Avian influenza virus (H5N1); effects of physico-chemical factors on its survival

Virology Journal Avian influenza virus (H5N1); effects of physico-chemical factors on its survival Muhammad Akbar Shahid 2 Muhammad Abubakar 1 Sajid Hameed 0 Shamsul Hassan 2 0 University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur , Bahawalpur , Pakistan 1 National Veterinary Laboratory , Park road, Islamabad , Pakistan 2 Poultry Research Institute , Shamsabad, Murree road, Punjab, Rawalpindi , Pakistan Present study was performed to determine the effects of physical and chemical agents on infective potential of highly pathogenic avian influenza (HPAI) H5N1 (local strain) virus recently isolated in Pakistan during 2006 outbreak. H5N1 virus having titer 108.3 ELD50/ml was mixed with sterilized peptone water to get final dilution of 4HA units and then exposed to physical (temperature, pH and ultraviolet light) and chemical (formalin, phenol crystals, iodine crystals, CID 20, virkon-S, zeptin 10%, KEPCIDE 300, KEPCIDE 400, lifebuoy, surf excel and caustic soda) agents. Harvested amnio-allantoic fluid (AAF) from embryonated chicken eggs inoculated with H5N1 treated virus (0.2 ml/egg) was subjected to haemagglutination (HA) and haemagglutination inhibition (HI) tests. H5N1 virus lost infectivity after 30 min at 56C, after 1 day at 28C but remained viable for more than 100 days at 4C. Acidic pH (1, 3) and basic pH (11, 13) were virucidal after 6 h contact time; however virus retained infectivity at pH 5 (18 h), 7 and 9 (more than 24 h). UV light was proved ineffectual in inactivating virus completely even after 60 min. Soap (lifebuoy), detergent (surf excel) and alkali (caustic soda) destroyed infectivity after 5 min at 0.1, 0.2 and 0.3% dilution. All commercially available disinfectants inactivated virus at recommended concentrations. Results of present study would be helpful in implementing bio-security measures at farms/hatcheries levels in the wake of avian influenza virus (AIV) outbreak. - Introduction Poultry industry in Pakistan is facing various managemental problems along with infectious diseases including avian influenza (AI). This disease of highly pathogenic type was first reported in Pakistan in 1995, caused by subtype H7N3. Since then, various outbreaks of H7N3, H9N2 have been reported in various parts of the country which have inflicted heavy losses to the commercial poultry enterprises [[1,2] and [3]]. In February 2006, avian influenza virus (AIV) subtype H5N1 was for the first time found in two isolated commercial flocks in this country. Biosecurity measures, controlling poultry movements and inactivated vaccines were devised to combat the spread of newly introduced HPAIV H5N1 [4]. Avian influenza viruses by virtue of their infective potential pose a significant threat to human health. AIV subtypes, namely H5, H7 and H9, currently endemic in poultry in some regions of the world, have been shown capable of infecting humans [[5,6] and [7]]. Therefore, AI infections represent risk factors either for direct infection of humans from the avian host or for the consequences of genetic reassortment between a mammalian and an avian influenza virus, which could become the basis for a generation of a new pandemic virus for humans [8]. It is of crucial importance that AI infections in poultry are controlled to eradicate. International organizations have issued a list of recommendations aiming to control the AI in Asia [9]. The recommendations include implementation of risk reduction interventions such as restriction policies, stamping out, and under certain circumstances appropriate vaccination programmes. Secondary spread of AI is mainly caused through human-related activities such as the movement of staff, vehicles, equipment, and other fomites along with restocking of birds in establishments without following adequate biosecurity measures. It therefore implies that if disinfection of premises, footwear and clothing, vehicles, crates, farm equipment and other materials is not carried out properly, infection will persist in the avian population and the concurrent damage to the poultry industry and the public health threat will not be halted. For this reason, cleaning and disinfecting must be considered an essential part of AI control programmes. The possibility of reoccurrence of the AI outbreaks in Pakistan is still there because vaccination against the AIV is not rigorously practiced. This threat of the avian influenza has necessitated the pervasive use of disinfectants effective against wide range of viruses, bacteria and fungal spores. There is a wide variety of disinfectants available in market which are claimed to be effective against pathogens. The information about the efficacy of physical and the chemical (disinfectants) agents is scanty. This study, therefore, was designed to evaluate the efficacy of various physical (temperature, Ultraviolet light and pH), and chemical (commercially available disinfectants) agents against local strain of AIV H5N1. The results of this study would be helpful in implementing effective bio-security measures at the farm and hatcheries level. Methods Source of Virus Avian influenza virus was isolated from infected poultry flocks during recent AI outbreaks in and around Rawalpindi/Islamabad area of Pakistan during 2006 at Disease Section of Poultry Research Institute (PRI), Rawalpindi, Pakistan. Subtyping as H5 was performed by haemagglutination (HA) and haemagglutination inhibition (HI) tests using specific antiserum against H5N1 (Weybridge, UK) as described by Olsen et al. [10]. Molecular characterization as H5N1 was carried out at National Reference Laboratory for Poultry Diseases, Animal Sciences Institute, National Agricultural Research Centre, Islamabad, Pakistan. The virus cultivated in 911 day-old embryonated chicken eggs was subjected to virus titration by the method of Reed and Muench [11]. The amnioallantoic fluid (AAF) having virus titer of 108.3 ELD50/ml was stored in aliquot at -70C till further use. Treatment of AIV H5N1 with physico-chemical agents The preserved virus was cultivated in 9 to11-day-old embryonated chicken eggs. Harvested amnio allantoic (AAF) fluid was titrated on the basis of haemagglutination (HA) potential. Peptone water was prepared, autoclaved and incubated at 37C for 24 h to check sterility. AAF was diluted in peptone water to have 4 HA unit titer. It was divided into aliquots in sterilized glass vials with 4 ml each. Each vial with H5N1 virus suspension was exposed to 4, 28 and 56C, ultraviolet light, and different pH values (1, 3, 5, 7, 9, 11 and 13) for different time intervals. The disinfectants used for inactivation of the H5N1 virus included Formalin (Formaldehyde; Merck), Phenol crystals (Merck), Iodine crystals (Merck), CID 20 (CID LINES, Belgium), Virkon-S (Antec International, UK), Zeptin 10% (Nawan laboratories, Pakistan), KEPCIDE 300 (KEPRO B.V., Holland), and KEPCIDE 400 (KEPRO B.V., Holland), which were mixed with peptone water to (...truncated)