兔中枢神经内Orexin B免疫阳性神经纤维的分布

采用免疫组织化学方法研究了青紫蓝兔中枢神经系统（CNS）内orexin B免疫阳性纤维的分布。结果显示，orexin B免疫阳性神经纤维广泛分布于CNS内，在端脑分布于大脑皮质、隔核和杏仁核；在间脑分布于丘脑、下丘脑、上丘脑和垂体，主要分布于丘脑中线核和下丘脑；在中脑分布于中脑中央灰质、前丘、后丘、脚间核和中缝核；在脑桥分布于蓝斑、被盖背侧核和中缝核；在延髓分布于最后区、孤束核和迷走神经背侧运动核；在小脑和脊髓也有分布。兔CNS内orexin B免疫阳性纤维的分布与orexin A的基本相似，本试验结果为理解orexin B在兔体内的作用部位提供了形态学资料。     

类猪圆环病毒因子P1在自然感染仔猪体内的组织分布

选用3头自然感染仔猪和1头阴性对照猪,剖杀后采集心脏、肝脏、脾脏、肺脏、肾脏、胰脏、脑、空肠、扁桃体、胸腺、淋巴结、膀胱、性腺等组织,用荧光定量PCR方法检测病毒在组织中的分布及病毒载量.结果显示,类猪圆环病毒因子P1分布在仔猪的心脏、肝脏、肺脏、胰脏、脑和膀胱等组织,病毒载量以胰脏、脑和膀胱为最高,可达105拷贝/g以上；对照猪脏器中没有检测到病毒核酸.该研究为P1的诊断及致病机制奠定了基础.     

兔脑nNOS阳性神经元的分布和形态结构

用SABC免疫组织化学技术,观察了神经元型一氧化氮合酶（nNOS）阳性神经元在家兔脑内的分布和形态。结果显示,在家兔的大脑皮质、小脑、中脑、脑桥等处有较多的nNOS免疫阳性神经元分布,延髓分布较为稀少。nNOS免疫阳性神经元呈棕褐色,着色主要位于胞浆内,细胞核着色较淡,nNOS阳性纤维大多呈棕色串珠样。表明一氧化氮作为神经递质,可能与脑的调控功能有关。     

SABC法对伪狂犬病病毒鄂A株在仔猪体内的定位

运用免疫组织化学SABC法对人工感染伪狂犬病病毒的仔猪部分组织进行组织学观察。结果显示，在肺、大脑、小脑、脊髓、脊神经节、淋巴结、扁桃体、胸腺、脾、肝、肾、肾上腺、胃、肠等组织内均发现阳性细胞，尤其在肺、神经组织和淋巴组织内为多，病毒主要侵害上皮细胞、神经细胞和淋巴细胞，主要存在于被感染细胞的胞浆和胞核内，但以胞浆内为主。     

PDCoV感染对仔猪全身免疫器官的影响

旨在研究猪δ冠状病毒(porcine deltacoronavirus, PDCoV)感染对仔猪免疫系统的影响。选取10头5日龄健康仔猪随机分为空白对照组和PDCoV感染组(n=5),PDCoV感染72 h后剖解仔猪，采集仔猪的胸腺、扁桃体、脾脏和淋巴结(颈浅淋巴结、腹股沟淋巴结、髂下淋巴结、下颌淋巴结和肠系膜淋巴结),4%(g/L)的多聚甲醛溶液固定，苏木精-伊红染色观察免疫系统病理变化，免疫组织化学检测病毒分布。苏木精-伊红染色结果显示，PDCoV感染仔猪后脾和淋巴结都出现了明显的病理损伤。脾小梁增生；红髓增多，红髓和白髓界线不清、分布铁黄素颗粒；白髓萎缩，脾小结减少，淋巴细胞减少。淋巴结局部组织出现毛细血管充血或轻微出血，皮质和髓质界线不清，淋巴小结体积增大，数量增多，生发中心明显变大。免疫组织化学结果显示，仔猪肠系膜淋巴结有大量病毒分布，下颌淋巴结存在少量病毒，其余免疫组织无病毒分布。上述结果说明，PDCoV感染后可引起仔猪免疫器官产生病理损伤，降低仔猪的免疫功能，这为深入了解PDCoV致病机制和临床防控提供了理论依据。     

兔脑一氧化氮合酶(NOS)阳性神经元的形态、结构和分布

应用MADPH—d酶组织化学技术，对兔脑一氧化氮合酶（NOS）阳性神经元的形态、结构和分布进行了研究。结果显示：①NOS阳性神经元呈蓝色，细胞核不着色，突起染色很清晰；神经元胞体大多呈多角形、梭形，还有一些呈圆形或卵圆形等。②NOS阳性神经元几乎分布于家兔的各个脑区，包括大脑皮质、小脑、丘脑下部、中脑和脑桥。小脑分布最集中，而延髓分布较少。以上结果表明，NOS阳性神经元及其催化产生的一氧化氮（NO）与中枢神经系统的诸多功能有关。     

人工感染PRRSV JXA1株仔猪肾脏组织病理变化及病毒分布

为了解感染猪繁殖与呼吸综合征病毒(PRRSV)仔猪肾脏组织的主要病变和病毒主要分布的细胞类型,试验取人工感染PRRSV JXA1株的仔猪肾脏组织制作石蜡切片,采用HE染色法观察其病理变化,采用免疫组织化学方法检测病毒GP5蛋白和N蛋白在肾脏组织内的分布。结果表明,感染仔猪的肾脏组织呈现广泛性充血和出血,大量的炎症细胞浸润;感染仔猪肾脏组织的免疫组织化学染色,PRRSV GP5蛋白和N蛋白呈广泛性阳性着色,阳性细胞包括肾小球毛细血管内皮细胞、肾小管周围毛细血管内皮细胞、肾小管上皮细胞和肾间质等。说明PRRSV感染仔猪病毒粒子能够侵入肾脏组织,感染肾血管内皮细胞和肾小管上皮细胞等。     

伪狂犬病毒在潜伏感染猪体内的组织分布

潜伏感染是猪伪狂犬病防治和净化工作中的重要障碍之一。本研究用伪狂犬病毒（PRV）gG-/LacZ＋标记毒株感染经过PRV灭活疫苗免疫的PRV阴性仔猪,建立了猪伪狂犬病毒潜伏感染动物模型,再用地塞米松激活PRV在猪体内的潜伏感染。运用免疫组织化学SABC染色法研究了PRV在潜伏感染猪和潜伏感染激活猪体内部分组织的分布情况。结果显示,阳性细胞主要分布在神经系统的大脑、小脑、脑干、三叉神经和视神经以及非神经系统的扁桃体、肺和肾等部位。阳性细胞数量随着攻毒后时间的发展呈现减少的趋势,而注射地塞米松后,阳性细胞数量在上述组织中显著增加。     

兔脑NOS阳性神经元的总体分布和形态、结构的研究

为研究一氧化氮合酶（NOS）阳性神经元在各年龄段家兔脑内的分布规律及其衰老性变化，用NADPH-d组织化学技术，观察了一氧化氮合酶阳性神经元在家兔脑内的分布和形态。结果显示：在家兔的小脑、大脑皮质、丘脑下部、中脑、脑桥等处有较多的一氧化氮合酶阳性神经元分布，延髓分布极少。NADPH—d阳性神经元呈蓝色，细胞核不着色，突起染色都很清晰。表明一氧化氮与中枢神经系统的诸多功能有关。     

PCV2病毒载量与病理损伤的相关性

本试验选取11头6周龄健康仔猪(母源抗体呈阳性4头为A组;母源抗体呈阴性7头,随机选取其中4头为B组;剩余3头为C组,作为空白对照),A、B组进行人工攻毒,获得了实际生产中PCV2亚临床感染状态,采用荧光定量PCR与组织病理切片技术相结合,旨在探究机体各脏器(心、脾、肺、肾、淋巴结)组织中PCV2病毒载量与病理损伤的相关性。结果显示,A组以淋巴结、脾免疫器官的组织病毒载量最高,然而,B组,淋巴结、脾免疫器官的组织病毒载量反而偏低;组织病理学发现PCV2主要引起淋巴结、脾免疫器官的淋巴细胞坏死、损耗,肺脏间质性肺炎,肾脏间质性肾炎,心脏心肌炎等病变。相同脏器的组织病毒载量和病理损伤程度相关性统计结果表明:不同个体其相同脏器的组织病毒载量与病理损伤程度呈正相关性,而相同个体其不同的脏器之间组织病毒载量与病理损伤程度的规律性还有待进一步试验。本试验为PCV2的实践病理诊断,疫苗免疫效力评价,疾病防控提供理论参考。     

免疫组化法检测大鼠肝脏组织中的乙型肝炎病毒和戊型肝炎病毒

为探讨SD大鼠乙型肝炎病毒(HBV)和戊型肝炎病毒(HEV)的感染情况,本试验采集了53例SD大鼠肝脏,采用免疫组织化学染色、HE染色和Mallory三染色法对上述肝脏样品中的HBsAg、HBcAg和HEV进行了检测,同时对其病理形态学变化进行了观察。结果如下:在53例肝脏样品中有47例呈HBsAg阳性反应,阳性率为88.68%;有38例呈HBcAg阳性反应,阳性率为71.70%;HBV的双阳性率为66.15%,总阳性率为96.23%;而HEV的阳性率为100%。镜下HBsAg阳性反应物在肝细胞胞核、胞浆和胞膜上均有分布,而HBcAg阳性反应物主要分布于肝细胞胞核和胞浆内;HEV阳性反应物分布在肝细胞胞核、胞浆和小叶间胆管上皮中。组织病理学观察结果显示,在上述免疫组化阳性反应肝脏样品的HE染色切片中,均有不同程度的病理变化,主要表现为肝细胞固缩、变性、坏死、中央静脉淤血、淋巴细胞浸润、小叶间胆管增生。Mallory三色染色结果证明有22%的肝脏样品有纤维结缔组织增生。     

The high prevalence of hepatitis E virus infection in wild boars in Ibaraki Prefecture,Japan

Hepatitis E virus (HEV) is known as a causative agent of zoonosis and food poisoning. Pigs and some species of wild animals, including wild boar, are known to be a reservoir of HEV. In this study, we investigated the situation regarding HEV infection in wild boars in Ibaraki Prefecture, Japan. Serum, liver and feces samples from 68 animals were collected, and the presence or absence of HEV genomic RNA and HEV antibodies were analyzed. The viral genome was detected in samples from 7 (10.3%) animals, with all HEVs classified as genotype 3, subtype 3b. HEV antibodies were detected in samples from 28 (41%) animals. This report demonstrates for the first time the high prevalence of HEV infection in wild boars in Ibaraki Prefecture.     

Genotype 3 hepatitis E has been widespread in pig farms of Shanghai suburbs

Hepatitis E virus (HEV) genotype 3 was first identified in swine raised on a Shanghai suburban pig farm in late 2006. To accurately determine the prevalence of HEV infections among Shanghai pig farms, 426 pig fecal samples were collected from 37 pig farms located in all 10 Shanghai suburban districts and tested for the presence of HEV RNA using RT-PCR. Genetic analysis based on an amplified 150-bp ORF2 fragment revealed 111 samples to be HEV positive, and the prevalence of HEV infection within the different districts varied between 0 and 41.7%. Thirty-two samples were sequenced, and phylogenetic analysis indicated that 10 isolates belonged to HEV genotype 4 and were most closely related to 3 human and 2 swine HEV strains, all of which had originally been isolated from Asian countries including Japan and China. The remaining 22 isolates belonged to genotype 3 and were most closely related to a strain of swine HEV, US-SW, isolated from pigs in the United States. Our data indicated that genotype 3 HEV was widespread among suburban Shanghai pig farms although further study is required to determine the source and zoonotic nature of the virus.     

2015年-2016年度南宁部分地区猪戊型肝炎病毒基因型分析

为了解广西猪群中戊型肝炎病毒(HEV)基因型,利用套式RT-PCR用对南宁周边猪场采集到的104份新鲜猪粪便进行HEV检测、分离并成功扩增和克隆了11株阳性毒株的ORF2基因部分保守片段.序列测定结果表明,11株HEV ORF2序列自身核苷酸同源性为97.7%~100%,推导编码的氨基酸同源性为97.9%~100%;与4型HEV代表毒株Ch-S-1、Ch-T11、swGX40等的核苷酸同源性为84.6%~90.6%,而与其他各型之间的同源性较低.系统发育进化树结果表明,11株HEV广西株为基因4型,其中,10株HEV为4a亚型,1株毒株为4e亚型.结果表明,广西猪群中流行的HEV均为基因4型,且以4a亚型为优势流行毒株.     

Detection of hepatitis E virus RNA in saliva for diagnosis of acute infection

Diagnosis of acute hepatitis E virus (HEV) infection is established by detection of anti‐HEV IgM antibodies by ELISA or by amplification of serum viral RNA. Here, we evaluate the diagnostic value of testing HEV RNA in saliva to identify patients with acute HEV infection. Prospective proof‐of‐concept study including patients with acute hepatitis. Whole blood and neat saliva samples were obtained from all patients. Saliva samples were processed and analysed for HEV RNA by RT‐PCR within 2 hr after collection. A total of 34 patients with acute hepatitis and 12 healthy donors were included in the study. HEV RNA in serum was confirmed by RT‐PCR in eight of these patients (23.5%; 95% CI: 12.2%–40.2%). HEV was isolated in the saliva of eight of 34 patients (23.5%; 95% CI: 12.2%–40.2%). All patients with HEV RNA amplified in saliva had detectable HEV RNA in serum. HEV was isolated neither in the saliva of any of the 26 patients without detectable HEV RNA in serum nor in healthy donors. Our study suggests that acute HEV infection could be diagnosed by assessing viral load in saliva.     

The study of seroprevalence of hepatitis E virus and an investigation into the lifestyle behaviours of the aborigines in Malaysia

Malaysia is a non‐endemic country for hepatitis E virus (HEV) infection. However, seroprevalence as high as 50% among samples of aboriginal people were reported over two decades ago. A total of 207 samples collected from seven aboriginal villages in rural settlements across two states in Malaysia were analysed for anti‐HEV IgG and IgM by an enzyme‐linked immunoassay. Following the detection of anti‐HEV seroprevalence, we organized health outreach to inform and educate the community. Qualitative interviews were conducted with individuals tested positive for anti‐HEV antibodies. Data derived from interviews and observations were used to investigate possible lifestyle behaviours associated with HEV infection. Anti‐HEV IgG was detected in six samples (5.9%) from the village of Dusun Kubur. Qualitative inquiry and observation study revealed poor dietary and household hygiene, contaminated food and water, contact with animal faeces, unsanitary and domestic waste disposal, and wildlife reservoirs could be the contributing factors for transmission and acquisition of HEV infection. Investigation during health outreach is important to provide insights for future empirical research and implementation for improvement of lifestyle behaviours among the aborigines. Managing the risk of HEV infection in the aborigines may reduce the risk of HEV transmission to the local communities.     

Prevalence of hepatitis E virus in swine under different breeding environment and abattoir in Beijing, China

This study was to investigate the prevalence of HEV in pig herds under different breeding environment and in abattoirs located in Beijing, China. In total 638 sera samples and 114 liver samples were collected for a serological survey and a RT-PCR assay, respectively. The average prevalence rate of HEV in pig herds in Beijing suburb were 47.5-100%. Seropositive rate was 76.6% for pig herds of large-scale and 90% for family-scale farms. For sera samples collected from abattoir, 127 samples (78.4%) were found to be positive. Among four liver samples (3.5%) that positive for HEV RNA detection, two strains of HEV has been identified. the two detected HEV sequences shared 99.1% nucleotide sequence identity with each other, and 76.4-81.0%, 76.5-76.8%, 75.7-79.0% and 85.2-96.4% with related strains representing genotype 1, 2, 3, and 4, respectively. Further phylogenetic analysis revealed two HEV sequences belonged to genotype 4 and exhibited a high identity with strain JKO-ChiSai98C (95.4 and 95.7%), and with strains 87, 277 and 292 (96.1-96.4%) isolated from patients with sporadic acute hepatitis E in Beijing, China. The results of this study indicated that the prevalence of HEV in the pig herds were quite high. Additional public-health concerns might be placed on pork safety.     

Detection of HEV in naturally infected swine from central Argentina by an optimized HEV/MS2 duplex RT‐qPCR

Hepatitis E virus (HEV) is currently considered as a global health concern due to the recognition of its zoonotic transmission to humans, mainly from swine, and its association with the development of severe cases of hepatitis in human risk populations. The lack of updated data on HEV state of infection in swineherds of Argentina, and the necessity of robust technologies for its detection in complex biological samples, positions HEV as an emerging issue in public health. Here, we have optimized a RT‐qPCR with internal control for a more precise and accurate HEV RNA detection in swine stool samples. We implemented this optimized molecular tool to analyse the current epidemiological scenario of HEV infection in swine from the core region of commercial activity of Argentina. A total of 135 stool samples were collected from 16 different farms and tested for HEV presence, resulting in 11 positive cases (8.1%). Phylogenetic analysis demonstrated that all of them correspond to HEV genotype 3 and that different subtypes circulate in the region. Moreover, two of the detected strains presented a high nucleotide similarity with a previously identified isolate from human sewage discharges, suggesting the zoonotic transmission of HEV to humans. Collectively, this work provides a better understanding of HEV epidemiology in Argentina while contributes to the improvement of HEV detection technologies.