Establishment of inflammatory model induced by Pseudorabies virus infection in mice

BackgroundPseudorabies virus (PRV) infection leads to high mortality in swine. Despite extensive efforts, effective treatments against PRV infection are limited. Furthermore, the inflammatory response induced by PRV strain GXLB-2013 is unclear.ObjectivesOur study aimed to investigate the inflammatory response induced by PRV strain GXLB-2013, establish an inflammation model to elucidate the pathogenesis of PRV infection further, and develop effective drugs against PRV infection.MethodsKunming mice were infected intramuscularly with medium, LPS, and different doses of PRV-GXLB-2013. Viral spread and histopathological damage to brain, spleen, and lung were determined at 7 days post-infection (dpi). Immune organ indices, levels of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines, as well as levels of activity of COX-2 and iNOS were determined at 4, 7, and 14 dpi.ResultsAt 10⁵–10⁶ TCID₅₀ PRV produced obviously neurological symptoms and 100% mortality in mice. Viral antigens were detectable in kidney, heart, lung, liver, spleen, and brain. In addition, inflammatory injuries were apparent in brain, spleen, and lung of PRV-infected mice. Moreover, PRV induced increases in immune organ indices, ROS and NO levels, activity of COX-2 and iNOS, and the content of key pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor-α, interferon-γ and MCP-1. Among the tested doses, 10² TCID₅₀ of PRV produced a significant inflammatory mediator increase.ConclusionsAn inflammatory model induced by PRV infection was established in mice, and 10² TCID₅₀ PRV was considered as the best concentration for the establishment of the model.     

Evaluation of two enzyme-linked immunosorbent assays for serodiagnosis of Aleutian mink disease virus infection in mink

Background

Aleutian disease in mink is caused by infection with Aleutian mink disease virus (AMDV). In Sweden, the infection most commonly causes classical Aleutian disease in which the immune system fails to neutralize the virus and the infection becomes persistent. Diagnosis of AMDV infection is based on serological methods that detect virus-specific antibodies. Traditionally counterimmunoelectrophoresis (CIEP) has been the preferred method, but in order to enable automation interest has been paid to other antibody detecting systems. Recently, at least two different ELISA systems that detect antibodies to AMDV have been manufactured; one is based on an in vitro grown AMDV as antigen, and the other system is based on the AMDV capsid protein VP2 as antigen. The aim of this study was to evaluate the two ELISA systems for detection of antibodies to AMDV using CIEP as the gold standard.

Results

When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the ELISA with the VP2 antigen had a sensitivity of 99.7% and a specificity of 98.3% compared to CIEP (n = 364). Analysis of samples with the AMDV-G antigen based ELISA employing an assay cut-off value based on the negative control samples, as suggested by the manufacturer, resulted in a sensitivity of 54.3% and a specificity of 93.2% with reference to CIEP as the gold standard (n = 359). When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the AMDV-G ELISA had a sensitivity of 37.6% and a specificity of 98.3%.

Conclusions

The ELISA system based on VP2 antigen had high sensitivity and specificity, and was concluded to be an alternative to the CIEP as a diagnostic tool for AMDV antibodies. In contrast, the AMDV-G ELISA suffered from low sensitivity when compared to CIEP.     

Monitoring chronic infection with a field strain of Aleutian mink disease virus

Aleutian mink disease virus (AMDV) readily spread within farmed mink and causes chronic infections with significant impacts for welfare and economy. In the present study a currently circulating Danish AMDV strain was used to induce chronic experimental infection of farmed mink.PCR was used to detect viral DNA in full blood, organs, faeces and oro-nasal swabs weekly for the first 8 weeks and then biweekly for another 16 weeks after AMDV challenge inoculation of wild type mink. The mink (n = 29) was infected and seroconverted 2–3 weeks after AMDV inoculation and AMDV antibodies persisted during the maximum experimental period of 24 weeks. Viraemia and faecal excretion of viral DNA was detected in the mink (n = 29) at various and intermittent time intervals. Excretion of viral DNA in oro-nasal swabs was detected for 1–8 weeks in 21 mink. This highlights the risk of transmitting AMDV between infected farms.PCR was successfully used to detect viral DNA in organs 8, 16 and 24 weeks after AMDV inoculation with only minor differences between these weeks which is of diagnostic interest.This AMDV challenge model was also used to mimic natural infection of susceptible sapphire mink. Four of 6 sapphire mink were infected indirectly via the AMDV inoculated wild type mink whereas the other 2 sapphire mink remained uninfected.     

猪伪狂犬病病毒gD基因的研究进展

本文重点介绍伪狂犬病病毒(PRV)中最主要的免疫糖蛋白的编码基因gD基因,对其在PRV基因结构中的位置,病毒传播中所起的作用和人们利用其生物学特性在疫苗研究中取得的进展进行了比较详细的综述。     

某规模猪场猪伪狂犬病的诊断

本文针对一规模猪场哺乳仔猪发病死亡情况,根据该场发病仔猪的临床表现、解剖病变,结合PCR诊断、家兔接种实验以及病毒分离,诊断为伪狂犬病毒野毒感染,最后对该猪场发病的原因以及防控对策进行了讨论分析。     

禽流感病毒的致病性和免疫性研究进展

介绍了禽流感病毒的病原、基因结构及其对禽类、人类的致病机理和危害，探讨了感染病毒后宿主的反应、机体的体液免疫和细胞免疫，在一定程度上概述了禽流感病毒的固有特点和规律，为进一步研究打下了基础。     

The pathogenesis and immunology of bluetongue virus infection of ruminants

Bluetongue (BLU) virus is transmitted from infected to susceptible ruminants by hematophagous vector midges (Culicoides species). Cattle are important reservoir hosts of the virus because infection typically is asymptomatic and characterized by prolonged cell associated viremia, and because at least some species of insect vector preferentially feed on cattle. Interaction of BLU virus with the cell membrane of erythrocytes in infected cattle likely facilitates both prolonged viremia as well as infection of the insect vector. BLU disease is most common in sheep and some wildlife species. A variety of host, agent and environmental factors clearly can influence expression of disease in these species. The pathogenesis of BLU virus infection of cattle and sheep is remarkably similar, thus the basis for expression of disease in sheep but not cattle remains to be firmly established. Some difference in susceptibility of endothelial cells to infection in the two species is one potential explanation.

Ruminants develop a variety of antiviral responses after BLU virus infection. Antibodies to outer capsid protein VP2 are responsible for virus neutralization, and confer resistance to reinfection with the homologous serotype of BLU virus. Antibodies to epitopes on proteins which are common to all viruses of the BLU serogroup form the basis of current diagnostic serologic tests. Cell mediated responses have been incompletely characterized, in part because BLU virus replicates within dividing lymphocytes and virus-mediated cytolysis inhibits in vitro blastogenesis. Immunological competence of ruminants to BLU virus arises prior to midgestation, and suggestions that persistent immune tolerant BLU virus infection occurs after in utero exposure of cattle have not been substantiated and are not consistent with recent findings.     

新城疫病毒感染绵羊诱导免疫应答反应的实验研究

为探索新城疫病毒(NDV)作为羊病毒病疫苗载体的可行性,验证NDV感染绵羊的可能性,本研究采用NDV Clone-30株分别通过滴鼻和气管灌注两种途径接种绵羊,接种后观察临床症状,检测接种动物排毒情况,通过血凝抑制试验检测接种绵羊特异性血凝抑制抗体,ELISA试验检测接种绵羊血清特异性IgG抗体,微量中和试验检测接种绵羊血清中和抗体.研究结果表明,NDV在绵羊体内是非致病性的,并且通过两种接种途径在绵羊体内均可以产生血凝抑制抗体、NDV特异性IgG抗体和中和抗体反应.本研究结果表明NDV有可能作为宿主限制性疫苗载体用于预防无特效疫苗的羊类疾病,且与气管灌注接种途径比,滴鼻接种途径相对安全.     

The response of normal and iron anemic calves to nasal infection with an attenuated strain of parainfluenza-3 virus.

Calves with experimentally induced iron deficiency anemia and normal calves, both groups deprived of colostrum, were exposed to intranasal instillation of an attenuated parainfluenza-3 virus strain.The calves became infected, but there was no difference in the clinical picture between the 2 groups of calves. Neither was there a difference in the humoral or local immune response to parainfluenza-3 virus.     

Prime-boost immunization with HA/C3d DNA followed by a recombinant pseudorabies virus boost enhanced protective immunity against H3N2 swine influenza virus in mice

DNA and recombinant virus vaccines against swine influenza virus (SIV) have been pursued with promising results, but induce poor immunogenicity. This study evaluated the effects of a vaccine regimen in mice including priming with three DNA vaccines expressing soluble HA (sHA), complete HA (tmHA), or sHA fused with three copies murine C3d (sHA-mC3d3) and boosting with recombinant pseudorabies virus expressing HA (rPRV-HA). Immune responses were monitored by ELISA, HI assays, and virus neutralization. Protective efficacy was evaluated by virus isolation from lungs, distribution in tissues, and pathology following challenge with H3N2 SIV. Priming with sHA-mC3d3 and boosting with rPRV-HA induced higher levels of HA-specific antibodies and yielded the most effective protection. This finding implied that priming with a DNA vaccine expressing C3d fused with antigen and boosting with a recombinant vector vaccine is an effective way to induce protective humoral immunity and prevent some infectious diseases.     

Enhancing immune responses to inactivated foot-and-mouth virus vaccine by a polysaccharide adjuvant of aqueous extracts from Artemisia rupestris L.

BackgroundNew-generation adjuvants for foot-and-mouth disease virus (FMDV) vaccines can improve the efficacy of existing vaccines. Chinese medicinal herb polysaccharide possesses better promoting effects.ObjectivesIn this study, the aqueous extract from Artemisia rupestris L. (AEAR), an immunoregulatory crude polysaccharide, was utilized as the adjuvant of inactivated FMDV vaccine to explore their immune regulation roles.MethodsThe mice in each group were subcutaneously injected with different vaccine formulations containing inactivated FMDV antigen adjuvanted with three doses (low, medium, and high) of AEAR or AEAR with ISA-206 adjuvant for 2 times respectively in 1 and 14 days. The variations of antibody level, lymphocyte count, and cytokine secretion in 14 to 42 days after first vaccination were monitored. Then cytotoxic T lymphocyte (CTL) response and antibody duration were measured after the second vaccination.ResultsAEAR significantly induced FMDV-specific antibody titers and lymphocyte activation. AEAR at a medium dose stimulated Th1/Th2-type response through interleukin-4 and interferon-γ secreted by CD4⁺ T cells. Effective T lymphocyte counts were significantly elevated by AEAR. Importantly, the efficient CTL response was remarkably provoked by AEAR. Furthermore, AEAR at a low dose and ISA-206 adjuvant also synergistically promoted immune responses more significantly in immunized mice than those injected with only ISA-206 adjuvant and the stable antibody duration without body weight loss was 6 months.ConclusionsThese findings suggested that AEAR had potential utility as a polysaccharide adjuvant for FMDV vaccines.     

Mucin biosynthesis in the bovine goblet cell induced by Cooperia oncophora infection

Mucin hypersecretion is considered to be one of the most common components of the immune response to gastrointestinal nematode infection. However, investigations have not been conducted in the Cattle–Cooperia oncophora system to verify the findings largely derived from murine models. In this study, we examined the expression of seven mucins and seven enzymes in the mucin biosynthesis pathway involved in O-linked glycosylation in the bovine small intestine including goblet cells enriched using laser capture microdissection during a primary C. oncophora infection. At the mRNA level, MUC2 expression was significantly higher in both lamina propria and goblet cells at 28 days post-infection compared to the naïve control. MUC5B expression at the mRNA level was also higher in lamina propria at 28 dpi. Expression of MUC1, MUC4, MUC5AC, and MUC6 was extremely low or not detectable in goblet cells, columnar epithelial cells, and lamina propria from both naïve control and infected animals. Among the seven enzymes involved in post-translational O-linked glycosylation of mucins, GCNT3, which may represent one of the key rate-limiting steps in mucin biosynthesis, was up-regulated in goblet cells, columnar epithelial cells, lamina propria, and gross small intestine tissue during the course of infection. Western blot analysis revealed that MUC2 glycoprotein was strongly induced by infection in both gross small intestine tissue and its mucosal layer. In contrast, the higher MUC5B protein expression was observed only in the mucosal layer. Immunohistochemistry provided further evidence of the mucin glycoprotein production and localization. Our results provided insight into regulation of mucin biosynthesis in various cell types in the bovine small intestine during gastrointestinal nematode infection and will facilitate our understanding of mucins and their role in immune response against parasitic nematodes.     

Role of polymorphonuclear neutrophils (PMNs) and NK cells in the protection conferred by different vaccines against Chlamydophila abortus infection

Ovine enzootic abortion (OEA) is caused by Chlamydophila abortus, an intracellular bacterium which acts by infecting the placenta, causing abortion in the last term of gestation. The main prevention strategy against OEA is the vaccination of flocks. An effective vaccine against C. abortus must induce a Th1-like specific immune response, which is characterized by the early production of IFN-gamma and the activation of CD8(+)T cells. Moreover, vaccine effectiveness could be modulated by the functioning of the innate immunity. The purpose of this study was to ascertain how polymorphonuclear neutrophils (PMNs) and NK cells might influence vaccine-induced protection. The live attenuated 1B vaccine and two inactivated experimental vaccines, adjuvated with aluminium hydroxide (AH) or QS-21 (QS), were used in PMN-depleted or NK cell-depleted mice. For PMN depletion, RB6-8C5 monoclonal antibody, which recognizes GR1(+) receptors (Robben, P.M., LaRegina, M., Kuziel, W.A., Sibley, L.D. 2005. Recruitment of Gr-1(+) monocytes is essential for control of acute toxoplasmosis. The Journal of Experimental Medicine 201, 1761-1769.) was used, while for NK cell-depletion the anti-asialo GM1 polyclonal antibody was used. The depletion of PMNs caused 100% mortality in non-vaccinated mice (NV) and 60% mortality in the AH-vaccinated mice by day 10 p.i., while both groups showed a significant increase in their bacterial burden in the liver by day 4 p.i. The depletion of NK cells caused mortality only in the NV group (50% by day 10 p.i.), although this group and the 1B vaccinated mice showed an increased bacterial burden in the liver at day 4 p.i. Our results suggest that the importance of PMNs in inactivated vaccines depends on the adjuvant chosen. The results also demonstrated that the importance of NK cells is greater in live vaccines than in inactivated vaccines.     

Intestinal mucosal immune response against virulent duck enteritis virus infection in ducklings

Duck virus enteritis (DVE) is an acute and contagious herpes virus infection of duck, geese and swans with high morbidity and mortality. The development of specific mucosal immune system against duck enteritis virus (DEV) infection for ducks has been hindered by a lack of knowledge concerning the purification of immunoglobulin A (IgA) of duck. In the present work, the method for purification of duck immunoglobulin A was developed, and the induction of intestinal mucosal immune responses against DEV was studied by orally infected ducklings with virulent DEV. The results showed that a continuous increased DEV DNA levels were observed in blood and various organs examined of orally infected ducklings throughout the infection, which was accompanied by the development of infection in ducklings from mild progressed to severe pathological lesions. Furthermore, a marked increased level of DEV-specific IgA and IgG antibodies in bile, serum and the intestinal tract, as well as the density of IgA⁺ cells in intestine were detected between 1 and 12 days p.i., followed by a drastic reduction of the antibody levels and the density of IgA⁺ cells at 15 days p.i. The results indicate that the DVE infection can stimulate both IgA-dominated antibody immune responses in the intestinal tract, and IgG-dominated antibody systemic immunity in the serum of ducklings orally inoculated with virulent DEV. The severe lesions of the villus epithelial cells and the lymphoid organs can suppress the intestinal mucosal immune responses.     

Pseudorabies virus infection in hunting dogs in Oita,Japan: Report from a prefecture free from Aujeszky’s disease in domestic pigs

We isolated two pseudorabies virus (PRV) isolates (designated OT-1 and OT-2) from two hunting dogs exhibiting neurological manifestations after eating the flesh of wild boar hunted in Oita prefecture, Kyushu Island, Japan. The isolates corresponded to a previously reported PRV (MY-1 strain) isolated from a hunting dog in neighboring Miyazaki prefecture, and it clustered into genotype II based on the glycoprotein C sequence. Our results suggest that this common PRV strain may have been maintained in wild boars on Kyushu Island even though domestic pigs in this area have attained an Aujeszky’s disease-free status.     

Tembusu virus infection in Cherry Valley ducks: The effect of age at infection

Three groups of Cherry Valley ducks at 5 day, 2 week and 5 week of age were intranasally infected with the WFCL strain of Tembusu virus (TMUV) to investigate the effect of host age on the outcome of TMUV infection. For each age group, clinical signs, gross and microscopic lesions, viral copy numbers in tissues and serum neutralizing antibody titers were recorded. Age-related differences in the resistance to TMUV infection were observed with younger ducks being more susceptible. Some ducks infected at 5 day and 2 week of age developed severe clinical signs, including severe neurological dysfunction and death. However, subclinical signs and no mortality were observed in ducks infected at 5 week of age. A decline in the severity of gross and microscopic lesions was observed as ducks mature. Systemic infections were established in the three age groups post challenge. Higher viral copy numbers in the tissues, especially in vital organs such as the brain and the heart, were developed in the ducks infected at 5 day of age than older ducks, correlating with the severity of clinical signs and lesions in the tissues. Furthermore, ducks infected at 5 week of age developed significantly higher serum neutralizing antibody titers than ducks infected at 5 day of age as determined by serum neutralization test. Therefore, age-related differences in the resistance to TMUV infection should be considered when studying the pathogenicity, pathogenesis, formulation of the vaccination and therapy strategies of TMUV infection in ducks.     

Feline immunodeficiency virus infection: a valuable model to study HIV-1 associated encephalitis

Feline immunodeficiency virus (FIV), like human immunodeficiency virus (HIV)-1, is a neurotropic lentivirus and is associated with neuropathology in natural and experimental infections. FIV enters the brain early following experimental infection, and virus has been proposed to enter the brain via the blood–brain barrier and blood–CSF barrier, within infected lymphocytes and monocytes/macrophages. However the entry of cell-free virus or the direct infection of brain endothelial cells and astrocytes of the blood–brain barrier may also contribute to CNS infection. This review explores the role played by the FIV model in the elucidation of mechanism of lentiviral entry to the brain and viral interactions with the CNS, particularly in relation to lymphotropic lentiviruses.