Phylogenetic Analysis of TTV (TorqueTeno Virus) in Iraqi Woman Suffering of Failure Kidney Disease
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Indian Journal of Forensic Medicine & Toxicology, January-March 2021, Vol. 15, No. 1 1659
Phylogenetic Analysis of TTV (TorqueTeno Virus) in Iraqi
Woman Suffering of Failure Kidney Disease
YasameenHasan Ali1, Tariq M Quasim2, DBEL Wahab R.Alshaikly3
1
Assistant Professor College of Nursing, 2Tutor Department of pharmacy, Al-Rasheed University College
3
Prof,
Abstract
There are still very few studies of the Torque Tenovirus infection rate between hemodialysis patients in
Iraq. Thus, the aim of this study was to assess the frequency of TTV viremia in hemodialysis Iraqi patients.
This study has carried out in Baghdad city from October 2019 to February 2020 and included 150 patients
with kidney failure disease undergoing hemodialysis and 50 control subjects .Blood samples have been
obtained for serological TTV detection. The result shows PCR has been used to detect TTV, isolates have
been identified through N22 region sequencing and phylogenetic analysis has also been performed. Of the
150 patients, 2 (1 male and 1 female) were TTV positive.The results shows in ( isolate 14) substitution
four Transversion A>C, C>G,A>T, and T>A three Transition T>C,A>G, and T>C of Specific region within
TTV(N-22) and showed 94% identified with a standard in Gene Bank while having 99% identified with
a standard in Gene Bank with (isolate 17) substitution fiveTransversion A>T,A>C,T>A, A>Tand C>G
threeTransition A>G,A>G and T>C . The phylogenetic analysis showed that identical among themselves
and the world with 100% compatibility values.
Key words: kidney failure disease,Torque Teno virus (TTV), PCR- Sequencing.
Introduction may be appliances, surfaces and staff of various origins
in haemodialysis units(9).Besides HCV and HBV, TTV
The Torque Teno virus (TTV) was initially isolated
are among the frequently transmitted viruses in patients
from a post-transfusional non-A-G hepatitis patient in
with haemodialysis, the frequency of which varies
Japan (1). The virus is an omnipresent negative stranded
greatly contingent on virological, demographic and
DNA virus which has been listed as an Anelloviridae
clinical factors (10).Thus the aim of this study were to
family member (2). Though considered a hepatotropic
assess the frequency of TTV viremia in hemodialysis
virus, with different transmission modes (3).TTV is
Iraqi patients.
classified as intosevengenogroups including many
genotypes according to sequence analysis (4), of Materialsand Methods
which genotype 1 is the most prevalent .The virus is
spread globally according to geographic location with This study has carried out in Baghdad city from
variable seropositivity levels (5,6). TTV usually infects October 2019 to February 2020 and included 150 patients
patients exposed to blood transfusion as patients with with kidney failure disease undergoing hemodialysis and
thalassemia and haemodialysis,also intravenous users 50 control subjects who were apparently healthy. Five ml
of the drug (7).The virus has been rendered pathogenic of whole blood was collected from each subject. blood
and is also considered a virus flora, but its significance sample was allowed to clot naturally then centrifuged at
stems from its capability to demonstrate the combined (3000 rpm) for 5 minutes and serum was separated from
capacity from the innate and acquired immunity (8). packed RBCs. Serum was divided into 2 parts in sterile
Despite the innumerable utility of haemodialysis in Eppendorftubes which then stored in deep freeze at
chronic renal failure patients, it has been attributed as -20°C, one part was used for DNA extraction from TTV
a high danger setting for blood-borne infections, that , and the second part for detection of TTV by ELISA.1660 Indian Journal of Forensic Medicine & Toxicology, January-March 2021, Vol. 15, No. 1
Detection of TorqueTeno virus by ELISA using PCR with forward primer 5’-GTC AAG GGG
CAA TTC GGG CWC-3’ and reverse primer 5’-GTC
Detection ofTTV were done firstly by ELISA (Cat.
TGG CCC CAC TCA CTT TCG-3’. The program of
No: MBS9313728, MyBioSource, USA).
PCR as seen in table (1), PCR products were verified in
Amplification of TTV by using PCR 1.5% agarose gel.
Specific region within TTV(N-22) amplified by
Table (1): PCR program that was applied in the thermocycler devices.
Phylogenetic analysis
Two TTV isolates from this samples were utilized for phylogenetic analysis to establish the genetic relationship
between Iraqi human TTV strains and the NCBI data base strains. Using the MEGA6 software, the nucleotide
sequences were aligned based on the amino acid sequences to prevent nonsense from being introduced.
Results
The distribution of the samples according to gender showed insignificant distribution ( χ2=5.9017 ,P>0.206),
the negative samples showed 63 females and 85 males while the two positive samples were divided equally into one
male sample and one female sample. The control samples were 23 males and 27 females.
Table (2): samples distribution according to gender.
Groups Female Male
Control 27 23
TTV negative 63 85
TTV positive 1 1
Chi-square 5.9017
p-value 0.206
The results of ELISA test showed 21 samples were positive (out of the total 150 samples). ELISA test has low
Specificity of results (31.58%). The percentage of TTV positivity were different after the PCR test were done as
shown in table (3).Indian Journal of Forensic Medicine & Toxicology, January-March 2021, Vol. 15, No. 1 1661
Table(3):Comparison between ELISA and PCR techniques.
Specificity Total collected
of ELISA percentage positive samples Tests samples for
% patients
14% 21 ELISA
31.58% 150
1.30% 2 PCR
The results of aligning the sequences results (GGC>GGG).Those variations resulted after aligning
ofTTV are illustrated in table (4) which showing the sequence of our isolates to the reference sequence
the variation of each sample. Each sample showed (ID: AF123933.1) which showed the highest similarity
different variations. The first sample showed 7 to our resulted sequence NCBI.
variations which are; at nucleotide 112 (TTT>TTA),
The phylogenetic tree diagrammatic by Molecular
at nucleotide 178 (AAC>ACC)missense, a nonsense
Evolutionary Genetics Analysis (MEGA) software
at nucleotide 182 (AAT>AAC), at nucleotide 194
version 6.0 the phylogenetic trees of these species are
(GGC>GGG), at nucleotide 196 (TAC>TCC), at
shown in Figure (1).These alignmentsappeared the
nucleotide 200 (GTA>GTG) and at nucleotide 206
TTV between Iraq and others in the world by Specific
(TAT>TAC). The other sample showed 8variations at
region within TTV(N-22) for translating specific
which are; at nucleotide 50 (AAC>GAC), at nucleotide
region .Hierarchical cluster analysis determine the
127 (TAC>TCC), at nucleotide 123 (AAC>GAC),
following clusters including TTV Iraqi isolate(14,17)
at nucleotide 176 (AAC>ACC), at nucleotide
the identical100 % it is close to Egypt(ID:KY750545)
179(ATC>ACC), at nucleotide 198 (TTT>TTA), at
the identical 100 % .
nucleotide 201 (TAC >TTC) and at nucleotide 219
Figure 1: Neighbor-joining tree from TTV sequence alignment.1662 Indian Journal of Forensic Medicine & Toxicology, January-March 2021, Vol. 15, No. 1
Discussion polymerase chain reaction diagnostic techniques is
rapid, easy, inexpensive protocol becoming the most
The patients with chronic renal failure suffering
commonly utilized of all molecular genetics ways for
from hemodialysis (HD)are at risk of TTV infection (11).
detecting important genes.Irshad’s analysis found that
Data on TTV infection in peritoneal dialysis patients
Torque teno virus DNA genotype 1 was the principal
was very rare; these results Indicated the prevalence
genotype of TTV DNA in patients with hemodialysis
of TTV in a population with continuous ambulatory (21). In contrast, genotype 2 was reported to be more
peritoneal dialysis is close to that of healthy controls
(12). At worldwide distribution, torque teno virus was pathogenic than other genotypes (22, 23). Likewise, a
study carried out in Turkey to examine the N22 region
found to be more widespread in men (13). Spandoleet al
(14) reported that the distribution from TTV in males and found that genotypes 1 and 2 were the most common
in the different groups investigated and the only one
females did not notice any difference, this agreement
identified in the population of blood donors (24). Torque
with our study. At the other hand, Mohamed et al (15)
teno virus has been spread worldwide, with a high
recorded that TTV-positive patients were 66.7 % and
prevalence of TTV infection in various populations
33.3 % respectively in age groups 20-40 and 40-60 years,
including liver disease patients, HIV patients, drug users
3.7 % TTV-positive DNA were males compared to 6.1 %
and healthy individuals (25). Irshad et al (18) reported that
of females with no noticeable difference between males
PCR fragment molecular and phylogenetic analyzes
and females .Al-Hamdaniet al (16) found that 29.2 % from
detected that TTV could be divided into many genotypes
thalassemia patients with predominance of males over
found worldwide, without any direct connection to the
females were detected with TTV (64.4 % vs. 35.6 %).
geographic distribution of diseases. This disparity in
Takemoto et al (17) reported that patients with dialysis
the prevalence of TTVs in various geographic areas
were mostly males (55 %) with a mean age of 53.8 years.
can be explained by the presence of different roads of
The current study showed the relationship between TTV
this virus spreading across different geographic areas.
infection and patients suffering from hemodialysis in a
Castro et al (23) represented a molecular epidemiological
population of kidney failure. The discovery of TTV and
study examining a large number of Brazilian isolates
its parenteral mode of transmission resulted in the idea
and reported that TTV was identified among 11.9% of
of its potential inclusion in a category of viral hepatitis
Brazilian blood donors through the study of the TTV
that is not A-G (18).Sequence analysis shows that there
genome ORF-1 region close to that found between
is a high degree of genetic variation in the TTV genome
German and Japanese blood donors.
and can be categorized into at least six main genotypes
(groups 1–6) and multiple subtypes (1a, 1b, 2a and 2b) Conflict of Interest: There is no conflict of interest
(19, 20). Direct UTR region amplicon sequencing revealed
among the authors.
extensive mix-infection of different TTV strains
within the infected person. The divergence among Funding: Self
various detecting regions indicatedthat TV is due to the
Ethical Clearance: This study is ethically approved
coexistence of multiple phylogenetic groups(4). In the
by the Institutional ethical Committee.
current study the sera samples from failer kidney disease
patients and control were were assayed for the existence References
from TTV-DNA utilizing PCR and amplifying specific
1. Nishizawa T, Okamoto H, Konishi K, Yoshizawa
zone of TT genome. To determine the phylogenetic
H, Miyakawa Y, Mayumi M. A novel DNA virus
relationship of Iraqi isolated strains among human TTV,
(TTV) associated with elevated transaminase
sequences from GenBank and isolates from the present
levels in posttransfusion hepatitis of unknown
study were included for phylogenetic analysis. The etiology. Biochemical and biophysical research
findings of the ELISA technique showed that 21 samples communications. 1997 Dec 8;241(1):92-7.
were positive (out of a total of 150 samples), has low
2. King AM, Lefkowitz E, Adams MJ, Carstens
Specificity specificity (31.58%) as compared with the EB, editors. Virus taxonomy: ninth report of the
PCR diagnosis of the Sanger Sequencing technique International Committee on Taxonomy of Viruses.
for Torque teno virus with high results specificity. TheIndian Journal of Forensic Medicine & Toxicology, January-March 2021, Vol. 15, No. 1 1663
Elsevier; 2011 Nov 10. donors and haemodialysis patients in southern
3. Focosi D, Antonelli G, Pistello M, Maggi F. Brazil. Memórias do Instituto Oswaldo Cruz. 2012
Torquetenovirus: the human virome from bench to Aug;107(5):684-6.
bedside. Clinical Microbiology and Infection. 2016 14. Spandole-Dinu S, Cimponeriu DG, Crăciun
Jul 1;22(7):589-93. AM, Radu I, Nica S, Toma M, Alexiu OA, Iorga
4. Hsiao KL, Wang LY, Lin CL, Liu HF. New CS, Berca LM, Nica R. Prevalence of human
phylogenetic groups of torque teno virus identified anelloviruses in Romanian healthy subjects and
in eastern Taiwan indigenes. PloS one. 2016 Feb patients with common pathologies. BMC infectious
22;11(2):e0149901. diseases. 2018 Dec;18(1):334.
5. Spandole S, Cimponeriu D, Berca LM, Mihăescu 15. Almoshrf IA, El Hussein AR, Elkhidir IM, Enan KA.
G. Human anelloviruses: an update of molecular, Molecular Detection of Torque Teno Virus among
epidemiological and clinical aspects. Archives of HIV Seropositive Patients in Khartoum, Sudan.
virology. 2015 Apr 1;160(4):893-908. Microbiology Research Journal International. 2018
Mar 28:1-6.
6. Amen N, Maria M, Azam M, Aziz A, Qamar R,
Bostan NJ. Low Seroprevalence of torque teno 16. Al-Hamdani AH, Al-Jabali IN, Aufi IM. Torque
virus in hcv positive patients and phylogenetic Teno Virus (TTV) infection and genotypes in
analysis from Pakistani isolates. Trop Biomed. Iraqi thalassemia patients. International Journal for
2018 Mar 1;35:205-. Sciences and Technology. 2014 Sep;143(1734):1-
5.
7. Akbari H, Piroozmand A, Dadgostar E, Nikoueinejad
H, Chitsazian Z, Einollahi B, Mahabadi JA. 17. Takemoto AY, Okubo P, Saito PK, Yamakawa
Prevalence of Transfusion-transmitted Virus (TTV) RH, Watanabe MA, Veríssimo da Silva Junior W,
Infection and its Association with Renal Post- Borelli SD, Bedendo J. Torque teno virus among
transplantation Complications in Iran. International dialysis and renal-transplant patients. Brazilian
journal of organ transplantation medicine. Journal of Microbiology. 2015 Mar;46(1):307-11.
2018;9(3):126. 18. Irshad M, Mandal K, Singh S, Agarwal SK. Torque
8. Chen T, Väisänen E, Mattila PS, Hedman K, teno virus infection in hemodialysis patients in
Söderlund-Venermo M. Antigenic diversity and North India. International urology and nephrology.
seroprevalences of Torque teno viruses in children 2010 Dec 1;42(4):1077-83.
and adults by ORF2-based immunoassays. Journal 19. El-taher SM, Fouad NA, Fouad MA, Mahedy AW,
of General Virology. 2013 Feb 1;94(2):409-17. Elnazi AK. Transfusion-transmitted virus infection
9. Zuckerman M. Surveillance and control of blood- in hemodialysis patients in Arar, Saudi Arabia:
borne virus infections in haemodialysis units. Prevalence, predictors and genotyping. Saudi
Journal of Hospital Infection. 2002 Jan 1;50(1):1-5. Journal of Kidney Diseases and Transplantation.
2015 Nov 1;26(6):1215.
10. Oliveira KB. Torque teno virus: a ubiquitous virus.
Revista brasileira de hematologia e hemoterapia. 20. Chattopadhyay S, Rao S, Das BC, Singh NP, Kar
2015 Dec;37(6):357-8. P. Prevalence of transfusion‐transmitted virus
infection in patients on maintenance hemodialysis
11. Jalali H, Mahdavi MR, Zaeromali N. Torque
from New Delhi, India. Hemodialysis International.
Teno Virus (TTV) Among β-Thalassemia and
2005 Oct;9(4):362-6.
Haemodialysis Patients in Mazandaran Province
(North of Iran). International journal of molecular 21. Irshad M, Joshi YK, Sharma Y, Dhar I. Transfusion
and cellular medicine. 2017;6(1):56. transmitted virus: a review on its molecular
characteristics and role in medicine. World
12. Hsu BG, Wang LY, Hu CT, Wang CH, Fang
journal of gastroenterology: WJG. 2006 Aug
TC, Lin HH. TT virus infection in patients on
28;12(32):5122.
peritoneal dialysis in Taiwan. Renal failure. 2007
Jan 1;29(5):553-7. 22. Mankotia DS, Irshad M. Development of an
immunoassay for detection of torque Teno virus
13. Massaú A, Martins C, Nachtigal GC, Araújo
(TTV) antibodies using the N22 expression
AB, Rossetti ML, Niel C, Silva CM. The high
product from TTV genotype 2. Intervirology.
prevalence of Torque teno virus DNA in blood1664 Indian Journal of Forensic Medicine & Toxicology, January-March 2021, Vol. 15, No. 1
2017;60(5):207-16. Anatolia, Turkey. Japanese journal of infectious
23. de Castro Amarante MF, Kashima S, Covas DT. diseases. 2005 Aug 1;58(4):222.
TT virus (TTV) genotyping in blood donors and 25. Rinonce HT, Yano Y, Utsumi T, Heriyanto DS,
multiple transfused patients in Brazil. Virus genes. Anggorowati N, Widasari DI, Ghozali A, Utoro
2007 Dec 1;35(3):503-9. T, Lusida MI, Prasanto H, Hayashi Y. Prevalence
24. Kalkan A, Ozdarendeli A, Bulut Y, Saral Y, Ozden and genotypic distribution of GB virus C and
M, Kelestimur N, Toraman ZA. Prevalence and torque teno virus among patients undergoing
genotypic distribution of hepatitis GB-C/HG and hemodialysis. Molecular Medicine Reports. 2017
TT viruses in blood donors, mentally retarded May 1;15(5):2843-52.
children and four groups of patients in easternYou can also read
