Detection and Molecular Characterization of Double-Stranded RNA Viruses in Philippine Trichomonas Vaginalis Isolates Windell L

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ORIGINAL ARTICLE Detection and molecular characterization of double-stranded RNA viruses in Philippine Trichomonas vaginalis isolates Windell L. Rivera a,b,*, Christine Aubrey C. Justo a,b, Mary Ann Cielo V. Relucio-San Diego a,b, Lorenz M. Loyola b

a Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City, 1101, Philippines b Molecular Protozoology Laboratory, Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City, 1101, Philippines

Received 15 July 2014; received in revised form 5 July 2015; accepted 23 July 2015

Available online ---

KEYWORDS Abstract Background/Purpose: The flagellated protozoon Trichomonas vaginalis that para- Female sex sitizes the urogenital tract of humans was reported to harbor double-stranded RNA (dsRNA) vi- worker (FSW); ruses. These viruses, identified as Trichomonas vaginalis virus (TVV), belong to the genus Philippines; Trichomonasvirus of the family Totiviridae. Four species, formally recognized by the Interna- Trichomonas tional Committee on Taxonomy of Viruses (ICTV), have been reported and distinguished by vaginalis virus/ pairwise comparisons of the sequences of genes coding for major capsid protein (CP) and Trichomonasvirus RNA-dependent RNA polymerase (RdRp). Methods: Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the complimentary DNA of target virus genes coding for CP and RdRp. Sequence analyses confirmed the identity of the TVV isolates from T. vaginalis cultures. Results: A total of 35 dsRNA viruses were identified from 18 (19%) T. vaginalis isolates. Multiple TVV species were observed in six of the 18 T. vaginalis cultures. Phylogenetic analyses show monophyly in TVV1 and TVV2 whereas TVV3 and TVV4 appear paraphyletic. The phylogeny of Philippine Trichomonasvirus reflects the global distribution of its host. Conclusion: This is the first study in the Philippines and one of the two reports worldwide to detect the four TVVs and their concurrent infection in a single T. vaginalis isolate. Copyright ª 2015, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. All rights reserved.

* Corresponding author. Tel./fax: þ63 29205471. E-mail address: [email protected] (W.L. Rivera).

Introduction low birth weight in infants,11,12 neonatal ocular and respiratory infections,13,14 and higher probability of contracting Trichomoniasis, caused by the flagellated protozoon Tri- viruses such as the human papillomavirus and the human- 15,16 chomonas vaginalis, is the most widespread nonviral sexu- immunodeficiency virus (HIV). ally transmitted infection (STI) in the world.1 Reports have No studies on TVV have been performed in the shown that T. vaginalis can be infected with mono- Philippines. In this study, T. vaginalis from vaginal swab segmented dsRNA viruses called Trichomonas vaginalis samples of female sex workers (FSWs) attending a social virus (TVV). TVV is a member of the Totiviridae family hygiene clinic in Angeles City, Pampanga, Philippines were under the distinct genus Trichomonasvirus.2,3 The Interna- screened for the presence of TVV. This study is the first tional Committee on Taxonomy of Viruses (ICTV) recognizes report of the detection and characterization of TVV in the four Trichomonasvirus species named as TVV1, TVV2, TVV3, Philippines. and TVV4.4 TVV is globally prevalent with reports as high as 82% in South Africa,5 75% in Baltimore, MD, USA,6 and 55% in Methods Cuba.7 The presence of TVV is important in the typing of T. vaginalis8 and has been suggested to upregulate the Collection of samples proinflammatory response in simultaneous trichomoniasis and bacterial vaginosis.9 Trichomoniasis may occur asymp- This study was approved by the Ethics Review Committee of tomatically but severe cases manifest a variety of symp- the Philippine Department of Health. FSWs attending a toms including malodorous discharge, pruritus, dysuria, and weekly medical check-up in a social hygiene clinic in dyspareunia in women and may lead to urethritis and Angeles City, Pampanga, Philippines (Figure 1) were prostatitis in men.10 Chronic infections have been reported informed and had given written consent prior to providing to lead to cervical erosion, premature labor, premature vaginal swabs. Certified medical personnel assisted in the rupture of the placental membrane, high infant mortality, collection of swab samples conducted from July 2013 to

Figure 1. Map of the collection site in Angeles City, Pampanga, Philippines.

Table 1 BLAST results of Trichomonas vaginalis virus (TVV) isolates in the Philippines. Isolate Genbank acc. no. BLASTn BLASTx Description % Description % (reference) Identity (reference) Identity 14B KJ883444 TVV1 (U08999) 90 RdRp (AET81012) 92 KJ883456 TVV2 (AF127178) 86 RdRp (AAF29445) 89 KJ883464 TVV3 (HQ607515) 85 CP (NP_659389) 69 KJ883467 TVV4 (HQ607526) 88 CP (AED99797) 93 15B KJ883445 TVV1 (U08999) 90 RdRp (AET81012) 92 KJ883457 TVV2 (AF127178) 86 RdRp (AAF29445) 88 KJ883465 TVV3 (HQ607515) 88 CP (AED99799) 85 KJ883468 TVV4 (HQ607526) 84 CP (AED99795) 82 77B KJ883446 TVV1 (U08999) 84 RdRp (ABF57713) 87 80B KJ883447 TVV1 (U08999) 84 RdRp (ABF57713) 88 148B KJ883448 TVV1 (U08999) 90 RdRp (AET81012) 92 319B KJ883449 TVV1 (U08999) 88 RdRp (AAA62868) 89 KJ883458 TVV2 (AF127178) 86 RdRp (AAF29445) 90 KJ883462 TVV3 (HQ607515) 88 CP (AED99799) 85 KJ883469 TVV4 (HQ607526) 88 CP (AED99797) 93 535B KJ883450 TVV1 (U08999) 89 RdRp (AET81012) 92 KJ883459 TVV2 (AF127178) 87 RdRp (AAF29445) 89 KJ883472 TVV4 (HQ607526) 85 CP (AED99797) 80 564B KJ883451 TVV1 (HQ607513) 81 RdRp (AED99812) 85 KJ883460 TVV2 (AF127178) 86 RdRp (AAF29445) 90 KJ883463 TVV3 (HQ607525) 88 CP (AED99801) 78 KJ883470 TVV4 (HQ607526) 89 CP (AED99797) 95 766B KJ883452 TVV1 (HQ607516) 90 RdRp (AED99818) 98 KJ883461 TVV2 (HQ607514) 91 RdRp (AED99808) 93 KJ883466 TVV3 (HQ607515) 87 CP (AED99801) 79 KJ883471 TVV4 (HQ607526) 89 CP (AED99793) 97 773B KJ883453 TVV1 (HQ607516) 91 RdRp (AED99818) 97 779B KJ883454 TVV1 (HQ607516) 91 RdRp (AED99818) 97 780B KJ883455 TVV1 (HQ607516) 90 RdRp (AED99818) 96 Cp Z capsid protein; RdRp Z RNA-dependent RNA polymerase.

November 2013. The swab samples were placed in tubes TVV2F2461-TVV2, TVV3F61-TVV3R482, and TVV4F1338- containing BI-S-33 medium17 supplemented with 10% heat- TVV4R1834.18 The RT-PCR reaction mixture (50 mL) consists inactivated horse serum, 500 mg/mL streptomycin and of 1X Q-buffer, 400mM of each nucleoside triphosphate 500 U/mL penicillin. The samples were then transported to (dNTP), 0.6 mM of each primer, 2 mL Qiagen OneStep RT-PCR the laboratory within 24 hours and incubated at 37C for enzyme mix, and 1 mL of template from the total cellular 24e72 hours. Examination under the light microscope was RNA extract. Thermal cycling conditions were as follows: RT carried out every 24 hours to verify the growth of T. vagi- at 45C for 30 minutes, initial PCR activation at 95C for 15 nalis. The samples positive for T. vaginalis were sub- minutes, 35 cycles of 94C for 10 seconds, 50C for 1 min- cultured and axenized by continuous passage in the same ute, and 68C for 4 minutes; and a ﬁnal extension of 68C medium under similar conditions. for 10 minutes. The ampliﬁed cDNA were visualized under UV light in a 1.5% agarose gel stained with SYBR Safe stain (Invitrogen, Life Technologies, CA, USA). The expected Detection of TVV through reverse transcription sizes of amplicons for TVV1, TVV2, TVV3 and TVV4 were 569 polymerase chain reaction (RT-PCR) base pairs (bp), 625 bp, 437 bp, and 514 bp, respectively.

The extraction of total RNA from axenized cultures of T. vaginalis grown for 24 hours was done using TRIzol reagent Nucleotide sequence analyses (Ambion, Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol. Species-specific primers and Samples presenting an amplicon of approximately 500 bp the OneStep RT-PCR kit (Qiagen GmbH, Hilden, Germany) were purified using QIAquick Gel Extraction kit (Qiagen were used to amplify the complementary DNA (cDNA) of the GmbH, Hilden, Germany). PCR products were sent to Mac- target virus genes. The speciesespecific primer pairs used rogen Inc., Seoul, South Korea for direct sequencing. The in the detection of TVV include TVV1F2875-TVV1R3443, nucleotide sequences were processed using BioEdit

was measured using the Xia test in DAMBE v5.2.1 (http:// Table 2 Summary of the prevalence of Trichomonas dambe.bio.uottawa.ca.). Model selection for building vaginalis virus (TVV) in Philippine T. vaginalis (n Z 96). phylogenetic tree was carried out using jModelTest Prevalence, Ampliﬁed Amplicon v2.1.4.20 Maximum parsimony (MP) and neighbor-joining % (actual count) gene/region size (bp) (NJ) trees were created using PAUP* and maximum likeli- Single TVV 13 (12) hood (ML) trees were generated from PhyML 3.0 online Multiple TVV 6 (6) (http://www.atgc-montpellier.fr/phyml) with 1000 boot- TVV1 13 (12) RdRp 505e568 strap replicates. Bayesian inference (BI) was done using 21 TVV2 13 (12) RdRp 542e603 MrBayes v3.1.2. Tree editing with bootstrap and posterior 22 TVV3 5 (5) 50 UTR, CP 305e314 probability values was done in TreeGraph 2.0.47. Sub- TVV4 6 (6) CP 488e468 mission of the sequences to Genbank was done using Sequin Application v13.05 (http://www.ncbi.nlm.nih.gov/Sequin/ Cp Z capsid protein; RdRp Z RNA-dependent RNA polymerase; ). The nucleotide sequences and predicted amino acid se- UTR Z untranslated region. quences of the Philippine TVV isolates were deposited and are available in Genbank through accession numbers KJ883444eKJ883472 (Table 1). v7.2.5.19 BLASTn conﬁrmed the identity of the TVV isolates and BLASTx provided the predicted amino acid and frame used for the protein translation in Sequin Application Results v13.70 (http://www.ncbi.nlm.nih.gov/Sequin/). Pairwise comparisons of the nucleotide and amino acid sequences Philippine T. vaginalis isolates infected with TVV were carried out using the program EMBOSS Needle (http:// www.ebi.ac.uk/Tools/psa/emboss_needle/). Alignment of A total of 772 vaginal swabs were collected from FSWs. T. sequences with reference isolates was performed by visual vaginalis was observed in 96 (12%) samples through culture inspection and using ClustalW of BioEdit v7.2.5.19 The method. Eighteen (19%) of the 96 T. vaginalis isolates were saturation of substitution of the sequences for phylogeny infected with a total of 35 dsRNA viruses. Table 2 shows the

Figure 2. Multiple TVV-infection in representative Philippine T. vaginalis isolates. The four species of TVV were identiﬁed through RT-PCR. The negative control is a no template control and the marker used is KAPA Universal Ladder with fragments ranging from 100 bp to 10 kbp. The arrow indicates the 500 bp fragment.

prevalence of TVV in the T. vaginalis isolates. The gel proﬁle of representative isolates is shown in Figure 2.

Nucleotide sequence analyses of TVV isolates

Direct sequencing and homology search in BLASTn conﬁrmed the identity of the detected dsRNA viruses through RT-PCR. A cut-off value of < 50% identity23 in both nucleotide and amino acid sequences was adapted. Tables 1 and 3 present the BLAST results and pairwise comparisons of the identiﬁed Philippine TVV, respectively. 78 66 78 100 93 89 Phylogenetic analyses of TVV isolates

Phylogenetic analyses of the Philippine TVV isolates were performed using partial nucleotide sequences of the dsRNA viruses. Separate phylogenetic trees for each TVV species (Figure 3) were constructed, as different regions of the viral genome have been ampliﬁed (Table 2). ML trees as well as MP show monophyletic classiﬁcation of the TVV1 and the TVV2 isolates. Paraphyly was observed in the ML

ddddd tree of TVV3 and TVV4 isolates, but MP values indicate a monophyletic assembly of the isolates. NJ bootstrap and BI was adapted. posterior probability values further support the classiﬁca- 23 tion of the identiﬁed TVV isolates.

Discussion a 50% identity

< Angeles City is a highly urbanized city in the Philippines that serves as a priority area in the Philippines for STI control and HIV prevention. Prostitution has been prevalent in the city since the early days and programs to reduce STI/HIV/ AIDS by government and nongovernment organizations were 24 ddddddddddddddddd ddddddddddddddddd ddddddddddddddddd ddddddddddddddddd ddddddddddddddddd ddddddddddddddddd

virus (TVV) isolates. implemented and continuously developed. To prevent transmission of sexual diseases, FSWs working in licensed ). A cut-off value of establishments are required to have a weekly medical check-up in the local social hygiene clinic. The prevalence of T. vaginalis in the Philippines is increasing. The T. vaginalis prevalence in this study (12%) is consistent with previous reports of increasing T. vaginalis infection in the country. In 2002, T. vaginalis was detected through wet

Trichomonas vaginalis mount/culture method in 3.18% of 2267 women, without vaginal bleeding, attending selected health facilities across the Philippines.25 In 2008, T. vaginalis was detected by PCR in 6.81% of 969 sex workers attending social hygiene clinics across the Philippines.26 In 2010, the prevalence rate of

TVV1 TVV2trichomoniasis detected TVV3 by PCR among 377 TVV4 FSWs in Angeles City was 9.55%.24 Eighteen of the 96 samples positive for T. vaginalis were infected with TVV. The presence of TVV, as well as metronidazole susceptibility has been found to differ 8

http://www.ebi.ac.uk/Tools/psa/emboss_needle/ signiﬁcantly in the two types of T. vaginalis. The prevalence of TVV suggests that Philippine T. vaginalis is mainly type 2, which is typically free of TVV and resistant to metronidazole. More study is needed to conﬁrm the type of T. vaginalis in the Philippines.

Pairwise identity scores of the Philippine A total of 35 viruses were identiﬁed in 18 T. vaginalis 94 9398 80 99 79 83 94 83 100 98 93 92 100 74 76 72 77 72 73 72 73 63 67 89 100 89 100 100 89 89 100 84 95 76 85 51 100 100 89 80 99 82 100 83 95 88 99 10081 79 82 81 100 99 97 78 94 75 99 78 78 75 76 75 74 75 74 71 66 100 65 100 90 100 94 86 51 100 96 86 83 83 100 88 76 76 76 84 84 96 100 81 76 79 77 74 74 74 64 83 8381 78 8280 80 77 8080 83 77 73 8070 81 77 73 71 78 80 77 67 76 82 80 66 77 100 82 70 77 83 76 83 67 79 100 80 74 71 80 92 93 74 71 100 93 92 66 100 100 87 88 100 98 85 85 98 87 85 85 87 86 100 78 98 100 87 86 88 100 41 81 58 81 44 100 44 75 44 100 95 79 92 95 76 92 82 100 80 91 95 100 14B 15B 77B 80B 148B 319B 535B 564B 766B 773B 779B 780B 14B 15B 319B 535B 564B 766B 14B 15B 319B 564B 766B 14B 15B 319B 535B 564B 766B 100 99 78 81 100 95 98 78 76 75 75 66 100 100 89 100 94 85 100 83 85 77 89 100 88 99 83 87 85 100 99 81 84 100 95 98 77 76 75 75 66 cultures, with multiple TVVs detected in six of the cultures (Table 2). Single infection of T. vaginalis with either TVV1 Identity scores (%) of the nucleotide sequences are highlighted and shown above and right of the 100% diagonal and those of the

a or TVV2 is consistent with the higher detection of TVV1 and 319B 535B Table 3 predicted amino acid areprogram shown EMBOSS left Needle and ( below. Pairwise comparison of the nucleotide and amino acid sequences was done using the 14B 15B 77B 80B 148B 564B 766B 773B 779B 780B TVV2 than the remaining two TVV species.27 TVV has been

Figure 3. ML trees of the four dsRNA viruses [(AeD) TVV1eTVV4, respectively] belonging to the genus Trichomonasvirus. Se- quences were derived from the amplified cDNA of Philippine dsRNA viruses (in bold letters) using species-specific primers in RT-PCR. The optimum model of nucleotide substitution for constructing the phylogeny of TVV1, TVV2, and TVV3 is GTRþG and HKYþG for TVV4. Indicated at the end of the nodes are the posterior probability values from BI and bootstrap values from ML, NJ and MP, respectively. Posterior probability values lower than 0.95 and bootstrap values lower than 75% are not shown. The outgroup Eimeria brunetti RNA virus 1 (EbV1) is a member of the Totiviridae family. found to promote inflammatory reaction in an in vitro Concurrent TVV infection, with at least three TVVs, was hosteparasiteeendosymbiontebacteria environment. TVV recorded in six T. vaginalis sample cultures. The presence and the protozoon’s surface lipophosphoglycan play of multiple TVVs in a single culture may be due to a mixture important roles in upregulating inflammatory reaction in of parasites infected with different TVVs or due to con- simultaneous trichomoniasis and bacterial vaginosis.9 current infection of TVVs in a single parasite. Having mul- Additionally, it may cause persistent infection of T. vagi- tiple sex partners and low condom use25 may have nalis in humans by altering the surface expression of the contributed to the detection of multiple TVV in T. vaginalis highly immunogenic protein P27028,29 and cysteine pro- culture derived from one FSW. A pool of parasites infected teinases.30 In agreement with previous reports of TVV- with distinct TVV species transmitted by and to different associated cytopathology,30,31 it was observed during this individuals may increase the prevalence of multiple TVV study that TVV-infected T. vaginalis was harder to maintain infections. The occurrence of multiple TVVs in a single T. in culture than the uninfected ones except for one isolate vaginalis is supported by other studies.32,33,18 The coexis- (564B) harboring four TVVs. tence of different viral species in a single host has also been

Please cite this article in press as: Rivera WL, et al., Detection and molecular characterization of double-stranded RNA viruses in Phil- ippine Trichomonas vaginalis isolates, Journal of Microbiology, Immunology and Infection (2015), http://dx.doi.org/10.1016/ j.jmii.2015.07.016 + MODEL dsRNA viruses in Philippine T. vaginalis isolates 7 observed in the genera Totivirus and Victorivirus.18 The 2. Goodman RP, Ghabrial SA, Fichorova RN, Nibert ML. Tricho- nonlytic life cycle, intracellular transmission and well- monasvirus: a new genus of protozoan viruses in the family adapted nature of the virus causing few deleterious ef- Totiviridae. Arch Virol 2011;156:171e9. fects and largely noncytopathic, persistent infection in its 3. Fraga J, Rojas L, Sariego I, Fernandez-Calienes A. Genetic host2 may contribute to the occurrence of stable concur- characterization of three Cuban Trichomonas vaginalis virus. Phylogeny of Totiviridae family. 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