Repository of Human Blood Derivative Biospecimens in Biobank: Technical Implications.

Human biorepositories are collection of biological samples and health information from a large number of participants generally in the cohort studies. The main purpose of established biobanks is organization of biomedical research for upgrading the knowledge of human disorders from cancer to infectious and rare disease. The studies of generation relationships and understanding the preclinical stages of ageing are also from the solution of bitobank. This review overview the significance and storage condition of biospecimens including whole blood, red blood cells (RBC), buffy coat, plasma, serum, DNA and RNA that derived from blood in human biobanks. These biological samples provide valuable information on the prevalence of germline mutations, epigenetic modifications or interaction between genes and proteins in associated with the development of certain types of disease. The quality of biospecimen in biobanks is a powerful tool for valid identification of biomarkers. Therefore optimum qualities of human biological samples in long time storage that have been assessed in several studies also indicate in this review.

INTRODUCTION
Biobanks are carefully characterized to arrange for real answers in scientific and clinical research for diagnosis and treatment of a range of life-threatening diseases. The establishment of biobanks most often offers the volunteer-based sample for conducting the research. 1,2 Development of this new field of biorepository and biospecimen science could increase upcoming issues related to human genome, transcriptome, and proteome. 3 The public health policy has convinced the governments to generate population biobanks for diagnostic and etiopathogenesis studies over the past two decades. 4 Along these lines, there are a growing number of national biobanks for collecting data and samples from a population or subpopulation for providing largescale research resources. 5,6 Initially repositories had taken small rules in which biospecimens were kept through laboratory notebook and limited to a few freezers. Today's technological developments support specimens' annotation and establishment and therefore accelerate the arrangement of hands-on biobanks. Moreover using web services for computerized biobank expand communication with clients and opened collaborations between the public and private divisions. These services 61 also guarantee acquire of biological material and help international research for improving human health. 1,7 Therefore creations of biobank lead to accumulation of repositories of biological samples from clinical sources into the central database. 3,8 Cohort studies encompass a prospective design with long-term follow-up with a large number of participants. Genomic sequencing approaches become routine in research laboratories that assist researches in human blood DNA or RNA and studies of similarities of three-generation and identification of preclinical stages of ageing at earlier age. 3,5 Scientists have sought to integrate relationship of genetic and biomarkers data with lifestyle and environmental factors by obtaining biospecimens from participants in cohort studies to solve the important questions on human health. 6,9 They also arrange opportunities for biomarkers discovery to outline subgroups of diseases and the therapeutic activities that anticipate developing patient management. [10][11][12] Biomarkers represent the biological characteristic from physiological state, pathogenic processes, or pharmacologic responses to a therapeutic intervention. 11 Evaluation of biomarkers is useful approaches for early detection of disease, and identification of predictive and prognostic factors in disease management. Recent developments in areas of gene-expression microarrays, proteomics, and metabonomics with combining molecular profiling data from multiple omics lead to the discovery and validation of molecular biomarkers in medical research. Studies of biomarkers are non-invasive strategies that are afforded by keeping biospecimens in biobank. 11,12 Along these lines, large pharmaceutical companies, universities, and state organizations attempt funding for biobanks undertake comprehensive benefits in analysis of metabolite, biomarkers, minerals and relevant immunoprotective factors related to health and diseases. 3,11 Peripheral blood, urine, hair and nail and purified DNA as well as biofluids (breast milk, semen and saliva), are important samples in the human biobank. It should be careful that people who join in research studies related to biobank are usually not the same to those who do not take part and mostly are in healthier condition therefore to remain valid studies belong to biobanks caution need to considered in keeping eligible prevalence of participants with cohort studies. 2,[13][14][15] Therefore, this review looks at studies that investigated the status of biobanking for their potential in using of blood derivative biospecimen and the importance of high quality storage of these samples.

Types of Biospecimen in Human Biobank
Blood, urine and tumour tissue are common biological samples in biobanks that donate by participant under a separate consent form and are critical for biomedical research. However more progressively wide-ranging of biospecimen types including hair, nail clippings, saliva, faeces and breast milk have also might be collected and processed according to guidelines. 6,13,16 Demographic associated data from life style, medical history and medications, and possible causes of exposure data manage by trained research staff to create a resource of scientific database. Samples do not labeled with individual names and they are identifiable by only a unique code. 6 Sometimes collected samples link to the clinical trials or public health studies therefore the privacy of general public is crucial concern in management of any biobank. Maintain of samples quality and protections of individual rights are very critical thus biobank managers are responsible for potential staff with perfect understanding of the facts of given biological samples in to a biobank. This helps the researchers' access to biobanks when they are in need of specimen for their research studies. Biobanks work as vital resource for worldwide scientists and facilitates to find particular collections of biospecimens. 15 In following we talk about the potential significance of human blood derivative biospecimens in biobank such as whole blood, red blood cells (RBC), buffy coat, plasma, serum, and purified nucleic acids.

Significance of Whole Blood and Blood Cells in Long Term Human Biobanks
From human biospecimens, the whole blood and buffy coat are essential to biobanking efforts and are willing to share among international biobank. Whole blood is typically collective with an anticoagulant and consists of RBCs, white blood cells (WBCs), and platelets which suspend in plasma. Equally, the part of anticoagulated blood sample that consist of the white blood cells and platelets termed buffy coat. Both are the main source for cellular nucleic acids and achieving optimal quality and quantity of germline DNA and constructing DNA biobank. 1,17 DNA/RNA biorepositories are potentially required for the comprehensive analysis of functional genomics that give details for characteristic and expression of whole sets of human genes. 9,17 Prominent request of whole blood and buffy coat in biobanks are exploration of copynumber variations (CNVs) or chromosomal regions in association with non-Mendelian diseases 18 as well as molecular mechanisms including epigenetic modifications or interaction between genes and proteins that play a major role in the risk of developing some diseases. 19 DNA is the main informative molecule that leading the protein synthesis activity in a particular cell. Crystal structures of coiled coils DNA indicate that their geometries can vary according to sequence or the presence of stabilizers such as proteins or small molecules. 20 Likewise, protein-RNA interaction is required for the stability of the supercoiled RNA. 21 One important concern in long term storage of buffy coat specimens is stage-specific transcriptome studies by using gene expression array and RNA sequencing analyses in a great number of infectious and noninfectious diseases. 3 Epigenomics related studies are another field of genomics approach in biobank medicine. DNA methylations play an important role in silencing tumor suppressor genes during cancer development. 18 Furthermore, red blood cells (RBCs) reveal unique protein expression profile in the states of health and disease. During maturation they lost all cellular organelles and consequently are not capable for proteins substitute. RBCs inherited disorders with heterogeneous clinical presentation either in membrane structural combination or function of membrane transporters that characterize them for comprehensive membrane proteomics research from transfusion medicine to infectious diseases. 22,23

Storage-Keeping Quality for Human Blood Cells
Blood is the most collective biospecimens in human biobank as a source of DNA and RNA. Anticoagulated blood is prerequisite in purification of genomic or mitochondrial DNA and RNA molecules (tRNA, mRNA, miRNA) as well as plasma derived cell-free circulating nucleic acid molecules. Among the most commonly used anticoagulants, EDTA used for a variety of DNA based studies and protein assays, citrate is more appropriate in WBC culture, however heparin despite the advantage for RNA and DNA yields is not recommended as it could inhibit PCR. 24,25 The yields of extracted DNA and RNA from whole blood or buffy coat specimens are determined by blood storage condition and the keeping quality of biospecimens in long term.
RNA is the most easily degradable part of a biological sample in biological contexts by abundant and ubiquitous RNAses that make it a major problem in biobank. 21,26 WHO-IARC biobank suggests nitrogen storage below -130°C as optimal temperature for long term preservance of biomolecules such as RNA. 27 Samples storage at -140°C by liquid nitrogen keep the RNA in intact and functional state in a period of more than 50 months as biochemical activity that influence intracellular contents are assumed to be inactive. [28][29][30] Currently numerous centers use −80 °C temperature freezing for simple and safe storage compare to liquid nitrogen storage. 6,31 Although some studies have indicated for 5' transcript tags RNA fragmentation after five years storage at −80 °C 32 however, recent study showed that long-term freezing in -80°C does not adversely affect to the RNA extracted value from the deposited biospecimen. 33 The impact of thawing on RNA integrity and gene expression in fresh frozen biospecimens has also been evaluated. Minimal RNA degradation was detected after half hour of thawing in unfixed samples. 34 RNA integrity is kept with RNase-free handling however, addition of commercially available RNase inhibitor such as RNAshell or RNAstable give accurate real-time PCR results and protect the value of the biospecimens for other assays. 26,29 Tissues incubated in RNAlater efficiently prevent RNA degradation. 26,29 Biosources of DNA in biobank is an integral part for clinical research. It has been shown that storing the blood sample at -80 °C does not adversely emotional impact on the quality of extracted DNA. 31 To keep the biospecimens reliability from multiple freeze-thaw cycles, protection and stabilization of DNA at room temperature (RT) endorsed in biobanking. It also eliminates the costs associated with freezer storage and reduces the maintenance costs for biobanks. 20,31 In this way, DNAshell and RNAshell make sure the safe storage of blood spots at room temperature for long periods of time and DNA molecules are appropriate for common downstream analysis. 35 Stable dry-state storage of blood is desirable to reduce required storage space and to diminish electrical and the budgets for biospecimens shipping. DNA purified from various commercially available dry-state stabilization matrices run successfully in molecular applications. 17,35 Formalin-free preservatives show approval but fluctuating resources to preserve DNA and RNA, though RNA is degraded during paraffin embedding process. 36 Purified DNA is stable at 4 °C for numerous weeks and for months at −20 °C. 15,31 However, purified DNA and RNA are more stable at −80 °C in aqueous buffers or nuclease-free water for longterm storage. 31,34,37 Blood cells without dimethylsulphoxide (DMSO) are unable to get along viable because cells will rupture if frozen. However storage of RBCs is recommended at −80 °C for cell membrane proteomics research. 22 Complete descriptions of blood derived biospecimens in biobanks are explained in Table 1.

Technical Importance of Serum and Plasma in Human Biobanks
Plasma is the liquid part of normal unclotted blood that suspending red and white cells as well as platelets and need anticoagulants like EDTA for its separation. 24 In UK Biobank, anti-coagulants EDTA and lithium-heparin are used for high-quality plasma. 25 Serum forms when blood cells and clotting proteins remove from blood by centrifugation. Plasma contributes up to about 55% of the body's total blood volume. This difference in processing is the basis of some diversity such as metabolites between serum and plasma. Throughout the coagulation, platelets proteins such as proinflammatory cytokines and metabolites like sphingosine-1-phosphate diffuse within serum. 38,39 Therefore the advantages of serum for some metabolite are on debate. Human plasma and serum are most common materials in biological and clinical studies and could practice interchangeably for a wide variety of substrates like enzymes, hormones and cell free DNA or RNA for diagnosis and monitoring of disease. 9,40 However a number of studies have demonstrated the different level of metabolites between plasma and serum. 41 More than 4000 compounds that are annotated in human metabolite databases 42 and plasma is preferable matrices in clinical chemistry investigations for metabolic disease such as diabetes. 43 Moreover higher serum levels of lysophosphatidylcholine and diacylglycerol releasing in coagulation process, prefer plasma in study of lipid metabolite. 44 There are a wide range of proteins in size and structure with a concentration of 60-80 mg per ml that is highly correlated between serum and plasma. Nonetheless because of lacking coagulant protein, the assessment of proteins in serum is more valuable in clinical diagnosis and monitoring of a range of diseases. Recent advance in proteomics and bioinformatics procedures improved the number of up proteins to 10,000 in serum though most of which present at very low relative abundances. 41 The study of serum protein profile supportively evaluates the state of health or disease and help to manufacture the design of proper drugs in interaction with host cells. Inflammation is the common cause in serum protein alterations mostly by change in expression of the acute phase response. Albumin, prealbumin, apo B, apo A-1, retinol-binding proteins (RBP) and transferrin are acute phase hepatic proteins that usually decreases while C-reactive protein (CRP) and serum amyloid A (SAA) with marked increases and complement factors C3 and C4, ceruloplas-min, fibrinogen, plasminogen, haptoglubin, alpha 1-antitrypsin/ acid glycoprotein/chymotrypsin, and ferritin with more modest increases relate to inflammation that could confound serum protein interpretations. 41,45,46 Any disequilibrium in serum proteins points to the presence of infectious diseases, inflammation, electrolyte disorders, autoimmune disease or tumors. 47,48 All known resources of proteins in plasma and serum based on Mass spectrometry derived data are available in Plasma Proteome Database (PPD) that allows users in identifying of proteins or peptides compared to known plasma proteins. 10 Tumor-associated DNA in patients with cancer divide into DNA from cells in the blood and circulating cell free DNA from the source of tumor cells. The studies of free DNA are sensitive and specific for cancer diagnosis and follow-up therapy and improve by great development in PCR-based methods, mutations detection and DNA methylation analysis. 40 The advantages of serum or plasma for these kinds of studies remain to determine, however it was reported that serum has greater yield of free DNA or RNA due to the release of nuclear acids from damaged blood cells throughout coagulation. 49

Serum or Plasma Stability in Long Time Storage
Previous studies have indicated that the values of most analytes are comparable in mammalian serum and plasma when the blood is taken in standardize way and separated from the cells in 2 hours. Hemolyzed samples convince significant differences in potassium, phosphorus, albumin, and lactate dehydrogenase. Appropriate blood collection tubes with and without anticoagulant are accessible commercially. 50 Clotted blood need to collect either into glass or coated plastic tubes to inhibit sticking the clot to the container walls. Gel-barrier tubes are frequently used with no significant effect on metabolites concentration. 51 They are preferable because of the easy administration of blood collection and separation of serum in the period of shipment. Samples for biochemical analysis need to separate as quickly as possible to prevent ongoing metabolism of cellular constituents as well as hemolysis and leakage of analytes between the plasma or serum and cellular compartments. 52 Despite many research, the ideal storage of serum and plasma specimens for long time in human biobanks remains to determine. Previous report showed that time and temperature has the major effect on the analyte stability. 53 Freeze-drying increase maximum storage time and the numbers of time to brought samples to room temperature. However, storage at -80˚ C or liquid nitrogen showed the adequate stability of different biomolecules in serum and plasma. Repeated freeze/thaw essentially need to avoid because of degradation of proteins and nucleic acids, however sometimes is unavoidable due to a limited amount of aliquots. Most lipid metabolites concentration in serum or plasma decreases by freeze-thawing however recent study prove that in two freeze-thaw cycles from 159 investigated metabolites most are stable except for methionine sulfoxide. 54,55 Table 2describes briefly the best condition for storage and potential use of serum and plasma.

CONCLUSIONS
In summary, the scientific developments in human health that have been occurring over the past few years indicate the need for suitable biological material for the conduct of research. Biobanks support researchers' access for knowledge generation by a large numbers of biological well documented samples that selected with appropriate scientific criteria. The worth of biobanks will depend on the management of data related to phenotyping of donors and processing and storage of biospecimen. The potential of blood for biobanking appears to be excellent. It includes a variety of blood derivative biospecimens such as serum, plasma, buffy coat and red blood cells. The obtaining, storage, and quality valuation of these biospecimens need the standards operating procedures for further studies that has been discussed in this review.


Development of a DNA microarray platform for the detection of viruses transmitted by small mammals and arthropods

Khan, M. J. Development of a DNA microarray platform for the detection of viruses transmitted by small mammals and arthropods. 197p. Thesis (Doctoral). School of Pharmaceutical Sciences of Ribeirao Preto – University of Sao Paulo, 2015. Human activities have being responsible for the global environmental changes, resulting in an increase number of incident of vectorand rodent-borne diseases worldwide. Rodents and arthropods-borne viruses are important globally emerging and re-emerging viruses and most of them are RNA viruses. Efficient and early diagnosis of these infections are very important to prevent their spread, to improve clinical management of the patients, as wells to protect livestock and domestic animals. Currently, available diagnostic methods such as immunoassays, polymerase chain reaction and virus isolation can detect only one or few viruses in a single assay. The DNA microarray platform has emerged as diagnostic tool suitable for high throughput screening of pathogenic agents. The aim of this study was to develop a DNA microarray platform (RoboArboVirusChip) for detecting rodentand arthropod-borne viruses, which belong to seven families: Bunyaviridae (genera Orthobunyavirus, Nairovirus and Phlebovirus), Flaviviridae (genus Flavivirus), Togaviridae (genus Alphavirus), Reoviridae (genera Orbivirus, Seadornavirus and Coltvivirus), Rhabdoviridae (genera Vesiculovirus and Ephemerovirus), and Asfarviridae (genus Asfarvirus). Specific oligonucleotide probes of 60-mer (n=4209) targeting 412 virus species and generic probes of 25-35-mer (n=87) targeting viruses at the genus level were designed. A total of 17 reference viruses belonging to the Bunyaviridae, Flaviviridae, Rhabdoviridae and Togaviridae families were used to standardize RoboArboVirusChip. All reference viruses were specifically detected without any cross hybridization; however, the generic probes were not able to identify the viruses at the genus level. The RoboArboVirusChip was able to specifically identify four viruses contained in three different mixtures: i) virus of different families, ii) virus of the Flavivirus genus, and iii) the Dengue virus (DENV) serotypes. The four DENV serotypes were use to evaluate the sensitivity of the RoboArboVirusChip, which was able to detect a minimum of 25 RNA copies/mL of the viruses, confirming its high sensitivity. The applicability of the RoboArboVirusChip to detect viruses in clinical samples was tested with serum samples obtained from dengue suspected cases (four positive cases and 40 negative cases). DENV was detected in the four positive serum samples, while in the 40 negative serum samples, it was not detected any virus. The results obtained in this study suggest that the RoboArboVirusChip platform could be a useful tool for early diagnosis of robovirus and arbovirus infections during epidemic outbreaks, helping in the rapid implementation of disease containment strategies.

Roboviruses (Rodent-borne viruses) belong to two different families:
Arenaviridae (Mammarenavirus and Reptarenavirus genera) and Bunyaviridae (genera Hantavirus) (Gegúndez and Lledó, 2005). Approximately, ten mammarenaviruses are known to cause disease in human through natural or laboratory acquired infection. The clinical symptoms of four viruses (Guanarito, Junin, Lassa and Machupo) are mimic in several ways. Reptarenaviruses are Snake-infecting new arenavirus genera causes' fatal disease called Boid inclusion body disease (BIBD). There is no evidence of these viruses causing disease in human (Hetzel et al., 2014). About twenty Hantaviruses are responsible for in Asia and Europe "Hemorrhagic fever with Renal Syndrome" (HFRS) and in Americas Hantavirus Cardiopulmonary Syndrome (HCPS) (Zapata and Salvato, 2013;Kruger et al., 2014). They infect humans by the inhalation of aerosols contain virus, through ingestion of contaminated stored food or due to consumption contaminated rodents meat (Ter Meulen et al., 1996) (Jones et al., 2008;Weaver and Reisen, 2010).
There are currently more than 550 arboviruses have been identified, mostly belong to families above mentioned. More than 150 viruses are responsible for flu-like to fatal disease in humans and 50 viruses pathogenic animals (Gao et al., 2010;Cleton et al., 2012;Gan and Leo, 2014;Hubálek et al., 2014). For an example, only dengue (DENV) virus infections are at present roughly to cause over 100,000,000 clinical cases every year specific a re-emergence throughout the tropical and subtropical countries.
Approximately 17 million human's deaths every year reported throughout the world. The arboviral infections are rapidly spreading and this is major public health issue. There are not specific and effective treatments are available for the roboviruses and arboviruses with deal for those symptoms supportive therapy has been used with exception of Yellow fever (YFV) and Japanese encephalitis (JEV) vaccines (Cleton et al., 2012;Bhatt et al., 2013;Dash et al., 2013). Mainly, the medically important bugs: Mosquitoes, ticks, midges and sandflies, are known vectors responsible for the inoculation of arboviruses during blood feed, through contaminated mouthparts they enter in the body of host (humans and animals). The viruses first amplify within the tissues salivary gland of arthropods to produce high concentration of virus and are then passed on to host (Gubler, 2001). Other, non-vector transmission through laboratory associated, organ transplantation, mother to child and breast feeding (Descloux, 2013). These viruses are mostly causing zoonosis in the wild and are maintained in nature in complex cycles involving birds, rodents, marsupials, primates and sloths as reservoirs and wild mosquitoes, which suck the blood of these animals as vectors. Among the animal reservoir, mostly migratory birds, are infected, and are important carriers of arboviruses to distant sites (Meslin, 1997). The introduction of humans or animals viremic at these sites could lead to the emergence of urban cycles. Regarding robovirus, their emergence is associated with environmental changes caused by intensive agriculture causing increased food availability, an increase in the population of wild rodents, including attracting these animals to living next to man, who is mainly virally infected, inhalation of fecal and urinary components excreted by these infected animals and some important factors are responsible for the arboviruses will continue to emerge in new regions: landscape composition, deforestation, floods, increasing surface temperatures, urbanization, travel and commerce like animals and their products (Zeier et al., 2005;Dash et al., 2013).

Background of Roboviruses:-
The Robovirus term refers to Rodent-borne viruses (viruses transmitted by rodents) belonging to two families: 1. Family Arenaviridae (two genera Mammarenvirus and Reptarenavirus)

Family Bunyaviridae (only genus Hantavirus)
Both families of viruses are transmitted from rodents (natural reservoir host) to human beings (Gegúndez and Lledó, 2005).
Note: The genus Hantavirus we discussed in arbovirus section.

Taxonomy
The first arenavirus prototype LCMV was isolated in 1933 by doing serial passage of SLEV virus infected material from 42 years old dead human body, in monkey's brain. Arenaviruses are the group of viruses that represents the family Arenaviridae and was named after the sandy (Latin, arenosus) appearance of the ribosomes originally seen in thin sections of virions (Armstrong, 1934).
The family Arenaviridae consists of two proposed genera Mammarenvirus and Reptarenavirus (Radoshitzky et al., 2015). The Mammarenavirus (referred to "mammals and arenaviruses") for the genus for all currently known mammalian viruses, which is currently subdivided into two major groups, New World (Tacaribe virus) and Old World  Table 1).

Reservoir host in nature:-
Each virus is associated with either one species or a few closely related rodents with the exception of Tacaribe virus, which natural reservoir is bat. The New World rodents (rats and mice) belong to family Muridae, subfamily Sigmodontinae, while the Old World rodents belong to family Muridae, subfamily Murinae. Taken together, these types of rodents are located across the greater proportion of the earth's landmass, including Europe, Asia, Africa, and the Americas. One notable exception is Tacaribe virus, found in Trinidad in the Caribbean region, which was isolated from bats and mosquitoes during a rabies surveillance in Trinidad from, 1956-1958(Downs et al., 1963. In 2012, several arenaviruses have been isolated from non-poisonous snakes: Boa constrictors (Boa constrictor), an annulated tree snake (Corallus annulatus) and python with inclusion body disease-associated arenaviruses (BIBDAV) (Stenglein et al., 2012).
More recently, the re-isolation of the arenavirus from pool of one hundred host-seeking lone star ticks (Amblyomma americanus) was collected in a Florida in 2012 (Sayler et al., 2014).

Transmission to Rodents:
The rodents normally infected with arenaviruses; however, they are asymptomatic, with chronic viremia and viruria, thus spreading viruses to other noninfected rodents and other mammals. Infected reservoir host sheds virus into the environment via urine, feces and saliva. Some New World arenaviruses transmitted among adult rodents, via fighting and inflicting bites. In addition, Old World arenaviruses can pass from mother rodents to their offspring during pregnancy (Vitullo et al., 1987;Vitullo and Merani, 1988). New rodents in each host species infected with virus once, in many cases only in limited part of the geographical localization.

Transmission to Humans:
Arenaviruses infect humans by inhalation of aerosolized particles, through ingestion of contaminated stored food or consumption of their contaminated meat. (Ter Meulen et al., 1996). However, there are some risk factors involved in the virus spread; for example, human infection is associated with agricultural work, as the infected rodents prefers a field or agricultural habitat. In areas where the rodent species habitat includes human homes or other buildings, infection occurs in domestic settings. This may occur in a variety of ways. Person-to-person transmission is associated with direct contact with the blood, saliva, semen, stool, tissues, urine, vomits, and other secretions or via inhalation of aerosolized virus from clinical specimens. Patient infected with Lassa virus may shed virus in urine and semen for several weeks after recovery, and it is documented that sexual activity has resulted in secondary transmission (Mccormick et al., 1987).
Mother to child transmission is well evidenced and may result in severe congenital malformation or death of the fetus or newborn (Cj, 2000;).   (Cheng et al., 2015;Rathbun et al., 2015).

Antiviral drugs:
Currently, only licensed partially effective drug nucleoside inhibitor ribavirin is available for treatment of arenavirus infection associated with side effects (Cheng et al., 2015).

Taxonomy of genus Reptarenavirus
In the 1970's, inclusion body disease first detected in the United States, where it was affecting many species of snakes in zoo and private museum. Based on cross-react by western blot with anti-Junín virus antibodies, comparison weak cross reactivity was noted with anti-Machupo virus and anti-LCMV antibodies. The snake viruses do not able to react with antisera from patients with Bolivian hemorrhagic fever in ELISA. These new snake viruses may be hold the Old World and New World complexes of this family (Mj., 2012;Stenglein et al., 2012;Hetzel et al., 2013). A total seven viruses identified in the genus, three confirm species and four of them are tentative (Table 3).

Reservoir host in nature:
Not yet been confirmed (Hepojoki et al., 2015).

Transmission:
Snakes consume of rodents and bats, and both can be infected by arenaviruses and filoviruses (Zapata and Salvato, 2013).

Disease in Human:
To date, no disease was reported in human (Hepojoki, J.2015).

Treatment:
Not treatment available

Arbovirus:
The origin of "Arbovirus" for first time was using in laboratory jargon at in early 1940s from the joke like "you can't see it, smell it, touch it, or culture it, but it can kill you". The Cuban Dr. Carlos Finlay, first time proposed mosquito-vector theory in 1881 and the US Army Yellow fever commission head Major Walter Reed discovered first arbovirus, Yellow fever virus in 1901 (Russell, 1934;Reeves, 2001;Chaves-Carballo, 2005). In 1930, six viruses (African swine fever, Blue tongue, Louping ill, Nairobi sheep disease, Vesicular stomatitis and Yellow fever) were known caused disease in human and transmitted by arthropods and today more than 550 known.
Arboviruses (an acronym for "arthropod-borne viruses") are one of the most important emergence and re-emergence agents nearly worldwide. They cause a wide range of infections in humans and domestic animals (Jones et al., 2008). They are able to replicate and transmitted by medically important bugs such as mosquitoes, ticks, midges and sand flies, are known vectors responsible for the inoculation of arboviruses during blood feed (Gubler, 2001). These arboviruses must replicate biologically in the arthropod vector prior to transmission (Weaver, 1997). As, mechanically transmitted, without replication in the vector, Biological transmission can be vertical, involving the passage of the virus from an infected female vector to both male and female offspring. Horizontal transmission can be venereal, from a vertically infected male directly to a female vector, as well as oral from a female vector to a vertebrate host via the saliva during blood feeding. The latter horizontal mode of transmission is most common for the majority of arboviruses and involves infection of the vector alimentary tract following a viremic blood meal, dissemination of the virus in the vector, and virus replication in the salivary glands, followed by the injection of infectious saliva during blood feeding (Weaver and Reisen, 2010).

Taxonomy:
The arboviruses belong to seven taxonomic families: Bunyaviridae (genera

Genus Orthobunyavirus: History and taxonomy
To avoid confusion during discussion of any virus belonging to the family Bunyaviridae, the original Bunyavirus genus name it was changed to Orthobunyavirus in 1995 (Regenmortel, 2000). This genus is the largest genus within the family Bunyaviridae, with more than 170 viruses. Currently 48 viruses are species recognized by the ICTV, includes 122 viruses strain/ isolates, which is distributed throughout the world (Elliott, 2014). Most of the viruses are distributed into 18 different serogroups or left unclassified (Table 5). However, the historical term "serogroup" is not more useful for the viral taxonomy (Ladner et al., 2014).

Arthropod vectors:
The mosquitoes are main vector for this genus; mosquitoes transmit one hundred thirty seven viruses (137), sandflies (commonly referred to as " culicoid midges ") seven (7), mosquitoes and sandflies both are responsible for seven (7) virus, through ticks four (4) and for fifteen (15) viruses vectors are still unknown (Table 5 and suppementary   Table 3).

Human disease:
At least 30 orthrbunyaviruses are responsible for flu-like to hemorrhagic fever disease and 140 viruses non-pathogenic for humans. A total 23 viruses, eight from Bunyamwera group, seven from group C, three from California group, two form Guama group, one from each Bwamba, Simbu and Wyeomyia groups are responsible for flu-like illness. Encephalitis viruses belong mainly to three groups: California 4, Bunyamwera 1 and one from Wyeomyia. Garissa virus (Ngari complex) that belongs to Bunyamwera group causes hemorrhagic fever.

Animal disease:
There are eight orthrobunyaviruses responsible for disease in animals. Two viruses from California group; La Cross virus caused encephalitis in dogs (Black et al., 1994) and Snowshoe hare virus known for equine encephalitis (Heath et al., 1989). The same number from Bunyamwera group; Cache Valley virus agent of an embryonic and fetal death of sheep, possibility of for all ruminants (Edwards, 1994) and Main Drain virus as a cause of equine encephalomyelitis (Emmons et al., 1983). Four viruses from Simbu group affects livestock's ; Akabane virus is an exotic agent for severe congenital defects (Kirkland, 2002), Aino virus is associated with fetal defects in sheep and cattle (Noda et al., 1998), Schmallenberg virus manifesting the CNS of sheep and cattle (Wu et al., 2014) and Shuni virus causes of neurological disorders in horses (Van Eeden et al., 2014).

Epidemiology
Viruses of the Orthobunyaviruses are reported worldwide (Lowen and Elliott, 2005).

Vaccine:
No licensed vaccines are available for orthobunyaviruses those extremely effecting humans (Hollidge et al., 2012).

Genus Phleboviruses: History and taxonomy
The genera Phlebovirus name came from Phlebotomine sandflies because most of virus members are transmitted by phlebotomine except Uukuniemi and Bhanja serogroups (Tesh, 1988). The genera Phlebovirus contains more than 80 viruses, with 28 confirmed classified into 9 antigenic complexes groups and 32 as tentative and unclassified complexes.Before 1991, Uukuniemi-like group was in a separate genus.

Animal disease:
Rift Valley fever causes severe disease with more than 70% mortality rate in young animals like lamb and calf. The goat and buffaloes are also face problem with 10% mortality. Other Bhanja virus also pathogenic for the same animals and causes fever and meningoencephalitis.

Genus Nairovirus: History and Taxonomy
The genus Nairovirus is named for Nairobi sheep disease (NSD), which was originally isolated near Nairobi, Kenya in 1910 as acute hemorrhagic gastroenteritis cases of sheep (Davies et al., 1978). There are thirty-six viruses, seven species confirmed and 29 strains. The genus Nairovirus is divided into seven serogroups (Table 7).

Arthropod vectors:
The ticks are main vector for Nairoviruses; thirty-two viruses are use ticks as main vector, one virus is involved with ticks and sandflies and the vector of three viruses still not confirmed.

Human disease
Only three viruses from this genus have been identified as human pathogens, two viruses belong to Nairobi sheep disease virus serogroup causes flu-like symptoms and third Crimean-Congo hemorrhagic fever virus severe fatal with up to 50% mortality (Flusin et al., 2011).

Animal disease
Gunjan virus also known as Nairobi sheep disease virus an important disease of ruminants and other Soldado virus can affect young seabirds (Hubálek et al., 2014).

History and taxonomy of genus Hantavirus:
The  (Table 8).

Rodent's vectors:
Hantaviruses are transmitted by various species of rodents (Khan and Khan, 2003

Animal disease
Not reported.

Family Flaviviridae; History and classification
In 1881s, The Cuban Dr. Carlos Finlay first time suggested that mosquitoes were responsible to propagation of Yellow fever virus (an alternative name "vomito negro") in vertebrates host. The Walter Reed Yellow Fever Commission proved this hypothesis around 1900 (Chiong, 1989). This family currently includes four different genera.  (Table 10).  Also, non-vector-borne four flaviviruses have been isolated from bats and rodents.

Human disease:
Twenty-one flaviviruses are responsible for encephalitis and two viruses for hemorrhagic fever and 15 viruses are causes flu-like symptoms in humans (  (Hubálek et al., 2014).

Treatment:
No antiviral drugs are approved for use against the flaviviruses (Leyssen et al., 2008).

Vaccine
Licensed vaccines are available for YEV and JEV to protect human and WNV lineage 1 are for horses in the United States and in Europe (Hills et al., 2014;Beck and Barrett, 2015).

Family Reoviridae; History and classification
Sabin first introduced family Reoviridae name derived from Respiratory Enteric Orphan viruses in 1959 and originally these viruses are classified as echo virus 10 (Urbano and Urbano, 1994 (Dennehy, 2015) Seadornavirus Arthropods Humans and Insects (Attoui et al., 2005) Viruses belong to three genera; Coltivirus, Orbivirus and Seadornavirus transmitted by arthropods and infect animals are consider arboviruses.

History and taxonomy of genus coltivirus:
The genus Coltivirus name has been derived from Colorado tick fever virus (Urbano and Urbano, 1994). Currently, genus contains two species (Table 12). Viruses maintains in many animals like chipmunks, deer, squirrels, wild mice, wood rats, wild rabbits , rodents and sheep (Attoui et al., 2005).

Human disease:
Colorado ticks fever virus causes an acute systemic fever illness and Eaych virus

Animal disease:
Not reported yet.

Epidemiology
Colorado tick fever virus reported in USA and Eaych virus in Europe and Asia (Attoui et al., 1998;Charrel et al., 2004).

Treatment:
No antiviral treatment is available against coltiviruses except supportive care (Romero and Simonsen, 2008).

Arthropod vectors:
Transmitted by range of hematophagus arthropods; mainly, 68 viruses by sandflies, 55 through ticks, mosquitoes responsible for 33 and 10 viruses with unknown vectors.

Vaccine:
Vaccines are available against two notable orbiviruses African swine fever virus and Bluetongue virus (Paton and Taylor, 2011).

Arthropod vectors:
All three viruses transmitted by mosquitoes (Table 14).

Human disease:
Only Banna virus is causes encephalitis in human and other two viruses are nonpathogenic (Table 14) 1.

Treatment:
No specific treatment except relief of fever and pain (Attoui et al., 2005).

Genus Ephemerovirus; History and taxonomy
The origin of Bovine Ephemero Fever virus was first noticed as an infectious agent in East Africa in mid-nineteenth century (Nandi and Negi, 1999). The genus Ephemerovirus is one of eleven currently recognized genera in the family Rhabdoviridae. This genus divided into two serogroups with eight viruses, The BEFV group contains six virus five species and one tentative. Two viruses represented from second group MALV (Table 16). Sandflies also additional vector in BEFV group. Two vectors one from each group (Mosquitoes and sandflies) and two vectors still unknown from BEFV group (Table 16).

Human disease
Viruses belong to this genus not pathogenic for humans (Table 16).

Animal disease
The Bovine ephemeral fever virus causes three days sickness in cattle and water buffaloes, dengue fever of cattle, stick sickness and bovine epizootic fever and second virus from this genus Kotonkan virus manifest an ephemeral fever-like illness in cattle (Tomori et al., 1974;Doherty, 1977;Nandi and Negi, 1999).

Epidemiology:
Bovine ephemeral fever is present throughout Africa, Middle East, northern Australia and Southeast Asia. Other virus from this genus Kotonkan distribute in Nigeria (Africa) (Hubálek et al., 2014).

Treatment
There is not any specific treatment available.

Vaccine:
Only an attenuated vaccine has been using in Australia and Japan against BEFV within genus (Uren et al., 1994).

Genus Tibrovirus; History and taxonomy
This genus is name as an adapted from the species Tibrogargan (TIBV) and it was first isolated in 1976 from a pool of sandflies in Australia (Queensland). The genus Tibrovirus newly recognized with two species, Tibrogargan virus (TIBV) and Coastal Plains virus (CPV) (Lauck et al., 2015) (Table 17 and sup Table 13). Usually, both viruses are transmitted via biting of sandflies (TableXX).

Human disease:
None of responsible for disease in human (Lauck et al., 2015).

Treatment
Not required.

Genus Vesiculovirus; History and taxonomy
In 1916, first Vesiculovirus or Vesicular Stomatitis Virus (VSV) was reported in United States an epidemic in livestock (Hanson, 1952). The genus Vesiculovirus have total 27 viruses, nine of them are confirmed species and eighteen viruses are tentative (Table 18 and sup Table 14).

Human disease:
A total six viruses causes flu-like symptoms in humans and other 21 viruses nonpathogenic for human (Table 18). are pathogenic for cattle, equids cause tongue vesicles and fever causes (Letchworth et al., 1999).

Epidemiology
Vesiculoviruses are present only in the Americas especially south Mexico, Central America and some regions of South America (Hubálek et al., 2014).

Treatment
No specific treatment still available against Vesiculoviruses (Jadi et al., 2011).

Family Togaviridae; History and classification
This family name come from the Roman Cloak (in Latin = toga) for this reason called togaviruses. The family togaviridae is categorized into two genera: Alphavirus and Rubivirus. The Rubivirus genus is containing of a single member its Rubella virus (also called German measles) mode of transmission by droplet spread or direct contacts (Jose et al., 2009;Mangala Prasad et al., 2013).

Alphavirus genus; History and taxonomy
In 1930, first Alphavirus was isolated as Western Equine Encephalitis virus by Dr. Karl Friedrich Meyer with very interesting story. During encephalitis in horse at San Joaquin Valley (California, USA), Scientist team tried to get head of horse symptomatic for encephalitis and owner of horse threatened team " I won't sell the horse, and you try to do anything to the horse I shoot you". But, Dr. Meyer deal with former wife when he was asleep in exchange horse head for USD 20, the team successfully isolated virus from the brain tissue (Sabin, 1980). The genus Alphavirus generally referred to; the viruses responsible for rheumatic disease in humans (Old World) and viruses especially associated with encephalitis disease in the Americas (New World) (Suhrbier et al., 2012). The Alphavirus genus consists of about 36 different viruses divided antigenically into seven serogroups and some viruses are still unclassified they not have their yet confirm serogroup status (Table 19).

Arthropod vectors:
The mosquitoes are main vector for the genus Alphavirus with approximately 31 viruses and 5 viruses are transmitted through unknown vectors (Table 19).

Vertebrate reservoir hosts:
The vertebrate reservoir hosts are generally mammals, birds and primates (Weaver and Barrett, 2004).

Human disease:
Twenties viruses are non-pathogenic for humans from all serogroups except  (Table 19).

Animal disease:
There are nine exotic Alphaviruses significant on health of animals, the members within the sero-complex Western equine encephalitis virus is contains four viruses; Buggy Creek virus has little impact on house sparrows (O'brien and Brown, 2011), Highland J virus cause encephalitis in horses and recognized pathogenic for various avian species . Sindbis virus cases encephalitis in horses and notable virus in this group Western Equine Encephalitis virus encephalomyelitis (Calisher et al., 1986). One viruses from Eastern Equine Encephalitis (EEEV) serogroup and one Venezuelan Equine Encephalitis (VEEV) causes encephalitis in equines (Zacks and Paessler, 2010). From Semiliki forest virus group; two viruses Getah virus pathogenic for racehorse and causes fever and other semiliki forest virus responsible for encephalitis in horses (Robin et al., 1974;Fukunaga et al., 2000) . One virus from Middelburg serocomplex (Middelburg virus) also responsible for encephalitis in equines (Attoui et al., 2007).

Treatment:
Currently, there are no licensed specific treatment available except symptoms supportive drugs for any Alphavirus (Phillips et al., 2013).

Vaccine:
We are still waiting licensed vaccines for humans use, but some (EEEV, VEEV and WEEV) unlicensed vaccines are available to protect at-high risk laboratory personnel (Hoke, 2005). For the veterinary use, vaccines (EEEV,VEEV and WEEV) are available for routine use to control infection in endemic areas (Barber et al., 1978).

Family Asfarviridae; History and classification:
In 1910, R.E Montgomery discovered first African swine fever virus (African swine fever and related viruses) from an ill pig in Kenya (at Kabete, Kiambu County). It was observed that the occurrence of cases appear when domestic pigs come into close contact with wildlife species, specially warthogs (Costard et al., 2009). There is a unique genus Asfarvirus in family Asfarviridae.

Genus Asfarvirus: History and taxonomy
This genus has only one species and its called African swine fever virus (Table   20).

Vaccine
There is no effective vaccine available for African swine fever (Penrith et al., 2013).

Genome of Roboviruses and Arboviruses
All these viral families have a RNA genome except of the family Asfarviridae that contains DNA (Table 21). Therefore, RNA viruses replication include high mutation and short replication times in compare to DNA viruses. RNA viruses are able to jump from its original host into a new host species and adapt to changing new environmental conditions.

Diagnosis of viral infections:
Historically, the diagnosis of viral infections includes virus isolation in cell culture, the direct immunofluorescence and enzyme immunoassays methods. Recently, the polymerase chain reaction (PCR) has revolutionized the diagnosis of viral infections by increasing the detection sensitivity and enabling the detection of several viruses in parallel. However, the multiple steps necessary for performing conventional PCR make it too laborious and prone of contamination with the amplification product, generating false positives results. This problem was solved with the development of the real-time PCR. This method is based on DNA amplification in addition to a fluorescent agent that is detected in the same thermal cycler that the amplification occurs. This method requires no manipulation after amplification and is therefore called a closed system. The main advantages of this homogeneous system are: reducing the time required for the test the possibility of viral load and low risk of contamination with the amplified product (Böhland, 1992). However, the technology currently available for real-time PCR allows detection of only a few viruses in a single test. To solve this limitation, DNA microarray platform are been develop for the detection of several viral agents (Palacios et al., 2007).
This method consists of detecting the viral genome by means of DNA probes, which are fixed on silica plates. The microarray platform has the potential to detect multiple viral agents. The number of viral agents that can be detected with the microarray platform far exceeds any other method currently available hundreds of thousands of probes can be printed on a sheet of 70 x 20 mm to detect a large number of viral agents.

JUSTIFICATION
Several viruses transmitted by rodents and arthropods cause permanent threat to human health; in addition, others new viruses could become emergent viruses. Therefore, there is a great interest in the development of method for early diagnosis of infection with these viruses. Traditional diagnostic methods can detect only one or few viruses in a single assay, turning the final diagnosis very slow and expensive when several assays are needed. The DNA microarray platform has emerged recently as a high-throughput method that can be use to screen up to thousand of pathogens in a single assay, providing a early and cheaper final diagnosis.

GENERAL:
To develop a DNA microarray platform for the detection of viruses transmitted by rodents and arthropods
2) To prepare a control DNA microarray slide by using published probes.
3) To evaluate the RoboArboVirusChip and control slides by using laboratory reference viruses.

Viral reference Strains:
The viral reference strains were available in a repository at the Center for Research in Virology (CPV), Faculty of Medicine of Ribeirao Preto (FMRP), Universityof Sao Paulo. A total 21 known viruses were used in this study (Table 22). tentative or strains with sequences available in GenBank and we obtained total 2144 viral sequences from viruses mentioned above (Table 23). (S-2), VP5 (S-6) and NS3 (S-10) segments. The sequences of the probes obtained were analyzed with the BLAST program (http://www.ncbi.nlm.nih.gov/) and those with >95% identity with the virus in question was selected.
For selection of generic probes, sequences of representative viruses within each genus were aligned. These alignments were used to select generic 20-35-mer probes from conserved genomic regions as mentioned above.

Preparation of RoboArboVirusChip
The slide was drawn using the Agilent eArray program (Https://Earray.Chem.Agilent.Com/Earray/). The slide was designed in such a manner that Agilent added the necessary repetitions of each probe together with the positive and negative controls sequences by default to reach 15,000 probes per array. A slide containing eight arrays with 15,000 probes were designed.

Control slide (GreeneChipVr):
The control slides were used for compare reference viruses with

Preparation of viral stocks
All viral infections were performed by using C6/36 or Vero E6 cell line, which

Confirmation of viral infection by indirect immunofluorescence
The viral infected and normal cells (C6/36 or Vero E6) were harvested and cells washed 3 times with 1XPBS by centrifugation at 1000 rpm for 10 minutes at 4ºC, then cells were re-suspend in 1XPBS and 20µl was dispensed onto immunofluorescent glass slides and allowed to air-dry at room temperature. Cells were fixed onto a slide and place in a coplin jar for 10 minutes containing 100% cold acetone, then the cells were

The cDNA synthesis
The reaction mixture for cDNA synthesis had a total volume 20µl, contained

Visualization of DNA:
An aliquot of 10µl of the amplification products were evaluated by electrophoresis, loaded on 1.8% agarose gel (Pronadisa,Spain) in 40 mM Tris-Acetate-1mM EDTA (TAE) buffer. The bands of DNA were achieved through gel stained with GelRed and visualized with UV light.

Determination of the viral infectivity titer by plaque assay
A total 15 viruses were titrated by Plaque assay (Burleson, 1992). VERO E6 cells

PCR (qRT-PCR)
Viral RNA detection was carried out by real-time RT-PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, USA), as previously described (Dos Santos et al., 2008). Briefly, the 25-µL reaction mixture contained 0.

cDNA synthesis (Reverse Transcription):-
The synthesis of cDNA was performed using a random primer eight nucleotides . The mixture was incubated at 25°C for 10 minutes followed by incubation at 37°C for 2 hours and finally at 85°C for 5 minutes.

The polymerase chain reaction (PCR) amplification
The PCR mixture contained in a ratio of 1:9 primers SIAN (5 'GTA GCA CGT TTC CCA GGT CTC 3'), the latter recognizes a specific region of primer SIA.

Purification of PCR product
PCR products were purified with Wizard SV Gel and PCR Clean-Up System (Progmega, USA) following the manufacturer's recommendations. The PCR product was eluted with 15µl of RNase /DNase free water, before proceeding with the labeling reaction.

Addition of the poly -T tail:
The PCR products were attached to a poly-T tail. To this new product, 10 µl of the modified PCR product was added to 6.5 µl RNase/DNase free water and incubated at 95°C for 2 minutes for opening the double-stranded DNA, the product was then transferred in ice for 2 minutes. Then, 8.5µl of a mixture containing 2.5µl of 10X Tailing buffer, 0.8 mM dTTP, (Genisphere, USA) and 2µl of the enzyme terminal deoxynucleotidyl transferase (Genisphere, USA) were added. The reaction mixture was incubated at 37°C for 30 minutes and subjected immediately to a connection process.

Ligation of 3 DNA capture sequence
The DNA with the poly-T tail (25µl) was heated at 95°C for 10 minutes and immediately transferred in ice for 2 minutes. Subsequently the mixture was added to 5µl link (Cy3) and 2U of T4 DNA ligase in each sample, which was then incubated at 25°C for 30 minutes. In order to stop the reaction, EDTA was added to obtain a final concentration of 300 mM. The mixture was then homogenized and the volume was adjusted with 1X TE buffer to a final volume of 50µl.

Purification of Tagged DNA
The tagged DNA was purified with Wizard SV Gel and PCR Clean-Up System (Progmega, USA) following the manufacturer's recommendations. The PCR product was eluted with 15µl of RNase/DNase free water.

DNA Hybridization
The hybridization buffers (Genisphere, USA) were thawed, heated at 65°C for 10 minutes, homogenized by vortexing and subjected to a brief centrifugation before use. A mix containing 10µl of nuclease free water (Genisphere, USA) and 20µl of 2X hybridization buffer (Genisphere, USA) were added to 10µl of the PCR product. This mixture was heated at 80°C for 10 minutes and then added onto the gasket (Agilent, USA) and subsequently the microarray slide (RoboArboVirusChip) was mounted on the gasket hybridization chamber and incubated overnight at 65°C in a hybridization oven with a rotation of 15 rpm.

First Wash Post cDNA Hybridization
After hybridization, slides were washed three times to remove non-hybridized PCR products. The wash solution (2X SSC, 0.2% SDS,) was first heated to 65°C. The gasket and the RoboArboChip were removed and washed for 15 minutes in a pre-heated buffer (2X SSC, 0.2% SDS). Subsequently, the slide was washed for 15 minutes at room temperature in 2X SSC and finally for 15 minutes with 0.2 X SSC. The slide was then transferred to a 50 ml tube and centrifuged at 1000 rpm for 2 minutes for drying.

Hybridization of 3DNA dendrimers
For this second hybridization, 2,5µl of capture reagent of 3DNA dendrimer were mixed with 20µl of 2X hybridization buffer (Genisphere, USA) and 17.5µl RNase/DNase free water to a final volume of 40µl. The mixture was incubated at 80°C for 10 minutes and then applied to the array. The RoboArboChip was again mounted on the hybridization chamber and incubated at 65°C in a hybridization oven with a rotation of 15 rpm for 4 hours. This was followed by washing the slide in the dark as mentioned above.

Slide scan and data analysis
The slide was scanned at 532 nm using the Axon GenePix 4000B scanner (Molecular Devices, USA) with 10µ pixel resolution. The obtained image was analyzed with software GenePix Pro 7 to obtain data of fluorescence intensity of each point.
Briefly, the GenePix Array List "gal file", containing the list of probes, was open generating the feature grid, which was positioned on the microarray image and then the fluorescence intensity of spots were obtained. The obtained data were analyzed with the program DetectiV (Watson et al., 2007). Two kinds of files were used, one containing the probes ID and intensities and the other containing the probes annotation file, which has information about species, genus and family of each probes. Probe intensity values were log2 transformed. The mean intensity of the negative control was calculated and used to divide the intensity of the probes for normalization. In case a virus is not present in the sample, it is expected the probes value for this virus to be equal to zero. To analyze if the probes intensity was significantly different from zero, a t-test was carried out. Results were first filtered such that groups had a normalized log2 ratio greater than or equal to 1 (a ratio of two to the control) and then sorted by p-value.

Estimated the sensitivity of RoboArboVirusChip and comparison with control slide and qReal-time PCR
The sensitivity of RoboArboVirusChip was compare with control slide. A control slide was designed using sequences available on the slide GreeneChip database Gene Expression Omnibus (GEO) database. The platform selected was GPL10319, which have 45,000 probes. However, the virus species corresponding to each probe is not indicated. For the identification of viral species, each probe was checked by BLAST. To prepare this slide in the same format of eight arrays with 15,000 probes / array, probes corresponding to various controls were removed. Remaining mostly, those corresponding to the virus. In this experiment, we were selected DENV-4 titrated stocks. AxyPrep viral RNA/DNA Miniprep Kit (Axigen, USA) purified total RNA. The RNA were diluted in 10 folds serial dilutions and dilutions 10 -3 to 10 -8 (4.2x10 8 to 4.2x10 1 PFU/mL) were used for both slides. All steps same as mention above follow for microarray experiment.

Detection of mixture of viruses
We used viral RNA of twelve viruses divided in to three pools to evaluate the ability of the RoboArboVirusChip to detect mixture of viruses in a single sample. First pool was mix of viruses represented from three families; BSQV and SLEV (Flaviviridae), PIRYV (Rhabdoviridae) and MAYV (Togaviridae). Second pool was purely belong to flaviviruses: ROCV, SLEV, YFV and DENV-2 and third pool included was the four DENV serotypes: DENV-1, DENV-2, DENV-3 and DENV-4. All viral RNAs derived from cell culture were purified separately and 2.5 µl from each virus were mixed and used for cDNA synthesis. The PCR amplification and microarray experiment were performed as mention above.

Clinical serum samples
Dengue positive (n=4) and negaive (n=40) serum samples from dengue suspected cases confirmed by RT-PCR were included in the study. All these samples were collected during 2006 -2012 from deferent primary health care centers at Ribierao Preto and stored in the serum repository of the Virologoy Laboratory at the FCFRP-USP. This study was approved by the Ethical Committee of the FCFRP-USP (CEP/FCFRP nº 313).

Preparation of viral stocks
A total 15 viruses were propagated in C6/36 mosquito cell line for the preparation of viral stocks, which were used for the validation, Lower limits of detection and pools of the RoboArboVirusChip.

Confirmation of viral infection by indirect immunofluorescence
The infected mosquito C6/36 cells with all viruses were confirmed by indirect immunofluorescence and RT-PCR as illustrated in Figures 2 and 3, respectively.

Determination of viral titer by plaque assay
Viral titer of stocks supernatant was determined by the plaque assay and this supernatant was used for the lower limit of detection of the RoboAboVirusChip (Table   24). specific viral oligonucleotide probes targeting 239 viruses were designed. In addition, we designed a control slide based on the GreeneChipVr probes list deposited in the GEO data base (GPL10319). Both RoboArboVirusChip and GreeneChipVr were prepared in a format of eight arrays per slide.

Implementation of microarray method: Control slide
In the first microarray experiment, we evaluated the performance of the control slide with cDNA and PCR products of four viruses: DENV-2, DENV-3, YFV and BUSV. The cDNA and PCR products were hybridized against the probes contained in the slide arryas. The PCR known products were obtained with primers that amplify a specific a genomic region. The slide was scanned to obtain fluorescence intensity image ( Figure 4). Figure 5 shows the presence of fluorescence in several spots within the arrays, but also the presence of artifact, which might be due to problem during the washing steps. The feature data from the scanned image was extracted and analyzed with the program GenePix Pro7. Spots with artifacts were excluded from the analysis. The visualization of fluorescence intensity and significant analysis was carried out with the DetectiV program. Figures 6-13 show the visualization of spots fluorescence intensity grouped by family, genus and species levels. While Table 22-29 show the significant analysis of probes for each virus target sorted by p-values.        Figure 8. Visualization of probes fluorescence intensity of control slide hybridized with DENV-2 (10 µL cDNA). Data were sorted by family, genus and species levels.      Figure 11. Visualization of probes fluorescence intensity of control slide hybridized with DENV-2 (5 µL cDNA). Data were sorted by family, genus and species levels.  Figure 12. Visualization of probes fluorescence intensity of control slide hybridized with YFV (5 µL cDNA). Data were sorted by family, genus and species levels. The control slide was able to detect DENV-2 and DENV-3, but not YFV neither BUSV. In addition, several cross-hybridization were observed. We did not found any difference between cDNA or PCR products.

Microarray experiments with control and RoboArboVirusChip v1 slides
A second experiment was performed with the control and RoboArboVirusChip slides, including agitation during the washing steps to reduce the production of artifacts.
The following viruses were analyzed with both control and RoboArboVirusChip slides:            Figure 19. Visualization of probes fluorescence intensity of control slide hybridized with ILHV. Data were sorted by family, genus and species levels.       The control slide was able to detect SLEV, YFV, ILHV, DENV-4, DENV-3, and DENV-2, but not OROV neither BUSV. After analysis of the probes included in the control slide, we found that no probes were included for BUSQV. In addition, several probes with cross-hybridization were found in both experiments using this slide. We concluded that this slide is not matching the standard as a control slide; that is why we decided not to use farther in this study.

RoboArboVirusChip second version (V2)
To prepare RoboArboVirusChip V2, first we removed probes belonging to viruses mentioned above. In addition,         The results showed that all analyzed viruses were specifically detected; although cross-hybridization was also observed in this version.

Comparison of cDNA and PCR products as target for the RoboArboVirusChip v2
The virus load found in clinical samples, especially in serum samples are lower than the viral load found in the supernatant of cell cultures. Therefore, the inclusion of virus genomic amplification by PCR would improve the sensitivity of the RoboArboVirusChip v2 when using serum samples. To test this hypothesis, we have compared the sensitivity of RoboArboVirusChip v2 to detect cDNA and one step and two steps RT-PCR products of YFV (2.5x10 11 PFU/mL) and DENV-2 (2.5x10 8 PFU/mL). In this experiment we have used cDNA and a one-step RT-PCR products obtained from virus RNAs diluted 1:10 and 1:1000 (Table 49). The RT-PCR was performed using random primers as described in Material and Methods. The RoboArboVirusChip v2 was able to detect the viruses using PCR products, and the two steps RT-PCR was the better one. Therefore, we decided to use the two steps RT-PCR in the farther next all experiments.

Detection of mixture of viruses by the RoboArboVirusChip v2
Infection with more than one virus is a likely event that can be seen in human, especially for arboviruses. In this experiment, we have evaluated the ability of RoboArboVirusChip to detect mixture of viruses in a single sample, we used two pools of viral RNA of eight flaviviruses, each pool contained four viral RNAs: 1) ROCV, SLEV, YFV and DENV-2; and 2) DENV-1, DENV-2, DENV-3 and DENV-4. The microarray experiment and data analysis were performed as mention above (Table 55). The RoboArboVirusChip v2 was able to specifically detect all viruses within the two pools. However, some probes cross-hybridized with non-target viruses.

Design of the RoboArboVirusChip v3
We found some probes from the following viruses with cross hybridization in the  Table 1).

Detection of mixture of viruses by the RoboArboVirusChip v3
We tested the ability of this version to detect the mixture of viruses in a single sample. Two pools, one containing RNAs of viruses from three families, Togaviridae       The RoboArboVirusChip v3 was able to specifically detect all the viruses within the two pools.

Analysis of clinical samples with the RoboArboVirusChip v3
Serum samples collected form DENV infected (n=4) and uninfected (n=40) patients were used to determine the applicability of the RoboArboVirusChip v3 for detecting virus in serum samples. A pool of four serum samples from uninfected patients were analyzed per array, all of which showed a negative result in the microarray assay (Table 79). While all serum samples of DENV infected patients (DENV-1=1, DENV-2=1, DENV-3, and DENV-4=1), analyzed in independent arrays, were specifically detected (Table 80).

DISCUSSION
The differential diagnosis of infectious disease is a great challenge in clinical medicine, public and veterinary health, especially during epidemic outbreaks. The initial sign and symptoms caused by roboviruses and arboviruses infecting humans are very similar, therefore specific laboratory test are necessary for the final diagnosis. In this study we have developed a microarray platform that can be used in the surveillance of all roboviruses and arboviruses with sequences deposited in the GenBank.
The early identification of viruses during epidemic outbreaks helps the heath authorities to implement the more appropriated disease containment strategies.

Conventional methods of virus identification include immunoassays, PCRs and virus
isolation; however these methods can detect only one or few viruses in a single assay, turning the final diagnosis laborious, time consuming and expensive when several assays are needed (Elnifro et al., 2000). The DNA microarray platform developed in this study can be used to screen up to 412 viruses in a single assay, allowing an early identification of an outbreak causative agent. We have evaluated this platform with 17 reference viruses and all of them were readily detected and identified by specific hybridization to the appropriate oligonucleotides probes. Unfortunately, the genus specific probes were not able to detect the virus at the genus level, suggesting this platform could be not use for new virus discovery.
The RoboArboVirusChip designed in this study contain up to 10 probes for 412 viruses with sequences deposited in the GenBank up to February 2012. However, new probes designed based on new sequences deposited in the GenBank for those viruses with less than 10 probes will be updated and tested in the future. To our knowledge, this is only DNA microarray platform containing probes aiming the specific identification of roboviruses and arboviruse. Although the DNA microarray platform called GreenechipVr was designed to detect all the vertebrate viruses (1710 viruses, including roboviruses and arboviruses) (Palacios, 2007) (3 viruses). Therefore, our platform has a better capability to detect roboviruses and arboviruse than the GreenechipVr. In addition, GreenechipVr includes less than 10 probes for several roboviruses and arboviruses, suggesting that is less sensitive to detect those viruses when compared to the RoboArboVirusChip, which contain at least 10 probes for most of those viruses.
Although our platform is the only one targeting roboviruses and arboviruses, some studies in the literature describe the development of DNA microarray tools containing probes for arboviruses. For example, there are DNA microarray platforms designed to identify: Dengue virus serotypes (Putonti et al., 2006), seven flaviviruses pathogenic for human (Nordström et al., 2005), flaviviruses causing infections of the central nervous system (Korimbocus et al., 2005), and seven European bunyaviruses causing encephalitis or meningo-encephalitis (Hasib et al., 2011). In addition, the ArboChip3.0 was designed to detect 33 flaviviruses pathogenic for human (Grubaugh, Petz, et al., 2013), and then updated to the ArboChip5.1 version to detect 144 RNA viruses transmitted by mosquitoes from four arbovirus genera (Alphavirus, Flavivirus, Orthobunyavirus and Phlebovirus) (Grubaugh, Mcmenamy, et al., 2013). All those studies targeted less number of arboviruses when compared to our RoboArboVirusChip.
In addition, our platform target all pathogenic and non-pathogenic arboviruses for human and livestocks. In some cases, non-pathogenic viruses easily can turn emergent viruses.
For example, Rio Mamore virus was previously considered non-pathogenic for human but recently fatal Hantavirus Pulmonary Syndrome (HPS) cases have been reported in Peru, French Guiana and Brazilian Amazon regions associated with this virus (Casapía et al., 2012;Matheus et al., 2012;De Oliveira et al., 2014).
Other sutides in the literature describe the development of DNA microarray tools targeting roboviruses. In one such study, 47 probes targeting only Lassa virus were designed (Olschläger and Günther, 2012 (Bhatt et al., 2013).
Infection with any of the four viral serotypes (DENV-1 to -4) can be asymptomatic or lead to disease with clinical symptoms ranging from an undifferentiated and self-limiting fever to a severe dengue disease, which are mostly characterized by plasma leakage and hypovolemic shock that in some cases can be fatal (Who, 2009). Secondary infection with a second virus serotype is a risk factor for the development of severe form of the disease (Halstead, 1970). Therefore, epidemiological surveillance is of great interest to the implement control of the spread of new introduced serotypes. The RoboArboVirusChip was able to clearly differentiate the four DENV serotypes, suggesting that it can be used in the surveillance of DENV serotypes circulation.
Co-infection with more than one virus type is a common event for arboviruses in hyper-endemic regions. Co-infection with more than one DENV serotypes and DENV/Chikungunya virus have been described in the literature (Figueiredo et al., 2011;Sulca et al., 2013;Cnops et al., 2014;Parreira et al., 2014). [PIRYV]). Therefore, the RoboArboVirusChip could be used to identify co-infection with different viruses.
The RoboArboVirusChip was able to detect a minimum of 21-67 RNA copies/mL of DENV-1 to -4, which is similar to the sensitivity described by other DNA microarray platforms (Grubaugh, Mcmenamy, et al., 2013;Huang et al., 2013), and to the sensitivity of a real-time RT-PCR describes by our group and which has been used to detect DENV in serum and saliva samples (Dos Santos et al., 2008;Poloni et al., 2010;Muller et al., 2014). These data suggest that the RoboArboVirusChip could be use to analyze clinical samples, which was confirmed by the detection of DENV in four clinical samples.
Finally, we have searched for roboviruses and other arboviruses in serum of 40 DENV negative patients but no virus was detected. May be other viruses different from roboviruses and arboviruses or even other pathogens, mimicking dengue symptoms, were the causative agents of the diseases in those patients, that is why we did not detected any virus with the RoboArboVirusChip. The dengue negative cases were collected during an epidemic outbreak in 2006 in Ribeirao Preto. The peak of dengue occurred between April and June, which coincided with the appearance of influenza cases that can be confused with dengue (CVE, 2006). In this sense, in a study performed in Puerto Rico in 2009, 31 of 289 dengue suspected cases was confirmed with dengue and most of the other were infected with influenza virus (n=136) (Zarocostas, 2009).
Another study performed in Nicaragua in 2005-2009, analyzing 124 dengue suspected cases but negative by DNA microarray and new generation sequencing methods, detected mainly herpesviruses but not arbovirus or robovirus (Yozwiak et al., 2012).
Although there is circulation of other arboviruses during dengue outbreaks, this event seems to be not common (Mondini et al., 2007;Terzian et al., 2011).


extfieldsofstudy:
Biology
Medicine
Computer Science

s2fieldsofstudy:
Biology
Computer Science

text:
dbPSP: a curated database for protein phosphorylation sites in prokaryotes

As one of the most important post-translational modifications, phosphorylation is highly involved in almost all of biological processes through temporally and spatially modifying substrate proteins. Recently, phosphorylation in prokaryotes attracted much attention for its critical roles in various cellular processes such as signal transduction. Thus, an integrative data resource of the prokaryotic phosphorylation will be useful for further analysis. In this study, we presented a curated database of phosphorylation sites in prokaryotes (dbPSP, Database URL: http://dbpsp.biocuckoo.org) for 96 prokaryotic organisms, which belong to 11 phyla in two domains including bacteria and archaea. From the scientific literature, we manually collected experimentally identified phosphorylation sites on seven types of residues, including serine, threonine, tyrosine, aspartic acid, histidine, cysteine and arginine. In total, the dbPSP database contains 7391 phosphorylation sites in 3750 prokaryotic proteins. With the dataset, the sequence preferences of the phosphorylation sites and functional annotations of the phosphoproteins were analyzed, while the results shows that there were obvious differences among the phosphorylation in bacteria, archaea and eukaryotes. All the phosphorylation sites were annotated with original references and other descriptions in the database, which could be easily accessed through user-friendly website interface including various search and browse options. Taken together, the dbPSP database provides a comprehensive data resource for further studies of protein phosphorylation in prokaryotes. Database URL: http://dbpsp.biocuckoo.org

Introduction
As one of the most ubiquitous and important protein posttranslational modifications (PTMs), the reversible protein phosphorylation was involved in almost all biological processes (1,2). Phosphorylation was catalysed by a protein kinase through transferring a phosphate moiety from adenosine triphosphates (ATPs) to the acceptor residue in the substrate (2). Phosphorylation in eukaryotes was extensively studied during the past decades since 1932 (3), and most of the identified phosphorylation acceptor residues were serine (Ser), threonines (Thr) and tyrosines (Tyr) (4,5). Protein phosphorylation had been regarded as a biological process exclusively in eukaryotes until the first evidence of the phosphorylation in bacteria, which was identified in isocitrate dehydrogenase from Escherichia coil by Garnak and Reeves (6) in 1979, while protein phosphorylation in archaea was reported in the extreme halophilic archaeon Halobacterium salinarum by Spudich and Stoeckenius (7) in 1980. Subsequently, phosphorylation in prokaryotes were extended to other residues such as histidine (His) (8), aspartic acid (Asp) (9) and cysteine (Cys) (10). It was found that His/Asp phosphorylation plays critical roles in various cellular processes such as two-component system based signaling transduction (11), while Ser/Thr/Tyr phosphorylation in prokaryotes attracted more and more attention recently (12).
Recently, rapid progresses in high-throughput (HTP) mass spectrometry based proteomic technologies greatly advanced the identification of phosphorylation sites (13,14). Numerous studies have been carried out to profile the phosphorylation events and advance the phosphoproteome techniques to a state-of-the-art stage (13,14). For example, recently Sharma et al. (15) (20) profiled 121 arginine (Arg) phosphorylation sites in 87 proteins from B. subtilis in vivo. These leading studies made great contributions to expanding the understanding of molecular mechanisms and functional roles for phosphorylation in prokaryotes.
As the discoveries accumulated, the collection and maintenance of the identified phosphorylation sites became an urgent issue to be solved. Previously, a number of comprehensive databases for phosphorylation sites were constructed (21), while most of which were focused on eukaryotes. Databases such as Phosphorylation Site Database (22), SysPTM 2.0 (23), PHOSIDA (24), dbPTM 3.0 (25) and UniProt (26) have collected the prokaryotic phosphorylation sites. However, only a limited proportion of the identified prokaryotic phosphoproteins and sites were covered. In this study, we developed and presented the database of phosphorylation sites in prokaryotes (dbPSP). Totally, 7391 phosphorylation sites on seven types of phosphorylated residues including serine (Ser), threonine (Thr), tyrosine (Tyr), aspartic acid (Asp), histidine (His), cysteine (Cys) and arginine (Arg) in 3750 prokaryotic proteins from 96 organisms in 11 phyla were manually curated from the published literature. On the basis of the datasets, we analysed the sequence preferences of the phosphorylation sites and functional annotations of the phosphoproteins among eukaryotes, bacteria and archaea, while the results show that there were obvious differences among phosphorylation in the three domains of life. Taken together, the dbPSP database could serve as a comprehensive data resource for further studies of protein phosphorylation in prokaryotes.

Construction and content
The construction of database dbPSP was summarized as a diagram in Figure 1A. We searched PubMed (http://www. ncbi.nlm.nih.gov/pubmed) with keywords including 'bacteria phosphorylation', 'archaea phosphorylation' and 'archaebacteria phosphorylation' (1 March 2014). All the retrieved 16 658 articles were manually reviewed and checked by domain experts to collect the experimentally identified prokaryotic phosphorylation sites. The curated phosphorylated residues were explicitly mapped to UniProt proteomes sequences (Release 2014_06) (26), while the annotations and cross references of phosphoproteins were also retrieved from UniProt database and integrated into the database. The references which identified phosphorylation sites were also provided in the dbPSP database.
Besides manual curation from literatures, the prokaryotic phosphorylation sites in public databases were also collected. From databases including PHOSPHORYLATION SITE DATABASE (22), SysPTM 2.0 (23), PHOSIDA (24), dbPTM 3.0 (25) and UniProt (26), 1400, 348, 305, 186 and 176 phosphorylation sites were retrieved, respectively (Table 1). These datasets were cross-checked with our manually collected dataset and integrated into dbPSP database. In total, 7391 non-redundant phosphorylation sites among seven types of residues were found in 3750 substrates from 11 phyla were provided in the database, which present a comprehensive data resource for prokaryotic phosphorylation. In total, 7171 and 209 sites were identified by HTP and low-throughput studies, respectively. Various annotations such as protein names, gene names, keywords, functional descriptions and sequence annotations from the UniProt database (26) were retrieved to annotate the collected phosphoproteins.
With the abundant phosphorylation sites, the distributions for different residue types and species were summarized, while the results were presented in Figure 2. It was observed that phosphorylated serine, tyrosine, and threonine occupied 36.65%, 29.59% and 29.41% of modified residues, respectively ( Figure 2A). The known phosphorylation sites on aspartic acid, histidine, cysteine, and arginine were limited and need further studies to explore (Figure 2A). In the dbPSP database, the phosphorylation sites were collected from 96 prokaryotic organisms in 11 phyla. The distribution of species at the phyla level was presented in Figure 2B. The phylum Crenarchaeota and Proteobacteria have the most substrates with the most proportions of 39.43% and 24.10%, respectively ( Figure 2B), while phosphorylation sites in Thermotogae and Chlamydiae/Verrucomicrobia group were limited.

Usage
To provide convenient usage, the database was developed in a user-friendly manner, while browse and search options were provided to access the information of prokaryotic phosphorylation sites in the database. Since the phosphorylation sites are identified in different residues and various species, two browse options including 'Browse by residue types' ( Figure 3A) and 'Browse by phyla' (Figure 3B) were developed in the database. Here, the serine hydroxymethyltransferase in E. coli (strain K12) was selected as an example to describe the usage of browse and search options. In the 'Browse by residue types', the phosphorylated residues are shown in diagrams ( Figure 3A). By clicking the diagram of tyrosine phosphorylation, the distribution of tyrosine phosphorylated phosphoproteins in various organisms is returned ( Figure 3A). Then the tyrosine phosphorylated phosphoproteins in Proteobacteria could be listed in a tabular format with 'UniProt Accession', 'Name/ Alias' by clicking the link of 'Proteobacteria' (Figure 3C). In the option of 'Browse by phyla' (Figure 3B), the 11 phyla in two domains including bacteria and archaea are listed for users to browse the phosphoproteins ( Figure 3C). Through clicking on the figure of 'Proteobacteria', the distribution of phosphoproteins for different modification residue types is shown ( Figure 3C). Then the list of tyrosine phosphorylated phosphoproteins could be retrieved after clicking the link 'Tyrosine', while the detailed information for specific phosphoproteins is provided by clicking protein entry ( Figure 3D).
Besides browse options, the web interface provides four search options including simple search ( Figure 4A     the keyword 'glyA' in the 'Gene Name' area, the results will be generated in a tabular format with 'UniProt Accession', 'Name/Alias' ( Figure 4A). Alternatively, users can use the 'Advanced Search' with three search terms specified in different areas and combined with three operators of 'and', 'or' and 'exclude', which could reduce the potential hits and provide highly related results ( Figure 4B). Furthermore, 'Batch Search' is designed for retrieving multiple phosphoproteins with a list of keywords ( Figure 4C). Finally, 'Blast Search' is implemented in the database to find homologous proteins with a protein sequence in Fasta Format. The NCBI BLAST package (27) is employed search related sequences ( Figure 4D).

Discussion
As one of most important protein PTMs, prokaryotic protein phosphorylation was critical for numerous cellular processes through modification of various types of residues (28,29). After the first discovery of phosphorylation events in prokaryotes, a large number of substrates and sites have been identified to dissect the molecular mechanisms and functional roles of phosphorylation. Although previously various databases were developed to maintain the known phosphorylation sites, most of these databases were focused on eukaryotes. In this regard, an integrated and comprehensive database for prokaryotic phosphorylation is urgently needed. In this study, we presented a manually curated and comprehensive database of dbPSP, which aimed to maintain known phosphorylation sites from various organisms in prokaryotes. Previously, numerous studies on eukaryotes indicated that phosphorylation was mediated by linear motifs (5,30). With the dataset collected in this study, we analysed the sequence preferences and motifs for Ser/Thr phosphorylation in bacteria ( Figure 5A), archaea ( Figure 5B) and eukaryotes ( Figure 5C), while 10 092 eukaryotic Ser/Thr phosphorylation sites from phospho.ELM database were employed for comparison (31). As the sequence preferences illustrated by WebLogo (32), alanine and lysine has high frequencies around the phosphorylation sites in bacteria ( Figure 5A) and archaea ( Figure 5B), respectively, there were abundant serine and glutamic acid around the phosphorylated residues ( Figure 5C). To further dissect the differences, pLogo was employed to pairwisely compare the sequence preferences ( Figure 5D-F) (33). It was observed that positively charged residues including arginine and lysine were enriched around phosphorylated Ser/Thr in archaea than bacteria ( Figure 5D) and eukaryotes ( Figure 5F), while proline were over-presented in þ1 position of the phosphorylation sites in eukaryotes than bacteria ( Figure 5E) and archaea ( Figure 5F). Taken together, obvious differences were observed among the sequence preferences of phosphorylation sites in the three domains of organisms.
Furthermore, with the comprehensive phosphorylation datasets in the dbPSP database, we tried to analyze the functional annotations of phosphoproteins in prokaryotes with the examples of E. coli (strain K12) (E. Coli k12) and Sulfolobus acidocaldarius, which contained the most identified phosphoproteins and sites in bacteria and archaea, respectively. The gene ontology (GO) (31 March 2012) association files were downloaded from the The Gene Ontology Annotation (GOA) database at the European Bioinformatics Institute (EBI) (http://www.ebi.ac.uk/goa) (34) and the complete proteomes were retrieved from AmiPro Database (26). With hypergeometric distribution (35), we statistically analysed the enriched biological processes, molecular functions and cellular components for phosphoproteins in E. Coli k12 ( Figure 6A, Pvalue < 10 À9 ) and S. acidocaldarius ( Figure 6B, P-value < 10 À2 ). It was observed that translation (GO:0006412) was the intensively enriched biological process in phosphoproteins from E. Coli k12 ( Figure 6A), while translationrelated annotations of tRNA aminoacylation for protein translation (GO:0006418) and regulation of translational fidelity (GO:0006450) were also over-presented in phosphoproteins from S. acidocaldarius ( Figure 6). For molecular functions, phosphoproteins from E. Coli k12 and S. acidocaldarius both enriched annotations of nucleotide binding (GO:0000166) ( Figure 6). Furthermore, phosphoproteins from E. Coli k12 over-presented other molecular functions including structural constituent of ribosome (GO:0003735), rRNA binding (GO:0019843), protein binding (GO:0005515), magnesium ion binding (GO:0000287), identical protein binding (GO:0042802) and RNA binding (GO:0003723) ( Figure 6A), while phosphoproteins from S. acidocaldarius enriched aminoacyl-tRNA ligase activity (GO:0004812), ligase activity (GO:0016874), aminoacyl-tRNA editing activity (GO:0002161), nucleic acid binding (GO:0003676) and ATP binding (GO:0005524) ( Figure 6B). In addition, a handful of cellular components were over-presented in phosphoproteins from E. Coli k12 ( Figure 6A), while no enrichment was observed in for S. acidocaldarius.
Taken together, in this study the dbPSP database was developed to maintain the experimentally identified phosphorylation sites in prokaryotes. We anticipated that such database could provide a useful resource for further studies and understanding of phosphorylation in prokaryotes. Conflict of interest. None declared.