The First Detection of Co-Infection of Double-Stranded RNA Virus 1, 2 and 3 in Iranian Isolates of Trichomonas vaginalis

Background: The Totiviridae family includes a number of double-stranded RNA viruses that can infect Trichomonas vaginalis. Some T. vaginalis isolates are infected with one or more double-stranded RNA (dsRNA) viruses. In this study, different strains of double-stranded RNA virus in Iranian isolates of T. vaginalis were evaluated for the first time in Iran. Methods: Vaginal swabs were collected from 1550 participants who were referred to hospitals associated with Iran University of Medical Sciences, Tehran, Iran from June to November 2018. T. vaginalis isolates were cultured in Diamond’s modified medium. After the extraction of nucleic acids using a DNA/RNA extraction kit, RT-PCR was performed and PCR products were purified and sequenced. Results: In general 9 (0.6%) isolates were confirmed as T. vaginalis among 1550 collected vaginal samples. Among 9 isolates of T. vaginalis, three of them were infected with TVV1. One isolate has multiple infections with T. vaginalis virus (TVV1, TVV2 and TVV3) as coinfection. The nucleotide BLAST indicated that the T. vaginalis virus 1(TVV1) isolates were most closely related to TVV1-OC5, TVV1-UR1-1.The T. vaginalis virus 2 (TVV2) sequence had also a similarity with TVV2-UR1-1, TVV2-UR1 and TVV2-OC3. The sequence of T. vaginalis virus 3(TVV3) had similarity with TVV3-OC5, TVV3-UR1-1 and TVV3-UR1. Conclusion: Three dsRNA viruses T. vaginalis virus (TVV1, TVV2 and TVV3) were detected using RT-PCR in T. vaginalis Iranian isolates. The coinfection of TVV1, TVV2 and TVV3 in one isolate of T.vaginalis was observed for the first time in Iran.


Virus;
Trichomonas vaginalis; Double-stranded RNA virus; Iran Introduction richomonas vaginalis is a protozoan parasite that resides in the human genitourinary tract and causes trichomoniasis (1). Patients with trichomoniasis have a wide range of clinical symptoms ranging from vaginitis, cervicitis, urethritis and irritation to asymptomatic presentation (2).
Trichomoniasis are associated with premature delivery, low birth weight and cervical cancer, it also increases the risk of infection by human immunodeficiency virus (HIV) (3)(4)(5). The first line treatment for trichomoniasis is metronidazole; however, resistance to metronidazole was observed in some occasions (6). The prevalence rate of trichomoniasis in Iran ranges from 2 to 8% and likely up to 30% among high-risk populations (2). Pathogenesis and virulence factor of T.vaginalis is still unknown (1).
These parasites are infected with one or more double-stranded RNA (dsRNA) viruses (TVVs) from genus Trichomonas virus and Totiviridae family (7). The virus has been formed in the cytoplasm close to the plasma membrane or near the Golgi complex. Viruses have different sizes (33-200 nm) and shapes (filamentous, spherical, and cylindrical particles) (8).
Totiviridae is a family that has 5 genera including Leishmania virus, Giardia virus, Totivirus, Victoria virus and Trichomonas virus. Different species of T. vaginalis virus (TVV) have been identified including TVV1, TVV2, TVV3, and TVV4 according to genome sequences and phylogenetic analysis (1,9).
Double-stranded RNA virus genome encodes a viral capsid protein (CP) and a viral RNA-dependent RNA polymerase (RdRp) in 2 overlaps of open reading frames (ORFs) (1). The biological significance of T. vaginalis infection by TVVs remains poorly understood. It seems that TVV infection is generally a noncytopathic phenomenon that leads to stable and permanent infections. Viruses can be clas-sified as either cytopathic meaning that cells are killed during the infection, or noncytopathic meaning that their replication programs are benign by stopping the process of proapoptotic mechanisms (10). Infections of the protozoan host occurs without an extracellular transmission phase (11) and transmission of TVV just happens through cell division and also during mating (9,12).
T. vaginalis infection by TVVs could modulate the pathogenicity of T. vaginalis infections by expression of a highly immunogenic T. vaginalis protein, P270 (13) and also the expression of cysteine proteinases that are assumed to be virulence factors (14). Genotype of T. vaginalis infected with TVV-1 viruses was evaluated by PCR-RFLP in a study (15).
The cDNA sequences of only TVV1 strains in CP region to date have been reported in Iran. Based on these sequences, phylogenetic analysis has been performed according to some new TVV1 sequences in comparison with different TVVs in the family of Totiviridae (16). In this study, different strains of dsRNA virus in Iranian isolates of T. vaginalis were evaluated for the first time in Iran.

Study population
Overall, 1550 vaginal samples were collected from women who were referred to hospitals associated with Iran University of Medical Sciences (IUMS), Tehran, Iran from June to November 2018.

Ethical considerations
This study was approved by the Ethics Committee of Iran University of Medical Sciences in accordance with Helsinki Declaration and guidelines (IR.IUMS.REC.1397.1383) and all the participants taking part in the study were in agreement with informed consent T

Collection of the specimens
Vaginal swabs were collected from each participant. At first, wet mount samples with sterile phosphate-buffered saline(PBS; pH 7.3 ± 0.1) were prepared and observed using light microscope with 10X and then 40X magnification and then another swab was inoculated into a culture tube at 37°C in Diamond's TYM medium with 10% heat-inactivated calf serum, 100 U/mL penicillin and 30 mg/mL streptomycin sulphate. Finally T.vaginalis culture was harvested in late log phase and centrifuged at 2000 g for 15 min and pellets were stored at -70°C and isolates were confirmed as T.vaginalis by PCR (15,16).

RNA extraction
T. vaginalis samples were washed with sterile PBS and centrifuged at 4°C and 5000 g, for 10 min. The RNA was extracted using the Pure Viral Nucleic Acid Kit Roche (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions as previously described (16) .The reversetranscription polymerase chain reaction (RT-PCR) was performed in order to detect TVV RNA in T. vaginalis positive cases.
cDNA synthesis of RNA was performed at 42°C for 30 min in a 20 µL reaction mixture containing 0.5 µg of total RNA, 20 pmol of random hexamer, 4 µL of 5x reverse transcriptase reaction buffer, 125 µmol dNTPs, 104 U Moloney Murine Leukemia Virus reverse transcriptase (Fermentas GmbH, St. Leon-Rot, Germany), 19.2 U RNase inhibitor (Fermentas GmbH) and 1 µL of diethylpyrocarbonate treated water and finally heated at 70°C for 10 min in order to inactivate the reverse transcriptase (16).

PCR amplification
PCR was performed using specific primers described before (1,17) in a 25 µL mixture containing the template (3 µL of RT reaction), 2.5 U Taq DNA polymerase, 2.5 µL of 1 x PCR buffer, 20 pmol of each primer, 100 µmol dNTPs and 1.5 mmol MgCl2.Amplification was performed as fol-lows: initial denaturing for 5 min at 95°C; 40 cycles of 94°C for 30 s, 51°C for 1 min and 72 °C for 40 s; extension at 72°C for 5 min and finally the PCR products were electrophoresed . Expected bands were found for TVV1 at 569 bp, TVV2 at 625 bp, TVV3 at 438 bp, and TVV4 at 514 bp.

PCR sequencing
A second round of RT-PCR was done for more confirmation and PCR products were purified using the High Pure PCR Product Purification Kit (Roche Diagnostic, Mannheim, Germany) according to the manufacturer's instructions and were used for direct sequencing using the dye termination method and an ABI 3730xl sequencer. The new sequences of amplified TVV with RT-PCR method was analyzed using genius version 10.1, blasted and compared with those corresponding to different TVV1-TVV3 (dsRNA viruses belonging to the genus Trichomonas virus of family Totiviridae) taken from the Gen-Bank database. The protein sequences were determined using the program ExPASy (ca.expasy.org/tools/pi_tool)
Among 1550 vaginal samples collected, 9 isolates were confirmed as T.vaginalis using actin gene primers. The mean age of the women was 31 ± 4.34 years (ranging from 18 to 45 years). The result of PCR amplification for T. vaginalis isolates was evaluated by agarose gel electrophoresis and a band of 1100 bp was observed in (Fig. 1). The T. vaginalis isolates were examined by the designed PCR for TVV1, TVV2 and TVV3 and TVV4. In this study, among 9 T.vaginalis isolates, three of them were infected with TVV1; one of them was co-infected with TVV1, TVV2 and TVV3.

Discussion
Parasitic protozoa, T. vaginalis can be infected by one or more subtypes of dsRNA viruses (1). TVV is transmitted vertically during the T. vaginalis binary fission (5); however it is lost through long term passages (18).
In this study, we identified three TVVs including TVV1, TVV2, and TVV3 detected from Iranian isolates of T. vaginalis using genome sequencing and molecular analysis with other sequences of different TVVs. Furthermore, T. vaginalis was detected by PCR in 9 of 1550 women attending hospitals associated with Iran University of Medical Sciences, Tehran, Iran. In general, 33.3% of T. vaginalis isolates were infected with dsRNA viruses. Among 9 T. vaginalis isolates, three of them were infected with TVV1 that one of them was infected with TVV1, TVV2 and TVV3 at the same time. So, there was one isolate harboring three TVVs, including TVV1, TVV2 and TVV3. Direct sequencing and nucleotide and amino acids BLAST search confirmed the identity of these dsRNA viruses using RT-PCR.
In the previous study, infection to just TVV1 in T.vaginalis isolates has been reported in Iran. Phylogenetic analysis confirmed that the Iranian isolates were related to TVV1-OC5, TVV1-UR1 (16). The presence of multiple TVVs in a single isolate of T.vaginalis has been reported by some studies (1,19,20). The multiple viral infections in a single host has also been reported in the genera Totivirus and Victorivirus (1). The presence of several TVVs in a single culture may be due to the presence of multiple parasites that were infected with different TVVs or because of synchronous infection of TVVs in a single parasite (21).
Moreover, 44 % T. vaginalis isolates were infected with dsRNA virus from different geographic origins (22). The frequency of TVV infection among T.vaginalis isolates has been reported as 55% in Cuba (23) and 50% in USA (24). The TVV infection rate of 81.9% was reported in T. vaginalis isolates from South Africa (7).
In contrast, two studies conducted in Korea and Tehran reported that only 13.6% (25) and 17.39% of the T.vaginalis isolates were infected with TVVs respectively; however only TVV1 was identified in T. vaginalis isolates from Iran (26).
Goodman et al conducted a study on T. vaginalis isolates and reported that TVV-1 was the most prevalent (77.5 %) of them, followed by TVV-2 and TVV-3 (30%). The less frequent TVVs were related to TVV-4 (10 %). The result of the study also demonstrated that one isolate of T. vaginalis could be infected by several TVV species (1). Among three T. vaginalis isolates, TVV-2 and TVV-1 were identified and TVV species were associated with two TVV2 and one TVV1 (27).
Rivera et al reported different subtypes of TVV in T. vaginalis isolates. In this study among 18 T. vaginalis isolates, 35 dsRNA viruses were detected. Various TVV were identified in six of the 18 T. vaginalis samples. Phylogenetic analyses in this study proposed monophyly in TVV1 and TVV2, but TVV3 and TVV4 appear paraphyletic (21).
In a study, 8 out of 40 (20 %) T. vaginalis isolates were infected with dsRNA viruses. TVV2 was the most prevalent species that was identified in 5 out of 8 (62.5%), followed by TVV4 in 3 out of 8 (37.5%). All isolates infected with TVV were detected from the symptomatic patients. A significant association between the presence of dsRNA viral infection of T. vaginalis isolates and symptoms like vaginal discharge, erythema and dysuria was found (P<0.01) (17).
Although the result of some studies showed there was no relation between the ages of patients and the viral infection (17,26), a significant relation between age and TVV infection has been found in the older age of individuals in a study (28).
In the present study, there was no relation between the ages of patients and the viral in-fection. Although symptoms like vaginal discharge and erythema were found in women infected with TVVs, there was no significant association between the presence of dsRNA viral infection of T. vaginalis isolates and symptoms.
The presence of TVV has also been associated the expression of T. vaginalis virulence factors. Although this infection makes upregulation of a highly immunogenic protein, P270 (13), the expression of surface P270 was just noticeable in T. vaginalis cells infected with TVV2 and TVV3, suggesting that the transcription of the P270 gene is likely regulated by viral factors common in type 2 and type 3 viruses (29).
In the current study, infection with all of the three TVV1, TVV2 and TVV3 species of Trichomonas virus were identified in Iran and TVV2 and TVV3 species of Trichomonas virus were reported for the first time. BLAST comparisons of the partial nucleotide sequences and amino acids confirmed the viruses as T. vaginalis virus.

Conclusion
This research is the first report of the T. vaginalis infection by TVV2 and TVV3 in Iran. The prevalence of TVV subtypes is noticeable in T. vaginalis in Iran and three dsRNA viruses TVV1-TVV2 and TVV3 were detected using RT-PCR in T. vaginalis isolates. Single and multiple infections to different subtypes of TVV in T. vaginalis isolates were observed in this study.