G. Koch

Location of antigenic sites defined by neutralizing monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide

Neutralizing monoclonal antibodies directed against five antigenic sites on the spike (S) S1 glycopolypeptide of avian infectious bronchitis virus (IBV) were used to select neutralization-resistant variants of the virus. By comparing the nucleotide sequence of such variants with the sequence of the IBV parent strain, we located five antigenic sites on the amino acid sequence of the S1 glycopolypeptide. The variants had mutations within three regions corresponding to amino acid residues 24 to 61, 132 to 149 and 291 to 398 of the S1 glycopolypeptide. The location of three overlapping antigenic sites on the IBV spike protein was similar to the location of antigenic sites on the spike protein of other coronaviruses.

Differentiation of virulent and non-virulent strains of Newcastle disease virus within 24 hours by polymerase chain reaction.

Fast diagnosis of Newcastle disease is a prerequisite for confining outbreaks. Diagnosis implies the differentation of virulent and non-virulent Newcastle disease viruses (NDV). However, conventional methods, i.e. isolation of the virus and determination of the intracerebal pathogenicity index, take at least 5 days. Therefore, we investigated whether diagnosis can be performed by using the reverse transcrip-tase-polymerase chain reaction (RT-PCR) on RNA isolated directly from tissue homogenate. Two oligonucleotide primers, representing the sequence at the cleavage site of the F protein of either virulent or non-virulent NDV strains, respectively, were used to differentiate NDV. Using the RT-PCR we were able to differentiate 15 NDV reference strains, 11 of which were virulent and 14 non-virulent. The RT-PCR was further validated by using homogenate of brain, trachea, lung and spleen from 12 chicken flocks and one turkey flock suspected of Newcastle disease. The RT-PCR detected virulent NDV in samples of seven flocks and non-virulent NDV in two out of three flocks in agreement with conventional methods. However the RT-PCR failed to detect virus in 1/3 flocks from which non-virulent virus was isolated. The results are discussed. We conclude that the RT-PCR described can be used to confirm diagnosis of Newcastle disease within 24 h using RNA isolated directly from tissue homogenate.

Antigenicity of the peplomer protein of infectious bronchitis virus

Abstract To study the antigenic structure of the peplomer protein of the avian coronavirus infectious bronchitis virus, fragments from the peplomer gene were generated by restriction-enzyme cleavage or by limited DNase digestion and inserted in the Escherichia coli expression plasrnid pEX (Stanley and Luzio, 1984). The antigenicity of the expression products was tested using a number of polyclonal antisera and monoclonal antibodies. The polyclonal antisera recognized different sets of epitopes in the 1162-residue sequence. The N-terminal region of one of the two subunits, S2, was recognized by all polyclonal sera and by two monoclonal antibodies. This clearly immunodominant region contains at least two adjacent or overlapping epitopes, one of which has been localized within 18 residues. The epitopes found as antigenic pEX expression products do not coincide with the regions in the S1 subunit that have been found to contain hypervariable sequences. We suggest that these regions constitute conformation dependent neutralization epitopes that cannot be detected in the pEX system. The relevance of our finclings for vaccine development is discussed.

A cross-sectional serological survey of the Dutch commercial poultry population for the presence of low pathogenic avian influenza virus infections

After the discovery of poultry infected with highly pathogenic avian influenza (HPAI) virus of subtype H7N7 in the central area of the Netherlands on 28 February 2003, the hypothesis was put forward that an outbreak of the low pathogenic (LP) variant of H7N7 had preceded, unnoticed, the occurrence of the HPAI virus. Consequently, a cross-sectional serological survey of the Dutch poultry population was executed in the second week of March 2003. The basic requirements set were detection of a 5% prevalence of flocks exposed to LPAI virus with 95% confidence within the production type stratification level within each province in the Netherlands. Because of supposed higher risk of avian influenza infections in ducks, turkeys and free-range poultry, all the commercial flocks of these production types present in the Netherlands were sampled. The serological screening of 28018 sera from 1193 randomly selected poultry farms, located outside surveillance zones showed that LPAI H7 virus infections had occurred on three neighbouring farms all located in the southwest of the Netherlands. No antibodies against the neuraminidase N7 subtype were detected in the sera of these farms, indicating that the subtype was different from the HPAI H7N7 subtype that caused the avian influenza epidemic in 2003. In addition, evidence of infections with non-H5 or non-H7 subtypes of influenza A virus were obtained in two other farms located in the northeast and the southeast of the Netherlands. It was concluded that the HPAI subtype H7N7 outbreak was most likely not preceded by a significant circulation of a LPAI subtype H7N7 virus. Based on the Dutch experience, recommendations are made to detect avian influenza infections faster in the future.

Leukocyte subpopulations in kidney and trachea of chickens infected with infectious bronchitis virus.

Using immunohistochemical methods, we studied the nephropathogenicity of the infectious bronchitis virus (IBV)-strain V1648- and the leukocyte phenotypes in the pathological lesions in the kidneys and the trachea formed after inoculation with this virus strain. One-day-old WLA chickens were intravenously inoculated, and after 5, 7 and 11 days their kidneys, trachea and lungs were removed. Monoclonal antibodies were used to detect viral antigen, and lymphoid and non-lymphoid cell populations. In serial sections, the detection of the viral antigen was correlated to the phenotypes of the cells. At days 5 and 7 after inoculation, viral antigen was detected in the epithelium and the interstitium of the kidney tubuli and in the epithelium of the trachea. The infiltrated cells in these tissues were mainly of the T cell phenotype. The cellular immune reaction was correlated with the detection of viral antigen.

Detection by immunofluorescent assay of serotype-specific and group-specific antigens of infectious bronchitis virus in tracheas of broilers with respiratory problems.

The performances of seven immunofluorescent assays (IFAs) for infectious bronchitis virus (IBV) were examined on 115 trachea samples collected from 60 broiler flocks with clinical respiratory distress. Whether IBV strains could be serotyped directly on trachea sections by IFAs was examined using four different serotype-specific monoclonal antibodies (MAbs). Two group-specific IFAs using two different group-specific MAbs, were compared with a conventional IFA using a chicken hyperimmune anti-IBV serum. The use of the six MAbs in the IFA showed, in contrast to the use of the hyperimmune serum, no or only faint non-specific staining. Although the sensitivities of the two group-specific IFAs using MAbs were not higher (P> 0.05, power 80%) than the sensitivity of the IFA using hyperimmune serum, the interpretation of the staining of the first two IFAs was easier. Seventeen of the 41 isolated IBV strains could be typed by IFA using the serotype-specific MAbs. Serotyping by IFA was possible in about 70% of the tracheas that stained positive with group-specific MAbs or hyperimmune serum and from which IBV was isolated. Use of serotype-specific IFAs is a new and very fast way of diagnosing IBV infections including serotyping, providing enough time to adjust the vaccination programme for the next broiler flock.

Linear Neutralizing Epitopes on the Peplomer Protein of Coronaviruses

Three years ago, we reported a comparison of the primary structures of the S peplomer proteins of three coronaviruses - mouse hepatitis virus (MHV, strain A59), infectious bronchitis virus (IBV, strain M41), and feline infectious peritonitis virus (FIPV, strain 79-1146) - which represent the three antigenic clusters in the Coronavirus family (De Groot et al., 1987a, b). A periodicity in the C-terminal part of the S sequence indicated the presence of a coiled-coil structure, which forms the stalk of the peplomer. The nonconserved N-terminal sequence probably forms the bulbous part of the peplomer.

Inactivation of chicken anaemia virus in chickens by heating and fermentation.

The transmission of pathogenic microorganisms such as viruses by the use of animal products in animal feed constitutes a potential risk to the health of livestock. To reduce the risk, it is necessary to understand the survival of viruses during the processing of animal products to feed-stuffs. Since chicken anaemia virus (CAV) is very resistant to inactivation, we used it as a model for the inactivation of pathogenic viruses during treatment of animal products. It is concluded that fermentation of CAV viraemic tissue did not affect the inactivation of CAV, however, heating at a core temperature of 95 degrees C for 30 min or 100 degrees C for 10 min is sufficient to inactivate CAV. Compared with the conditions for inactivation reported in the literature for other pathogenic viruses, our treatment is more stringent. CAV viraemic chickens are thus suitable as a model to test the heat inactivation of pathogenic viruses.

Detection of specific IgM antibodies to infectious bronchitis virus by an antibody-capture ELISA

The results of a new antibody-capture ELISA (alpha-IgM-IBV ELISA), specific for IgM directed against Infectious Bronchitis Virus (IBV) show that this assay is a useful tool for diagnosing IBV infections. The data include individual results of the alpha-IgM-IBV ELISA in sequential SPF chicken sera after vaccination with H120 and challenge with M41, the specificity is based on results of 499 SPF sera, and the sensitivity on sera from experimentally vaccinated and challenged birds. Also reported are ELISA results on 168 field sera originating from seven broiler flocks (24 samples per flock) collected during the acute phase of an IBV infection. The alpha-IBV-IgM ELISA results obtained with field sera from broiler flocks infected by Massachusetts, D207, D212 or an unidentified serotype of IBV indicated that the ELISA, in which only M41-strain was used as antigen, was able to detect IgM responses to IBV serotypes other than Massachusetts. The specificity of the alpha-IBV-IgM ELISA was 99%, the sensitivity based on the experimental vaccination was 83 to 100%, depending on the day post vaccination. The sensitivity, based on the experimental challenge, was 83%, while the sensitivity, based on 168 field sera, was 93.5%. The IgM responses were rapid and transient and therefore indicative for acute IBV infections.

Studies on the pathogenesis of chicken infectious anaemia virus infection in six-week-old SPF chickens.

The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated intramuscularly with graded doses of Cux-1 strain (10(6)-10(2) TCID50/chicken). Viraemia, virus shedding, development of virus neutralizing (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, polymerase chain reaction (PCR), immunocytochemistry (IP) and in situ hybridization until postinfection day (PID) 28. In 6-week-old chickens infected with high doses of CAV, viraemia and VN antibodies could be detected 4 PID and onward without virus shedding or contact transmission to sentinel birds. However, virus shedding and contact transmission were demonstrated in one-day-old infected chickens. In the 6-week-old groups infected with lower doses, VN antibodies developed by PID 14, transient viraemia and virus shedding were detected. The thymus cortex of all 1-day-old inoculated chickens stained with VP3-specific mAb. Cells with positive in situ hybridization signal were fewer and scattered throughout the thymus tissue of the one-day-old inoculated chickens as compared to IP-positive cells. These results suggest that early immune response induced by high doses of CAV in 6-week-old chickens curtails viral replication and prevents virus shedding.