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The human parechovirus belongs to the Picornaviridae family. It is one of the most important viral pathogen which is capable of causing many infectious diseases in small children. There are totally 14 types of human parechoviruses present in the environment. Since it s known to cause several infectious diseases it is important to study the nature and analyse the sequence of the virus through various molecular biology techniques. This paper gives the basic information about the detection of the virus from a clinical sample with the help of various molecular techniques. From the given sample the RNA was isolated and analysed by agarose gel electrophoresis. RT-PCR was carried out and the product was further studied to confirm the origin of the pathogen in the given sample. Analysis showed the presence of the human parechovirus (HPeV) in the sample which was said to cause diarrhoea in small children. To identify the sequence of the inserted DNA TA cloning was performed .The DNA was introduced into the competent E.Coli cells along with the pGEM®-T easy vector and Taq polymerase. The purified RT-PCR product and EcoRI digested DNA was isolated from the blue and white colonies respectively and agarose gel electrophoresis was carried out to identify the size of the vector and the inserted DNA sequence. The sequence alignment was done and the genetic tree was mapped.
Parechovirus are a group of viral pathogens which belong to the family Picornaviridae. This family consist of five genera, enteroviruses, rhinoviruses, hepatoviruses, foot and mouth disease virus and poliovirus. Nucleotide sequencing results showed that these viruses are genetically distinct from other viruses from the same family. Because of this reason these viruses have been classified into a separate genus in the family. There are 14 types of human Parechovirus: HPeV 1 to HPeV 14. The sequencing is done in the region of virus encoding one of the structural proteins VP1. The human Parechovirus causes mild gastrointestinal or respiratory illness. It usually affects small children in the age group of 2-5 years. This virus also causes myocarditis and encephalitis.
The Parechovirus is a single standard RNA genome, 7100-8500 nucleotides long. It is packaged in to a icosahedral capcid made of each capsid protein VP1 through VP4. It contains a viral at the 5' end of the RNA instead of the methylated nucleotide cap structure. The virus has four distinct domains: a 5' untranslated region (5' UTR), leading to a single open reading frame. A3' UTR is present in the downstream of the open reading frame and also consists of a poly (A) tract. In RT-PCR using Parechovirus-specific primers followed by sequencing offers a quick method of detecting par echoviruses in clinical material, and identifying which type is present. The type of unknown virus can be found out in a sample containing HPEV where its VP1 encoding sequence is similar to the viruses of the same type.
Materials and Methods:
Isolation of RNA from the given sample
The HPeV RNA was isolated using a commercial kit- QIAamp viral RNA mini kit which is produced by QIAgen and all the procedures were performed as described in Stanway, 2009.The isolated RNA was analysed by agarose gel electrophoresis by taking an electronic copy of the gel with the help of the gel documentation instrument (stanway 2009).since the amount of the viral RNA was very small ,it is difficult to identify the RNA in the gel, but the cellular RNA was clearly visible ,indicated the successful isolation of the RNA.
RT-PCR and agarose gel analysis
A DNA sample was isolated and then it was amplified with the help of RT-PCR for the detection of the pathogen. RT-PCR products were analysed by agarose gel electrophoresis. The gel was analysed for the detection of the virus.
TA cloning of RT-PCR products
Cloning allows the specific region of the DNA to be produce in large quantities. It makes use of a DNA fragment of interest (the given sample) and a vector (pGEM®-T easy).this vector is capable of replicating inside a cell. The cell which is used to increase the amount of the inserted DNA is usually an E.Coli. the vector is obtained from promega. http://www.promega.com/tbs/. The PCR product is purified and then it was ligated with the pGEM®-T easy vector. The ligation mixture is then transferred into a compete cell plate which contains ampicillin and Xgal.
Plasmid DNA isolation
This was carried out with the help of QIAgen QIAprep spin miniprep kit. The procedure was followed step by step as mentioned in stanway, 2009.
Restriction endonuclease digestion
The DNA was digested with the restriction enzyme EcoRI. The inserted DNA was released and the size was measured from the gel keeping the standard size marker obtained from the 1kb ladder.
DNA sequence analysis
Finally the sequence of the inserted DNA was analysed using basic bioinformatics tools. As a result the type of the HPeV virus was found
The RNA was isolated from given faecal sample with the help of QIAgen virus RNA isolation kit ( http://www.qiagen.com/ ).The agarose gel electrophoresis was carried out and results showed that the RNA isolation was successful. The RNA bands of different sizes were observed as a smear on the gel. The size of the nucleic acid was estimated by comparing with the standards of known size.
Figure1: Agarose gel electrophoresis showing Parechovirus in the isolated RNA.
The isolated RNA was then converted into DNA by the reverse transcriptase mechanism. RT-PCR was carried out using single tube kit. A positive result was confirmed by the presence of HPeV with a band of the expected size (948bp).
Figure 2: RT-PCR agarose gel electrophoresis.
The RT-PCR product and the pGEM®-T easy vector were ligated together. It is then introduce in to a Ecoli cell to get large amount of vector. The Ecoli cells were then disseminated on selective agar plates. We failed to get result containing both the blue and white colonies instead only white colonies were present. The absence of the white colonies was due to a technical error in the bacterium used. In the previously done experiments A mixture of blue and white colonies were seen on the E plate (ligating reaction containing vector and RT-PCR DNA), the C-plate was observed only with blue colonies this is because of there was no insert DNA to ligate in the vector and the W-plate had no colonies since there was no DNA added to the plate.
The plasmid DNA was then isolated from the white colonies using a alkaline lysis procedure. After the plasmid DNA isolation the plasmid was digested with restriction enzyme EcoR1. From the map of pGEM®-T Easy, the EcoR1 restriction sites are observed on the either side of TA cloning site. Digestion of pGEM®-T with EcoR1 showed a band referring to the inserted DNA on the agarose gel and the gel also showed another band corresponding to the vector in lane 2 and 3 respectively (fig 3).
The isolated DNA from the white colony was then sequenced in both orientations using gene service. Using Basic Local Alignment Search Tool (BLAST), the orientation of T7 and SP6 sequence were determined. Later the indirect sequence was modified and compared with all eight sequences of HPeV( figure 4). The results showed that, the HPeV in the original fecal sample belong to HPeV1.
Figure 5 : comparison of HPeV with all eight serotype.
The sequence analysis which was carried out on the inserted DNA revealed that the original faecal sample contained a Human Parechovirus type 1.HPeV was confirmed through the RT-PCR technique. This technique is carried out with the help of HPeV specific primers, OL993 and OL994.the primer has the capacity to recognize a semi conserved sequence present in VP1.as a result a positive band of the DNA was observed in the agarose gel.
The vector which is used for the TA cloning is a high copy number vector called as pGEM®-T Easy .the vector consists of T7 and SP6 RNA polymerase promoter regions. T7 RNA polymerase promoter is a self dependent RNA polymerase. It is known for its high specificity for the associated promoter sequence. This promoter is generally used for the generation of the template for in vitro translation and also used for generating the RNA processing substrate.
The vector pGEM®-T Easy contains a recognition sites for EcoRI and NotI flanking the insertion site. Therefore with just a single restriction enzyme the desired inserted DNA can be removed. The T7 and the SP6 promoter region has a multiple cloning region which is present in the α-peptide coding region of the enzyme β-galactosidase.this allows the direct identification of the recombinant clones by colour screening (blue/white colonies) on the media plate, through the process of insertion inactivation of the α-peptide.the
The blue/white colony screening depends on the disruption of the LacZ gene. The LacZ gene consists of a multiple cloning site (MCS) and an enzyme β-galactosidase. The MCS can be cleaved with different restriction enzymes. So the foreign DNA can be inserted into the LacZ α gene thus there is no production of functional β-galactosidase.the hydrolysis of the colourless Xgal in the presence of the enzyme β-galactosidase causes the appearance of the blue colour colonies in the plate (colonies with vector, no insert).the appearance of the white colonies indicate the insertion of the foreign DNA into the plasmid. As the growth medium contains an antibiotic Ampicillin, it suppresses the growth of the bacterial colonies. Since the vector pGEM®-T Easy contains an Ampicillin resistant gene in it, it allows for the successful growth of the transformed bacteria to survive in spite of the antibiotic. However insertion of the PCR product inactivates the β-galactosidase which results in the formation of the white colonies instead of blue colour colonies.
Even though the provided sample confirmed the presence of the viral RNA, the E plate had no white colonies (2009 data) ,this may be due to the loss of the DNA during the purification process. The final agarose gel electrophoresis (figure 3 ) showed a band of the digested DNA vector and the inserted DNA fragment . the lane 1 shows the DNA marker, lane 2 and 3 show two bands (isolation from white colonies) .the above band shows the vector of approximate size of around 3000 kbp and the second band shows the inserted DNS of the desired size 948 bp.lane 5 shows a vector which is isolated from the blue colony and the lane 4 shows only the vector which seem to have appeared as a very faint band . this may be due to some error in the digestion process. It may be also caused if the white colony received only a minute piece of DNA to be ligated into the vector. This may result in the blocking of the expression of the DNA.
The type of the virus was identified on the basis of sequence of the virus which encodes one of the structural proteins VP1.the sequencing of the sample was done with the help of several bioinformatics tools such as BLAST, ClustalW, Chromas, etc.the inserted DNA was sequenced from both the ends in two different sequence reactions. When the RT-PCR product was cloned into the vector, the insert can be of either orientation to know which sequence is in the direct orientation and which sequence is in the reverse orientation, BLAST program was performed.
to check whether a particular sequence is in direct or in reverse orientation we can make use of a bioinformatics tool called as orientation checker.
In our case the SP6 sequence was in reverse orientation to convert into the direct orientation we made use of the reverse transcriptase tool. (http://bioinformatics.org/sms/rev_comp.html). Both the sequences were then compared using a program ClustalW.the sequence was then edited to the necessary size and then it was analysed with the different VP1 sequence of the eight HPeV types. A genetic tree was then produced using jalview. The nearest distance of the DNA with our original sequence was found to be HPev1. (Figure 5) the HPeV1 has a P1 amino acid sequence of completely sequenced HPeV. This result concluded that the cause of diarrhoeas in small children was due to the presence of the HPeV1.this result led to the direct detection of the disease.
Figure 5: genetic tree showing the average distance from the virus present in the original sample.
To improve the knowledge in the field of the HPeV more research can be carried out in all the other virus types. This may give us a wide range of selection of methods in the field of pathogen detections. The HPev1 which was detected from the given faecal sample was found to have link with many other disease. It has the capability of causing disruption to the replication of the virus. It is known to have a 2 stem loop structure and a pseudo knot .damage cause to any of these structures may hinder the process of viral replication. Advance research in the particular virus HPeV1 showed that they resembled the other type of virus in many ways. This particular analysis of the virus has given us a insight into the family of the HPeV and has broadened the prospective for us to probe the other molecular techniques.