View of Mutation of Torque Teno Virus among Women with Urinary Tract Infection in Diyala Governorate

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Mutation of Torque Teno Virus among Women with Urinary Tract Infection in Diyala Governorate

Nedhal Mahmood Kaleefah¹ andAreejAtiyah Hussein²

1Department of Microbiology, College of Medicine, University of Diyala, Iraq

2 PhD, Department of Microbiology, College of Medicine, University of Diyala, Iraq

*Corresponding Author:Nedhal Mahmood Kaleefah, Email: [email protected].

ABSTRACT

Background: Torque Teno Virus (TTV) shows a high prevalence in infected and healthy individuals globally.

The generation of many TTV variants is driven by the high mutation rate, which is closer to the RNA virus. The cause and mechanism of great genomic variation are unknown as ORF1 is the most extended encoded region in the genome of TTV with the highest rate of genetic mutations.Objectives:To determine the infection rateof Torque Teno Virus and types of mutations in women with urinary tract infections in Diyala Governorate.Methods:A cross-sectional study was performed between the 20^thof September 2020 to the 20^th of January 2021 for 100 women diagnosed with urinary tract infections (50 pregnant and 50 non-pregnant women).

Their ages ranged from 17-77 years old of women admitted to Al-Batool Teaching Hospital for Maternity and Children, Women Emergency Unit and Unit of Urological Consultation at Baqubah Teaching Hospital in Diyala Governorate. The DNA extraction,was performed on the collected urine samples and followed with genes amplification with specific primers using a nested polymerase chain reaction and finally the phylogenic analysis was done to determine the genotype of TTV.Results:Torque TenoVirus DNA was detected in (8%). About 12.50% of positive women for TTV had UTI history during childhood, while 7(87.50%) without UTI history during childhood. Three of the positive samples (37.50%) who were positive to TTV had UTI before pregnancy compared to the rest 5(62.50%) of TTV positive infection in women without UTI infection before pregnancy.

Statistical analysis revealed significant differences during childhood while non-significant before pregnancy.Genetically there are many substitution mutations were shown in local isolates such as isolate (1) in five positionsC\G,G\A,C\T,C\G, A\G, isolate (2)in three positionsG\A,T\A,A\G, isolate (3) did not show any mutation, isolate (4)in four positionsT\A,A\C,A\G,T\A, isolate (5) in three positions A\C,T\A,A\G, isolate (6)in three positions G\A,C\G,G\C, isolate (7) in two positions A\G,C\G and isolate (8) in three positions A\G,A\G,G\A.Conclusion:Transversion and transition mutation of amino acid occurred in most local isolates in two, three or five positions after alignment with some GenBank isolates, except isolate number three which was showed 100% similarity with Egyptian isolate.

Key Words: Torque Teno Virus,Urinary Tract Infections, PCR-Sequencing,Mutation, phylogenetic tree.

INTRODUCTION

Pregnancy can enhance the risk of urinary tract infections from the end of the first trimester through mechanical compression and progesterone release (1)The hormonal and mechanical changes in the urinary tract make women more vulnerable, starting from 6 weeks through 24 weeks (2). Higher maternal complications among teenage pregnant women represent an important health problem with social and medical impact (3). Very old patients with urinary tract infections had a higher risk of developing urosepsis shock than younger patients (4).

Risk factors for recurrent urinary tract infections include frequent sexual intercourse (three or more acts of intercourse per week), spermicide, and a new sex partner. A history of the first urinary tract infection before the age of 15 years increases one’s risk of recurrent urinary tract infections(5).

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Torque Teno Viruses are small (~30 nm in diameter), icosahedral virions that lack an envelope with a circular, negative-sense, single-stranded DNA genome (6). Detection of Torque Teno Virus infection in 11% of healthy individuals suggests transmission through routes other than blood and injection (e.g., faecal-oral route), causing food and water contamination (7).

The virus is undergoing divergent evolution as very high sequence diversity was found in the ORF1 gene (8). A comprehensive study of codon usage analysis of the ORF1, which encodes the viral capsid protein, was undertaken for the first time to reveal its evolutionary history (9). The capsid is assembled from 60 copies of the capsid protein, viral protein (VP1 and VP2) is a phosphatase and VP3 (also called apoptin), which induces apoptosis only in cancer cells (10).

The origin of this extreme diversity is unclear but may be due in part to the known high mutation rates of single-stranded DNA viruses (11). Multiple processes determine viral mutation rates, including polymerase intrinsic fidelity, replication mode, 3′ exonuclease activity, spontaneous nucleic acid damage, access to post-replicative repair, editing by host-encoded deaminases, imbalances in nucleotide pools, template sequence context, and template structure (12). Genetic variation is a necessity of all biological systems. Viruses use all known mechanisms of variation; mutation, several forms of recombination, and segment reassortment in the case of viruses with a segmented genome (13).

To our knowledge there is no molecular study about this virus in Diyala Governorate so the present study design to determine the infection rate of Torque Teno Virus and types of mutations in women with urinary tract infections, using the nested conventional polymerase reaction technique in Diyala Governorate.

MATERIAL AND METHODS

The study was performed between the 20^thof September 2020 to the 20^th of January 2021 for 100 women diagnosed with urinary tract infections (50 pregnant and 50 non-pregnant women). Their ages ranged from 17-77 years of women admitted to Al-Batool Teaching Hospital for Maternity and Children, Women's Emergency Unit and Unit of Urological Consultation at Baqubah Teaching Hospital in Diyala Governorate.

Microscopic Examination of Urine and Cultivation positive Samples

The general flow test of the urine samples in the present study was conducted according to the presence of purulent cells, blood cells, and other approved indicators that indicate inflammation and samples containing more than ten purulent cells (14(.Routine urine culture microbiology was performed on Blood agar and MacConkey agar (15).

Molecular Detectionof Torque Teno Virus DNA Extraction of Torque Teno Virus DNA

ZR viral DNA extraction kits (Cat. No. D3015 and D3016, Epigenetics - USA) are used to extract viral DNA directlyfrom a the collected urine samples.

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Torque Teno Virus Genome Amplification by Nested PCR.

Primers for Torque Teno Virus

Two pairs of primers were used to amplify a TTV gene fragment, NG059, NG061 (16), as summarized in Table 1 and 2.

Table 1: The Specific Primer NG059 of the Gene.

Primer * Sequence Tm (ᵒC) GC (%) Product Size

Forward 5'- ACAGACAGAGGAGAAGGCAACATG- 3' 58.8 50 271

base pair

Reverse 5'-CTGGCATTTTACCATTTCCAAAGTT- 3' 54.7 36

*Integrated DNA technology, Canada.

Table 2: The Specific Primer NG061 of the Gene.

Primer* Sequence Tm (ᵒC) GC (%) Product Size

Forward 5'-GGCAACATGYTRTGGATAGACTGG-3' 56.1 45.8 271

base pair

Reverse 5'-CTGGCATTTTACCATTTCCAAAGTT-3' 51.8 36.4

*Integrated DNA technology, Canada.

Principle of Nested PCR

A standard PCR has been performed to amplify the N-22 region (NG059, NG061 gene) (17). An amplified pitch (271bp) with the aid of reverse and forward primer and a PCR reaction mix was performed at (25μl) total volumes, as shown in Table 3.

Table 3: Components of Nested Polymerase Chain Reaction.

No. Components Final Concentration

1 Taq PCR PreMix 12.5µl

2 Forward primer 10 picomols/µl (1µl)

3 Reverse primer 10 picomols/µl (1µl)

4 PCR Product (PCR 1) external 1.5µl

5 Deionized water 9 µl

6 Final volume 25µl

Thermocycler was programmed to amplify the genome by MultiGeneOptiMax thermal cycler gradient, as shown in Tables 4.

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Table 4: The Thermal Cycling Condition for DNA Amplification Specific Primer NG059 and NG061 of the Gene (First and second Run).

Agarose Gel

Electrophoresis

Agarose Gel was prepare according to (18). Three µl of the processor loading buffer have been mixed with 5 µl of the supposed DNA to be electrophoresis (loading dye). After the mixing process, the process of loadingis to the holes of the gel.

DNA Sequencing and Phylogenetic Analysis

The PCR products sequencing was carried out by sending the PCR DNA products with their specific primers in a freezer bag to Macrogen company in Korea, https://dna.macrogen.com. The sequencing study was designed between the standard genes and local isolates in the basic local alignment search tool (BLAST), available at the National Center of Biotechnology Information (NCBI) online http://www.ncbi.nlm.nih.gov. The evolutionary analysis was conducted using MEGA6 (Molecular Evolutionary Genetics Analysis software version 6 software).

RESULTS

Detection of TTV

In this study only 8% of TTV virus infections was detected among (50 pregnant and 50 non-pregnant women) with urinary tract infection from different regions in Diyala Governorate. Whaere as the rest samples show negativeresult (92%), as shown in Figure 1.

No. Steps Temperature Time Cycles

1 Pre-Denaturation 94 ℃ 5 Minutes 1 Cycle

2 Denaturation 94 ℃ 1 Second

50 Cycles

3 Annealing 52 ℃ 1 Second

4 Extension 72 ℃ 1 Second

5 Final extension 72 ℃ 7 Minutes 1 Cycle

6 Holding 4℃ -

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Figure 1: Gel Electrophoresis of The Second-Round PCR, Amplification for (NG061). M: DNA ladder (100-1000 plus), 3 and 4 samples were positive while other samples were negative. The product (271 bp) was electrophoresis on 1% agarose at 7 volt/cm². TBE buffer 1X for 1:30 hours, stained by red safe stain and illustrated under UV light.

Distribution of Torque Teno Virus Infection According to the History of Urinary Tract Infection among the Study Population

Concerning the history of urinary tract infection among the study population, Table (6) showed one case (12.50%) positive to TTV that UTI history during childhood, while 7(87.50%) without UTI history during childhood. Three (37.50%) who were positive to TTV had UTI before pregnancy compared to 5(62.50%) of TTV positive infection in women without UTI infection before pregnancy. Statistical analysis revealed significant differences during childhood while non-significant before pregnancy.

Table 6: Distribution of Torque Teno virus Infection According to the History of Urinary Tract Infection among the Study Population.

History of UTI PositiveNo% NegativeNo%

During Childhood

Yes 1(12.50%) 25(27.17%)

No 7(87.50%) 67(72.83%)

Total 8(100%) 92(100%)

Chi-Square (χ²) 4.5* 19.174 **

Before pregnancy

Yes 3(37.50%) 53(57.61%)

No 5(62.50%) 39(42.39%)

Chi-Square (χ²) 0.500 NS 2.130 NS

* (P≤0.05), ** (P≤0.01).

NS= Non-significant

Alignment of TTV Sequenceswith a Reference Sequence

The DNA sequences of result of eight local isolates of the TTV displayed a significant alignment with Torque Teno Virus strain NA-MU 15 ORF1 gene, partial CDS (ID: KY750543.1) length: 269Number of Matches: 1.

Pairwise sequences alignment of all eight isolates were performed on the amplicon of ORF1 region (271 bp) compared to the reference isolates. The nucleotide sequence on ORF1 was produced from the amplification of

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

271 bp 1000 bp

300 bp 200 bp 100 bp

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two genes that were put on Blast tool, translated to a specific amino acid and produced a significant alignment with ORF1 partial Torque Teno Virus sequence (ID: AVV62254.1) length: 89 Number of matches: 1, using the GenBank library in NCBI site (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All local isolates were registered in GenBank under ID (MW513364.1, MW513365.1, MW513366.1, MW513367.1, MW513368.1, MW513369.1, MW513370.1 and MW513371.1) as shown in (Table 7).

Table 7: Variation in Nucleotides and amino Acid in TTV ORF Genes (Capsid Proteins) (NG059) and (NG061) in Local Isolates.

Source: TorqueTeno Virus \ ORF Gene No. of

isolate

Variation position Nucleotide change

Diversity in Amino Acid Sequence ID with compare

Identities

1 Transversion 66 TGC\TGG Cysteine\ Tryptophan ID: KY750543.1 98%

Transition 114 GAG\GAA Glutamic acid\ Glutamic acid Transition 153 CAC\CAT Histidine\ Histidine

Transversion 219 CTC\CTG Leucine\ Leucine Transition 231 GTA\GTG Valine\ Valine

2 Transition 198 CTG\CTA Leucine\ Leucine ID: KY750543.1 99%

Transversion 199 ATA\TTA Leucine\ Isoleucine Transition 231 GTA\GTG Valine\ Valine

3 --- ID: KY750543.1 100%

4 Transversion 102 GGT\GGA Glycine\ Glycine ID: KY750543.1 98%

Transversion 196 ATA\CTA Isoleucine\ Asparagine Transition 208 AAC\GAC Asparagine\ Aspartic acid Transversion 255 GGT\GGA Glycine\ Glycine

5 Transversion 150 GAA\GAC Glutamic acid\ Aspartic acid ID: KY750543.1 99%

Transversion 199 TTA\ATA Leucine\ Isoleucine Transition 231 GTA\GTG Valine\ Valine

6 Transition 114 GAG\GAA Glutamic acid\ Glutamic acid ID: KY750543.1 99%

Transversion 219 CTC\CTG Leucine\ Leucine Transversion 264 ATG\ATC Methionine\ Isoleucine

7 Transition 147 ATA\ATG Isoleucine\ Methionine ID: KY750543.1 99%

Transversion 214 CCC\GCC Proline\ Alanine

8 Transition 144 ATA\ATG Isoleucine\ Methionine ID: KY750543.1 99%

Transition 208 AAC\GAC Asparagine\ Aspartic acid Transition 261 ATG\ATA Methionine\ Isoleucine

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Phylogenetic Tree

The most common approaches that constructed the phylogenetic tree were constructed based onthe neighbour- joining method using NCBI and MEGA 6 software for eight local isolates and 17 reference isolates (Table 8). In the current study, the phylogenetic tree results analysis according to the closest revealed that the local isolate No.

6 showed clustered with the reference isolates (ID: KY750543.1 Egypt, ID: AF397741.1 USA, ID: AJ402241.1, England, ID: AF146809.1 Australia and ID: AY256672.1Saudi Arabia). Isolate No.1 showed very closed to ID:

AF212332.1Italian and ID: DQ665287.1 Brazilian isolates. The isolates number (3 and 7), (2 and 5) and (4 and 8) were very closely related to each other and closed to ID: GQ179967.1Iran isolates. Finally,These results confirmed that they are in P-distance, as shownin Figure (2).

Table 8: Compatibility Local Isolates with Reference Isolate from GenBank.

No. Accession number Country Source Compatibility

1. ID: KY750543.1 Egypt Torque teno virus 98%

2. ID: AY256672.1 Saudi Arabia Torque teno virus 98%

3. ID: AF397741.1 USA Torque teno virus 98%

4. ID: AJ402241.1 Italy Torque teno virus 98%

5. ID: AF185129.1 United Kingdom Torque teno virus 97%

6. ID: AF146809.1 England Torque teno virus 97%

7. ID: AF268450.1 Turkey: Istanbul Torque teno virus 97%

8. ID: AF212332.1 Australia Torque teno virus 97%

9. ID: DQ665287.1 Brazil Torque teno virus 97%

10. ID: AF108853.1 Spain Torque teno virus 96%

11. ID: AF241474.1 Argentina Torque teno virus 96%

12. ID: GQ179963.1 Iran Torque teno virus 94%

13. ID: AJ571653.1 Portugal Torque teno virus 94%

14. ID: AF123969.1 Japan Torque teno virus 92%

15. ID: AF222052.1 France Torque teno virus 92%

16. ID: GQ179967.1 Iran: Isfahan Torque teno virus 93%

17. ID: AJ309728.1 Poland Torque teno virus 91%

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Figure 2: Phylogenetic Tree of TTV (ORF1 Region) The tree was built using Mega 6 software with the neighbor-joining method of the sequences of ORF1 in eight TTV isolates. the phylogenic tree includes reference isolates isolatesobtained from GenBank. The green triangle demonstrates recent isolates.

DISCUSSION

The infection rate of TTV in the present study was 8% depending on urine samples taken from women with urinary tract infection based on microscopic examination and culture who were admitted to Al- Batool Teaching Hospital for Maternity and Children, Women's Emergency Unit and Urological Consultation Unit at Baqubah Teaching Hospital in Diyala Governorate. There is a limit information available and few studies that conducted to investigate TTV in Iraq. This rate is relatively low compared to several Iraqi studies that revealed a variation in TTV infection rates in different populations, such as15% among hemodialysis patients in Kirkuk Governorate (19), 23.3% among healthy blood donors versus 30.8% of the HCV-positive patients and 89.2% of the HBV-positive patients in Baghdad city (20),29.2% among thalassemia patientsin Baghdad city (21),and recently high rates in oral carcinoma patients (43.33% in saliva and 40% in tumor biopsy) than in controls (11% in saliva and 18.33% in tumor biopsy) in Baghdad city (22).

This study's rate is relatively high compared to other Iraqi studies, such as the study by (23) who found only2% TTV infection among patients with hemoglobinopathies and hematological malignancies in Basrah Governorate. Also, (24) detected the virus of the 150 patients, 2 (1 male and 1 female) were TTV positive in Iraqi women suffering of failure kidney disease in Baghdad city.

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The results showed a significant correlation between TTV and UTI history prevalence during childhood while non-significant before pregnancy in the current study. The result showed that 7(87.50%) women had no UTI history during childhood while 1(12.5%) woman had a UTI history during childhood. Also, 5(62.50%) women were positive to TTV infection. However, they had no history of UTI before pregnancy and 3(37.50%) of women had a history of UTI before pregnancy and were positive to TTV infection. Little is known about the urinary tract's unknown viruses that potentially result in transmission (25). The study has been suggested that TTV infection is associated with many diseases. However, there is no direct evidence of links between infection and specific clinical diseases or possible candidates for holistic monitoring (26). The explanation for this variation may be due to the notion that the TTV virus is present in the healthy and diseased population (27).

After nucleotides sequence, a significant alignment with Torque Teno Virus strain NA-MU 15 ORF1 gene, partial cds with accession number (ID: KY750543.1) length: 269 were produced by using NCBI and MEGA 6 software. The sequences analysis results showed many mutations (transition and transversion) at TTV genome-related byORF1-N22 region except the local isolate (No. 3) that was 100% identical with reference isolates, such as isolate (1) in five positions C\G, G\A, C\T, C\G, A\G, isolate (2) in three positions G\A, T\A, A\G, isolate (4) in four positions T\A, A\C, A\G, T\A, isolate (5) in three positions A\C, T\A, A\G, isolate (6) in three positions G\A, C\G, G\C, isolate (7) in two positions A\G, C\G and isolate (8) in three positions A\G, A\G, G\A. Except isolate number three, which was showing 100% similarity with Egyptian isolate. The explanation is that all eight isolates were nucleotides sequence analysis highly identities with this reference isolate ranging between 98% to 100%. Also, high identition ranged between 96% to 100% with amino acids sequence analysis of all local isolates with this reference isolate.

The neighbour-joining method was used to construct the phylogenetic tree according to (28) using MEGA 6 and NCBI software. According to the closestisolate (No. 6) closed to Egyptian isolate ID:

KY750543.1 and American isolate ID: AF397741.1. Also, England isolates ID: AF146809.1, Australian isolates ID: AF212332.1 and Saudi Arabian isolate ID: AY256672.1. Local isolate (No. 1) clustered with Italian isolate ID: AJ402241.1and Brazilian isolate ID: DQ665287.1, while local isolates (No.3) showed very closely to isolate (No. 7), isolate (No.2) and isolate (No.5) are shown very closely and isolate (No.4) clustered with isolate (No.8) and all these six isolates closed to Iranian isolates with accession number ID: GQ179967.1. All isolates high compatibility with isolates revealed by (29) who reported TTV a high prevalence among children with thalassemia and non-thalassemic with genotypes 1 and 2 being the most prevalent in Egypt.This related with phylogenetic, ordination, and evolutionary diversity analyses indicated that TTV is transmitted readily between humans across the country's geography and between various species of animal domesticates (30).

Likewise, the current study agreed with Iraqi studies interested in the phylogenic study to TTV, such as (22), (23)and (24) found that sequence analysis showed a mutation in the genome for TTV corresponding to ORF1 (N22). While unlike Brazilian study, they found that the prevalence of TTV in healthy individuals was 69.0% using ORF (N22) primers(31). The N22 regions sequence used to detect TTV DNA and classify various genotypes (32). The local isolates may have multiple recombination and mutations in the Iraqi population. This causes the generation of a large number of TTV variants driven

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by the high mutation rate, which is closer to the RNA virus (33), as well as, ORF1 is the longest encoded region in the genome of TTV with the highest rate of genetic mutations (27), and intragenomic rearrangement generating open reading frames which could not be deducted from the genomic sequence (34).

The virus is undergoing divergent evolution as very high sequence diversity was found in the ORF1 gene(35). Amplification ORF1(N22 region) give high diversity strains when used in phylogenetic analysis (36). Some causes suggested a higher than expected rate of TTV mutation. It may be due to the presence of a hypervariable in ORF1 (37). Therefore, variation of the great genomic is occurring due to hypermutation or recombination (38).Results of the study done by (39) showed that hypermutated TTV exists in healthy individuals and HBV carriers and that TTV genomes were susceptible to immune reaction directed to HBV by interacting with APOBEC3 proteins. The variation of TTV may be due to the virus having a single strand genome and the small size make it more tend to mutate, adding to many immunity reactions to facilitate immune evasion.

CONCLUSION

Transversion and transition mutation of amino acid occurred in most local isolates in two, three or five positions after alignment with some GenBank isolates, except isolate number three which was showed 100% similarity with Egyptian isolate.

ACKNOWLEDGEMENT

The authors would like to thank all staff at Al-Batool Teaching Hospital for Maternity and Children, Women's Emergency Unit and Unit of Urological Consultation at Baqubah Teaching Hospital in Diyala Governorate for their help during samples collection to perform this study and thankful for all patients were included in this study.

REFERENCES

1. Konapala LA, Vasanthi V, Raj KK and Vinodkumar M. Pregnancy and hormonal effects on urinary tract infections in women: A Scoping Review, Int. J. Pharm. Sci. Rev. Res., 2018;5 (10):407-420.

2. KumarA. Urinary tract infection. In Adel E, Prabha S, and Soad AZH (eds.). Infections in pregnancy: An evidence-based approach, United Kingdom, Cambridge University Press,2019;Pp: 129-133.

3. Abd Al-Hussein BA, Sawsan GJ, and Mazinm GA. Maternal complications of teenage pregnancy in two teaching hospitals in Baghdad, Iraqi J. Community Med., 2019;32(2): 42-48.

4. Hsiao CY, Chen TH, YiCL, MengCH, Peir HH, and Ming CW. Risk factors for urosepsis shock in hospitalized patients aged over 80 years with urinary tract infection, Ann. Transl. Med., 2020;8(7): 1-13.

5. Emery J. A 25-Year-Old Woman with Recurrent Urinary Tract Infections. In D. Chelmow, N. Karjane, H.

Ricciotti, and A. Young (Eds.), Office Gynecology: A case-based approach, Cambridge: Cambridge University Press,2019; Pp: 269-271.

6. Ghosh S, Alyssa K, Yashpal SM, and Nobumichi K. Torque Teno Virus. In: Malik Y., Singh R., Yadav M. (eds).

Emerging and transboundary animal viruses, Livestock Diseases and Management, Springer, Singapore, 2020;Pp:

111-122.

7. Taheri M, Mohammad M, Jamal S, Ramin Y, and NeginN. Higher frequency of transfusion-transmitted virus (TTV) in HIV patients in comparison with healthy blood donors, Int. J. High Risk Behav. Addict., 2017; 6(3):1-6.

8. Amen N, Maria M, Azam M, Aziz A, Qamar R and Bostan N. Low Seroprevalence of Torque Teno Virus in HCV positive patients and phylogenetic analysis from Pakistani isolates, Trop. Biomed., 2018;35(1): 205-20.

9. Li G, Wenyan Z, Ruyi W, Gang X, Shilei W, Xiang J, Ningning W, Shuo S, and JiyongZ. Genetic analysis and evolutionary changes of the Torque Teno Sus Virus, Int. J. Mol. Sci., 2019; 20(12):2881.

(11)

10. Payne S. Other small DNA viruses, in: viruses from understanding to investigation, United States, academic press, 2017; Pp: 243-246.

11. DuffyS, Laura ASand Edward CH. Rates of evolutionary change in viruses: patterns and determinants, Nat Rev Genet., 2008; 9(4): 267-276.

12. SanjuánR and Pilar DC. Mechanisms of viral mutation, Cell. Mol. Life Sci.,2016; 73(23):4433-4448.

13. Domingo E. Molecular basis of genetic variation of viruses: Error-prone replication. Virus as Populations, 2020;

Pp: 35–71.

14. Procop GW, Deirdre LC, Geraldine SH, William MJ, Elmer WK, Paul CS and Gail LW. Koneman'scolour atlas and textbook of diagnostic microbiology, China, Jones and Bartlett Publishers, 2017; Pp: 71-75.

15. Doern CD. Pocket guide to clinical microbiology, 4th edition, Washington, ASM Press, 2018; Pp:142-149.

16. Okamoto H, Tsutomu N, Naomi K, Masato U, Hiroki I, Hisao I, Yuzo M, and Makoto M. Molecular cloning and characterization of a novel DNA virus (TTV) associated with post-transfusion hepatitis of unknown etiology, Hepatol. Res., 1998;10:1-16.

17. Irshad M, Kishore M, Shiwani S, and Agarwal SK. Torque Teno Virus infection in hemodialysis patients in North India, Int. Urol. Nephrol., 2010;42(4):1077-1083.

18. Drabik A, Bodzoń-Kułakowska A, and Silberring J. Gel electrophoresis, In Ciborowski, P., and Silberring, J.

(eds). Proteomic profiling and analytical chemistry, 2nd edition, Elsevier, 2016; Pp: 115-143.

19. Wahid NM, and Israa SH. Torque Teno Virus (TTV) as a risk factor in the hemodialysis process in Kirkuk, Indian J. Forensic Med. Toxicol., 2019; 13(4):1425-1431.

20. Khudair EA, Arwa MA, and Nawal MF. Detection of TTV antigen in patients with hepatitis HBV and HCV, Iraqi J. Med., 2019;17(1): 43-49.

21. Al-Jabali IN, and Arwa HA. Torque Teno Virus (TTV) infection and genotypes in Iraqi thalassemia patients, M.Sc., Thesis, College of Medicine, Al-Mustansiriya University, Iraq,2014.

22. Abed RM, Haidar AS, and Ali AR. Detection and genotyping of Torque Teno Virus in saliva and tumour biopsies from patients with oral cancer, Ann. Trop. Med. Public Health, 2020; 23:231-412.

23. Al-Hmudi HA. Molecular detection of Torque Teno Virus (TTV) infection of patients with haemoglobinopathies and haematological malignancies, Bas. J. Sci.,2019; 45(A2):44-53.

24. Ali Y, Tariq MQ, and Wahab RA. Phylogenetic analysis of TTV (TorqueTeno Virus) in Iraqi woman suffering of failure kidney disease, Indian J. Forensic Med. Toxicol., 2021;15(1):1659-1664.

25. SchreiberPW, Verena K, Kerstin H, Stefan S, Osvaldo Z, Amandeep K, Cornelia B, Michael G, Andrea Z,Riccarda C,Jürg B, Hans HH, ThomasFM, Nicolas JM, Alexandra T and Michael H. Metagenomic virome sequencing in living donor and recipient kidney transplant pairs revealed JC polyomavirus transmission, Clin Infect Dis., 2019; 69(6):987-994.

26. Strassl R, Konstantin D, Susanne RR, Harald H, Irene G, Johannes PK, RalfS, Helmuth H, Martin S, Farsad AE, Željko K, Roman RS, Elisabeth PS, Georg AB, and Gregor B. Torque Teno Virus for risk stratification of acute biopsy-proven alloreactivity in kidney transplant recipients, J. Infect. Dis., 2019; 219(12):1934-1939.

27. Bostan N, Nabghe, A and Habib B. Current and future prospects of Torque Teno Virus, J. Vaccines Vaccin, 2013;1:1-9.

28. Saitou N and Nei M. The neighbour-joining method: a new method for reconstructing phylogenetic trees, Mol.

Biol. Evol., 1987; 4(4):406-25.

29. Hassuna NA, Eman N, Medhat AF and Suzan MOM. Phylogenetic analysis of Torque Teno Virus in thalassemic children in Egypt, Intervirology, 2017; 60(3):102-108.

30. Sarairah H, Salwa B, and Waleed G. The molecular epidemiology and phylogeny of Torque Teno Virus (TTV) in Jordan, Viruses, 2020; 12(2): 1-18.

31. Mazzola JC, Patrícia KS, Roger HY, Maria AEW, Waldir VSJ, Alessandra CGM, and Sueli DB. Prevalence of Torque Teno Virus in healthy donors of Paraná State, Southern Brazil, Rev. Bras. Hematol. Hemoter., 2015;

37(5): 336-340.

32. Prescott LE, and Simmonds P. Global distribution of transfusion tranmissted virus, N. Engl. J. Med., 1998; 339:

776.

33. Sanjuán R, and Domingo-Calap P. Mechanisms of viral mutation, Cell. Mol. Life Sci., 2016; 73(23), 4433-4448.

34. Leppik L, Gunst K, Lehtinen M, Dillner J, Streker K and De Villiers EM. In vivo and in vitro intragenomic rearrangement of TT viruses, J. Virol., 2007; 81(17):9346-9356.

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35. Amen N, Maria M, Azam M, Aziz A, Qamar R, and Bostan N. Low Seroprevalence of Torque Teno Virus in HCV positive patients and phylogenetic analysis from Pakistani isolates, Trop. Biomed., 2018; 35(1): 205-20.

36. Hassuna NA, Medhat AF, Shrouk O, Wael MAE, Rasha FA and Reham AI. High frequency of Torque Teno Virus (TTV) among Egyptian hemodialysis patients, Afr. J. Microbiol. Res., 2019; 13(28):619-625.

37. Jelcic I, Agnes HW, Andreas H, Harald ZH, Ethel-Michele D. Isolation of multiple TT virus genotypes from spleen biopsy tissue from a Hodgkin's disease patient: genome reorganization and diversity in the hypervariable region, J. Virol., 2004; 78(14):7498-7507.

38. Manni F, Antonella R, Elisabetta, C, Giorgio Band Dario D. Detecting recombination in TT virus: a phylogenetic approach, J. Mol. Evol., 2002; 55(5):563-572.

39. Tsuge M, Chiemi N, Rie A, Miyuki M, Kana K, Sachi T, Hiromi A, Fukiko M, Shosuke K, Tsuyoshi H, Takashi K, Daiki M, Nobuhiko H, Michio I, Shoichi T, C Nelson H, Kazuaki C. G to A hypermutation of TT virus,Virus Res., 2010; 149(2):211-216.