Prevalence of natural virus infections in laboratory mice and rats used in Canada

Results of serological tests carried out over a period of 6 years to detect the presence of antibodies against 14 indigenous viruses in mice and rats used in 32 Canadian institutions are reported. Close to 20,000 individual sera were tested by the complement fixation or the hemagglutination inhibition technics. In order of mouse colony prevalence the six most common viruses present were pneumonia virus of mice, mouse hepatitis virus, rat virus, minute virus of mice, Sendai, and Theiler's mouse encephalomyelitis viruses. The most common viruses present in rat colonies were minute virus of mice, K virus, coronaviruses (rat coronavirus or sialodacryoadenitis virus), rat virus, H-1, pneumonia virus of mice, Theiler's mouse encephalomyelitis viruses, Sendai, and reovirus 3.     

Application of dried whole blood collected on filter paper disks to ELISA for the detection of Sendai virus and mouse hepatitis virus antibodies in mice.

This study was undertaken to simplify the preparation procedures for test specimens by applying whole blood collected on filter paper disks. The results of ELISA obtained using specimens collected in this way for the detection of Sendai virus and mouse hepatitis virus antibodies in mice were comparable to those for ordinary ELISA using serum samples.     

Evaluation of mouse thymic virus antibody detection techniques

Three serologic test methods for detection of serum antibodies to mouse thymic virus (MTV) were compared, including enzyme-linked immunosorbent assay (ELISA), complement fixation (CF) test and indirect fluorescent antibody (IFA) test. Serum was collected at regular intervals from CD-1 mice inoculated intraperitoneally with approximately 200 infectious doses of MTV, and from uninoculated mice placed in cages with the inoculated animals. The inoculated mice became MTV antibody-positive by all three assay methods at 15 days post inoculation, while the cage-contacts were seropositive by all methods at 30 days after contact. Although the incidence of positive results was similar by all methods, titers measured by ELISA were substantially higher than those measured by CF and IFA tests. Because MTV can cause persistent infections that adversely affect the suitability of mice for research, it is recommended that testing for antibodies to this virus be performed routinely.     

Production of idiotypic and anti-idiotypic antibodies by BALB/c mice in response to immunizations with glucagon, vasopressin, or insulin: supporting evidence for the network concept

After immunizations with glucagon or vasopressin, either conjugated to keyhole limpet hemocyanin or adsorbed to polyvinylpyrrolidone, both anti-hormone and anti-receptor activities were detectable in the serum of injected mice. Anti-hormone activity was identified by ELISA techniques; anti-receptor activity, by determining the ability of serum samples to compete with labeled hormone for glucagon or vasopressin receptors on rat liver plasma membranes. Anti-receptor activity appeared only after the peak anti-hormone response to each immunogen had been established, and required intensive immunizations (six to nine monthly injections). The presence of anti-idiotypic antibodies in serum samples containing glucagon or vasopressin anti-receptor activity was confirmed by demonstrating selective binding of such samples to corresponding rabbit idiotypic antibodies. Serum from mice immunized with insulin also contained anti-hormone activity, as determined by ELISA, and anti-receptor activity, as determined by noting insulin-mimicking properties in stimulating glucose transport in rat adipocytes. The anti-insulin receptor activity developed after only one boost with the hormone. These results are consistent with Jerne's network hypothesis in that the glucagon, vasopressin, and insulin anti-receptor activity may be attributed to antibodies produced in mice as part of an idiotypic-anti-idiotypic network.     

The advantage of enzyme-linked immunosorbent assay (ELISA) as a method of microbiological monitoring for rat virus (RV).

An enzyme-linked immunosorbent assay (ELISA) was tested to detect antibodies against rat virus (RV). The purified ELISA antigens were prepared from rat embryonic cells infected with RV-13 (prototype strain) and UT-2 (Japanese isolate), respectively. Western blotting analysis confirmed that both of these antigens had three structural polypeptides (81 K, 61 K, and 59 K). Eleven laboratory and wild rat colonies in Japan were tested for rat virus contamination, serologically. No significant differences in the sero-positive ratio and the distributions of ELISA titers were demonstrated in the ELISA, using antigens from RV-13 and UT-2. ELISA was more sensitive and specific for detecting antibodies against RV from rat serum rather than hemagglutination inhibition (HI) test. This study also confirmed that the RV contaminated widely in colonies of laboratory and wild rats in Japan, and suggested that RV would have to be checked during the microbiological monitoring of laboratory rats.     

Diagnosis of murine infections in relation to test methods employed

Comparative investigations of Sendai virus, pneumonia virus of mice (PVM), mouse encephalomyelitis virus (mouse polio), minute virus of mice (MVM), and reovirus type 3 (Reo 3) infected murine colonies revealed a 30% higher incidence of positive sera when enzyme-linked immunosorbent assay (ELISA) was employed instead of hemagglutination inhibition (HI) tests. Equivalent sensitivity as in the ELISA was obtained when the same sera were investigated by indirect immunofluorescence (IF) tests. The virus purification techniques described resulted in highly suitable antigens for all indirect ELISA established. Since IIF requires no purified antigens, this test is recommended as an alternative to ELISA as well as to HI and complement fixation (CF) tests for laboratories lacking the necessary equipment for high speed centrifugation. A high incidence of false positive HI reactions was found particularly in Reo 3 routine serology. An updated survey of seromonitoring showed that European murine colonies appeared to be infected far less with Reo 3 if ELISA or IIF tests were employed. During 1982-1984, only 13% of the mouse colonies screened possessed Reo 3 positive sera whereas no natural Reo 3 infection was found in rat colonies. Mouse hepatitis virus (MHV) and the coronaviruses of rats exhibited the highest incidence in murine colonies. A total of 60% of mouse and 41% of rat colonies were found to be infected by these viruses. In comparison with earlier serological surveys, the relative incidence of other murine infections was similar. Antibodies against Bacillus piliformis (Tyzzer's disease) were detected by the IIF test in 41% of the rat colonies screened.     

丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCVHVR1模拟表位融合蛋白。用Westernblot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况。皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应。结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35)。融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCVHVR1合成肽发生交叉反应。本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值。     

A quantitative immunofluorescence test for detection of serum antibody to Sendai virus in mice

A simple, semi-automated immunofluorescence assay, (TRACK XI), was developed for the detection and quantitation of circulating antibodies to Sendai virus in mice. The assay was validated by selecting Sendai virus-free and naturally infected mice from six different colonies and testing each serum in both ELISA and quantitative immunofluorescence (QIF) assays. The QIF test utilizes Sendai viral proteins immobilized within a dried colloid gel and permits serum antibody quantitation in 30 minutes. Using a dedicated fluorometer, antibody titers in test sera are calculated automatically from a three-point best fit calibration line. The QIF test gave 92.9% agreement with the ELISA and proved to be a reproducible, accurate and convenient assay for the quantitative measurement of serum antibody to Sendai virus in mice.     

Studies on the development of an ELISA kit for microbiological monitoring. 1. Evaluation of the reliability of the prototype kit by field tests

The prototype of an ELISA kit using protein A as the second reaction reagent for mice and anti-rat IgG for rats was prepared for seromonitoring of the Sendai virus and mouse hepatitis virus (MHV)/sialodacryoadenitis virus (SDAV)/Parker's rat coronavirus (PCV) infections. The respective antigen strains and protein concentrations were Sendai virus MN strain, 2 micrograms/ml and MHV Nu-67 strain, 5 micrograms/ml. The reliability of this prototype kit was investigated in two field tests performed on a total of 10,094 mouse and rat sera from 147 institutions. The results indicated that the two types of kits for the two species of animals were highly specific, but it is necessary to increase the detection sensitivity of the MHV antigen for the MHV antibody of mice and SDAV/PCV antibodies of rats.     

Ten-year long monitoring of laboratory mouse and rat colonies in French facilities: a retrospective study

From 1988 to 1997, a total of 69 mouse colonies and 36 rat colonies were examined for the presence of antibodies to 14 indigenous viruses of mice and rats. Among mouse viruses, high positivity rates were observed with mouse hepatitis virus (MHV), Theiler's encephalomyelitis virus (THEMV), minute virus of mice (MVM), Sendai virus and pneumonia virus of mice (PVM); the prevalence rates were high in rats with Khilam's rat virus (KRV), THEMV, Toolan's H-1 virus, Sendai virus, Parker's rat coronavirus (RCV/SDA) and PVM. During the last decade, the prevalence of some agents such as MHV, Sendai virus, THEMV, PVM and MVM has apparently decreased although they were still present in 1997 (except for PVM). Another point is the constant increase of colonies found free of viruses through this decade, demonstrating the efforts of the French research community to increase the quality of hygiene in laboratory animals.     

抗登革病毒NS1单抗的制备及检测NS1方法的建立

制备抗登革病毒NS1蛋白单克隆抗体,建立检测NS1的ELISA方法。表达1~4型登革病毒NS1蛋白,将1型NS1蛋白纯化后免疫BALB/c小鼠,通过杂交瘤技术制备单克隆抗体。经ELISA、Western blotting、间接免疫荧光筛选和鉴定单克隆抗体,进行纯化和HRP标记。通过鉴定每两株单抗之间是否存在竞争作用,选择非竞争单抗组合并建立NS1捕获法ELISA。结果获得7株高滴度抗NS1单抗,捕获法ELISA可以检出10ng/mL NS1。原核表达登革病毒NS1蛋白制备的单抗可以和天然病毒抗原反应,NS1捕获法ELISA可以用于登革病毒感染检测。     

Results of using the ELISA method in studying tick-borne encephalitis and rabies

The results of using the indirect variant of ELISA for the study of serum samples from humans and white mice for the presence of antibodies to tick-borne encephalitis virus and rabies virus are presented. The high sensitivity and specificity of this method have been confirmed.     

Comparison of enzyme-linked immunosorbent and virus neutralization assays for the serology of reovirus infections in laboratory animals

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of reovirus-specific IgM and IgG serum antibodies in rodents, detecting reovirus group reactive antibodies, was compared with reovirus types 1, 2 or 3 specific virus neutralization (VN) assays. To this end, serum samples were collected from specific pathogen-free (SPF) BALB/c RIVM mice, after experimental infection with any of the three mammalian reovirus serotypes. The majority (seven out of twelve) of the reovirus type 1-infected and one third (two out of six) of the reovirus type 3-infected mice died within 15 days after infection, whereas all (six out of six) of the reovirus type 2-infected animals survived. Using reovirus type 2 antigen in the ELISA, reovirus-specific IgM antibodies could be demonstrated within 1 week after infection in all the mice infected with reovirus types 2 or 3 and in the majority of the mice infected with type 1. Similarly, reovirus-specific IgG could be detected within 3 weeks in all the surviving mice infected with reovirus types 2 or 3 and within 5 weeks in all surviving mice infected with reovirus type 1. These results coincided well with the data obtained in the VN assays, in which all the infected animals also could be detected within 1 week after infection. As expected, titers were detected most rapidly and to the highest levels in the homologous VN assays. Given the sensitivity and the specificity of the ELISA system demonstrated in this paper and its suitability for incorporation in automated systems, the ELISA system should be considered valuable for the routine serologic diagnosis of reovirus infections in rodents.     

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive ELISA and for antigen capture in a sandwich ELISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by Western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from Egyptian or South African lineages. These monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.     

Adjuvant effects of dimethyl dioctadecyl ammonium bromide, complete Freund's adjuvant and aluminium hydroxide on neutralizing antibody, antibody-isotype and delayed-type hypersensitivity responses to Semliki Forest virus in mice.

Outbred mice were inoculated subcutaneously with inactivated Semliki Forest virus (SFV) in saline and combinations of the virus with complete Freund's adjuvant (CFA) aluminium hydroxide (Al) and dimethyl dioctadecyl ammonium bromide (DDA). The immune response was evaluated for delayed-type hypersensitivity, for total ELISA antibodies and antibody-isotypes and for neutralizing antibodies. DDA was the most efficient adjuvant in inducing DTH, CFA the second and Al induced a DTH response that was only slightly higher (statistically not significant) than that induced by the inactivated virus without adjuvants. All adjuvants enhanced the production of ELISA antibodies to similar levels. However, the levels of neutralizing antibodies induced were low in mice which were inoculated with the inactivated SFV alone or mixtures of the virus with Al. DDA induced high levels of neutralizing antibodies and CFA induced intermediate levels. The pattern of antibody-isotypes induced by DDA and CFA was different from the pattern induced by the inactivated virus or by the virus mixed with Al: DDA and CFA induced low amounts of IgG1 antibodies and relatively higher amounts of IgG2a and IgG2b antibodies while the inactivated virus and the mixture of the virus with Al induced higher proportions of IgG1 antibodies. In sera from convalescent mice the majority of antibody activity resided in the IgG2a and IgG2b immunoglobulin subclasses, while IgG1 antibodies were undetectable.     

Effect of Serum Heat-Inactivation and Dilution on Detection of Anti-WNV Antibodies in Mice by West Nile Virus E-protein Microsphere Immunoassay

Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.     

Replication-competent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (RSV) antigens protect mice against RSV challenge

Foreign glycoproteins expressed in recombinant vesicular stomatitis virus (VSV) can elicit specific and protective immunity in the mouse model. We have previously demonstrated the expression of respiratory syncytial virus (RSV) G (attachment) and F (fusion) glycoprotein genes in recombinant VSV. In this study, we demonstrate the expression of RSV F and G glycoproteins in attenuated, nonpropagating VSVs which lack the VSV G gene (VSVDeltaG) and the incorporation of these RSV proteins into recombinant virions. We also show that intranasal vaccination of mice with nondefective VSV recombinants expressing RSV G (VSV-RSV G) or RSV F (VSV-RSV F) elicited RSV-specific antibodies in serum (by enzyme-linked immunosorbent assay [ELISA]) as well as neutralizing antibodies to RSV and afford complete protection against RSV challenge. In contrast, VSVDeltaG-RSV F induced detectable serum antibodies to RSV by ELISA, but no detectable neutralizing antibodies, yet it still protected from RSV challenge. VSVDeltaG-RSV G failed to induce any detectable serum (by ELISA) or neutralizing antibodies and failed to protect from RSV challenge. The attenuated, nonpropagating VSVDeltaG-RSV F is a particularly attractive candidate for a live attenuated recombinant RSV vaccine.     

免疫A型呼吸道合胞病毒G75-225蛋白的小鼠对病毒感染的抗性

目的:评价A型呼吸道合胞病毒(RSV)G75-225蛋白(G151)与二硫键异构酶(DsbA)的重组蛋白抗原DsbA-G151的免疫活性。方法:采用PCR方法从A型RSVG蛋白扩增G151基因片段,插入表达载体pET-DsbA,经E.coli表达、亲和层析纯化制备DsbA-G151蛋白;将其免疫BALB/c小鼠后获得相应的抗血清,利用ELISA方法、保护性实验检测蛋白免疫活性。结果:构建了表达载体pET-DsbA-G151,表达、纯化获得了重组蛋白DsbA-G151。ELISA检测表明,DsbA-G151能在小鼠体内产生高滴度的特异性IgG;保护性实验显示该蛋白能有效保护BALB/c小鼠不被RSV感染。结论:经ELISA检测、保护性实验,表明DsbA-G151具有良好的免疫原性。     

表达尼帕病毒G囊膜糖蛋白重组牛痘病毒的研究

采用牛痘病毒WR株,构建了表达哺乳动物密码子优化的NiV G蛋白基因的重组病毒rWR-NiV-G。Westernblot证实大小为66kDa的重组G蛋白在rWR-NiV-G感染的Hela细胞中获得表达;采用兔抗NiV高免血清间接免疫荧光检测重组痘病毒表达G蛋白显示出良好的特异免疫反应原性。rWR-NiV-G感染NiV敏感的BHK细胞系,并与NiV融合蛋白F共同表达,可形成强烈细胞融合现象。rWR-NiV-G感染免疫BALB/c小鼠,可诱导显著的NiV G蛋白特异体液免疫反应。以原核表达NiV G蛋白片段为包被抗原,间接ELISA检测rWR-NiV-G感染免疫小鼠血清中的G蛋白特异抗体,具有良好的敏感性和特异性。同时,rWR-NiV-G感染免疫小鼠血清中的G蛋白特异抗体可有效中和NiV囊膜蛋白F和G介导的伪型VSV重组病毒侵入NiV易感宿主细胞的感染性。结果表明,重组牛痘病毒表达的NiV G蛋白有良好的免疫原性和生物学活性功能,为进一步深入研究NiV G蛋白生物学功能、免疫原性及重组活载体疫苗研究奠定了重要基础。