EPIDEMIOLOGY OF INFLUENZA VIRUS H5N1 IN ISLAMABAD CAPITAL TERRITORY BY ZAHIDA FATIMA (2005-VA-246) A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN EPIDEMIOLOGY AND PUBLIC HEALTH UNIVERSITY OF VETERINARY & ANIMAL SCIENCES, LAHORE (2015) To The Controller of Examinations, University of Veterinary and Animal Sciences, Lahore. We, the supervisory committee, certify that the contents and form of the thesis, submitted by ZAHIDA FATIMA, Regd. No. 2005-VA-246 been found satisfactory and recommend that it be processed for the evaluation by the External Examiner(s) for the award of the degree. PROF. DR. MUHAMMAD ATHAR KHAN _______________________ SUPERVISOR DR. KHALID NAEEM _______________________ CO-SUPERVISOR PROF.DR. MANSOOR UD DIN AHMAD _______________________ MEMBER PROF.DR. KHUSI MUHAMMAD ______________________ MEMBER DEDICATED TO MY LATE FATHER MAY HIS SOUL REST IN BEST PEACE (AAMEEN) i () In the name of Allah the most magnificent and the most beneficent. All praise for ALLAH All Mighty who has the control and command of each and every thing. It is He who has sent down to you, [O Muhammad], the Book; in it are verses [that are] precise - they are the foundation of the Book - and others unspecific. As for those in whose hearts is deviation [from truth], they will follow that of it which is unspecific, seeking discord and seeking an interpretation [suitable to them]. And no one knows its [true] interpretation except Allah. But those firms in knowledge say, "We believe in it. All [of it] is from our Lord." And no one will be reminded except those of understanding. ii ACKNOWLEDGEMENTS First of all I would like to thanks Allah Almighty and like to express my sincere and humble gratitude to Almighty whose blessing, help and guidance has been a real source of all my achievements in my life. This dissertation would not have been possible without the guidance and the help of several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. I would like to admit that I completed this project due to my loving parents who pray for my success and my husband, sisters and brothers for their efforts, love, support and encouragement. I wish to express my heartiest gratitude to my supervisor and my great mentor DR. MUHAMMAD ATHAR KHAN who introduced me to the new dimensions of knowledge of Epidemiology and public health and for his full co-operation throughout the course work and research work. I find it hard to imagine that anyone could be a more sincere, kind and better research advisor than what he has been to me throughout my phD. I’ve been most fortunate to have the guidance of PROF .DR. MANSOOR Ud DIN AHMAD, Department of Epidemiology and Public Health, University of Veterinary Sciences Lahore. I would like to record my gratitude for his kind supervision as a member in my supervisory committee, sympathetic attitude, constructive advices, and scientific discussions. And heartily thankful to Dr Khalid Naeem,CSO,ASI, NARC Islamabad, who helped a lot in all the aspects of research work, without his generous cooperation it was not possible for me to continue. He is the person who directed me in the true sense towards adopting the subject of Epidemiology as my future endeavors of my professional life. I wish to pay my gratitude to DR. KHUSHI MUHAMMAD, Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, as member in my supervisory committee for his support, encouragement, ever helping behavior and valuable guidance. iii I wish to express my sincere thanks to all my supervisory committee. It was a great honor for me to work under the guidance of Dr Muhammad Hassan Mushtaq and his sincere efforts for me. Above all, I must acknowledge NRLPD, Animal health, NARC, my work place that offered a scientific environment for carrying out this work. Special thanks to Zubair Anwar, Amjad Khan,Luqman Sohail, Dr. Jaamal, Dr. Asad Shah, Dr. Anum Hadi and my colleagues at Animal health who are always great help and source of encouragement and moral support throughout my PhD. ZAHIDA FATIMA iv DEDICATION (I) ACKNOWLEDGMENT (iii) LIST Of FIGURES (vi) LIST Of TABLES (vii) LIST OF ABBREVIATIONS (viii) TABLE OF CONTENTS S. NO. CHAPTERS PAGE NO. 1 1 INTRODUCTION 8 2 REVIEW OF LITERATURE 51 3 MATERIALS AND METHODS 69 4 RESULTS 92 5 DISCUSSION 101 6 SUMMARY 103 7 LITERATURE CITED v LIST OF FIGURES TITLE PAGE FIGURE NO. NO. 2.1 Important events of avian influenza in Pakistan 13 2.2 Country distribution of H5N1 in poultry in 1996 15 2.3 Epidemiological process in Asian H5N1 epidemics 18 2.4 Emergence of A/goose-guangdong/1/96-like H5N1 virus 19 2.5 Transmission pattern of influenza virus 21 2.6 Vehicles of transmission of AIV 22 4.1 Trends of mortality, morbidity and case fatality in layers 71 4.2 Trends of mortality, morbidity and case fatality in breeder 73 4.3 Rapid response team working in HPAI infected form 74 4.4 Trends of mortality, morbidity and case fatality in broiler 75 4.5 Spatial distribution of outbreaks in study areas 77 4.6 GMT of H5, H7, H9 87 vi LIST OF TABLES TABLE NO TITLE Page # 2.1 Major outbreaks in 2006 16 2.2 Inhibitors of AI 38 3.1 Data of administrative units of study area 51 3.2 Census of birds in study area 52 3.3 No. of samples taken from LBM 59 4.1 Outbreaks in layers in 2006-2008 70 4.2 Outbreaks of H5N1 72 4.3 Outbreaks in broiler birds 74 4.4 Case control age and case description 76 4.5 Univariate analysis of associated risk factors 78 4.6 Serology of H5N1 81 4.7 Serology of H7 83 4.8 Serology of H9 85 4.9 Samples taken from LBM 89 vii LIST OF ABBREVIATION AIV AVINA INFLUENZA VIRUS HA HAEMAGGLUTININ NS NON STRUCTURAL NEP NUCLEAR EXPORT PROTEIN LPAIV LOW PATHOGENIC AVIAN INFLUENZA VIRUS HPAIV HIGH PATHOGENIC AVIAN INFLUENZA VIRUS ICT ISLAMABAD CAPITAL TERRITORY LBM LIVE BIRD MARKET RT-PCR REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION NP NUCLEOPROTEIN NARC NATIONAL AGRICULTURAL RESEARCH COUNCIL WHO WORLD HEALTH ORGANIZATION CDC CONVENTIONAL DENDRITIC CELLS NOS NITRIC OXIDE SYTHETASE TIV TRIVALENT INACTIVATED NI NURAMINIDASE INHIBITION HI HAEMAGGLUTINATION INHIBITION PCR POLYMERASE CHAIN REACTION GMT GEOMETRIC MEAN TITER SAARC SOUTH ASIAN ASSOCIATION FOR REGIONAL COOPERATION SARS SEVERE ACUTE RSPIRATORTY SYNDROM viii CHAPTER 1 INTRODUCTION Influenza remains the most studied viruses in the scientific community throughout world (WHO, 2013) Avian influenza viruses have been isolated are found almost everywhere in the wild bird population, but most importantly in wild aquatic birds. The virus is the cause of disease in poultry at various levels of severity. There are two pathogenic strains, depending on the severity of diseases caused by virus, albeit viruses may switch between the two types due to mutation or re assortment of genes. The term “influenza” refers to as epidemics of acute, fast circulating respiratory or oral infections in human beings caused basically by various types of Orthomyxoviruses (Anonymous, 2006). The AIV viruses have been isolated from multiple hosts like avians, equines, swines, seals and human beings and many other type of hosts (Webster et al., 2008). Infection of AI viruses in poultry causes very high economic losses to the poultry farmers in Pakistan. One of the AIV types (H5N1) has been reported from many Asian, African and European poultry producing countries and this virus type has caused significant number of human infections most of which have been reported as fatal.(WHO,2013). The scientists suggest that this AIV type has the potential to mutate and emerge into a new form by developing necessary receptors for causing human to human infections by breaking species barriers. (Kilbourne et al., 2006; Lindstrom et al., 2004). Although sharing many symptoms with other respiratory infections,AIV influenza in addition causes unanticipated onset of three-day fever, including muscle pain and a degree of weakness or dizziness with the severity of other symptoms in the humans. In poultry farming, domestic poultry infection by avian influenza viruses (AIV) 1 Introduction generally bring forth syndromes which range from general infection to respiratory abnormalities and loss in egg production to severity in infection with almost 100% mortality (Bahlet al., 1977) 1.2 Types of Influenza The Influenza virus is divided into type A, B and C based on of host relationship in terms of virus antigenicity. An influenza virus is spherical or longitudinal in shape. These particles are enveloped. The viral genome is segmented and single stranded, negative sense RNA. All the virus types are differentiated on the basis of antigenic differences in their matrix and nucleoproteins. Amongst these AIV belongs to type A. It contains eight segments coding 11 proteins (Lamb et al., 1982). 1.2.1 Subtypes of Influenza Haemagglutinin (HA) and the neuraminidase (NA) transmembrane glycoproteins are main determinants of influenza A and B viruses’ antigenicity. These can provoke a subtype-specific immune response. Influenza A virus gather into sixteen H (H1 to H16) and nine N (N1 to N9) subtypes (Tong et al., 2012). 1.3 Natural hosts Wild birds, specifically species in order Charadriiformes (e.g tern, gulls) and anseriformes (e.g swans, ducks, geese), are reckoned the natural reservoir of subtypes of AIVs. These are widely distributed in these bird species all across the globe (Stallknecht 1990). In general natural hosts do not exhibit clinical signs of disease because of AIV type A viruses. Instead the viruses remain in an evolutionary equilibrium (Gorman 1991; Taubenberger 2005).