Immunological characterization of the Marin County strain of astrovirus

Summary Marin County virus (MCV) was isolated from a stool suspension and serially propagated in human embryonic kidney cell cultures. MCV particles in stool and cell-propagated virus stocks showed reactivity by immune electron microscopy (IEM) with rabbit antiserum to astrovirus type 5. MCV antigen was also detected in two MCV stool samples by enzyme immunoassay (EIA) with an astrovirus group-specific monoclonal antibody. Acute and convalescent sera from 3 of 3 MCV-infected patients showed seroconversion to cell-propagated MCV by EIA. Immunofluorescence of MCV propagated in cell culture showed positive reactivity with an astrovirus group specific monoclonal antibody and astrovirus type 5 antiserum, with some cross-reactivity with astrovirus type 1. Similar results were obtained with the prototype strain of astrovirus type 5. However, in plaque-reduction assays, both the prototype astrovirus type 5 and MCV were neutralized by type 5 antiserum only. We conclude that MCV can be serially propagated by techniques used for previously described astroviruses and is serotypically an astrovirus type 5.     

Detection of adenovirus types 40 and 41 in stool specimens by immune electron microscopy

An immune electron microscope (IEM) test was developed that allowed the direct detection of adenovirus type 40 (ad 40) or ad 41 in stools specimens. The polyclonal rabbit antisera used differentiated ad 40 and 41 from other ad serotypes but not from each other. The method was evaluated in a 13 month prospective study of stools from children with gastroenteritis. Seventy-two specimens found to contain ad by conventional electron microscope screening were retested by IEM. Results were typically obtained within 2 hr and showed that 55 (76%) viruses typed as ad 40/41. No ads were recovered from conventional virus isolation attempts on these specimens. Additionally, 39 of these 55 viruses were tested by restriction endonuclease analysis (REA) after growth in 293 cells, and results showed that all produced digest patterns typical of ad 40 (seven cases) or ad 41 (32 cases). Twenty-four percent (17/72) of viruses could not be typed by IEM; 9/17 (53%) yielded ads [ad 1 (1), ad 2 (4), ad 5 (1), ad 6 (1), ad 7 (2)] in routine culture, whereas REA identified the other eight as ad 2 (6), ad 1 (1), and ad 41 (1). The concordance between IEM and the reference methods was therefore 100% specificity and 97.5% sensitivity. The method described allows the clinically useful diagnosis of ad 40/41 infection to be rapidly made and will be a particularly useful technique in laboratories screening faeces by electron microscopy.     

Multicenter evaluation study on a new HEp2 ANA screening enzyme immune assay.

The COBAS Core HEp2 ANA enzyme immune assay (EIA) was evaluated in a precision and a clinical sample study in comparison to indirect immunofluorescence assay (IFA) on HEp2-cells. In the precision study the COBAS Core EIA yielded intraassay coefficient variations (CVs) mostly below 9%, and interassay CVs between 4.7% and 10.4%. When comparing the COBAS Core EIA to IFA, the results corresponded well in healthy subjects, systemic lupus erythematosus, mixed connective tissue disease and rheumatoid arthritis. In the case of Sj?gren's syndrome and scleroderma patients the COBAS Core EIA yielded a lower rate of positive results compared to IFA. This discrepancy may be explained by the lack of detection of autoantibodies to nuclear antigens that can be identified only by IFA due to their compartmentalization and higher localized antigen density in HEp2 cells. The discrepancies in the group of dermato/polymyositis patients are due to the fact that the EIA contains mainly nuclear antigens and was able to detect only antibodies against the cytoplasmic Jo1 antigen that was added to the HEp2 nuclear extract. Routine sera were also evaluated; good agreement was found in sera from patients attending tertiary reference centres for autoimmune diseases but a higher number of discrepancies was reported in sera from unselected populations.     

Some characteristics of three porcine adenoviruses

Summary Three strains of cloned porcine adenoviruses (25RCl, 6618Cl and A47C1) were shown to be serologically distinct in cross neutralization tests with specific antisera prepared in rabbits. They were not neutralized by reference antisera against the known human adenoviruses. Neutralizing antibodies to each of the three serotypes were demonstrated in samples of pig serum. Guinea pig, human 0, rat, monkey and mouse red cells were haemagglutinated by strain 25RCl, but fowl, ox, pig, rabbit and sheep cells were not haemagglutinated. Strains 6618Cl and A47Cl did not haemagglutinate these cells. Specific rabbit antiserum and normal pig sera contained HI antibodies to strain 25RCl adenovirus. Each of the three strains was resistant to ether and pH 4.0 buffer and growth in PK cells was inhibited by FUDR. In infected PK cells stained with haemotoxylin and eosin, typical basophilic central nuclear masses were produced by each strain of porcine adenovirus. In PK cells infected with strain 6618Cl multiple eosinophilic intranuclear inclusions in some cells were a prominent feature of the CPE. The provisional designation of strains 25RCl, 6618Cl and A47Cl as porcine adenovirus types 1, 2, and 3, respectively, is proposed.     

Isolation and propagation of enteric adenoviruses in HEp-2 cells.

Eighty-two stool samples from children with gastroenteritis in Canada, England, and Thailand which had been shown to contain adenovirus antigen (by a group-specific enzyme-linked immunosorbent assay) or adenovirus particles (by electron microscopy) or both, were tested for primary isolation of enteric adenoviruses in HEp-2 and Graham 293 cells. Graham 293 cells are known to support the replication of enteric adenovirus types (Ad40 and Ad41) on primary isolation, whereas HEp-2 cells reportedly do not. Of the 82 adenovirus isolates, 73 could be typed as Ad40 or Ad41 by type-specific monoclonal antibody enzyme-linked immunosorbent assay and by analysis of SmaI endonuclease digests. Of these 73, 30 (41%) could be isolated in HEp-2 cells, which included 43% (9/21) of those typed as Ad40 and 40% (21/52) of those typed as Ad41. On the basis of these results, the growth characteristics of adenoviruses in HEp-2 cell cultures, commonly used to distinguish enteric from nonenteric adenovirus types, are not valid for either diagnosis or epidemiological studies. For the samples studied here, use of these nondefinitive criteria would result in underestimation of the incidence of enteric adenoviruses in viral gastroenteritis.     

Candidate adenoviruses 40 and 41: fastidious adenoviruses from human infant stool

About 200 antigenically related adenoviruses were isolated from cases of infantile diarrhoea in the Netherlands and North-West Germany. The viruses were fastidious and failed to replicate serially in human diploid fibroblasts and in primary human embryonic kidney cells. A number of strains were established in HeLa, HEp-2, Graham (293), cynomolgus monkey kidney, and Chang conjunctival cells. The viruses were mammalian adenoviruses by the usual criteria. No relationship to the 39 known human adenovirus species was found, either by neutralization tests or by haemagglutination inhibition tests. Neutralization tests showed two distinct variants, represented by strains Tak and Dugan. The variants were identical in haemagglutination inhibition tests. DNA restriction enzyme analysis showed Tak and Dugan to have considerably different genomes, indicating that these variants should be classified as different species (Wadell et al, 1983). It is proposed that the variants should be called Mastadenovirus h 40 (with reference strains Dugan and Hovi X) and Mastadenovirus h 41 (with reference strain Tak). Neutralization and haemagglutination inhibition tests demonstrated that the viruses from Glasgow and Helsinki (Hovi X) described by Johansson et al [1980] and by Kidd and Madeley [1981] belong to these two adenovirus species.     

bcl-2重组腺病毒载体的构建

本研究目的在于构建 bcl-2的正义和反义重组腺病毒载体 ,以研究 bcl-2过度表达对周围神经损伤后脊髓前角运动神经元抗凋亡能力和对轴突再生的影响。为此 ,将正义和反义 bcl-2 c DNA克隆到腺病毒穿梭质粒 p Ad CMV,利用体内同源重组原理 ,用该重组质粒与 p JM17质粒共转染 2 93细胞 ,获得复制缺陷型重组腺病毒 Ad CMV/sense-bcl-2和 Ad CMV/antisense-bcl-2 ,其中 bcl-2 c DNA插入腺病毒基因组的 E1区 ,并由 CMV启动子控制。利用原位杂交、免疫组化反应鉴定重组腺病毒。结果显示 :从感染 Ad CMV/sense-bcl-2重组腺病毒的 2 93细胞中检测到 bcl-2的表达 ;重组腺病毒可感染 2 93细胞并在 2 93细胞内进行有效复制。从而表明本研究成功地构建了 bcl-2重组腺病毒 ,为进一步研究 bcl-2重组腺病毒的功能提供了条件     

Recognition of transplantation antigen in vitro: blocking of recognition structures by specific antibody

Recognition of cellular murine transplantation antigens by lymphoid cells could be blocked by antisera against recognition structures (RS). These antisera were provoked by the injection of parental strain lymph node cells in low doses into adult F₁ hybrids. The activity of anti-RS antisera was highly specific. Treatment of recognizing cells with a particular serum inhibited the reaction towards a given transplantation antigen but did not impair recognition of another histo-compatibility antigen. Anti-RS antisera could be neutralized only by absorption with lymphoid cells carrying the specific RS.     

Single bacteriocin typing scheme for the vibrio group of organisms.

A total of 743 strains of O-I "agglutinable" and 293 strains of O I "inagglutinable" Vibrio cholerae were subjected to bacteriocin typing based on deferred antagonism of eight indicator bacteria, including two strains of V. cholerae, by the method of Chakrabarty et al. (Infect. Immun. 1:293-299, 1970). A minor modification that was effected in the typing medium was replacement of iodoacetic acid by ammonium chloride (at a final concentration of 0.003%) which appeared to regulate bacteriocin production more accurately and increase the stability of the types. Of the agglutinable strains, 94% were found to be bacteriocinogenic and could be fitted into 11 of the earlier-reported types (Chakrabarty et al., Infect. Immun. 1:293-299, 1970), and 6 newer types were recognized. Likewise, 285 (90.7%) of the inagglutinable strains were found to be bacteriocinogenic and could be accommodated within 10 original and 8 newer types identified for the inagglutinable vibrios. Thirteen types were common to the two groups of vibrios. The single bacteriocin typing scheme appeared to be simple and adequate for both groups of organisms, and the producer as well as the indicator bacteria behaved remarkably stably in this typing scheme over many years.     

Isolation and identification of adenovirus recovered from the stool of children with diarrhoea in Lagos, Nigeria

In order to establish the role of adenovirus in gastroenteritis in Nigerian children, stool samples were collected from 138 young children with gastroenteritis and 29 other age-matched controls. The samples were inoculated into 6 different tissue culture cell lines and isolates with characteristic CPE were subjected to CFT confirmation of the presence of adenovirus antigen. All the samples were screened for adenovirus by a commercially available enzyme immunoassay (Biotrin Adenovirus Antigen EIA) for the presence of the group antigen. Of the 138 stool samples from children with diarrhoea screened by EIA, only 23 (16.7%) were positive, while 4 (13.8%) of the 29 controls were also found positive. A greater proportion of the adenovirus-positive cases were aged between 13 and 24 months. There was no difference in the prevalence of the infection between male and female. The fastidious, enteric adenoviruses of subgroup F were sought utilizing a second EIA (AdenoClone), and occurred in 3.6% of the samples from diarrhoeic children and was not detected in the control group. There was no significant difference between the clinical symptoms of children infected with adenovirus and those not infected with adenovirus. However, the source of drinking water had a significant effect on the frequency of stool per day. The infection occurred all year round except for April and there was no significant correlation with the climatic factors. This study implies that the fastidious adenovirus is important in the aetiology of diarrhoeal illness in Nigerian children.     

Choice of reference assay for the detection of rotavirus in fecal specimens: electron microscopy versus enzyme immunoassay.

Two previously demonstrated sensitive and specific enzyme immunoassays (EIAs) for rotavirus, one using polyclonal and monoclonal antisera (TestPack Rotavirus [TPK]; Abbott Laboratories) and the other using only monoclonal anti-rotavirus antibodies (Rotaclone [RTC]; Cambridge BioScience Corporation), were evaluated as potential reference assays for rotavirus testing in comparison with direct negative-staining electron microscopy (EM), the current laboratory standard. Two hundred and seven stool samples collected consecutively during the winter of 1989 from children with acute diarrhea admitted to a ward for infants from 0 to 2 years of age were tested by the EIAs and by EM. TPK specimens were read visually; RTC results were read spectrophotometrically. Specimens with discordant EIA and EM results were further evaluated by a fluorescent focus assay. Specimens positive by EM and those negative by EM but positive by fluorescent focus assay were considered to be positive for rotavirus. Of the 207 stools tested, 35 (17%) were positive for rotavirus by these criteria. EM had a sensitivity of only 80%. Specificities were 100% for RTC and EM and 89% for TPK. These findings indicate that EM, although very specific, is relatively insensitive compared with a highly sensitive monoclonal antibody-based EIA. An EIA with high sensitivity and specificity, such as RTC, is a more appropriate reference standard for rotavirus testing.     

人血管形成素-1重组腺病毒高效制备

目的：制备人血管形成素-1重组腺病毒,为基因转染心肌缺血区促血管新生研究作准备。方法：人血管形成素-1重组粘粒与DNA末端蛋白复合物混合后以磷酸钙共沉淀法转染293细胞。获得的重组腺病毒经扩增,并进行酶切鉴定。结果：酶切结果显示,重组粘粒中人血管形成素-1基因插入方向正确,所获重组腺病毒带有此基因。重组腺病毒滴度达5.6×10¹¹ pfu/L。结论：所获人血管形成素-1重组腺病毒为E₁、E₃缺陷型重组子,可用于动物体内基因治疗研究。     

Terminal dilution purification of adenovirus prototypes,and the antigenic relationship between types 4, 16 and 14

Two terminal dilution purifications in HEK cells were performed with adenovirus prototypes of types 1 to 7, 12, 16, 19 and 9-15, and one terminal dilution with a freshly isolated type 8 strain. The antisera prepared against viruses after terminal dilution neutralized the original and the purified virus to the same extent, indicating the antigenic purity of the respective prototypes. Cross-reactions in neutralization with other types within the subgroup were also identical. Only the prototype of type 16 (Ch 79) showed a cross-neutralization with type 4, while four type 16 wild strains showed cross-neutralization with type 14, but none with type 4. The prototype (CH 79) is a "prime strain" in relation to the type 16 wild strains.     

Serological comparison among various strains of equine infectious anemia virus

Summary The serological relationship between 8 strains of equine infectious anemia (EIA) virus was investigated by means of complement fixation and neutralization tests. All strains had a common complement fixing antigen, although there were some differences in the intensity of CF reaction in certain antigen-antiserum combinations.Cross neutralization tests revealed that all strains were only neutralized by homologous antisera. The respective antibody titers recorded ranged from 32 to 512. When horses were infected with the virus strains, neutralizing antibodies could be detected as early as 32 to 87 days after inoculation and maximum titers were demonstrable after 42 to 148 days. Of 34 horses infected with 6 strains of EIA virus, 33 horses had neutralizing antibody when they were tested 52 to 127 days after inoculation. Antibodies were only produced against the virus strains inoculated, and not against heterologous strains.     

Dual-label time-resolved fluoroimmunoassay for the simultaneous detection of adenovirus and rotavirus in faeces

Dual-label time-resolved fluoroimmunoassay (TR-FIA) was used for the simultaneous detection of adenovirus and rotavirus in stool specimens. A one-incubation 1 h assay was used. Altogether 169 stool specimens collected from hospitalized children with acute gastroenteritis were tested. The specimens were dispensed with europium and terbium labelled antibodies against adenovirus and rotavirus, respectively, to coated microtitration strip wells. The 11 specimens positive for adenovirus in the EIA were also found positive in the TR-FIA. Fifty-six specimens positive for rotavirus with the reference EIA and one additional specimen were positive with the dual-label TR-FIA. Although the simultaneous assay using terbium and europium chelates gave lower assay sensitivities for both adenovirus and rotavirus when compared to separate assays using only europium chelates, the sensitivities were found to be at least equal to those of conventional standard antispecies EIA. With this one-incubation dual-label TR-FIA the results were obtained in less than 1.5 h for two viruses.     

Detection of HIV envelope specific antibodies by immunoelectron microscopy and correlation with antibody titer and virus neutralizing activity

Human antisera positive for HIV were evaluated on HTLV-IIIB producing cells by two different immunoelectron microscopic (IEM) techniques. In preembedding immunoferritin IEM a heavy label was observed with early budding HIV. Under the same conditions cell released 'mature' particles were almost negative, which could be explained by the direct observation that most of the surface glycoprotein knobs are lost spontaneously during virus maturation. Using freshly infected cultures after agarose embedding, immunogold labelling of ultrathin cryosections allowed us to detect and differentiate internal core as well as virus envelope antigens. A good qualitative correlation between neutralization titers and IEM labelling intensity was observed. This type of immunocryoultramicrotomy appears to be useful for the detection of antigens in and on the virion. It might turn out valuable for the characterization of the env gp120 epitopes of HIV.     

Detection of respiratory syncytial virus antigen in nasal washings by Abbott TestPack enzyme immunoassay.

We compared the new Abbott TestPack (TP) respiratory syncytial virus (RSV) enzyme immunoassay (EIA) with cell culture and two commercial RSV EIAs (from Abbott Diagnostics and Kallestad Laboratories) by using split samples of fresh nasal washings from children with suspected RSV disease. Two tubes of HEp-2 cells were inoculated and observed for cytopathic effect for 14 days, and isolates were confirmed by immunofluorescence. The TP EIA was performed by following the manufacturer's instructions. Specimens positive by TP EIA but negative by culture were examined in a competitive inhibition (blocking) assay using the TP EIA, and rabbit anti-RSV serum. Of 218 specimens, 93 were positive by culture, 105 were positive by TP EIA, 80 were positive by the Abbott Diagnostics EIA, and 87 were positive by the Kallestad Laboratories EIA. The sensitivity, specificity, positive predictive value, and negative predictive value of the TP EIA were 92, 86, 81, and 93%, respectively. Of 20 apparently false-positive TP EIAs, 10 of 14 that were positive when retested were neutralized in the blocking assay, indicating that they were truly positive. The recalculated sensitivity, specificity, positive predictive value, and negative predictive value of the TP EIA were 92, 91, 90, and 93%, respectively. We conclude that the TP EIA is easy to perform, rapid (less than 0.5 h), and accurate.     

Enteric adenovirus infection among infants with diarrhea in rural Bangladesh.

A total of 4,409 stool specimens from infants less than 5 years of age seeking treatment for diarrhea in Matlab, Bangladesh, were tested for the presence of adenoviruses by using an enzyme immunoassay (EIA). EIA-positive stool samples were serotyped with monoclonal antibodies specific for adenovirus type 40 (Ad40) and Ad41 and group antigen, inoculated into Graham G293 cells, and retested by EIA. Of adenovirus-positive cultures, 125 (2.8%) specimens were confirmed as enteric adenoviruses (EAds), of which 51 (40.8%) were typed as Ad40 and 74 (59.2%) were typed as Ad41, and 12 of 4,409 (0.3%) were identified as nonenteric adenoviruses. A slight peak of incidence of EAd infection was observed in the cool, dry months, and an outbreak of Ad40 infections occurred in March 1988, when the detection rate of EAd reached 12.3%. Information on age, gender, and symptoms was available for 80 infants infected with adenovirus only. Age distribution was similar for types 40 and 41 and nonenteric adenovirus; the median ages were 11, 12, and 12 months, respectively. The ratio of males to females for the 80 infants varied according to serotype; Ad40 had the highest male/female ratio, 2.17. The symptoms experienced by the 80 children were similar for each adenovirus type. The most common clinical features of EAd infection were watery diarrhea (87.5%), more than eight loose bowel movements per day in the 24-h period prior to presentation (68.8%), with vomiting (80.0%), abdominal pain (76.3%), and low-grade fever (95.0%); these symptoms are significantly similar to symptoms of infants infected with group A rotavirus. EAd infection generally gave rise to mild to moderate dehydration, which is significantly similar to dehydration produced by infection with rotavirus.     

A crucial role for B cells in neuroinvasive scrapie.

Although prions are most efficiently propagated via intracerebral inoculation, peripheral administration has caused kuru [Gajdusek et al, 1966], iatrogenic Creutzfeldt-Jakob disease (CJD) [Gibbs et al, 1997], bovine spongiform encephalitis (BSE), and new variant CJD [Hill et al, 1997; Bruce et al, 1997]. Neurological disease after peripheral inoculation depends on prion expansion within cells of the lymphoreticular system (LRS) [Lasmezas et al. 1996; Wilesmith et al, 1992]. In order to identify the nature of the latter cells, we inoculated a panel of immune deficient mice with prions intraperitoneally. While defects affecting only T lymphocytes had no apparent effect, all mutations affecting differentiation and responses of B lymphocytes prevented development of clinical scrapie. Since absence of B cells and of antibodies correlates with severe defects in follicular dendritic cells (FDCs), the lack of any of these three components may prevent clinical scrapie. Yet, mice expressing immunoglobulins exclusively of the M subclass without detectable specificity for PrPc, and mice with differentiated B cells but lacking functional FDCs, developed scrapie after peripheral inoculation: therefore, differentiated B cells appear to play a crucial role in neuroinvasion of scrapie regardless of B-cell receptor specificity.