Localization of classical swine fever virus from chronically infected pigs by in situ hybridization and immunohistochemistry

Classical swine fever (CSF) virus (CSFV) nucleic acid and antigen were detected in 15 pigs with naturally occurring chronic CSF by in situ hybridization and immunohistochemistry. The most consistent and prominent microscopic lesions were perivascular mononuclear cell infiltration and gliosis in the central nervous system of pigs with chronic CSF. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the cytoplasm without background staining. A positive signal for both in situ hybridization and immunohistochemistry was detected in mononuclear cells and lymphocytes of lymphoid tissues. Viral nucleic acid was detected in some tissue sections in the absence of viral antigen. The in situ hybridization technique developed in this study was useful for the detection of CSFV RNA in tissues taken from chronically infected pigs and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.     

Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (PCV) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of PCV (which recognises both PCV-1 and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twenty-eight of the 30 Spanish pigs and all three American pigs had PCV in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyer's patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that PCV is present in tissues of almost all pigs affected by PDNS, and PCV has to be considered as a possible agent involved in the pathogenesis of the syndrome.     

Detection of in ovo-inoculated infectious bronchitis virus by immunohistochemistry and in situ hybridization with a riboprobe in epithelial cells of the lung and cloacal bursa

In situ hybridization and immunohistochemistry were utilized to identify tissues infected in ovo with infectious bronchitis virus (IBV). Chicken embryos were inoculated in ovo (chorioallantoic sac) with the Arkansas (Ark) serotype of IBV at 18 days of age. At 24, 48, 72, and 120 hr postinfection (HPI), bursa, lung, spleen, heart, and thymus were collected, fixed in 10% neutral buffered formalin, and paraffin embedded. The digoxigenin-labeled antisense S1 riboprobe detected viral mRNA in the cytoplasm of respiratory epithelial cells in the primary bronchus at 24, 48, and 72 HPI. Viral mRNA was detected in bursa samples collected at 48 hr. Immunohistochemistry detected viral antigens in epithelial cells of the parabronchi and bursal tissues at 24 and 48 hr, respectively. No viral mRNA or antigen was detected by in situ hybridization or immunohistochemistry, respectively, in heart, thymus, or spleen at any time after inoculation. On the basis of these data, IBV apparently initially infects lung tissue, then migrates to and infects cells of the bursa. These results indicate that in situ hybridization can be useful in detection of IBV-infected chickens and in understanding the pathogenesis and virulence of IBV infection.     

A pathogenesis study of foot-and-mouth disease in cattle, using in situ hybridization.

Eight calves were exposed in an aerosol chamber to nebulized foot-and-mouth disease virus. Two control animals were exposed in a similar manner to cell culture media only. Animals were euthanized at intervals and various tissues examined by in situ hybridization using a biotinylated RNA probe corresponding to a portion of the viral gene coding for the polymerase enzyme. By this technique large amounts of viral nucleic acid were found in coronary band, interdigital cleft and tongue as early as six hours postexposure, indicating a very rapid delivery from the portal of entry to the predilection sites for lesion development. This occurred well before the onset of viremia which by virus isolation was not detectable until 30 hours postexposure. The in situ hybridization signal in these tissues decreased in intensity and extent with time to focally positive areas, occasionally surrounding a vesicle. Other epidermal sites not normally thought of as sites for foot-and-mouth lesion development, such as carpus and eyelid, also had some viral nucleic acid detectable at various time intervals. In the lung by in situ hybridization, alveolar septa had viral nucleic acid early in infection (6-18 h postexposure) while later (36-96 h postexposure), the in situ hybridization signal was prominent in alveolar macrophages.     

Diagnostic investigation of chronic porcine reproductive and respiratory syndrome virus in a breeding herd of pigs

Forty-five sows and 15 boars were selected at random from a breeding herd known to be chronically infected with porcine reproductive and respiratory syndrome virus (PRRSV) and lymphoid, immune-privileged, and non-lymphoid/non-immune-privileged tissues were tested for the presence of the virus by PCR, virus isolation, and immunohistochemistry. The virus was isolated from the lateral retropharyngeal lymph node of one sow; the isolate was nucleic acid sequenced and determined to be of field origin, and it was inoculated into two PRRSV-naive pregnant sows (A and B) at 95 days of gestation. They were necropsied 14 days later and samples of maternal and fetal tissue and blood samples were collected. Sow A had 10 fresh, six partially autolysed, and two mummified fetuses, and sow B had six fresh and viable fetuses. Viral nucleic acid was detected by PCR in tissue pools from each sow and also from pooled fetal tissues, and the virus was isolated from fetal pools from sow A.     

寡核苷酸探针原位检测石蜡切片中PRRSV核酸

根据GenBank收录的美洲型猪繁殖与呼吸综合征病毒（PRRSV）ATCCVR-2332株ORF6和ORF7基因序列，用O1igo软件设计并合成大小为37bp的寡核苷酸探针，经生物素标记后，成功建立了原住检测石蜡组织切片中PRRSV核酸的方法。该探针能检测到56PgPRRSV核酸的RT—PCR产物DNA，能特异检测出PRRSV核酸及其PCR产物，而对猪瘟病毒（HCV）、猪细小病毒（PPV）、猪伪狂犬病病毒（PRV）、猪乙脑病毒（JEV）的核酸呈阴性反应。应用该方法检测PRRSVSC-1株人工感染的28日龄仔猪，在感染后7d即可在肺脏、肾脏、扁桃体、胸腺、肺门淋巴结、十二指肠和大脑检测到PRRSV核酸。该法可用于仔猪PRRSV感染的诊断和组织中核酸的定位及分布研究，也可用于甲醛固定组织的回顾性诊断。     

Detection of infectious bursal disease virus in experimentally infected chickens by in situ hybridization

In situ hybridization was used in a pathogenesis study of three vaccine pathotypes (Delaware variant A, D78, and BursaVac) of infectious bursal disease virus (IBDV). Tissues were excised (bursa, thymus, spleen, proventriculus, and cecal tonsils), fixed in formalin, and paraffin embedded at 12, 24, 48, 72, and 120 hr postinoculation (HPI). With an antisense VP2 gene probe, viral nucleic acid was detected in bursas from both D78- and BursaVac-infected chickens at 24, 48, 72, and 120 HPI. However, viral RNA was detected only in the Delaware variant A-infected birds at 72 HPI. Thymus and spleen were positive in the D78-infected birds at 48 HPI and in the BursaVac-inoculated group at 72 HPI. Viral nucleic acid was not present in detectable levels among any of the tissues tested at 12 HPI. However, by 24 hr, scattered positive lymphoid cells were visualized in the bursal follicles of chickens infected with D78 and BursaVac. In addition, low levels of viral nucleic acids were detected in the thymus and spleen among the D78- and BursaVac-infected birds. The sites of viral replication were consistent between the two vaccine-infected groups (D78 and BursaVac), whereas the chickens infected with Delaware variant A had limited IBDV replication in the bursa.     

Evidence for the localization of porcine reproductive and respiratory syndrome virus (PRRSV) antigen and RNA in ovarian follicles in gilts

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) infection in ovary was studied in sexually mature, cycling, nonsynchronized gilts infected with the PRRSV 16244B, a virulent field strain. Previous studies have shown that PRRSV can be isolated from ovaries and is transplacentally passed from gilts to the fetuses. The cause of infertility following PRRSV infection is not known. In this study, we identified the tropism of PRRSV in ovarian tissue from experimentally infected gilts in samples collected between 7 and 21 days postinfection (DPI). Tissues were collected and examined by virus isolation, in situ hybridization (ISH), immunohistochemistry (IHC), and double labeling to identify PRRSV-infected cell types. PRRSV was isolated in ovarian follicles at 7 days DPI. The IHC and ISH indicated that PRRSV-positive cells in ovaries were predominantly macrophages, which were numerous in atretic follicles. No evidence of infection and/or perpetuation of PRRSV in ova was observed, indicating that the female gonad is an unlikely site of persistence. No alteration of the normal ovarian architecture that would support a possible role of PRRSV infection in porcine female infertility was observed.     

Pathogenesis of Newcastle disease in chickens experimentally infected with viruses of different virulence

Groups of 4-week-old White Rock chickens were inoculated intraconjunctivally with nine isolates of Newcastle disease virus representing all pathotypes. Birds were monitored clinically and euthanatized sequentially, with collection of tissues for histopathologic examination and in situ hybridization using an anti-sense digoxigenin-labeled riboprobe corresponding to the sequence of the gene coding for the matrix protein. Disease was most severe with velogenic viscerotropic pathotypes and was characterized by acute systemic illness with extensive necrosis of lymphoid areas in the spleen and intestine. Viral nucleic acid was detected in multiple tissues but most prominently in macrophages associated with lymphoid tissue. Velogenic neurotropic isolates caused central nervous system disease despite minimal amounts of viral nucleic acid detected in neural tissue. Mesogenic and lentogenic pathotypes caused no overt disease; however, viral nucleic acid was present in myocardium and air sac epithelium following infection with these isolates. Compromise of air sac and myocardium may predispose mesogen- and lentogen-infected chickens to secondary infection and/or decreased meat and egg production.     

Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves.

Five diagnostic techniques performed on skin biopsies (shoulder region) and/or serum were compared for detection of bovine viral diarrhea virus infection in 224 calves 0-3 months of age, 23 calves older than 3 months but younger than 7 months, and 11 cattle older than 7 months. The diagnostic methods used were immunohistochemistry (IHC), 2 commercial antigen ELISAs, 1 commercial antibody ELISA, and real-time RT-PCR. Results of 249 out of 258 skin and serum samples were identical and correlated within the 3 antigen detection methods and the real-time RT-PCR used. Twenty-six of these 249 samples were BVDV-positive with all antigen detection methods and the real-time RT-PCR. Nine out of 258 samples yielding discordant results were additionally examined by RT-PCR, RT-PCR Reamplification (ReA), and antigen ELISA I on serum and by immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies. Virus isolation and genotyping was performed as well on these discordant samples. In 3 cases, transiently infected animals were identified. Two samples positive by real-time RT-PCR were interpreted as false positive and were ascribed to cross-contamination. The antigen ELISA II failed to detect 2 BVDV-positive calves due to the presence of maternal antibodies; the cause of 2 false-positive cases in this ELISA remained undetermined. Only persistently infected animals were identified in skin samples by IHC or antigen ELISA I. The 3 antigen detection methods and the real-time RT-PCR used in parallel had a high correlation rate (96.5%) and similar sensitivity and specificity values.     

Detection of a macrophage-specific antigen and the production of interferon gamma in chickens infected with Newcastle disease virus

Formalin-fixed, paraffin-embedded spleen and intestinal tissues were harvested at 2 days postinfection from 4-wk-old white rock chickens infected with five different strains of Newcastle disease virus (NDV). These tissues were examined for the presence of macrophage antigen expression, virus replication, and interferon gamma (IFN gamma) production. The five strains represented all three NDV pathotypes. Viral replication and IFN gamma, as determined by riboprobe in situ hybridization, were detected only in those chickens infected with velogenic viscerotropic NDV (VVNDV) strains. Macrophage antigen expression, an indicator of macrophage activation, was determined by immunohistochemistry with a macrophage-specific antibody, CVI-ChNL-68.1. Presence of macrophage antigen was most prominent in VVNDV-infected chickens. The distribution of this antigen within tissues was far more diffuse than the staining for viral mRNA. The presence of IFN gamma mRNA was detected in the spleen and intestinal lymphoid tissue of VVNDV-infected chickens. There was also increased macrophage antigen expression in the mesogen-infected birds, but it was less dramatic than in tissues from VVNDV-infected chickens. One of two lentogen-infected birds had evidence of increased macrophage antigen expression only in the spleen.     

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus)

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.     

禽流感病毒几种RT-PCR诊断技术

目前常采用琼脂扩散或间接 EL ISA监测禽流感的抗体水平 ,而针对抗原的检测一般采用病毒分离鉴定 ,这种方法需要的时间较长 ,不能对高致病禽流感做出迅速判定 ,因此 ,生产实践中迫切需要新的快速诊断方法。 RT-PCR就是一个很有开发、推广前景的快速诊断技术 ,特别是随着整个行业技术水平的提高 ,RT-PCR逐渐成为一种常规方法 ,可以应用于病原核酸的检测和亚型的鉴别 ,在禽流感病毒的快速诊断中越来越受到重视。依据其原理和技术特点的差异 ,RT-PCR技术又分为常规、巢式、荧光、多元 PCR和 PCR-RFL P、RRT-PCR等。本文对这些 RT-PCR技术在禽流感诊断中的应用做了简要论述。