Differentiation of influenza virus antigens using enzyme immunoassay]

Reported is a microprocess of enzyme immune assay in which slipformed air pockets of polyvinylchloride (PVC), as used in the pharmaceutical industry, are used as carriers of antigens or antibody. Two methods, the anti-globulin and the double-antibody methods, are based on antibody which had been coupled with alkaline phosphatase. Tests in which various sub-types of influenza virus were used have shown the double-antibody method to be a sensitive technique which can be successfully used in the differentiation of envelope antigens.     

Host-range barrier of influenza A viruses

Ample evidence suggests that all influenza viruses in mammals were probably derived from those in wild waterfowl at some time. In addition to those already established in mammals, the viruses have been transmitted to both mammals and to poultry from wild waterfowl and caused outbreaks in recent years. Experimentally, however, the viruses from one species of animals do not grow efficiently in other species. For example, human influenza viruses do not replicate in ducks or in horses, indicating their host range restriction. This paper reviews current knowledge on the host-range restriction of influenza viruses, focusing on the role of the hemagglutinin (HA).     

A competition enzyme immunoassay for brucellosis diagnosis

Two monoclonal antibodies (MAbs) conjugated with horseradish peroxidase were used independently in a competitive enzyme immunoassay (cEIA) to detect Brucella specific antibodies in 1120 sera from Brucella-free cattle, 61 from cattle known to be infected with B. abortus, and 207 sera from vaccinated calves. The results were compared to those obtained in the complement fixation test (CFT). The cEIA with both MAbs proved to be more sensitive than the CFT because no false-negative results were obtained. In addition, discrimination between sera from infected and vaccinated animals was more evident.     

Biology of influenza viruses

An attempt is made to present out of a multitude of available data those that will allow an insight into the biology of the influenza viruses, in order to contribute to the understanding of the present situation of influenza virus infections and the risk of pandemic influenza.This short overview is based on several reviews presented by others, some own reviews and statements, as well as some recent publications in scientific journals.     

Serological diagnosis of influenza A infections in the horse by enzyme immunoassay. Comparison with the complement fixation test

An enzyme immunoassay (EIA) using horseradish peroxidase and a type-specific antigen is described for the detection and quantitation of anti-influenza antibodies in the horse. Compared with the complement fixation (CF) test (using the same antigen), EIA proved to be superior with respect to sensitivity and reliability. The internal variation of EIA was low and thus small titres in EIA can be considered of diagnostic significance. However, no strict correlation with CF was observed. The use of an immunoconjugate against equine IgM in parallel with IgG would certainly improve the sensitivity of the test, especially in early stages of infection.     

Evolution of avian influenza viruses

Although influenza viruses can infect a wide variety of birds and mammals, the natural host of the virus is wild waterfowl, shorebirds, and gulls. When other species of animals, including chickens, turkeys, swine, horses, and humans, are infected with influenza viruses, they are considered aberrant hosts. The distinction between the normal and aberrant host is important when describing virus evolution in the different host groups. The evolutionary rate of influenza virus in the natural host reservoirs is believed to be slow, while in mammals the rate is much higher. The higher rate of evolution in mammals is thought to be a result of selective pressure on the virus to adapt to an aberrant host species. Chickens and turkey influenza virus isolates have previously and incorrectly been lumped together with wild waterfowl, gull, and shorebird influenza viruses when determining rates of evolutionary change. To determine mutational and evolutionary rates of a virus in any host species, two primary assumptions must be met: first, all isolates included in the analysis must have descended from a single introduction of the virus, and second, the outbreak must continue long enough to determine a trend. For poultry, three recent outbreaks of avian influenza meet these criteria, and the sequences of the hemagglutinin and nonstructural genes were compared. Sequences from all three outbreaks were compared to an avian influenza virus consensus sequence, which at the amino acid level is highly conserved for all the internal viral proteins. The consensus sequence also provides a common point of origin to compare all influenza viruses. The evolutionary rates determined for all three outbreaks were similar to what is observed in mammals, providing strong evidence of adaptation of influenza to the new host species, chickens and turkeys.     

Detection of bovine respiratory syncytial virus using a heterologous antigen-capture enzyme immunoassay

Based on the marked antigenic similarities that exist between antigens of the human and bovine strains of respiratory syncytial virus (RSV), an enzyme immunoassay (EIA) designed to detect human RSV was used to detect bovine RSV. The commercial test kit (RSV EIA) consists of a solid phase (beads) coated with a capture antiserum prepared against the Long strain of human RSV. The RSV EIA test was compared with the method of inoculation of cell cultures and fluorescent antibody (FA) staining of lung tissue for the detection of bovine RSV. Using a cell culture-propagated stock of strain 375 of bovine RSV, the threshold of sensitivity of the EIA test for the cattle strain of RSV was determined to be less than or equal to 10(2.3) CCID50/ml. In addition, RSV EIA detected the bovine RSV in nasal samples obtained from 3 experimentally inoculated cattle. The RSV EIA exhibited a sensitivity of greater than or equal to 80% during the period that shedding of infectious virus took place. All of the bovine RSV FA-positive lung samples (n = 37) were positive by the RSV EIA. Twenty-six of the remaining 214 bovine RSV FA-negative lung samples were positive by the RSV EIA. The RSV EIA was also used to test 137 nasal swabs obtained from cases of bovine respiratory disease. Of these, 38 tested positive by RSV EIA. All samples that tested positive by EIA were confirmed by blocking assays using hyperimmune serum anti-bovine RSV and a pool of monoclonal antibodies specific for that virus.     

A competitive enzyme immunoassay for the detection of bovine antibodies to Brucella abortus using monoclonal antibodies

A competitive enzyme immunoassay (EIA) for the detection of circulating bovine antibodies to Brucella abortus has been developed using horseradish peroxidase conjugated monoclonal antibodies (MAb) raised against B. abortus cell surface antigens. Antibodies present in the serum of either vaccinated or infected cattle can apparently displace the conjugated MAb from the lipopolysaccharide antigen (LPS) in a quantitatively different manner allowing an assessment of immune status of the animal. The results from a panel of sera from animals with a known status of vaccination or infection indicated that the test was more selective in the detection and discrimination of infected from uninfected or immunized animals, than conventional complement fixation, agglutination or indirect enzyme immunoassay procedures.     

Serologic detection of antibodies to Newcastle disease virus in water fowl using the hemagglutination inhibition test and enzyme immunoassay]

8410 samples from Moscovy duck, Pekin duck and geese were incorporated into examinations of antibodies against the Newcastle Disease virus. A new enzyme-immuno-assay (EIA) for antibody detection in Moscovy duck and Pekin duck was developed using purified antigen from NDV-strain "La Sota". The epidemiology as well as the relation of incidence of the Newcastle Disease in waterfowl was discussed.     

Differentiation of infectious bursal disease viruses by restriction enzyme analysis of RT-PCR amplified VP1 gene sequence

In order to differentiate infectious bursal disease virus (IBDV) isolates/strains, a quick method of RT-PCR followed by restriction enzyme analysis of VP1 gene sequence is being reported for the first time. A 480 bp fragment, comprising one of the RNA dependent RNA polymerase motifs of VP1 gene sequence of an Indian classical virus, an attenuated vaccine strain, Georgia and two Indian field isolates, genetically similar to reported very virulent strains of IBDV, was amplified by RT-PCR. Restriction enzyme digestion of PCR products with Taq1 enzyme generated distinct profile for field isolates, different from the classical and attenuated viruses, whereas restriction profile with BstNI restriction enzyme was similar in all the viruses, irrespective of the pathotype. Therefore, the present results suggest that Taq1 digestion can be taken up for the differentiation of field isolates from the classical and vaccine strains. The sequence analysis of VPI gene of reported very virulent IBD viruses from Europe and Japan, using 'MapDraw' programme of Lasergene software, revealed similar restriction enzyme profile as in Indian field isolates.     

流感病毒种内与种间传播的影响因素

A型流感病毒具有相对的宿主特异性,病毒在种内和种间的传播案例经常发生.对于某一特定宿主,病毒的感染受到诸多因素影响,包括病毒、宿主和环境因素等.本文对影响流感病毒种内和种间传播的因素进行概述.     

氟喹诺酮类药物多组份残留的酶免疫分析法

采用N-羟基琥珀酰亚胺(NHS)活性酯法制备诺氟沙星、环丙沙星、达氟沙星和沙拉沙星完全抗原,经紫外光谱扫描鉴定后,免疫小鼠,ELISA测定抗体交叉反应性,筛选簇特异性抗体,建立多组份ciELISA检测方法.紫外光谱扫描结果证实4种半抗原与载体蛋白发生共价结合,所制备的沙拉沙星与诺氟沙星抗血清效价都在1:64 000以上,环丙沙星和达氟沙星抗血清效价都在1 :32 000以上.交叉反应率测定结果表明,沙拉沙星抗血清对沙拉沙星、诺氟沙星、环丙沙星和恩诺沙星的交叉反应率依次为100.00%、92.68%、89.12%和83.56%;利用沙拉沙星抗血清建立了针对上述4种药物的多组份ciELISA检测方法,最低检测限依次为19.3、34.5、31.3、29.2 μg/L,大于5μg/L水平的添加回收率均大于83%.     

Biological properties of waterfowl-origin type A influenza viruses in chickens.

The replicative abilities and tissue tropism properties of 13 non-pathogenic or low-pathogenic waterfowl-origin type A influenza isolates recovered in 1986 were examined in chickens. Following intravenous challenge, reisolation of challenge virus was attempted from swabs of the luminal surfaces of the cloaca, jejunum, ileum, bursa, trachea, and air sacs and from swabs of bone marrow and liver tissues. Virus-isolation attempts were also accomplished on brain, thymus, spleen, pancreas, gonad, kidney, blood, and lung tissues. The overall frequency of influenza virus recovery for each experiment ranged from 3.1% to 49.3%. For all experiments combined, 58.3% of the kidney tissues and 62.9% of the cloacal swab samples collected on days 1 to 10 postinoculation were positive for challenge virus recovery. Virus titers up to 10(8.7) mean embryo infective dose per gram of kidney tissue were demonstrated in clinically normal chickens. Distinct biological variations and nephrotropism appear to exist among the corporate properties of virus populations making up each of the 13 waterfowl-origin type A influenza isolates.     

Detection of antibodies against Actinobacillus pleuropneumoniae serotype 5 using an inhibition enzyme immunoassay.

An inhibition enzyme immunoassay (EIA) for detection of antibodies against A. pleuropneumoniae serotype 5 (App-5) in pig sera, based on the inhibition of the binding of an App-5 specific monoclonal antibody was established. The monoclonal antibody (MAb 210-F11) was found to be directed against an epitope on the O-chain of App-5 LPS. In the inhibition EIA, highly purified App-5 LPS was used to coat microtitre plates. Serial dilutions of pig sera were added to the plates prior to the addition of the MAb 210-F11. The degree of binding of App-5 antibodies from pig sera was determined as the percentage inhibition of the MAb 210-F11. Pig serum from specific pathogen free (SPF) herds, from experimentally infected animals, and from acutely and chronically infected herds were tested. A serum dilution of 1/30 was found to be optimal, when using 50% inhibition as the discriminating inhibition percentage. No cross-reactivity was observed with serum from pigs infected with other App serotypes or bacteria isolated from the respiratory tract, such as A. suis and H. parasuis. The inhibition EIA will be used for surveillance of App-5 antibodies in SPF and conventional herds.