Sero epidemiological studies on porcine hemagglutinating encephalomyelitis virus in pigs in japan

Sero-epidemiological Studies on Porcine Hemagglutinating Encephalomyelitis Virus in Pigs in Japan （日本の豚群における豚血球凝集性脳脊髄炎ウイルスに 関する血清疫学的研究） 2020 The United Graduate School of Veterinary Sciences, Gifu University (Tokyo University of Agriculture and Technology) Chau Thi Huyen Trang Sero-epidemiological Studies on Porcine Hemagglutinating Encephalomyelitis Virus in Pigs in Japan （日本の豚群における豚血球凝集性脳脊髄炎ウイルスに 関する血清疫学的研究） The United Graduate School of Veterinary Sciences, Gifu University (Tokyo University of Agriculture and Technology) Chau Thi Huyen Trang TABLE OF CONTENTS GENERAL INTRODUCTION CHAPTER Seroprevalence against Porcine Hemagglutinating Encephalomyelitis Virus among sow pigs in six regions in Japan INTRODUCTION MATERIAL AND METHODS RESULTS 10 DISCUSSION .12 SUMMARY 15 CHAPTER Development of an indirect ELISA based on soluble antigen produced from virus-infected cells for detection of Porcine Hemagglutinating Encephalomyelitis Virus…………………………… ………… 27 INTRODUCTION 28 MATERIALS AND METHODS 30 RESULTS 33 DISCUSSION 36 SUMMARY 38 i CHAPTER Application of newly established ELISA for sero-epidemiological survey of PHEV infection and study of the relationship between antiPHEV antibody level of pigs of to months of age and sows in Japanese pig farms…………………… ………………………….….44 INTRODUCTION 45 MATERIALS AND METHODS 46 RESULTS 48 DISCUSSION 51 SUMMARY 55 GENERAL CONCLUSIONS 65 ACKNOWLEDGMENTS 69 REFERENCES 71 ABSTRACT 80 ii GENERAL INTRODUCTION Porcine hemagglutinating encephalomyelitis (PHE) is an acute, contagious disease in swine caused by the porcine hemagglutinating encephalomyelitis virus (PHEV) that mainly infects piglets under weeks of ages The virus was first reported in Canada in 1958 (49) and subsequently isolated from brains of suckling piglets with encephalomyelitis in 1962 (17) In 1969, PHEV was isolated in England from piglets showing vomiting, anorexia, and depression, but these piglets showed no clear signs of encephalomyelitis Surviving piglets remained stunted in growth, and the condition was therefore named “Vomiting and wasting disease” (VWD) (6) Since then, PHEV infection is found nearly worldwide Serological surveys of PHEV infection in the past has been reported in pigs throughout many countries in Europe (Switzerland, England, Belgium, Czech Republic, and Italy) (5, 26, 37, 42, 59), in Americas (United States of America [USA], Canada, and Argentina) (2, 11, 16, 21, 26, 55), in Asia (Japan, China, Taiwan and South Korea) (7, 13, 15, 21, 48) and in Australia (14) PHEV is an enveloped, single-stranded, positive-sense RNA virus that belongs to the genus of Betacoronavirus within the Coronaviridae family, subfamily Cornavirinae, order Nidovirales Together with bovine coronavirus (BCoV), human coronavirus (HCoV) strain OC43, equine, and canine respiratory coronaviruses, PHEV is a member of Betacoronavirus species (57) Porcine coronaviruses also include transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV) and porcine epidemic diarrhea virus (PEDV), which belong to the genus Alphacoronavirus, and porcine delta coronaviruses (PoDCV), which belong to the genus Deltacoronavirus These viruses often cause severe diarrhea, subclinical mild respiratory infections or enteric infections in piglets On the other hand, PHEV is a rare coronavirus that infect and propagate in neural cells (61) PHEV genome is approximately 30 kb in length, and it encodes major structural proteins: of which - the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins are similar as other coronavirus, and the hemagglutinin-esterase (HE) which only belongs to PHEV and members of Betacoronavirus group (51) The coronavirus N protein has been shown to be highly immunogenic and abundantly expressed during infection and therefore, it has been used for developing the serodiagnosis methods of the human and animal coronavirus infections such as PED, severe acute respiratory syndrome (SARS) and infectious bronchitis virus (IBV) (8, 25, 52) Pigs are the only species naturally susceptible to PHEV with the two clinical syndromes: VWD and acute encephalomyelitis in neonatal pigs (44) More recently, Lorbach JN et al (28) provided strong evidence for the role of PHEV as the respiratory pathogen in an acute outbreak influenza-like illness of pigs in Michigan, USA Although PHEV infections can occur in pigs of all ages, the infections are most serious in piglets which are less than weeks of age The mortality rates are reaching from 20% up to 100% (36, 46, 48) The clinical signs of acute encephalomyelitis caused by PHEV infection were observed in pigs >4 days of age and consisted of vomiting and wasting syndromes and neurologic signs; these symptoms gradually decreased in pigs of the upper weeks of age (46) Similar observations was described for VWD (33, 45, 63) Previously, a report of PHEV-surveillance in clinically ill pigs in South Korea revealed that the highest PHEV infection was found in suckling pigs (14.3%), followed by weaning pigs (6.5%) and growers pig (7%); no PHEV detection in finisher pigs (48) In Japan, the serological surveys of PHEV infection (1974-1988) revealed that approximately 50% to 82% of the swine sera were anti-PHEV antibody positive (19, 20, 22) In 1984, PHEV was first isolated in Japan from pigs affected with respiratory disease in Niigata and Osaka prefectures by Hirahara et al (18) On the other hand, there has been no report of typical outbreak of PHEV infection in Japan These results suggest that PHEV Japanese strain causes subclinical infection in pigs in Japan The standard assays for detecting PHEV antibody are the hemagglutination inhibition (HI) test and later the virus neutralization (VN) test (16, 42, 54) However, on the comparison of the effectiveness of these two tests on measuring the level of antibody against PHEV in sera of pigs, Sasaki et al (54) demonstrated that VN test is a more suitable method for sero-surveillances of PHEV infection than the HI test used previously They found that the PHEV neutralizing antibody titers did not correlate with titers obtained from HI testing Other serodiagnosis methods were also established for the simple and rapid detection of anti-PHEV antibodies such as the immunochromatographic strips that be easily-performed in the field without the requirement for expensive equipments (9, 10) However, these strips are expensive to use for domestic animals and are not commercially available currently More recently, an enzyme linked immunosorbent assay (ELISA) based on recombinant PHEV-S1 protein has been developed for the detection of anti-PHEV antibodies and used for seroprevalence of antibodies against PHEV in sow population in the United States (35) On the other hand, molecular tools including reverse transcription (RT)-PCR, nested PCR (46, 48, 56) and real-time RT-PCR assays have been established for detection viral RNA for direct and rapid diagnosis of PHEV infection (62) Previously, PHEV infection has not been considered as an important disease for commercial pig farming The reason may be that a number of pigs at commercial pig farms have antibodies to PHEV However, it has been proposed that PHEV infection should be considered as economically important disease for pig production because of the decrease of antibody positive pigs due to high gilt replacement rates and the popularization of specific pathogen-free pigs (36, 54) Besides that PHEV infection has been reported with sporadic outbreaks in recent years, and caused great economic losses in pig industry by VWD disease outbreaks For examples, over 500 weaned pigs were euthanized because of anorexia and wasting symptoms in an outbreak of a sow genetic nucleus herds in Canada (3); and total of 3,683 piglets were died of PHEV infection or were euthanized in an outbreak of vomiting, wasting, and encephalomyelitis syndrome in piglets in Argentina (46) However, until now, PHE has not been considered as an important swine infectious disease and PHEV is one of the least studied swine coronaviruses (36) Therefore, this study first aims to investigate the current status of PHEV infection in pig farms in Japan via serodiagnosis for detecting antibodies against PHEV The second aim is to develop a suitable diagnostic method that could be prepared easily with a rapid, simple, and low-cost for monitoring and managing PHEV infections circulating in pig’s population This present study included three chapters with the objectives as follows: In chapter 1, the seroprevalence against PHEV among sow pigs collected in 57 farms of six regions in Japan was investigated by the VN test In chapter 2, an indirect ELISA based on soluble antigen produced from virusinfected cells for the detection of PHEV was developed In chapter 3, the seroprevalence against PHEV among sows and pigs of to months of age in Japanese pig farms was conducted by the newly developed ELISA methods Also, the level and duration of maternal antibodies transferred from PHEV seropositive sows to their offspring were investigated CHAPTER Seroprevalence against Porcine Hemagglutinating Encephalomyelitis Virus among sow pigs in six regions in Japan ACKNOWLEDGEMENTS I would like to express my appreciation and thanks to my supervisor, Associate Professor Dr Hideki Hayashidani, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Japan for his considerable support and guidance throughout the course of my study I would also like to express my deepest gratitude and appreciation to my cosupervisor, Associate Professor Dr Takahide Taniguchi, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Japan for his dedicated supervision, advice, encouragement, and support throughout the course of my research and in transcription of this thesis Without his great guidance, this project would not have been possible My appreciation and gratitude are also extended to my co-supervisors, Professor Dr Hiroshi Asakura, National Institute of Health Science, Japan for his continuous guidance and suggestions throughout the course of my study I am deeply grateful to Institute of Animal Health, National Federation of Agricultural Co-operative Association, Japan, other companies and clinical veterinarians for help to collect swine sera samples I very much appreciate to my thesis committee members, Professor Dr Haruko Ogawa, Ohibiro University of Agriculture and Veterinary Medicine, Japan; Professor Dr Kenji Murakami, Iwate University, Japan; Professor Dr Mizutani Tetsuya, Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Japan; Professor Dr Naoto Ito, Gifu 69 University, Japan; Professor Dr Hiroshi Asakura, National Institute of Health Science, Japan for their insightful comments and helpful suggestions on my previous manuscript I would also like to express my special thanks to Professor Dr Arie Tsutomu, Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology, Japan; Ms Reiko Hitotsubashi, Japan International Cooperation Center (JICE); Assoc Prof Nguyen Trong Ngu, Assoc Prof Tran Ngoc Bich, Assoc Prof Nguyen Huu Hung, Assoc Prof Van Pham Dan Thuy, Dr Truong Thi Bich Van from Can Tho University, Vietnam for their advice, supports, and encouragements me in the completion of this project In addition, many thanks go to my friends and colleagues for their encouragements and sharing learning experiences I am also grateful to the Ministry of Education and Training of Vietnam (MOET) for supporting me with the scholarship and Can Tho University, where I work, for helping and supporting me throughout the course of my study in Japan Lastly, I dedicate this dissertation to my great family I want to express my deepest thanks to my adored parents, Mr CHAU VAN DONG and Mrs DUONG THI NO, to all my siblings for their infinity love, endless support, and spiritual encouragements 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age The mortality rates can be reaching up to 100% in piglets The clinical syndromes include vomiting and wasting disease (VWD) with or without encephalomyelitis PHEV infection is found nearly worldwide Serological surveys of PHEV infection have been reported in pigs throughout many countries of Europe (Switzerland, England, Belgium, Czech Republic, and Italy), Americas (USA, Canada, Argentina), Asia (Japan, China, Taiwan and South Korea), and Australia However, from 1988 up to the present, there have been no serological surveys of PHEV in commercial pig farms in Japan Therefore, the aims of this study were to investigate the current status of PHEV infection in Japanese pig farms by conducting the serum virus neutralization test and developing an indirect ELISA based on soluble antigen produced from virus-infected cells that could be used easily for monitoring and managing PHEV infections for mass screening of PHEV antibodies In Chapter 1, the current status of PHEV infection in regions of Japan, consisting of A (Hokkaido), B (Tohoku), C (Kanto and Koshin), D (Tokai and Hokuriku), E (Kinki, Chugoku, and Shikoku), and F (Kyushu and Okinawa) was investigated by using virus neutralization test A total of 425 sow sera collected from 57 farms was used for this study Results revealed that 76.7% (326/425) of sow sera were positive for PHEV infection High seropositive farms were detected at 84.2% (48/57) Antibodies levels were found at high NA titer (80.1%), followed by medium NA titer (18.1%), and low NA titer was only 1.8% 80 All farms in the location of C (Kanto and Koshin), E (Kinki, Chugoku, and Shikoku), and F regions (Kyushu and Okinawa) revealed seropositive against PHEV Noticeably, the anti-PHEV neutralizing antibodies had not detected in farms of A (Hokkaido), farms of B (Tohoku), and farms of D regions (Tokai and Hokuriku) There were no clinical signs of disease observed during the period time of collecting samples in this study To sum up, these results revealed that PHEV is currently circulating in pig farms in Japan; however, some farms appeared seronegative to PHEV infection in A, B, and D regions In Chapter 2, an indirect ELISA based on soluble antigen produced from virusinfected cells for detection of PHEV was established and validated by using 150 sera of sows and grower-finisher pigs that confirmed the virus-neutralizing antibody titer against PHEV The ELISA antigen was prepared from PHEV-infected FS-L3 cells by extraction using RBS buffer containing 0.2% NP-40 This developed ELISA showed a high sensitivity (95.4%) and specificity (96.9%) by receiver operating characteristic (ROC) analysis The area under the curve (AUC) was 0.996 and that revealed a high accuracy of this ELISA method The strong correlation between the results of indirect ELISA and VN test (r = 0.850, p