Suppression of hepatitis B virus replication in Tupaia hepatocytes by tumor necrosis factor alpha of Tupaia belangeri

Recently, Tupaia belangeri was used to study the full replication cycle of hepatitis B virus (HBV) in the primary hepatocyte cultures. Thus, the Tupaia model represents a suitable model to study the effects of cytokines on HBV infection. Here, Tupaia tumor necrosis factor-alpha (TNF-α) was molecularly cloned and expressed in mammalian cells. A test system for the biological activity of Tupaia TNF-α was established on the basis of its cytotoxic effect to the murine fibrosarcoma cell line L929. Recombinant Tupaia TNF-α was able to suppress HBV replication in primary Tupaia hepatocytes (PTH). However, the formation of HBV covalently closed circular DNA (cccDNA) and viral RNA was not completely prevented. Therefore, Tupaia TNF-α may contribute significantly to the control of HBV infection though it is not able to completely inhibit HBV replication alone. The characterization of this important cytokine allows further studies on its antiviral actions in the Tupaia model.     

犬乙肝病毒S基因遗传变异研究

应用人乙肝诊断试剂盒，对检测出来的“三阳（HBsAg,HBeAg,抗-HBc)犬的乙肝病料进行了病毒分离和形态学鉴定，并以人乙肝病毒S基因两侧的保守区域作为模板，采用PCR方法扩增出犬乙肝病毒部分基因，序列分析结果，其核苷酸序列为1157bp，编码385个氨基酸，遗传变异分析结果表明，在S基因保守区内有27个编码氨基酸与人的不同，其中16个为性质相反或不同的氨基酸置换，核苷酸和氨基酸序列同源性分别为96.5%和93.4%，系统发育进化树比较表明二者的遗传关系较远，揭示犬乙肝病毒与人乙型肝炎病毒S基因保守区之间存在很大差异；而与鸡，羊乙肝病毒S基因保守区序列相比较，遗传变异更大。     

从猪和牛检出类乙肝病毒抗原和抗体

从屠宰场随机采取健康猪和牛血清和肝脏进行人乙肝两对半检测。结果从肝脏均检出S抗原和e抗原 ;血清中则检出S抗体和C抗体。S抗原、e抗原和S抗体阳性率都很高 ,并有一部份为强阳性 ,从而确切地证实牛和猪的肝脏中存在着类乙肝病毒     

Changes in peripheral blood leucocytes of sheep experimentally infected with Mycoplasma agalactiae

Contagious agalactia is a serious disease of small ruminants affecting mainly mammary glands, joints and eyes. In sheep, the main aetiological agent is Mycoplasma agalactiae (Ma) whose abilities to persist in the target organs are known. Since there is no information on the effect of acute and chronic Ma infection on circulating leucocytes, the present study was designed to monitor granulocytes, monocytes, T and B lymphocytes, by flow cytometry, in female lactating sheep nasally infected with Ma. A profound depletion of leucocytes was observed from day 5 to day 34 post infection (p.i.). In particular, while the granulocytes returned to baseline levels by day 12 p.i., the monocytes remained significantly low until day 20 p.i. The infection caused a prolonged depletion of peripheral T lymphocytes (both CD4⁺ and CD8⁺) while B lymphocytes remained unaltered throughout the study. Mycoplasma agalactiae was detected by real-time PCR in several anatomical sites (ear, nose and milk) from day 2–5 p.i. until the end of the study (i.e., day 50 p.i.) while a transient bacteraemia was observed from day 5 to day 12 p.i. The leucopenia observed following intranasal Ma infection is likely due to leucocyte infiltration within the target organs.     

Hemorrhagic enteritis: virus distribution and sequential development of antibody in turkeys

Turkeys poults were inoculated intraperitoneally with hemorrhagic enteritis virus (HEV) at 4-1/2 weeks of age. Antibody response and sequential development of viral antigen in various tissues were monitored. An enzyme-linked immunosorbent assay (ELISA) was developed to study antibody production, and immunoperoxidase staining was used to determined sites of localization of the viral antigens in tissues. Results of ELISA and immunodiffusion tests were compared. ELISA detected antibody from day 3 post-infection (p.i.), and gel diffusion detected antibody from day 5 p.i. Peak ELISA antibody titer appeared from day 14 p.i. HEV antigen was detected from 2-6 days p.i. in the spleen, liver, intestine, kidney, and bone marrow; peak titers in the spleen were on day 3 p.i. Virus was not detected after day 6 p.i.     

Orthohepadnavirus infection in a neotropical bat (Platyrrhinus lineatus)

Hepatitis B virus (HBV) is the prototype of the Orthohepadnavirus genus and represents an important cause of chronic hepatitis, liver cirrhosis, and hepatic cancer in humans worldwide. To verify the occurrence and genetic variability of orthohepadnavirus among neotropical bats, we tested 81 liver samples of New World bats from São Paulo State, Southeastern Brazil, collected during 2012. PCR, sequencing, and phylogenetic analysis of Surface/Polymerase and Core viral genes confirmed the occurrence of the first isolate of bat orthohepadnavirus detected in South America. These results may contribute to subsequent studies of the origin, variability, host species, and evolution of bat orthohepadnaviruses in South America.     

屠宰猪肝脏中乙型肝炎病毒的检测

为了解屠宰猪体内乙型肝炎病毒感染情况,从河南、河北、北京和山东四个地区采集肝脏样品291例,应用免疫组化和PCR等方法对人乙型肝炎病毒相关抗原进行检测。结果表明,屠宰猪的肝脏中检测到乙型肝炎病毒的相关抗原,并且感染情况比较严重,为进一步研究猪肝脏中HBV致病性提供依据。     

Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L)

An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at ≥ 10^7.0, which corresponded with an increase in mortality. The kinetics of IFNα and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality.     

Bovine viral diarrhoea: Pathogenesis and diagnosis

Bovine viral diarrhoea virus (BVDV) is the most prevalent infectious disease of cattle. It causes financial losses from a variety of clinical manifestations and is the subject of a number of mitigation and eradication schemes around the world. The pathogenesis of BVDV infection is complex, with infection pre- and post-gestation leading to different outcomes. Infection of the dam during gestation results in fetal infection, which may lead to embryonic death, teratogenic effects or the birth of persistently infected (PI) calves. PI animals shed BVDV in their excretions and secretions throughout life and are the primary route of transmission of the virus. These animals can usually be readily detected by virus or viral antigen detection assays (RT-PCR, ELISA), except in the immediate post-natal period where colostral antibodies may mask virus presence. PI calves in utero (the ‘Trojan cow’ scenario) currently defy detection with available diagnostic tests, although dams carrying PI calves have been shown to have higher antibody levels than seropositive cows carrying non-PI calves.Acute infection with BVDV results in transient viraemia prior to seroconversion and can lead to reproductive dysfunction and immunosuppression leading to an increased incidence of secondary disease. Antibody assays readily detect virus exposure at the individual level and can also be used in pooled samples (serum and milk) to determine herd exposure or immunity. Diagnostic tests can be used to diagnose clinical cases, establish disease prevalence in groups and detect apparently normal but persistently infected animals. This review outlines the pathogenesis and pathology of BVD viral infection and uses this knowledge to select the best diagnostic tests for clinical diagnosis, monitoring, control and eradication efforts. Test methods, types of samples and problems areas of BVDV diagnosis are discussed.     

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 effects of sage extract feed supplementation on biochemical parameters, weight of internal organs and Salmonella counts in chickens

We investigated the effects of dietary addition of sage extract on the biochemical parameters, weight of some body organs and changes in the counts of Salmonella Enteritidis PT4 (SE) in experimentally infected chickens. The following diets were used: basal diet, basal diet with addition of an extract of Salvia officinalis L. (S), basal diet and SE, and basal diet and S and SE (SSE). Compared to the SE group, sage extract in the SSE group decreased activities of ALP and ALT and concentrations of glucose and bilirubin on the 4th day post inoculation (p.i.). However, on the 18th day p.i., lower levels of bilirubin and ALT activity only were detected. Addition of sage extract to the diets decreased the counts of Salmonella in the liver, spleen and caecum at both sampling times, along with lower production of mucus in the chickens' intestines. Our results suggest that the addition of sage extract to the diet could be effective in protecting SE-infected chickens.     

Biogenic amine levels in acute Babesia bovis infected cattle

Plasma and whole blood from splenectomized calves infected with B. bovis were assayed by fluorimetric techniques for histamine, 5-hydroxytryptamine (5-HT), noradrenaline and dopamine levels. In addition PCV, thrombocyte and parasite counts were also undertaken. Plasma histamine levels rose till day 4 post-infection (p.i.) and were still elevated on day 7 p.i. Wholw blood histamine levels were significantly higher on days 1, 4 and 6 p.i. Plasma 5-HT levels rose to peak levels on day 3 p.i. and were still significantly elevated on day 7 p.i. Whole blood 5-HT levels were significantly higher on days 1 and 2 p.i. but fell to subnormal levels terminally. Dopamine and noradrenaline levels for both whole blood and plasma were unaltered during the disease process. Thrombocyte levels initially rose, reaching maximum values on day 3 p.i. The level fell continuously below normal from day 4 to 7 p.i. The PCV fell continuously from day 1 p.i. The experimental animals suffered a severe and fatal syndrome, all dying 7 days p.i.     

Natural and experimental hepatitis E virus genotype 3 - infection in European wild boar is transmissible to domestic pigs

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E in humans in developing countries, but sporadic and autochthonous cases do also occur in industrialised countries. In Europe, food-borne zoonotic transmission of genotype 3 (gt3) has been associated with domestic pig and wild boar. However, little is known about the course of HEV infection in European wild boar and their role in HEV transmission to domestic pigs. To investigate the transmissibility and pathogenesis of wild boar-derived HEVgt3, we inoculated four wild boar and four miniature pigs intravenously. Using quantitative real-time RT-PCR viral RNA was detected in serum, faeces and in liver, spleen and lymph nodes. The antibody response evolved after fourteen days post inoculation. Histopathological findings included mild to moderate lymphoplasmacytic hepatitis which was more prominent in wild boar than in miniature pigs. By immunohistochemical methods, viral antigens were detected mainly in Kupffer cells and liver sinusoidal endothelial cells, partially associated with hepatic lesions, but also in spleen and lymph nodes. While clinical symptoms were subtle and gross pathology was inconspicuous, increased liver enzyme levels in serum indicated hepatocellular injury. As the faecal-oral route is supposed to be the most likely transmission route, we included four contact animals to prove horizontal transmission. Interestingly, HEVgt3-infection was also detected in wild boar and miniature pigs kept in contact to intravenously inoculated wild boar. Given the high virus loads and long duration of viral shedding, wild boar has to be considered as an important HEV reservoir and transmission host in Europe.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0121-8) contains supplementary material, which is available to authorized users.     

Toxocara canis in experimentally infected pigs: migratory pattern and tissue lesions

Fifteen Yorkshire female pigs were inoculated with 100,000 infective T. canis eggs. Three animals were used as uninfected controls. Groups of three infected pigs were euthanized by accepted methods on days 7, 14, 21, 28 and 126 p.i., respectively. Larvae were recovered from all animals included in each group slaughtered on days 7 and 14 p.i.; on day 21 p.i. from two pigs, on day 28 p.i. from one, and no larvae were found on day 126 p.i. Differences in the mean number of larvae per gram in lymph nodes, liver and lungs between slaughter days, were significant for livers on day 7 p.i. and for lungs on day 14 p.i. (P < 0.10). The decrease over time was significant in all the organs that previously had larvae. Larvae were not found in the other organs and tissues analysed. Macroscopical lesions were found in the liver, lungs and lymph nodes on days 7, 14, 21, and 28 p.i. The entire surface of the liver was covered with small white spots on day 7 p.i., on days 14 and 21 p.i. the spots were distinctly nodular and, in some places, individual lesions were confluent. Lesions had apparently started to heal on days 28 and 126 p.i. appearance was normal. Lymph nodes were enlarged and oedematous during the first 4 weeks and the lungs had small areas of consolidation visible all over the surface, but by day 126 p.i., no visible lesions could be seen. Microscopical lesions were observed in the liver on day 7 p.i., with a largely periportal hepatitis. Numerous eosinophils and lymphocytes were present. The typical granulomatous reaction was observed on days 14 and 21 p.i. with a central necrotic core and a narrow region of fibroblastic tissue. By day 28 p.i. lesions had almost disappeared and the number of eosinophils was fewer. There were fewer leukocytes and the fibrous tissue had disappeared from the liver on day 126 p.i. For the first 3 weeks, pictures of the lymph nodes and the lungs were characterised by the formation of a granuloma. In the center of the granuloma larvae were observed. The majority of the lesions had healed by day 126 p.i.     

Leukocyte-hepatocyte interaction in calicivirus infection: differences between rabbits that are resistant or susceptible to rabbit haemorrhagic disease (RHD)

Calicivirus infection is lethal for adult rabbits, whereas young rabbits (less than 8-weeks-old) are resistant to the same infectious agent. The virus replicates in the liver and causes a fulminant hepatitis in adult rabbits leading to rabbit haemorrhagic disease (RHD); this is in contrast with the mild and transient hepatitis observed in infected young rabbits. We have used electron microscopy to compare liver leukocyte infiltrates between young (resistant) and adult (susceptible) rabbits, 36-48 h after inoculation of the animals with caliciviruses. In adult rabbits, liver infiltrates were made up mostly of heterophils, and they were located near hepatocytes showing severe cellular damage. In contrast, liver leukocyte infiltrates of RHD-resistant young rabbits were dominated by lymphocytes that depicted membrane contacts with the cell surface of undamaged hepatocytes. We conclude that: (i) the cellular inflammatory response of the liver to calicivirus infection is different in rabbits that are susceptible (adult) or resistant (young) to RHD; (ii) leukocyte infiltration of the adult liver by heterophils is probably directed at the removal of dead hepatocytes, whereas the liver lymphocytic infiltration of young rabbits suggests the expression of viral antigens on the surface of liver cells of the RHD-resistant animals.     

Feeding Saccharomyces cerevisiae fermentation products lessens the severity of a viral–bacterial coinfection in preweaned calves

We have previously reported that supplementation with Saccharomyces cerevisiae fermentation products (SCFP) ameliorates clinical signs and lung pathology following experimental bovine respiratory syncytial virus (BRSV) infection in preweaned dairy calves. The objectives of this study were to determine the effect of SCFP supplementation on the metabolic and endocrine responses, and disease outcome of a viral–bacterial coinfection in preweaned calves. Twenty-seven, 1- to 2-d-old Holstein-Angus cross calves were enrolled in the study; one SCFP calf was removed from the trial during the pre-challenge phase due to complications from nephritis. Calves were assigned to two treatment groups: control or SCFP-treated, base milk replacer with 1 g/d SCFP (Smartcare, soluble formula) and calf starter top dressed with 5 g/d SCFP (NutriTek, insoluble formula). Calves were infected with BRSV on day 21, followed 6 d later by intratracheal inoculation with Pasteurella multocida (PM). Calves were euthanized on day 10 post-viral infection. Calves receiving SCFP had reduced thoracic ultrasonography scores on day 7 post-viral infection (P = 0.03) and a tendency toward reduced scores on day 10 post-viral infection (P = 0.09). Calves receiving SCFP also had less severe lung pathology scores at necropsy (P = 0.06). No differences between treatments were observed in lung viral loads (P = 0.48) or bacterial lung recovery (P = 0.34); however, there was a distinction in the lung location for PM recovery, with PM isolated more frequently from the cranial lobes in SCFP-treated calves, but more frequently from the caudal lobes of control calves. Calves treated with SCFP tended (P = 0.07) to have higher serum IL-6 concentrations following the coinfection. Calves treated with SCFP had lower concentrations of serum nonesterified fatty acids and beta-hydroxybutyric acid compared with controls following experimental challenge (P = 0.03 and P = 0.08, respectively), suggesting metabolic changes favoring growth and development. There were no differences between groups in gene expression of insulin receptor, insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), growth hormone receptor, or haptoglobin in the liver. Results from this study suggest that supplementing with SCFP may moderate the impact of a respiratory viral–bacterial coinfection on preweaned calves through metabolic and immune modifications.     

HMGB1与日本血吸虫病肝炎症和纤维化的相关性分析

旨在探究高迁移率族蛋白B1（high mobility group box 1,HMGB1）与日本血吸虫病肝炎症和纤维化的相关性,本研究将BALB/c小鼠随机分为空白对照组（n＝10）,感染后4周组、6周组和HMGB1抑制剂组（n＝5）,进行血常规及生化检测,观察肝病理学及Masson染色情况、荧光定量PCR检测HMGB1、IL-1β、TNF-α、IL-6、IFN-γ、α-SMA、Col1α1、TGF-β1、Smad2和Smad3的转录水平。结果显示：感染后4周,小鼠谷丙转氨酶（alanine aminotransferase,ALT）、肝组织HMGB1、α-SMA、TGF-β1、IL-6和 TNF-α的转录水平均显著或极显著升高（P<0.05或P < 0.01）;感染后6周,小鼠WBC、Neu、Mon、Eos、谷草转氨酶（aspartate aminotransferase,AST）和ALT极显著升高（P<0.01）,肝组织HMGB1、IL-1β、TNF-α、IL-6、IFN-γ、α-SMA、Col1α1、TGF-β1、Smad2和Smad3的转录水平均显著或极显著升高（P<0.05或P<0.01）;感染后6周小鼠肝虫卵聚积处伴有炎性细胞浸润和胶原纤维增生;HMGB1抑制剂可显著降低小鼠血清ALT值和肝组织HMGB1、α-SMA、TGF-β1、IL-6和TNF-α的转录水平（P<0.05）。综上表明,HMGB1与日本血吸虫病肝炎症和纤维化具有一定相关性。     

The migration of larval Toxocara canis in mice II. Post-intestinal migration in primary infections

Following oral infection of NIH mice with Toxocara canis embryonated eggs the L₂ pass the visceral phase of migration during the first week of infection. Larvae reach the liver and lungs and peak in number in these organs 2 and 3 days after infection, respectively. Larvae are then dispersed throughout the body and enter the myotropic—neurotropic phase by the 7th day of infection. Larvae injected directly into the brain are capable of migrating into the viscera and musculature. Considerable pathology occurs due to larval migrations, especially through the liver and lungs, and both acute and chronic disease are recorded. Studies of infections extending over a year show that the number of recoverable larvae declines gradually with periods of stable populations.On Days 3, 4 and 5 after infection, larvae were demonstrable in the faeces of infected mice. Prenatal infection was observed in a third of the offspring of mice infected the same day as conception.     

雏鸭人工感染鸭肝炎病毒后血液生化指标的测定

用Ⅰ型鸭肝炎病毒人工感染3日龄健康金定鸭,对接种后48 h后血清中血糖、谷丙转氨酶、谷草转氨酶等9项血液生化指标进行测定。结果显示,谷丙转氨酶含量较对照组极显著升高(P＜0.01);尿素氮含量显著升高(P＜0.05);葡萄糖、IgG含量与对照组相比有一定量的下降,但差异不显著(P＞0.05);谷草转氨酶、甘油三酯、尿酸、IgA和IgM含量在发病时略微升高(P＞0.05)。上述结果提示,雏鸭感染鸭病毒性肝炎后,肝脏和肾脏受到不同程度的损伤。