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«A DISSERTATION SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Sunil Kumar IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR ...»

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Characterization, diagnosis and environmental survival of turkey arthritis reoviruses

A DISSERTATION

SUBMITTED TO THE FACULTY OF

UNIVERSITY OF MINNESOTA

BY

Sunil Kumar

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Advisors: Drs. Sagar M. Goyal and Robert E. Porter June 2014 © Sunil Kumar 2014 Acknowledgements My graduate experience has been like no other endeavor I have ever pursued. It has been a time of great growth, life changes, and learning. I am so honored and appreciative to have had this opportunity. There are so many people that have helped me along the way, and I know that I could have not made it without them.

I would like to express my deep thanks to my advisors, Drs. Robert Porter and Sagar Goyal for proper guidance, motivation and support. Both of you leave upon me a strong impression of very kind and understanding persons and dedicated professors and researchers. Thank you for your confidence and for always going the extra mile for me. I have learnt many things about poultry diagnosis and pathology from Dr. Porter and have benefitted from Dr. Goyal’s vast expertise in virology research. In my opinion both of you are perfect match to supervise students on their journey to PhD. I thank Dr. Goyal for encouraging my research and for allowing me to grow as a research scientist as well as a diagnostician. Your advice and support have been invaluable. I am also very grateful to Dr. Porter for his scientific advice, insightful discussions and suggestions. I always enjoyed working with you and this experience and memories will continue with me throughout my professional career. I consider myself very fortunate for being given the opportunity to work under your supervision and hope that I could be as lively, enthusiastic, and energetic as both of you and someday be able to guide students like you have done.

I want to thank my committee members, Drs. Andre Ziegler, Hinh Ly, and Sally Noll. Thank you for your guidance and insightful advice not only for helping me to become a better scientist, but also for helping me to grow as an individual. You made my committee meetings something to be anticipated and memorable.

i I take this opportunity to thank past and present members of Dr. Goyal’s Lab.

Words cannot express how much I value the relationships I have built in this lab. I will never forget those days when I came for the first time to Dr. Goyal’s lab and the support, care and affection I got from Yogesh Chander and Martha Fuentes. Martha always took care of me as an elder sister and Yogesh as an elder brother. I also thank, Ashchalew, Hakan,Hamada, Harsha, Jinhai, Nader, Mostafa, Tamer and Zhili for providing me healthy and lively environment in the lab. I enjoyed collaborating with you and leaned much from your experiences.

I thank Dr. Patnayak for always being supportive and helping me whenever needed. I thank Drs. Armien, Phelps and Waltzek for sharing your ideas and experiences with me. I wish to continue working with you in collaboration. I am also appreciative to all the friends and colleagues I have made while in graduate school namely, Anil, Doug, Giordana, Jisun, Lucas, and Victor. I always enjoyed working with all of you and sharing our ideas. I hope our friendship is long lasting. I also thank the personnel of diagnostic lab: Wendy, Lotus and Rebecca for helping me during my thesis research.

I will forever be thankful to my former M.V.Sc. advisor, Dr. Naresh Jindal. He has been helpful in providing advice many times during my graduate school career. He was and remains my best role model for a scientist, mentor, and teacher. Dr. Jindal was the reason why I became a poultry diagnostician. Because of his trust in me and his kind support, I came to the US and decided to pursue a career in research. His enthusiasm and love for teaching and research, mainly in poultry, is contagious.

–  –  –

Diagnostic Laboratory for trusting me and supporting me. I also thank the staff of the College of Veterinary Medicine especially Lisa, Kate, Anna, Brenda, Julie and Natalie.

I am very thankful to families of Drs. Goyal, Patnayak, Vikram Verma, Anil Thachil and Sujata Sangwan who provide me family environment in the US. Many thanks to Susan, Steve, Mary Ann, and Dennis for your kind support, care and love.

My parents have been a constant source of support and love for me. No doubt that this PhD thesis is an outcome of a dream of my parents to add education in my family. I salute your decision and the struggles you faced to make this possible. Whatever I have achieved today and will achieve in future is all because of your dream. Special thanks to my wife and best friend, Deepika, who has decided to start life with me during such a difficult time of my PhD education. She has shown endless patience throughout the graduate school and takes care of me all the time. My sisters (Munni Devi and Rubi), niece (Neha, Sonia, Usha, and Saroj) and nephews (Kuldeep and Manjeet) have always provided their support and welcome me when I go home. Thanks to all my family, friends and relatives.





Finally I thank my God, for seeing me through all the difficulties. I have experienced His guidance on a daily basis.

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During the Spring and Summer of 2011, the University of Minnesota Veterinary Diagnostic Laboratory (MVDL) received 14 submissions of 12- to 18-week-old tom turkeys that were recumbent with wing tip bruises (“wing walkers”) and unilateral or bilateral swelling of the hock (tibiotarsal) joints. Gastrocnemius or digital flexor tendons were occasionally ruptured. A total of five turkey arthritis reoviruses (TARV-MN1 through TARV-MN5) were isolated in QT-35 cells and in embryonated chicken eggs from specific-pathogen-free chickens. The identity of the isolates was confirmed by electron microscopy, reverse transcription-polymerase chain reaction (RT-PCR) and gene sequence analysis. Additionally, blast analysis on the basis of 880bp nucleotide sequence of S4 gene confirmed all isolates as reovirus.

Phylogenetic analysis divided the five isolates into two subgroups: subgroup I containing TARV-MN1, TARV-MN2, TARV-MN3 and TARV-MN5 and subgroup II containing TARV-MN4. Isolates in subgroup I had a similarity of 97% to 100% with each other while subgroup II (TARV-MN4) had a similarity of only 89.2% with subgroup I viruses. This isolate showed 90% to 93% similarity with U.S. strains of turkey enteric reoviruses (TERVs) while the four isolates in subgroup I had 89% to 97.6% similarity with TERVs. These results indicate divergence within TARVs as well as between TARVs and TERVs, which needs to be confirmed by complete genome sequence analysis. Experimental studies to determine the role of TARV, TERV, and classical chicken reovirus (CRVs) in turkey arthritis have recently been completed. It has been found that TARVs are able to produce tenosynovitis in turkey poults but not TERV or CRV.

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isolates from a laboratory in Delaware. We characterized the S class gene segments of 12 TARVs and compared it with that of a TERV. Phylogenetic analysis of S2, S3 and S4 genome segments revealed grouping of all TARVs into two lineages while, on the basis of S1 genome segment, only one lineage was found. All TARVs had 95% to 100% nucleotide identity based on sigma C protein sequences (S1 segment) but varied from 90%-100%, 88.9%-100% and 88.7%-100% on the basis of S2, S3, and S4 genome segments, respectively. Point mutations as well as possible re-assortments were observed in TARVs throughout the S class.

We then did complete M class gene analysis of these 12 TARVs and included three more strains isolated in 2013 and 2014. Eight TERVs were isolated from fecal samples of turkeys and used for comparison. The aims of this study were to characterize turkey reovirus (TRV) based on complete M class genome segments, to determine genetic diversity within TARVs in comparison to TERVs and CRVs, and to find molecular markers that might be used to differentiate TARVs from TERVs. In this study, nt cut off values of 84%, 83% and 85% for the M1, M2 and M3 gene segments was proposed, generating 5, 7, and 3 genotypes, respectively. Phylogenetic analysis revealed point mutations and reassortments among TARVs, TERVs and CRVs.

On the basis of our results, we proposed M class genotype constellations (GCs) for avian reoviruses (including TARV, TERV, CRV, duck reovirus, and goose reovirus).

The TARVs and TERVs were divided into three GCs of which GC2 was unique for TARVs and TERVs only. The maximum number of GCs (n=7) was formed by CRVs of which GC1 and GC3 were shared with TARVs and TERVs indicating reassortment

–  –  –

identification of virus strains that can be used for developing universal vaccine(s) against avian reoviruses (ARVs).

Strains of TARVs (n=7) and TERVs (n=3) were further studied for L class genome segment sequences. All three L class gene segments of TARVs and TERVs and their encoded proteins λA, λB, and λC were similar in size to those of CRV reference strain S1133. The conserved motifs such as C2H2 zinc-binding motif and conserved polymerase region were present in λA and λB, respectively. In λC protein, a conserved motif for ATP/GTP-binding site and S-adenosyl-L-methionine (SAM)-binding pocket for methyl transferase were observed in TARVs and TERVs with only one substitution as compared to CRV.

We proposed a new GC system for classification of avian reoviruses based on nt identity cut-off values for each L class. Based on this new genotype classification, all ARVs were divided into six, seven and eight genotypes in L1, L2 and L3 genes, respectively. Interestingly TARVs and TERVs grouped with three CRVs (two arthritic strains from Taiwan and one enteritic strain from Japan) in genotype L1-I and formed a different genotypes (L2-I, L3-I) from CRVs in L2 and L3 genes. The maximum nucleotide divergence was observed in genotypes of L1 and L2 genes. However, lower divergence at the amino acid level indicated that changes were mostly of synonymous type. Compared to L1 and L2 genes, the nonsynonymous changes were more in L3 gene.

Point mutations and possible reassortments among TARVs, TERVs and CRVs were also observed.

–  –  –

reoviruses (both TARVs and TERVs). We developed a TaqMan real time RT-PCR (rRTPCR) assay for this purpose using a primer-probe set designed from the conserved region of the S4 segment of the turkey reovirus genome. The newly developed rRT-PCR was specific for the detection of turkey reoviruses. The detection limit of this assay was 10 genome copies per reaction. For TARV-MN4 strain of turkey arthritis reovirus, one 50% tissue culture infectious dose (TCID50) was equivalent to 11.6 + 0.2 genome copies. The highest coefficient of variation for intra-experimental and inter-experimental variability was 0.08 and 0.06, respectively, indicating the reproducibility of the assay. This new test should be useful for the rapid detection of both TARVs and TERVs.

Another objective of our study was to compare the survival of TARVs in the environment (poultry litter and drinking water) with that of TERVs and CRV. Virus survival was studied in both autoclaved and non-autoclaved poultry litter and drinking water at room temperature (~250C). Three TARV isolates (TARV-O’Neil, TARV-MN2, TARV-MN4), one TERV (TERV-MN1) and one CARV isolate were used in this study.

The viruses were propagated and titrated on QT-35 cells. In autoclaved de-chlorinated tap water all five viruses were able to survive for 9 to 13 weeks. In non-autoclaved water, all five viruses survived for 10 days. In autoclaved litter, the viruses survived for 6 to 8 weeks. In non-autoclaved litter, the survival was for 7 to 9 days only.

Another study was done to determine the efficacy of commonly used disinfectants in the turkey industry. We tested the antiviral efficacy of five disinfectants (Virocid, Keno X5, Synergize, One Stroke, and Tek Trol) against TARVs. For comparison, TERV and CARV were also included. At their recommended concentrations, all five

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Acknowledgements…………………………………………………………………...i Dedication……………………………………………….…………………………...iv Abstract……………………………………………………………………………...v Table of contents…………………………………………………………………….x List of tables………………………………………………………………………….xi List of figures………………………………………………………………………..xii CHAPTER 1: Introduction, literature review and objectives……………………………1 CHAPTER 2: Isolation and molecular characterization of turkey arthritis reovirus..…..26 CHAPTER 3: Characterization of S class gene segments of a newly isolated turkey arthritis reovirus………………………………………………………………….50 CHAPTER 4: Phylogenetic analysis, genomic diversity and molecular evolution of a newly isolated turkey arthritis reovirus based on M class gene segments….…..85 CHAPTER 5: Molecular Characterization of L class genome segments of a newly isolated turkey arthritis reovirus………………………………………….……..124 CHAPTER 6: A one step real-time RT-PCR for the detection of turkey reoviruses…..158 CHAPTER 7: Survival of newly isolated turkey arthritis reovirus in poultry litter and drinking water………………………………………………………....173 CHAPTER 8: Efficacy of five commonly used disinfectants against newly isolated turkey arthritis reovirus………………………………………………………188 CHAPTER 9: General discussion and conclusions…………………………………...197 REFERENCES……………………………………………………………….………..207

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Table 1.1 Proteins encoded by various genome segments of avian reovirus…………19 Table 3.



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