Pathological, ultrastructural, and immunohistochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (PEARS))

The pathogenicity and pathogenesis of Lelystad virus was studied in six 6-day-old SPF piglets. A third passage of the agent was propagated on porcine alveolar macrophages and intranasally inoculated into pigs. Pigs were killed at hours 24, 48, 60, and 72, and on days 6 and 8 after inoculation. From day 2 on pigs developed diffuse interstitial pneumonia with focal areas of catarrhal pneumonia, and from this day on splenic red pulp macrophages were enlarged and vacuolated. Lelystad virus was re-isolated from the lungs of infected pigs from day 2 after inoculation. Lelystad virus antigens were detected by immunohistochemical techniques in bronchiolar epithelium and alveolar cells, and in spleen cells of infected pigs from day 2 after inoculation. Ultrastructural examination of tissues by electron microscopy revealed degenerating alveolar macrophages and epithelial cells in lungs and nasal mucosa, with excessive vacuolation of the endoplasmic reticulum. Although the respiratory tract seems to be the target organ for this virus, macrophages in other organs, such as the spleen, can also be infected. This preference for macrophages may impair immunological defences.     

禽腺病毒的分离鉴定及LMH细胞适应毒的生物学特性研究

为了对采集自北京平谷某养殖场发病鸡中出现心包积液综合征症状的病料进行病毒分离和鉴定,将病料接种LMH细胞进行病毒分离,并在LMH上连续传代,取C5和C15代细胞毒分别接种3周龄SPF鸡进行致病性试验。根据分离病毒在LMH上的CPE特征、病毒血清中和试验、Hexon基因序列比对分析、SPF鸡致病性回归试验,表明从发病鸡病料中分离的一株病毒为血清4型禽腺病毒;病毒在LMH细胞盲传3代后,就能很好地适应细胞培养,出现禽腺病毒典型的CPE;培养至第10代,病毒在接种后72 h,病毒含量即可达到107.4TCID50/0.1m L的峰值滴度;接种细胞毒的SPF鸡出现4型禽腺病毒导致的典型临床症状和特异性剖检病理变化,两组接种鸡的死亡率分别为100%和70%。结果表明,研究分离到的4型禽腺病毒毒株对SPF鸡具有高致死率,但病毒经过细胞连续传代后对鸡的致死率降低。     

Studies on the multiplication of a porcine parvovirus

A porcine parvovirus has been characterized with regard to its replication in foetal porcine kidney cells and certain biophysical properties. Electron microscopy of infected cells at selected times postinfection revealed that porcine parvovirus replication took place within or near a series of granular intranuclear inclusions which may be contiguous with cellular heterochromatin. Developing virions were observed to aggregate into a nuclear-like amorphous mass which gradually disrupted as cellular integrity was lost. Purified virions were found to have a buoyant density in CsCl of 1.38 g/ml, while ‘empty’ particles has a buoyant density of 1.29 g/ml. The particle diameter was calculated to be approximately 22 nm.     

猪圆环病毒2型细胞培养适应毒株的培育和鉴定

从临床表现为仔猪断奶后多系统衰竭综合征（PMWS）淋巴组织病料，经聚合酶链式反应（PCR）证实为猪圆环病毒2型（PCV2）感染，采用无污染的猪肾细胞系（PK15）分离培养，并连续传代培育成一株细胞培养适应毒，命名为PCV2／LG株。分离毒株经细胞培养，于第25代后毒价显著升高，于第35代毒价可达10^5.6TCID 50/mL。采用免疫过氧化物酶单层细胞染色法（IPMA）、免疫电镜技术、分子克隆及核酸序列分析等鉴定表明，分离株感染细胞后病毒抗原主要分布在细胞核及细胞质中；病毒感染的阳性细胞呈散在分布，阳性细胞数可达50％以上；免疫电镜观察到与PCV2特异抗体结合形成的病毒免疫复合物呈实心小颗粒样粒子团，病毒粒子直径约为17nm；病毒抗原基因组由1768个核苷酸组成，与GenBank登录的8个PCV2基因组序列同源性达96．2％以上。用2mL的病毒细胞培养物（10^5.6TCID 50/mL）接种30日龄PCV2抗体阴性仔猪3头，可引起典型PMWS临床症状。本研究为进一步开展该病毒的致病性、疫苗免疫、诊断及分子生物学等研究奠定了基础。     

Comparative virulence of two porcine group-A rotavirus isolates in gnotobiotic pigs

The virulence of 2 porcine group-A rotavirus isolates was compared. Forty hysterotomy-derived 3-day-old gnotobiotic pigs were inoculated orally with 2 ml of intestinal homogenate containing either the Ohio State University (OSU) or the South Dakota State University (SDSU) strain of porcine rotavirus or were inoculated with medium only. Clinical signs of disease, body weight, distribution of viral antigen, fecal excretion of virus, and histologic lesions (observed by light and scanning electron microscopy) were determined. Morphometric measurements of villi and crypts were made. In pigs inoculated with OSU or SDSU strains, diarrhea began at postinoculation hours (PIH) 19 to 48 and PIH 24 to 54, respectively. None of the virus-infected pigs died as a consequence of infection and all had similar clinical signs of disease, body weight changes, and virus-shedding patterns, regardless of the strain of rotavirus with which they were infected. Microscopic findings in the small intestine of virus-infected pigs were similar, except that the SDSU strain caused more severe villus atrophy and villus fusion in the duodenum at PIH 72 and 168 than was associated with the OSU strain. Viral antigen in the small intestine of pigs infected with either virus was observed by use of immunofluorescence at PIH 24 and 72, but was seldom seen at PIH 168.     

Pathological,ultrastructural, and immunohistochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease (synonym: Porcine epidemic abortion and respiratory syndrome (PEARS))

Summary

The pathogenicity and pathogenesis of Lelystad virus was studied in six 6‐day‐old SPF piglets. A third passage of the agent was propagated on porcine alveolar macrophages and intranasally inoculated into pigs. Pigs were killed at hours 24, 48, 60, and 72, and on days 6 and 8 after inoculation. From day 2 on pigs developed diffuse interstitial pneumonia with focal areas of catarrhal pneumonia, and from this day on splenic red pulp macrophages were enlarged and vacuolated. Lelystad virus was re‐isolated from the lungs of infected pigs from day 2 after inoculation. Lelystad virus antigens were detected by immunohistochemical techniques in bronchiolar epithelium and alveolar cells, and in spleen cells of infected pigs from day 2 after inoculation. Ultrastructural examination of tissues by electron microscopy revealed degenerating alveolar macrophages and epithelial cells in lungs and nasal mucosa, with excessive vacuolation of the endoplasmic reticulum.

Although the respiratory tract seems to be the target organ for this virus, macrophages in other organs, such as the spleen, can also be infected. This preference for macrophages may impair immunological defences.     

Pseudorabies virus infections in explants of porcine nasal mucosa.

The spread of infection and the morphogenesis of three pseudorabies virus strains were studied in explants of porcine nasal mucosa. Virulent NIA-3 virus was compared with a deletion mutant 2.4N3A, and with a non-virulent Bartha virus. All three virus strains infected nasal epithelial cells. NIA-3 virus particles were enveloped mainly at the inner nuclear membrane; the virus rapidly invaded the stroma, causing widespread necrosis. In contrast, 2.4N3A virus particles were enveloped mainly at the endoplasmic reticulum and the infection spread more slowly. Bartha virus particles were enveloped mainly at the endoplasmic reticulum; the infection spread slowly and remained restricted to the epithelial cells. In situ DNA hybridisation showed an accumulation of Bartha virus DNA in the nucleus 24 hours after inoculation. In nasal mucosa viral virulence seemed directly related to the speed of replication and release of virus from infected cells.     

猪繁殖与呼吸综合征病毒在Marc-145细胞微载体上培养条件的研究

通过研究搅拌程序,细胞接种密度,微载体浓度与细胞生长的关系,探索Marc-145细胞在微载体上的生长条件,并测定猪繁殖与呼吸综合征病毒在微载体培养的细胞上的TCID50。结果表明:细胞接种密度为4.28×105 cells/mL时,病毒滴度可达到107.5TCID50,这说明利用微载体繁殖PRRSV是可行的,为猪繁殖与呼吸综合征疫苗的大规模生产奠定实验基础。     

Cell culture propagation of porcine rotavirus (reovirus-like agent).

Two isolates of porcine rotavirus (reovirus-like agent) were isolated and passaged in primary procine kidney cell cultures. Viral infectivity for cells was monitored by immunofluorescence because viral cytopathic effect was moderate. Successful passage of virus in cell culture required that viral suspensions obtained from infected cell cultures be treated with pancreatin prior to inoculation onto cell monolayers. Porcine rotavirus passage in cell culture also was accomplished, using trypsin treatments in lieu of pancreatin treatments. Porcine rotavirus passaged 10 times in cell culture infected gnotobiotic pigs and caused diarrhea. Gnotobiotic pigs that recovered from this infection were resistant to challenge exposure with porcine rotavirus but were susceptible to challenge exposure with transmissible gastroenteritis virus. As determined by immunofluorescent cross reactions, porcine rotavirus was found to be antigenically related to the human and bovine rotaviruses but not to reovirus type 3 or to transmissible gastroenteritis virus.     

腺病毒与慢病毒感染猪原代细胞的比较研究

本实验旨在探究腺病毒和慢病毒侵染猪原代细胞的最佳感染复数(MOI)和侵染时间,确定较优的侵染方式。实验选择体外分离培养猪骨骼肌卫星细胞、前体脂肪细胞和骨髓间充质干细胞后,分别用腺病毒(MOI=0、50、100、200、500、1 500)和慢病毒(MOI=0、30、50、80、100、300)感染细胞,每隔24 h在荧光显微镜下观察记录细胞中绿色荧光蛋白的表达情况。结果表明:当腺病毒MOI值为500、慢病毒MOI值为80,侵染4 d后,3种细胞荧光强度均较强且细胞形态完好。相比较而言,腺病毒能快速高效侵染猪骨骼肌卫星细胞;而对于猪前体脂肪细胞和猪骨髓间充质干细胞,2种病毒侵染速度相近,但慢病毒的侵染效率和荧光强度更高。因此,对于猪骨骼肌卫星细胞,选取腺病毒作为外源基因载体更为合适;对于猪前体脂肪细胞和猪骨髓间充质干细胞,慢病毒的侵染效果则更好。     

Comparison of the virulence of two isolates of porcine parvovirus in 72-day-old porcine fetuses.

Two strains of porcine parvovirus (PPV), designated Kresse and NADL-8, were compared for relative virulence in porcine fetuses. Strain Kresse was injected into the amniotic fluid of all fetuses of 1 uterine horn of each of 2 pregnant gilts at 72 days of gestation. Strain NADL-8 was administered similarly to fetuses of 4 other gilts at the same stage of gestation. All gilts were killed and necropsied 35 days later. Selected tissues of all fetuses were tested for infectious virus and viral antigen. Sera from live fetuses were tested for antibody to PPV. These tests confirmed that most fetuses exposed to PPV by intra-amniotic injection became infected. All of 11 fetuses exposed to strain Kresse by intra-amniotic injection were alive at the time of necropsy, and all appeared clinically normal. In contrast, 8 of 24 fetuses exposed similarly to strain NADL-8 were dead. Many of the fetuses from the uterine horns contralateral to the uterine horns inoculated with virus were infected after 72 days of gestational age by intrauterine spread of the virus. Four such fetuses, 3 infected with the NADL-8 strain and 1 infected with the Kresse strain, were dead at the time of necropsy. These findings were inconsistent with those of a previous report, which indicated that the Kresse strain of PPV was markedly more virulent than the NADL-8 strain of PPV for porcine fetuses. A possible reason for this apparent discrepancy is discussed.     

Effect of tilmicosin on chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages

OBJECTIVE: To evaluate chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages (AM) exposed to tilmicosin. ANIMALS: 12 healthy calves and 12 healthy pigs. PROCEDURES: Lungs were obtained immediately after euthanasia; AM were collected by means of bronchoalveolar lavage and density gradient centrifugation. Chemotactic activity was evaluated by exposing AM to lipopolysaccharide or macrophage inhibitory peptide during incubation with tilmicosin. Phagocytic activity was evaluated by incubating AM with tilmicosin for 24 hours and then with tilmicosin-resistant Salmonella serotype Typhimurium. Bactericidal activity was evaluated by incubating AM with tilmicosin (0, 10, or 20 microg/ml for bovine AM; 0 or 10 microg/ml or 10 microg/ml but washed free of tilmicosin for porcine AM) and then with Mannheimia haemolytica (bovine AM) or with Actinobacillus pleuropneumoniae or Pasteurella multocida (porcine AM). RESULTS: Tilmicosin had no significant effects on chemotactic or phagocytic activities of bovine or porcine AM. The time-course of bactericidal activity was best described by polynomial equations. Time to cessation of bacterial growth and area under the time versus bacterial number curve were significantly affected by incubation of AM with tilmicosin. CONCLUSIONS AND CLINICAL RELEVANCE: Results show that bactericidal activity of bovine and porcine AM was enhanced by tilmicosin, but not in proportion to the reported ability of AM to concentrate tilmicosin intracellularly. With or without exposure to tilmicosin, the time-course of bactericidal activity of bovine AM against M haemolytica and of porcine AM against A pleuropneumoniae or P multocida was too complex to be reduced to a simple linear equation.     

Preparing hemagglutinating antigen from isolates of infectious bronchitis virus

The hemagglutinating (HA) activity of 14 strains of infectious bronchitis virus (IBV) was investigated. The optimal conditions for IBV antigen preparation include inoculation of 10- or 11-day-old specific pathogen-free embryonated eggs and incubation for 30 hours at 37 C. Embryos were inoculated via the allantoic cavity with 0.1 ml of a low embryonic passage of the virus (10(7) to 10(8) EID50/ml). Allantoic fluid was harvested and pooled, and a 100-fold concentration of virus particles was achieved by centrifugation for 3 hours at 30,000 x g. Virus pellets were resuspended in Tris-hydrochloride buffer containing 3 units of phospholipase-C (type-1) enzyme/ml and incubated for 2 hours at 37 C. All IBV strains tested demonstrated positive HA activity with chicken red blood cells. The antigen was stored in liquid state or lyophilized at 4 C.     

The interferon sensitivity of selected porcine viruses.

The objective of this study was to compare the sensitivity of 11 porcine viruses to the antiviral effects of porcine interferon-alpha in serum from piglets which had been infected 19 h previously with transmissible gastroenteritis virus, and of porcine interferon-beta prepared in PK-15 cells by induction with polyinosinic:polycytidylic acid, in yield reduction assays in pig kidney cells which were treated with interferon before virus challenge, and both before and after virus challenge. The most sensitive virus to both types of interferon was vesicular stomatitis. A porcine isolate of bovine herpesvirus type 1, hemagglutinating encephalomyelitis virus and porcine enterovirus types 1 and 2 were also highly sensitive to interferon-alpha. There was little reduction in the yield of porcine parvovirus or porcine rotavirus, while swinepox, swine influenza and transmissible gastroenteritis viruses were intermediate in their sensitivity to interferon-alpha. In addition to vesicular stomatitis virus, porcine adenovirus type 3, swine influenza, hemagglutinating encephalomyelitis and porcine rotavirus were highly sensitive to interferon-beta, while swinepox, bovine herpesvirus type 1, porcine parvovirus, transmissible gastroenteritis and porcine enteroviruses were less sensitive than the above viruses to interferon-beta, although all showed significant reductions in virus yield.     

猪捷申病毒TaqMan实时定量RT-PCR方法的建立

为建立猪捷申病毒(PTV)的早期检测及定量分析方法,本研究基于PTV 11个血清型基因组5′端非编码区保守序列,设计引物和TaqMan探针,建立了检测PTV的TaqMan实时定量RT-PCR方法.应用该方法对PTV、猪细小病毒、猪繁殖与呼吸综合症病毒、猪圆环病毒2型、猪伪狂犬病毒以及猪瘟病毒进行特异性试验,结果除PTV为阳性外其它均为阴性;针对PTV最低可检测到10个拷贝;批内、批间重复试验的变异系数均小于3％.应用建立的方法与病毒分离方法分别对91份临床样品进行检测,检出率分别为79.12％和57.14％,两者的符合率是78.02％.经临床应用表明,该实时定量RT-PCR方法可为PTV的早期诊断及定量分析提供技术手段.     

表达猪流行性腹泻病毒S基因片段重组腺病毒的构建与免疫特性

利用RT-PCR、同源重组等技术构建了含有猪流行性腹泻病毒(PEDV)S基因上3个片段的重组腺病毒质粒pAd-S98～638,pAd-S1～638,pAd-S49838～1384,经PacI酶切后转染HEK-293A细胞,3次噬宽纯化分别获得了重组腺病毒rAd-s498～638,rAd-S1～638,rAd-S498～1384.重组腺病毒于HEK-293A细胞连续传代至20代效价稳定,TCID50分别为每毫升10-6.29、10-5.75、10-6.5.应用猪流行性腹泻病毒阳性血清进行间接免疫荧光抗体试验,在3个腺病毒感染的HEK-293A细胞的胞质见有清晰荧光.将3个重组腺病毒分别免疫小鼠,结果均诱导产生较强的体液免疫应答.结果表明这3个重组腺病毒对S基因的3个片段均可成功表达,并具有较好的免疫原性,为PEDV重组腺病毒活载体疫苗的研究奠定了基础.     

过表达MAT2A基因促进猪肌内脂肪细胞分化的研究

本研究通过构建腺病毒介导的体外超表达载体，探究腺苷甲硫氨酸转移酶2A（methionine adenosyltransferase 2A，MAT2A）基因在猪肌内脂肪细胞分化中的作用。根据GenBank中猪MAT2A基因mRNA序列（登录号：NM_001167650.1）设计引物，提取猪脂肪组织细胞总RNA并反转录获得cDNA，以此为模板进行PCR扩增并连接到pAdTrack-CMV腺病毒穿梭载体中，对重组质粒pAd-MAT2A进行测序鉴定；pAd-MAT2A载体经PacⅠ限制酶酶切线性化，经质粒大片段回收纯化后转染293A细胞进行病毒包装；采用实时荧光定量PCR检测MAT2A基因表达情况，并提取蛋白进行Western blotting分析，取分化第8天的细胞进行油红O染色。结果表明，穿梭载体pAdTrack-CMV-MAT2A构建成功，并能与骨架载体pAdEasy-1实现同源重组；腺病毒载体pAd-MAT2A转染293A细胞后，病毒滴度达到1E+6 PFU/mL，可满足侵染猪肌内脂肪细胞的需要。实时荧光定量PCR和Western blotting结果显示，MAT2A基因mRNA和蛋白水平均显著上调。油红O染色结果显示，过表达MAT2A基因可促进猪肌内脂肪细胞内脂滴聚积。结果表明，腺病毒介导的MAT2A基因过表达在猪肌内脂肪细胞中呈上调趋势，MAT2A基因可促进脂质积累。     

Survival of viruses in fermented edible waste material

The survival of selected viruses in fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Seven viruses, including pseudorabies, Newcastle disease, infectious canine hepatitis, avian infectious bronchitis, measles, vesicular stomatitis, and a porcine picornavirus were inoculated into a mixture of ground food waste (collected from a school lung program) containing Lactobacillus acidophilus. Mixtures were incubated at 5 C, 10 C, 20 C, and 30 C for 96 hours. Temperature, pH, and redox potential were monitored. Samples for virus isolation were obtained daily. Newcastle disease virus and infectious canine hepatitis virus survived the entire test period. The porcine picornavirus was inactivated at 30 C after 74 hours, but survived for the entire test period at the other temperatures. Pseudorabies virus was inactivated at 20 C and 30 C within 24 hours, but survived for 48 hours at 10 C and 96 hours at 5 C. Avian infectious bronchitis virus was inactivated at 20 C and 30 C within 24 hours, but survived 72 hours at 5 C and 10 C. Measles and vesicular stomatitis viruses were rapidly inactivated at all 4 temperatures.     

Retention of ingested porcine reproductive and respiratory syndrome virus in houseflies

OBJECTIVE: To evaluate retention of porcine reproductive and respiratory syndrome virus (PRRSV) in houseflies for various time frames and temperatures. SAMPLE POPULATION: Fifteen 2-week-old pigs, two 10-week-old pigs, and laboratory-cultivated houseflies. PROCEDURE: In an initial experiment, houseflies were exposed to PRRSV; housed at 15 degrees, 20 degrees, 25 degrees, and 30 degrees C; and tested at various time points. In a second experiment to determine dynamics of virus retention, houseflies were exposed to PRRSV and housed under controlled field conditions for 48 hours. Changes in the percentage of PRRSV-positive flies and virus load per fly were assessed over time, and detection of infective virus at 48 hours after exposure was measured. Finally, in a third experiment, virus loads were measured in houseflies allowed to feed on blood, oropharyngeal washings, and nasal washings obtained from experimentally infected pigs. RESULTS: In experiment 1, PRRSV retention in houseflies was proportional to temperature. In the second experiment, the percentage of PRRSV-positive houseflies and virus load per fly decreased over time; however, infective PRRSV was found in houseflies 48 hours after exposure. In experiment 3, PRRSV was detected in houseflies allowed to feed on all 3 porcine body fluids. CONCLUSIONS AND CLINICAL RELEVANCE: For the conditions of this study, houseflies did not support PRRSV replication. Therefore, retention of PRRSV in houseflies appears to be a function of initial virus load after ingestion and environmental temperature. These factors may impact the risk of insect-borne spread of PRRSV among farms.