virus (PRRSV) are more virulent/pathogenic than subtype 1 strains. This points to differences in the pathogenesis. In the present study, a new polarized nasal mucosa explant system was used to study the invasion of the low virulent subtype 1 PRRSV strain Lelystad (LV) and the highly virulent subtype 3 PRRSV strain Lena at the portal of entry. Different cell types of the monocytic lineage (alveolar macrophages (PAM), cultured blood monocytes and monocyte-derived dendritic cells (moDC)) were enclosed to examine replication kinetics of both strains in their putative target cells. At 0, 12, 24, 48 and 72 hours post inoculation (hpi), virus production was analyzed and the infected cells were quantified and identified. Lena replicated much more efficiently than LV in the nasal mucosa explants and to a lesser extent in PAM. Differences in replication were not found in monocytes and moDC. Confocal microscopy demonstrated that for LV, almost all viral antigen positive cells were CD163⁺Sialoadhesin (Sn)⁺, which were mainly located in the lamina propria of the respiratory mucosa. In Lena-infected nasal mucosa, CD163⁺Sn⁺, CD163⁺Sn^- and to a lesser extent CD163^-Sn^- monocytic subtypes were involved in infection. CD163⁺Sn^- cells were mostly located within or in the proximity of the epithelium. Our results show that, whereas LV replicates in a restricted subpopulation of CD163⁺Sn⁺ monocytic cells in the upper respiratory tract, Lena hijacks a broader range of subpopulations to spread within the mucosa. Replication in CD163⁺Sn^- cells suggests that an alternative entry receptor may contribute to the wider tropism of Lena.     

上海地区野鸟和家禽中PRRSV抗体的血清学调查

2005年3～5月，采用ELISA法对来自于上海地区的208份野鸟和357份家禽的血清样品进行了猪繁殖和呼吸综合征病毒（porcine reproductive andrespiratory syndrome virus，PRRSV）抗体的血清学调查。结果表明，受检的5种家禽存有不同程度的抗体阳性率，而野鸟的阳性样品集中于绿头鸭和麻雀。     

Terminology for classifying the porcine reproductive and respiratory syndrome virus (PRRSV) status of swine herds

Standardized terminology for the porcine reproductive and respiratory syndrome virus (PRRSV) status of swine herds is necessary to facilitate communication between veterinarians, swine producers, genetic companies, and other industry participants. It is also required for implementation of regional and national efforts towards PRRSV control and elimination. The purpose of this paper is to provide a herd classification system for describing the PRRSV status of herds, based upon a set of definitions reflecting the biology and ecology of PRRSV. The herd classification system was developed by a definitions committee formed jointly by the American Association of Swine Veterinarians (AASV) and the United States Department of Agriculture PRRS-Coordinated Agricultural Project, and was approved by the AASV Board of Directors on March 9, 2010. The committee included veterinarians from private practice and industry, researchers, and representatives from AASV and the National Pork Board. Breeding herds, with or without growing pigs on the same premises, are categorized as Positive Unstable (Category I), Positive Stable (Category II), Provisional Negative (Category III), or Negative (Category IV) on the basis of herd shedding and exposure status. Growing-pig herds are categorized as Positive or Negative. Recommended testing procedures and decision rules for herd classification are detailed.     

New insights into the genetic diversity of European porcine reproductive and respiratory syndrome virus (PRRSV)

The complete ORF5 sequences of 66 porcine reproductive and respiratory syndrome (PRRS) field virus strains (1991-2001) and three European modified live vaccine strains were determined, as well as ORFs 6 and 7 of 19 selected strains. The variability of the deduced ORF5 amino acid sequences was analysed using statistical process control (SPC), allowing for the objective assessment of variable and conserved regions. Four variable and four conserved regions as well as five hypervariable amino acid positions were defined. The effects of genetic variability on possible structural and functional properties were discussed with emphasis on immunogenic features. Phylogenetic analysis and pairwise comparison of the nucleotide sequences revealed that the genetic distances between the strains has greatly increased over time. The data do not support an evolutionary influence of the geographical location or the time of sample collection, nor of PRRSV vaccination on strain development. In contrast to other authors who tended to concentrate on the samples from either a common geographic origin or a short sampling period, we could not confirm geographically separate PRRSV clusters nor did we find evidence of positive selective pressure as measured by the ratio of synonymous to non-synonymous substitutions in ORF5, 6 or 7. Immunological implications and vaccination strategies are discussed.     

The effect of a killed porcine reproductive and respiratory syndrome virus (PRRSV) vaccine treatment on virus shedding in previously PRRSV infected pigs

Two experiments were conducted to investigate if virus shedding could be reduced following a killed porcine reproductive and respiratory syndrome virus (PRRSV) vaccination (KV) of PRRSV infected pigs. In experiment 1, PRRSV infected pigs were vaccinated with KV on days 14 and 28 following infection. Viremia and serum neutralizing (SN) antibody were compared to infected pigs with no KV. The second experiment was conducted in an identical manner. In addition to viremia and SN antibody, virus in oropharyngeal scrapings and interferon gamma (IFN-gamma) producing cells were monitored. Magnitude and duration of viremia were not different between KV vaccinated and non-vaccinated groups. No virus was detected in oropharyngeal scraping from any pig, nor was there a difference in the detection of viral RNA. In both experiments, however, increases in SN titer and number of IFN-gamma producing cells were observed. The SN titer was significantly higher in KV vaccinated groups than in non-vaccinated group on days 42 and 42-56 following infection in experiments 1 and 2, respectively. The number of IFN-gamma producing cells was slightly higher in KV vaccinated groups than in non-vaccinated group on days 42 and 63. These observations suggest that KV had no effect on virus shedding. However, previously infected pigs responded immunologically to KV, as demonstrated by increases in SN antibody titers and IFN-gamma producing cells.     

Interleukin-12 (IL-12) ameliorates the effects of porcine respiratory and reproductive syndrome virus (PRRSV) infection

Porcine respiratory and reproductive syndrome virus (PRRSV) disease, one of the most economically significant viral diseases in the swine industry, is characterized by miscarriages, premature farrowing, stillborn pigs, and respiratory disease associated with death and chronic poor performance of nursing and weaned pigs. Interleukin-12 (IL-12) is a key component in driving the development of cell-mediated immunity as well as stimulating interferon-gamma (IFN-gamma) production from T cells and natural killer cells. Although some studies have investigated the use of IL-12 as a vaccine adjuvant in swine, little is known about its effectiveness as a treatment against viral diseases in swine. The present study investigated whether recombinant porcine IL-12 (rpIL-12) enhances the immune response and thereby diminishes the effects of PRRSV infection in young pigs. Interestingly, in vitro experiments demonstrated that rpIL-12 is capable of inducing swine pulmonary alveolar macrophages (PAMs), the target cells of PRRSV, to produce IFN-gamma in a dose and time dependent manner. In addition, in vitro studies also revealed that rpIL-12 treatment was capable of significantly reducing PRRSV viral titers in PAMs. In vivo administration of rpIL-12 significantly decreased PRRSV titers in the lungs and blood of infected animals. Furthermore, treatment with rpIL-12 prevented significant growth retardation in PRRSV-infected animals. Finally, in response to viral antigen recall challenge, PAMs isolated from rpIL-12-treated/PRRSV-infected animals produced greater amounts of IFN-gamma and lesser amounts of interleukin-10 than PAMs isolated from non-rpIL-12-treated/PRRSV-infected animals. Taken together our data indicate that treatment with rpIL-12 may provide an effective approach to control or ameliorate PRRSV-induced disease in swine.     

Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged as an important cause of reproductive disorders and pneumonia in domestic pigs throughout the world. Acute cytocidal replication of PRRSV in alveolar lung macrophages causes the acute pneumonia; however, it remains largely unresolved whether there may also be a predisposition to longer-term local immunodeficiency in the PRRSV-convalescent lung. We applied various flow cytometric techniques to study the interplay between PRRSV replication and macrophage viability/function in pure cultures of porcine alveolar lung macrophages. Monitored by flow cytometric detection of intracellular PRRSV nucleocapsid protein, acute (24 h post infection) PRRSV replication did not impede the ability of alveolar macrophages to ingest fluorescently labelled Escherichia coli. At 48 h post infection, PRRSV-induced cytotoxicity (quantitated by flow analysis of cell size and membrane integrity) led to 40% reduction in the total number of phagocytozing cells. However, viable/uninfected macrophages in PRRSV-infected cultures exhibited normal phagocytic ability at 48 h, indicating that no soluble phagocytosis-suppressive mediators were induced by PRRSV infection in this system. In short, in our minimal system containing only a single cell type, phagocytosis-suppressive effects of PRRSV infection were detected, that acted at the culture level by reducing the total number of alveolar lung macrophages.     

Review: diagnostic methods for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) infections

This review describes methods that have been developed for the diagnose porcine reproductive and respiratory syndrome virus (PRRSV) infections. It summarizes the organs and tissues which should be sampled and the sampling times, and methods to detect viral RNA, viral antigens, and antibodies directed against PRRSV. The sensitivity, specificity, and limitations of the various tests are also described.     

反转录聚合酶链式反应检测猪繁殖与呼吸综合征持续性感染

猪繁殖与呼吸综合征感染的特点之一，是持续性的感染和病毒血症。本研究利用反转录滞酶链式反应检测了ＰＲＲＳＶ ＢＪ－４株单独感染ＳＰＦ仔猪和接种ＰＲＲＳＶ ＢＪ－４后再接种猪瘟疫苗不同时间的血清中病毒的存在，结果显示在感染２４h后的血甭样口 中就发现有病毒ＲＮＡ存在，病毒血症及少持续到感染后３７天，到５０天时已经消失，ＰＲＲＳＶ ＢＪ－４感染后再接种猪疫苗的仔猪的ＰＲＲＳＶ病毒血症没有受到影响。这些结果提供了ＰＲＲＳＶ持续感染的直接证据，解释了实际生产中通过引进临床正常但已经感染了猪繁殖与呼吸综合征病毒的猪群造成猪场内病毒的传和长期感染的存在，为采用合理的措施控制疾病提供了依据。     

检测猪繁殖与呼吸障碍综合征病毒抗体的两种ELISA方法的比较及其应用

采用检测猪繁殖与呼吸障碍综合征病毒(PRRSV)两种不同目标蛋白抗体的间接ELISA方法对来自10个规模猪场216份血清进行检测,结果显示,包被抗原分别为核蛋白(Nucleoprotein,N)和以GP5蛋白为主的膜蛋白(membrane pro-teins,G)的两种方法检测阳性率分别为83.8%和81.9%,符合率为92.6%,Kappa值为0.74,表明两种检测方法的检测结果在给定的临界点条件下抗体检测结果具有良好的一致性。通过数据标准化,调整阴阳性判定的临界点,发现两种检测方法所得出的阳性率变化趋势相似,但检测膜蛋白的G-ELISA抗体阳性率其降幅显著高于检测核蛋白的N-ELISA。本试验初步阐明了检测猪繁殖与呼吸障碍综合征病毒抗体的两种间接ELISA方法的特点,为科学使用提供依据。     

猪繁殖与呼吸综合征病毒与猪圆环病毒2型共感染猪组织中猪繁殖与呼吸综合征病毒抗原的分布

猪繁殖与呼吸综合征病毒(PRRSV)和猪圆环病毒2型(PCV2)同时感染猪50 d后,用免疫组化方法观察PRRSV抗原的分布.结果显示,单感染和共感染组在肺脏、脾脏、胸腺、扁桃体、颌下淋巴结、腹股沟淋巴结、肠系膜淋巴结、回肠和直肠中均可观察到PRRSV抗原阳性信号,且信号强度无显著差异;抗原信号的细胞分布范围也无差异,主要位于结缔组织的巨噬细胞、单核细胞、成纤维细胞、内皮细胞和淋巴细胞中.     

Effects of different us isolates of porcine reproductive and respiratory syndrome virus (PRRSV) on blood and bone marrow parameters of experimentally infected pigs

Seventy five-week-old, crossbred, caesarean-derived, colostrum-deprived pigs were randomly divided into five groups of 14 pigs and assigned one of five treatments: the intranasal inoculation of 1 (5.7) TCID50 of one of four plaque-purified isolates of porcine reproductive and respiratory syndrome virus (PRRSV) (VR2385, VR2431, ISU-984 and ISU-22), or uninfected cell culture and media. Haematological variables were measured for 21 days and bone marrow was analysed when the pigs were killed three, seven, 10, 21 or 28 days after the inoculation. The PRRSV-infected pigs had non-regenerative anaemia and markedly increased myeloid:erythroid ratios from three to 21 days after inoculation. There was a significant (P < 0.05) difference in the severity of the anaemia induced by the four PRRSV isolates; the most highly pneumovirulent strains (VR2385, ISU-984 and ISU-22) induced more severe anaemia than the least virulent isolate (VR2431). The anaemia induced by PRRSV was probably due to a direct or indirect effect on erythroid precursor cells in the bone marrow.     

Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV).

A double in situ hybridization method for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV) genomes in the same tissue section was applied to lung tissues from 9 pigs in which PRRSV and PCV coinfection had been previously demonstrated. Paraffin-embedded tissue sections were simultaneously hybridized with a digoxigenin-labeled antisense RNA probe for PRRSV and a fluorescein-labeled antisense RNA probe for PCV, and hybridization was detected with anti-digoxigenin alkaline phosphatase/fast red and anti-fluorescein peroxidase/diaminobenzidine, respectively. PRRSV and PCV genomes were identified in the same pulmonary cell types as reported previously in all 9 pigs. In all pigs, PCV-positive cells outnumbered PRRSV-positive cells. A small proportion of alveolar macrophages contained both PRRSV and PCV genomes.