Comparison of a new synthetic, peptide-derived, polyclonal antibody-based, immunohistochemical test with in situ hybridisation for the detection of swine hepatitis E virus in formalin-fixed, paraffin-embedded tissues

A synthetic, peptide-derived, polyclonal antibody-based, immunohistochemical test was developed to detect swine hepatitis E virus (HEV) and was compared with in situ hybridisation for the detection of HEV in formalin-fixed, paraffin-embedded tissues from experimentally infected pigs. Solid-phase peptide synthesis was used to generate peptides from swine HEV open reading frame 2, and the purified peptides were injected into rabbits to produce polyclonal antibodies. The specificity and sensitivity of the test were both 100%. Liver was most consistently positive for swine HEV antigen and RNA by immunohistochemistry and in situ hybridisation, respectively, but both were detected much less frequently in extrahepatic tissues such as lymph node, tonsil, spleen, and intestine. Swine HEV antigen and RNA showed a similar distribution in virus-infected hepatocytes in serial sections. The novel test developed in this study is suitable for consistently detecting swine HEV antigen in formalin-fixed, paraffin-embedded tissues.     

Prevalence and genotyping of hepatitis E virus in swine population in Korea between 1995 and 2004: a retrospective study

Hepatitis E virus (HEV) infections have been reported in pigs throughout the world but have only recently been recorded in Korean pigs. The aim of this study was to investigate whether HEV was present in archived porcine hepatic tissues collected between 1995 and 2004 using RT-PCR and immunohistochemistry and, if so, to determine the genotype of the isolates. Swine HEV was identified in the liver tissue of 42 pigs of 388 submissions (four pigs every year on average). The isolates showed genetic homology with swine and human HEV isolates identified in the United States and Japan (92.5-97%) and phylogenetic tree analysis indicated they belonged to genotype III. The study indicates that HEV is not a newly emerging virus in Korean pigs, but a pathogen that has existed in the country since at least 1995.     

Expression of Mx protein and interferon-alpha in pigs experimentally infected with swine influenza virus

Expression of Mx protein and interferon-alpha (IFN-alpha) was examined by immunohistochemistry in pigs experimentally infected with swine influenza virus. In infected pigs euthanatized at 1 day postinoculation (dpi), the lumen of bronchioles were filled with large numbers of mononuclear cells, small numbers of neutrophils, sloughing epithelial cells, and proteinaceous fluid. Lesions at 3 and 5 dpi were similar but less severe. Alveolar spaces were filled with neutrophils. By 7 and 10 dpi, microscopic lesions were resolved. The immunohistochemical signals for Mx protein and IFN-alpha antigen were confined to cells in areas that had hybridization signal for swine influenza virus. In situ hybridization and immunohistochemistry of serial sections of lung indicated that areas containing numerous swine influenza virus RNA-positive cells also have numerous Mx and IFN-alpha antigen-positive cells. Mean immunohistochemical scores for Mx protein-positive cells were correlated with mean immunohistochemical scores for IFN-alpha antigen-positive cells (r(s) = 0.8799, P < 0.05). These results indicated that Mx protein and IFN-alpha antigen were expressed in the lung from pigs experimentally infected with swine influenza virus, but their biological functions remain to be examined.     

Expression of myeloperoxidase in swine influenza virus (SIV)-infected neutrophils in lungs from pigs experimentally infected with SIV subtype H1N2

The expression of myeloperoxidase (MPO) was examined in the swine influenza virus (SIV)-infected neutrophils in the lungs of pigs experimentally infected with swine influenza virus (SIV) subtype H1N2 by immunohistochemistry. Five pigs each from the infected and non-infected group were euthanized 1, 3, 5, 7, and 10?days post-inoculation (dpi). Immunohistochemical reactivity was mainly seen in neutrophils. The score for pulmonary histopathological lesions correlated with the score for MPO immunohistochemical reactivity (r ( s )?=?0.962, P?相似文献   

Experimental infection of pregnant gilts with swine hepatitis E virus

To determine the effect of swine hepatitis E virus (HEV) infection on pregnant gilts, their fetuses, and offspring, 12 gilts were intravenously inoculated with swine HEV. Six gilts, who were not inoculated, served as controls. All inoculated gilts became actively infected and shed HEV in feces, but vertical transmission was not detected in the fetuses. There was no evidence of clinical disease in the gilts or their offspring. Mild multifocal lymphohistiocytic hepatitis was observed in 4 of 12 inoculated gilts. There was no significant effect of swine HEV on fetal size, fetal viability, or offspring birth weight or weight gain. The offspring acquired anti-HEV colostral antibodies but remained seronegative after the antibodies waned by 71 days of age. Swine HEV infection induced subclinical hepatitis in pregnant gilts, but had no effect on the gilts' reproductive performance, or the fetuses or offspring. Fulminant hepatitis associated with HEV infection was not reproduced in gilts.     

Pigs orally inoculated with swine hepatitis E virus are able to infect contact sentinels

The purpose of the present study was to explore the most likely natural route of infection of swine hepatitis E virus (HEV) by oral inoculation of pigs and to investigate the potential infection by direct contact exposure. A preliminary experiment was performed to assess the infectiousness of the bile used as source of virus. Once confirmed, 16 pigs were inoculated via oral drop with an HEV positive bile suspension containing 2 × 10⁵ genome equivalents per pig. Nine animals were kept as contact sentinels and 12 more pigs were used as negative controls. A number of pigs from the three groups were euthanized at 16, 32 and 64 days post-inoculation. From the HEV inoculated group, three pigs shed virus in faeces, two had virus RNA in bile at necropsy and two seroconverted. In the contact group, two animals showed presence of HEV RNA in bile. This study demonstrates that pigs orally inoculated with a single HEV dose got infection, although few animals had evidence of infection. Moreover, the virus was successfully transmitted to direct contact exposed pigs.     

Ultrastructural changes related to the lymph node haemorrhages in acute African swine fever

In order to determine the pathogenic mechanisms involved in lymph node haemorrhages in acute African swine fever ( ), eight pigs were inoculated with virus, strain Malawi'83. Lymph node haemorrhages were observed from three days post infection (dpi) onwards, coinciding with ASF virus replication in monocytes and macrophages adjacent to stimulated endothelial cells, phagocytic stimulation of capillary and small-vessel endothelial cells, increase in the number of fenestrations of endothelial cells, and endothelial cell loss, as well as clusters of blood cells and necrotic material beneath the endothelium. Vascular lumina were blocked by platelet plugs and fibrin microthrombi. These phenomena became more marked as the disease progressed. At five dpi, virus replication was also found in circulating neutrophils. At seven dpi, lesions were more intense and were accompanied by virus replication in sinus and capillary endothelial cells, and in other cell populations including pericytes, fibroblasts, smooth muscle fibres and reticular cells. The results obtained in this study suggest that lymph node haemorrhages are related to endothelial stimulation and the onset of disseminated intravascular coagulation. Virus replication in vessel wall cells occurs only in the final stages of the disease and plays a secondary role.     

猪戊型肝炎研究进展

猪戊型肝炎是一种新发现的传染病,其病原戊型肝炎病毒(HEV)是一种无囊膜的RNA病毒,基因组全长为7.2 kb~7.4 kb。研究发现,世界上许多国家的猪群中普遍存在较高的HEV抗体,也检测到猪戊型肝炎病毒,与猪接触的职业人群的血清抗HEV抗体均高于非职业人群,有些国家从猪群中分离到的HEV核苷酸序列与当地居民感染的HEV具有高度的同源性。据此认为猪戊型肝炎是一种人兽共患病,猪可能作为戊型肝炎的贮存宿主,在人类病毒性肝炎流行病学中具有重要的地位,对于公共卫生、异种器官移植和食品安全等构成潜在威胁。     

Lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) to establish a persistent infection is the principal contributing factor to the world-wide spread of the disease. Several studies have documented the course of viral infection in postnatally infected pigs; however, very little is known regarding sites of virus replication during persistent infection of pigs exposed to PRRSV in utero. In this study, virus replication and PRRSV-specific antibody were followed for several hundred days in a group of pigs derived from three sows infected at 90 days of gestation with PRRSV isolate VR-2332. Eighty-four percent of pigs were born viremic with a mortality of 54% within 21 days after birth. At approximately 60 days sera from pigs were negative for virus by virus isolation. Analysis of virus replication in the tissues of pigs randomly sacrificed between 63 and 132 days showed no evidence of virus in lung and other non-lymphoid organs. However, virus was easily recovered from tonsil and lymph nodes and in situ hybridization identified these tissues as sites of virus replication. Even though replication was at a low level, virus was easily transmitted to sentinel pigs. By 260 days pigs became seronegative and did not transmit virus to sentinel pigs. Sacrifice of remaining pigs after 300 days showed no evidence of virus in blood and tissues. This study shows that congenital PRRSV-infected pigs can support virus replication for an extended period during which virus replication is primarily restricted to tonsil and lymph nodes.     

猪血凝性脑脊髓炎病毒的血清学调查

应用血凝和血凝抑制试验检测了从辽宁省部分地区采集的猪血清中血凝性脑脊髓炎病毒（HEV）的抗体滴度。结果表明,368份血清样品中,有162份呈抗体阳性反应,阳性率高达44.0%。被采集血清的猪未表现出临床症状,说明该地区存在血凝性脑脊髓炎病毒的隐性感染。     

Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm

The objective of the present study was to determine the pattern of Hepatitis E virus (HEV) infection in a naturally infected, farrow-to-finish herd. For that purpose, a prospective study was conducted in randomly selected 19 sows and 45 piglets. Blood samples were collected from sows at 1 week post-farrowing and from piglets at 1, 3, 6, 9, 12, 15, 18 and 22 weeks of age. Furthermore 3 or 5 animals were necropsied at each bleeding day (but at 1 week of age), and serum, bile, liver, mesenteric lymph nodes and faeces taken. HEV IgG, IgM and IgA antibodies were determined in serum and viral RNA was analysed in all collected samples by semi-nested RT-PCR. Histopathological examination of mesenteric lymph nodes and liver was also conducted. From 13 analysed sows, 10 (76.9%) were positive to IgG, one to IgA (7.7%) and two to IgM (15.4%) antibodies specific to HEV. In piglets, IgG and IgA maternal antibodies lasted until 9 and 3 weeks of age, respectively. IgG seroconversion occurred by 15 weeks of age while IgM and IgA at 12. On individual basis, IgG was detectable until the end of the study while IgM and IgA antibody duration was of 4-7 weeks. HEV RNA was detected in serum at all analysed ages with the highest prevalence at 15 weeks of age. HEV was detected in faeces and lymph nodes for the first time at 9 weeks of age and peaked at 12 and 15 weeks of age. This peak coincided with the occurrence of hepatitis as well as with HEV detection in bile, liver, mesenteric lymph nodes and faeces, and also with highest IgG and IgM OD values at 15 weeks. Finally, different HEV sequences from this farm were obtained, which they clustered within 3 different groups, together with other Spanish sequences, all of them of genotype 3. Moreover, the present study also indicates that the same pig can be infected with at least two different strains of HEV during its productive life. This is the first study characterizing HEV infection in naturally infected pigs with chronological virus detection and its relationship with tissue lesions throughout the productive life of the animals.     

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.     

Hepatitis E virus chronic infection of swine co-infected with Porcine Reproductive and Respiratory Syndrome Virus

In developed countries, most of hepatitis E human cases are of zoonotic origin. Swine is a major hepatitis E virus (HEV) reservoir and foodborne transmissions after pork product consumption have been described. The risk for HEV-containing pig livers at slaughter time is related to the age at infection and to the virus shedding duration. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a virus that impairs the immune response; it is highly prevalent in pig production areas and suspected to influence HEV infection dynamics. The impact of PRRSV on the features of HEV infections was studied through an experimental HEV/PRRSV co-infection of specific-pathogen-free (SPF) pigs. The follow-up of the co-infected animals showed that HEV shedding was delayed by a factor of 1.9 in co-infected pigs compared to HEV-only infected pigs and specific immune response was delayed by a factor of 1.6. HEV shedding was significantly increased with co-infection and dramatically extended (48.6 versus 9.7 days for HEV only). The long-term HEV shedding was significantly correlated with the delayed humoral response in co-infected pigs. Direct transmission rate was estimated to be 4.7 times higher in case of co-infection than in HEV only infected pigs (0.70 and 0.15 per day respectively). HEV infection susceptibility was increased by a factor of 3.3, showing the major impact of PRRSV infection on HEV dynamics. Finally, HEV/PRRSV co-infection – frequently observed in pig herds – may lead to chronic HEV infection which may dramatically increase the risk of pig livers containing HEV at slaughter time.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0207-y) contains supplementary material, which is available to authorized users.     

Distribution of ASFV antigens in pig tissues experimentally infected with two different Spanish virus isolates.

Lymph nodes, spleen, liver, lung and kidney obtained from pigs experimentally infected with two African Swine Fever Virus (ASFV) isolates of differing virulence were fixed by perfusion with glutaraldehyde and embedded in paraffin. An immunoperoxidase technique using a polyclonal anti-ASFV serum was performed on tissue sections in order to detect ASFV antigen. The distribution of ASFV antigen in such infected organs is shown and the differences between both infections compared and discussed. Monocytes, macrophages, hepatocytes, endothelial cells, neutrophils and epithelial cells were found to contain ASFV antigens.     

The pathogenesis of vesicular exanthema of swine virus and San Miguel sea lion virus in swine

Vesicular exanthema of swine virus type A48 or San Miguel sea lion virus type 2, when inoculated intradermally into swine, resulted in fluid-filled vesicles at the sites of inoculation in the snout, coronary band, and tongue. Pigs that developed vesicles also had fevers. Secondary vesicle formation varied, depending on virus serotype. Viremia was found in one pig infected with San Miguel sea lion virus five days after infection. Virus was recovered from nasal-oral passages for up to five days after infection in both groups of pigs and from the gastrointestinal and urinary tracts of pigs infected with San Miguel sea lion virus. Neutralizing antibodies began to increase three days after inoculation and reached peak titers in seven to ten days. In the absence of secondary bacterial infection, healing was well advanced by ten days after inoculation. Lesions usually were limited to nonhaired portions of the integument and tongue. Individual epithelial cells became infected when a break in the skin allowed virus access to susceptible epithelial cells from either exogenous or endogenous sources. Individual infected cells ruptured and adjacent cells were infected, resulting in the formation of multiple microvesicles. Centrifugal coalescence of microvesicles led to formation of grossly visible macrovesicles. Lesions rarely developed from viral contamination of intact hair follicles. A mild virus-induced encephalitis was seen in pigs infected with vesicular exanthema of swine virus, and virus was recovered from brain tissue of pigs infected with San Miguel sea lion virus.     

Acute phase protein response during subclinical infection of pigs with H1N1 swine influenza virus

In the present study acute phase proteins (APPs) responses in pigs after subclinical infection with H1N1 swine influenza virus (SwH1N1) were evaluated. Fourteen 5 weeks old, seronegative piglets, both sexes were used. Ten of them were infected intranasally with SwH1N1. C-reactive protein (CRP), haptoglobin (Hp), serum amyloid A (SAA) and pig major acute phase protein (Pig-MAP) concentrations in serum were measured using commercial ELISAs. No significant clinical signs were observed in any of the infected pigs, however, all infected animals developed specific antibodies against SwH1N1 and viral shedding was observed from 2 to 5dpi. Only concentrations of Hp and SAA were significantly induced after infection, with mean maximum levels from days 1 to 2 post infection (dpi). The concentrations of CRP and Pig-MAP remained generally unchanged, however in half of infected pigs the concentration of CRP tended to increase at 1dpi (but without statistical significance). The results of our study confirmed that monitoring of APPs may be useful for detection of subclinically infected pigs. The use of SAA or Hp and Pig-MAP may be a valuable in combination [i.e. Hp (increased concentration) and Pig-MAP (unchanged concentration)] to detect subclinically SIV infected pigs, or to identify pigs actually producing a large amount of virus. Additional studies need to be done in order to confirm these findings.