Detection of highly pathogenic influenza and pandemic influenza virus in formalin fixed tissues by immunohistochemical methods

Abstract

Tissues infected with highly pathogenic avian influenza viruses such as H5N1 and H7N7 are normally required to be fixed in formalin or paraformaldehyde before examination in order to inactivate the virus. In this study commercially available monoclonal antibodies to the influenza nucleoprotein (NP) were evaluated in order to determine which antibodies would identify positive cells in tissues fixed in formalin or paraformaldehyde. An assessment of which antigen retrieval process would unmask antigens blocked by formalin fixation was also made. Of six commercially available monoclonal antibodies tested, only one (HB65, European Veterinary Laboratories) was able to identify all formalin fixed avian, swine and human influenza virus infected tissues, and this was after pronase induced epitope retrieval. This monoclonal antibody is recommended for routine diagnostic use for the detection of influenza A infected tissues that have been fixed in formalin or paraformaldehyde.

Introduction

Infections caused by influenza A viruses can lead to a significant morbidity and mortality in animal species. Some strains of avian H5 and H7 are highly pathogenic (HPAI) with outbreaks in poultry leading to a high mortality. Human infections with H5N1 continue to circulate in the Asian-pacific region with a high mortality (Fiebig et al., 2011) and human infections by LPAI of the H9 subtype have also been documented resulting in mild flu-like symptoms without fatality (Peiris et al., 1999).

There are a number of detection methods for the influenza in clinical and laboratory samples, such as rapid antigen testing, although virus culture and sub-typing using reference antisera still remain the gold standard. Molecular detection methods such as RT-PCR and sequencing are now available for rapid diagnosis but the immunohistochemical (IHC) method enables direct morphological localization of viral antigen in fresh and fixed tissues, and permits a correlation of virus replication with the corresponding pathological cellular changes. IHC results can be obtained within a reasonable time frame with a low cost of equipment and consumables.

Handling of unfixed tissues infected with H5N1 and H7N7 is dangerous, and usually performed in a biosafety category 3 laboratory, so most biosafety protocols insist on virus inactivation by formalin or paraformaldehyde. Therefore, the analysis of HPAI using frozen sections cannot be done routinely on unfixed tissues owing to safety concerns. Although it is more convenient and safe to handle specimens that have been rendered non-infectious by fixation, it is possible that antibodies for influenza antigens do not work on formalin fixed tissues. There is thus a need to develop a standard protocol which is optimized to detect viral antigen in fixed tissues. Furthermore, though many influenza reference centres have developed their own antibodies, the current study sought to optimize the detection of avian, seasonal and pandemic influenza viruses in formalin fixed paraffin embedded tissues by IHC method using commercially available monoclonal antibodies, and to evaluate a standard protocol with appropriate antibodies for the detection of these viruses.