(A suggested proposal)

Study of Human Coronaviruses isolated from Children in Kurdistan region causing Acute Respiratory Infection A suggested proposal from: Dlshad Abdullah Hasan PhD student College of Medicine Hawler Medical University Iraqi Kurdistan Region Email: dlshad2011@yahoo.com

Background: Viral infections are among the leading causes of respiratory disease in children, and respiratory infections are the most common infectious disease worldwide. The World Health Organization (WHO) continues to rank lower respiratory tract infections as the leading cause of burden of disease in the world.{1} Acute respiratory infection can be caused by several viruses, and coronaviruses are the most important among them. Coronaviruses are medically important respiratory and enteric pathogens of humans and a wide range of animals. Four human coronavirus strains have been described, which are associated with a spectrum of disease, from mild, febrile upper respiratory tract infection to severe illnesses, including croup, bronchiolitis, and pneumonia, with a wide geographic distribution , although there are differences in the frequency of detection of the four viruses in different parts of the world at different times. {4} The family Coronaviridae comprises two genera, Coronavirus and Torovirus, and is part of the order Nidovirales, which also contains the families Arteriviridae and Roniviridae.{6} The name Nidovirales (from the Latin word nidus, which means nest) refers to the 3 coterminal nested set of sub-genomic mRNAs that is produced during infection. The viral genomes which feature the largest genomes and the most complex genetic organization of all plus-strand RNA viruses.{2}. This genome is 2732 kb, and encode for 5` replicase polyproteins (ORF1a and ORF1b) and 3` structural proteins in the conserved order, 5`- spike (S)- envelope (E)- membrane (M)- nucleocapsid (N)-3`.{ 3,5}. The virions measure between (120- to 160-nm) in diameter, roughly spherical particles with a linear, non-segmented, capped, and polyadenylated positive-sense single-stranded RNA genome that is encapsidated in a helical nucleocapsid. The envelope is derived from intracellular membranes and contains a characteristic crown of widely spaced club-shaped spikes that are 12 to 24 nm long, giving rise to the virus' name (corona, Latin = crown). {6, 7} The coronavirus genome RNA is translated to express the two most 5` open reading frames (ORFs), ORF1a and ORF1b, occupying approximately two-thirds of the genome.{8}The 1

upstream ORF1a encodes a polyprotein of 450500 kDa, termed polyprotein pp1a, whereas ORF1a and ORF1b together encode pp1ab (750800 kDa). orf 1a and orf 1ab are extensively processed by orf 1a-encoded proteases to yield 15 -16 mature nonstructural (replicase) proteins. These non-structural proteins (nsp) assemble to form the membrane associated viral replication/transcription machinery in the cytoplasm of the infected cell. {9, 10} Some of these non-structural proteins encode proteins of essential functions, such as PLpro (nsp3), 3CLpro (nsp5), RNA-dependent RNA polymerase (Pol) (nsp12) and helicase (nsp13).{11} Interactions between viral proteins play pivotal roles in many processes during the viral infection cycle. Analysis of protein-protein interactions is essential to understand protein functions and the molecular mechanisms underlying biological processes. {12} The Coronaviridae family are divided into three groups, subdivided into subgroups, based initially on serologic, and more recently, on genetic analyses. With the identification of more distantly related viruses, the taxonomy of these viruses is likely to undergo further changes. Groups 1 and 2 consist of various mammalian CoVs, whereas avian viruses cluster in group 3. {13, 14} Shortly after the emergence of severe acute respiratory syndrome CoV (SARS-CoV) in 2003, group 2 CoVs were further divided into two subgroups, termed 2A and 2B {15} Coronaviruses are readily transmitted across species. This phenomenon was illustrated when the SARS-coronavirus crossed species from bats to intermediate hosts such as palm civets and then to humans. It also explains the large number of species, including humans, infected with viruses closely related to bovine coronavirus.

Aims of the study: The over all aims of the study are: 1-Screening all enrolled patients in Kudistan region for detection of new strains of Human coronaviruses. 2- Evaluation the prevalence of Human coronaviruses in hospitalized children patient with acute respiratory disease. 3-Proteomic assay like protein protein interactions for characterization role of some coronavirus proteins, e.g. replication, transcription

Design and Methods

1-Sample collection: The study will be include children aged under fifteen years admitting to the paediatric hospital with upper or lower respiratory tract infection and children with enteric fever. Nose , throat swab and nasopharyngeal samples with blood samples will be collect from enrolled patients. The period of sampling will be one years. Samples will stored at 70C until use. 2-Clinical data Clinical symptoms will be record and categorized depending on physician report into appropriate groups, like chronic respiratory condition, lower respiratory tract infection (LRTI), upper respiratory tract infection (URTI), other. 3- RNA extraction Viral RNAs will be extracted from specimens by using a QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) within 10 h of receipt of specimens. The eluted RNAs (templates for RT-PCR) will be stored immediately at (70C) until use. 4-Screening test: The patients samples will be screening for presence of all four Human coronaviruses strains by using ( Pancoronavirus RT-PCR assay).Screening of samples will be performed by amplifying with a suitable bp fragment of the polymerase gene and designing sets of primers to four Human coronviruses. PCR- products will be run on a polyacrylamide gel, stained with ethidium bromide, and visualized under UV-light. 5- RT-PCR for expression of any new gene identified in strains of coronaviruses and DNA sequencing. 6- In vitro expression and Protein Protein interactions. Cloning and expression of the Human coronavirus ORFs, and using Mammalian two-hybrid analysis (M2H): For protein expression we can use either Recombinant glutathione S-transferase (GST)-fusion
system or maltose-binding protein (MBP)-fusion. Proteins might be expressed in E. coli BL21 and DH5alpha, and tested for either in vitro interaction of for immunological assays as well. 7- Construction of a replication and transcription report system coronaviruses : By

applying ( Luciferase Reporter Gene Assay) . 8-Screening of infected lab animals by ELISA to test building of antibodies to antigenic groups of Human coronaviruses.

Highlighting some sides of study project:

I-Capacity of study to carry out : This project will be carried out (as split site PhD project) in collaboration between two Universities, under supervision of two supervisors from the each Universities. The capacity of our university in carrying out part of this work include : detection of viral agent by PCR methods, basic molecular assays like SDS and agarose gel electrophoresis, application of ELISA. The other parts of the work: like sequencing, gene expressing and protein - protein interaction study and other advance methods, can not be carried out in home University, and here the role and contribution of foreign supervisor are highlighting. This role coming from learning and training on these sophistic techniques in his laboratory. II-Medical important of study: Since the etiologic agent of SARS epidemic in 2003 was identified as a novel coronavirus, this has led to a significant increase in interest in study of coronaviruses infection in humans and animals. The medically important of this project comes from that we expect that the results of the project will lead to highlighting the clinical spectrums of the disease and general characteristics of Human coronaviruse to be compared with others Human coronaviruses emerged from other previously published studies in other countries. The significance of Human coronaviruses in the etiology of acute respiratory infection in children will be focused and the seasonal circulation and molecular epidemiology of the virus in region will recorded. We hope that these findings and other expected results of the study will gives new insight in understanding and management of the disease here in Kurdistan. Finally better therapies and prevention strategies are needed to decrease the burden of acute respiratory infection. In this context we hope through studying activities of some proteins and enzymes involved in genome replication of the virus, some additions will be make in this field. III-State of problem :

Human coronaviruses are medically important viruses that causes acute respiratory and enteric infection in humans and animals. Few data about the Human coronaviruses prevalence, circulation in community and molecular epidemiology in Iraq and Kurdistan are available. Furthermore, although there are many studies of other viruses but recording data about virological study of Human coronaviruses here are very limited, most previous data are coming from serological studies or study other viruses infection of the disease. One study carried out by H.J Hasony (1982) and another by KA.Albargish (1999) in Basrea , were used ELISA technique for detection of antibodies against Human coronaviruses in population of southern Iraq and Respiratory syncytial virus infection in children in Basra, respectively. This lacking to recorded data about the Human coronavieuses infection, make this study and similar studies necessary and good contribution in understanding and management of the disease in region. More than, the application of sophistic techniques of virology in this study will make the way open for entering such novel techniques in academic researches here in Kurdistan, especially through collaboration with advance institute in west. Kurdistan now is open country differ from tow decades ago, many different people from different areas comes here for work and other reasons, this may lead to bring new microorganisms or strains of them into the region. In this context new diseases or new etiologies may be emerging in our population. In sight of this situation, our project of studying Human coronaviruses, which they are members of sense positive RNA viruses. It is established scientifically that these viruses have high rats of mutation, and as a result of the unique mechanism of viral replication, Human coronaviruses have a high frequency of recombination. In addition to that coronaviruses have and will likely continue to cross species and cause disease in unrelated hosts. This disease may be mild or severe. Here coming more insight of significant reasons for this project to be carry out in Kurdistan. So through the application of this project and at the end of study we expect that we will recorded the molecular epidemiology and phylogenetic tree of Human coronaviruses. According to above reasons we expect that may be some variation in our results in comparison with previous results from other countries.

Reference:

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7-Kamps

Hoffmann SARS Reference - 10/2003-Flying Publisher Third Edition.

8- Isabelle Imbert1, Jean-Claude Guillemot1, Jean-Marie Bourhis, Ce cile Bussetta, Bruno Coutard, Marie-Pierre Egloff, Francois Ferron, Alexander E Gorbalenya and Bruno Canard. (2006) A second, non-canonical RNA-dependent RNA polymerase in SARS Coronavirus. The EMBO Journal 25; 49334942 9- Tobias Hertzig, Elke Scandella, Barbara Schelle, John Ziebuhr, Stuart G. Siddell, Burkhard Ludewig and Volker Thiel. (2004). Rapid identification of coronavirus replicase inhibitors using a selectable replicon RNA. Journal of General Virology 85; 17171725 10- Yujia Zhai, Fei Sun, Xuenei Li, Hai Parg, Xiaoling Xu, Mark Bartlan & Zihe Roa. 2005. Insight into SARS-CoV transcription and replication from the structure of the nsp7nsp hexadecamer. Nature Publishing Group. http:www.nature.com/nsmb.

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