Avian influenza virus detection: sensitivity comparison of various in vitro and in OVO methods

Introduction Highly pathogenic avian influenza (HPAI) is a markedly contagious viral disease of domestic chickens and turkeys, characterized by severe illness with high morbidity and mortality (Easterday et al. 1997). Human infections with the HPAI virus H5N1 frequently cause death since 1997 (Class et al., 1998, Subbarao et al., 1998). Two surface glycoproteins, the haemagglutinin (HA) and neuraminidase (NA) form the basis of antigenic subtyping of avian influenza viruses (AIVs) (Wright and Webster, 2001). Currently sixteen HA plus nine NA subtypes have thereby been classified, thus far (Fouchier et al. 2005). In response to continuing outbreak of avian influenza worldwide since the end of 2003, many countries strengthen surveillance studies in wild birds for early detection of avian influenza virus (AIV). To screen large number of samples rapidly, reliably, sensitively and specifically, an appropriate diagnostic test is a prime prerequisite. Different diagnostic tools are available and can be used, depending on the individual laboratory facilities. Among these, embryo inoculation or tissue cultures are the methods of choice for virus isolation. For the detection of viral nucleic acid, reverse transcriptase polymerase chain reaction (RT-PCR) is a commonly practiced method. Other techniques, including real time RT-PCR (rRT-PCR) and nucleic acid sequence based amplification assay (NASBA), are also developed to detect viral nucleic acid. Here we tested the sensitivity and specificity of rRT-PCR, NASBA-microplate detection method (NASBA-MDM) and compared them with fifty percent embryo infective dose (EID50) titer of virus. Additionally, through tissue culture infective dose fifty (TCID50), we compared sensitivity of chicken embryo fibroblast (CEF) and Madin-Darby canine kidney (MDCK) cell cultures to AIV. Materials and Methods Viruses Viruses, A/northern pintail/Aomori/395/04 H7N1 (H7N1) and A/northern pintail/Miyagi/258/05 H6N2 (H6N2) were taken from the repository of the Laboratory of Zoonosis, School of Veterinary Medicine, Kitasato University. These viruses (H7N1 and H6N2) were isolated from fecal materials of northern pintail (Anas acuta) through embryo inoculation and subtyped by RTPCR and haemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests (Jahangir et al. 2008). Stock viruses were cultured and titrated in 9-10 days old embryonated chicken egg to prepare working viruses. Sample preparation Viruses in allantoic fluid were mixed with tracheal swab. Tracheal swab was prepared as follows. Tracheas of chickens were collected from slaughter house, cut into small pieces and mixed with 3.5ml of brain heart infusion (BHI) broth. Stomaching was carried out with high speed for 2 minutes. Liquid (BHI-Tracheal mixture later referred to as tracheal swab) was collected to the maximum possible. The virus in allantoic fluid was subjected to ten-fold serial dilutions with this tracheal