Detection of HTLV-1 RNA in Plasma

HTLV-1 viral RNA is detected rarely in plasma of HTLV-1 infected subjects.

Maria Antonietta Demontis, Maaz Tahir Sadiq, Simon Golz and Graham P Taylor

Section of Infectious Diseases, Department of Medicine

Imperial College

Norfolk Place

London W2 1PG

Running head:Detection of HTLV-1 in plasma samples

Keywords: HTLV-1, detection, plasma, diagnosis, nested PCR, quantitative PCR, HTLV-1 RNA.

Abstract Word Count 223

Word Count – Body of Text 1893

Corresponding author:

Graham P. Taylor, Professor of Human Retrovirology, Infectious Diseases, Department of Medicine, St Mary's Campus, Imperial College Norfolk Place London W2 1PG

Telephone:020 7594 3910

Fax Number:020 7594 3910

E-Mail:

Abstract

Background: Plasma of patients infected with HTLV-1 is considered non-infectious but detection of HTLV-1 genomic RNA in plasma has been recently reported. The aim of this project was to detect and quantify HTLV-1 RNA in plasma and assess its potential value in diagnosis and prognosis.

Methods:RNA from one milliliter of plasma from 65subjects infected with HTLV-1 (27 asymptomatic carriers (AC), 17 patients with HTLV-1-associated myelopathy (HAM/TSP), 14 with Adult T-cell Leukaemia/Lymphoma (ATLL), two co-infected with HIV and five with other HTLV-1-associated disease, was extracted and reverse transcribed. HTLV-1 specific nested PCR was performed using primers to amplify the conserved Tax region. All samples were run in quadruplicate, nested PCR products were detected by gel electrophoresis.

Results:HTLV-1 RNA was detected in plasma from 18(28%) patients,always at a very low copy number (3-13 copies viral cDNA per milliliterof plasma). Mean values of HTLV-1 proviral load did not differ between patients in whom HTLV-1 RNA was detected and patients in whom it was not possible to detect HTLV-1 RNA in plasma.

Conclusions:HTLV-1 genomic RNA can be detected in the plasma of a minority of patients but not at a level or frequency to be useful clinically or diagnostically. Lack of transmission of HTLV-1 by plasma is due to the rare presence of HTLV-1 virions, regardless of any other factor.

Introduction

Human T-cell lymphotrophic virus (HTLV) was the first retrovirus to be associated with human disease [Poiesz et al.1980]. HTLV Type 1 (HTLV-1) is accepted to be the aetiological agent of adult T-cell leukemia/lymphoma (ATLL)[Yoshida et al. 1984; Takatsuki et al. 1985] and HTLV-1-associated myelopathy/Tropical spastic paraparesis(HAM/TSP) [Gessain et al. 1985] and is associated with a number of other inflammatory conditions[Vernant et al. 1987; Morgan et al 1989; Nishioka et al 1989].

HTLV-1 exists predominantly as cell-bound provirus. HTLV-1 virions are considered to be poorly infectious, requiring prolonged cell-cell contact for trasmission[Derse et al. 2001]. Additionally, already infected cells produce a very small number of free particles, of which only 1 in 105 are infectious[Fan et al 1992].

HTLV-1 RNA transfers from an infected to an uninfected cell by means of a virological synapse [Igakura et al. 2003].This structure, similar to immunological synapses and allowing for direct transmission of HTLV-1 viral particles across small intercellular spaces, has been observed ex vivo[Majorovits et al. 2008]. It is believed that most cell-to-cell transmission occurs via this mechanism[Bangham, 2003]. Whilst some degree of such ‘infectious spread’ must occur in vivo, HTLV-1 Tax-induced mitotic clonal expansion is considered to be a major contributor to the HTLV-1 infected cell burden[Wattel et al. 1996; Tanaka et al. 2005;].

It has long been observed that plasma from HTLV-1 infected patients is not infectious. Whether this is due to the absence of HTLV-1 virions from plasma or because they are non-infectious is not clear. Recently, Cabral et al[Cabral et al. 2012] reported the detection of HTLV-1 RNA in 8% of plasma samples from asymptomatic carriers (AC) and patientswith HAM/TSP. Given the ease of handling of plasma compared with peripheral blood mononuclear cells (PBMCs) the aims of this study are: 1)to determine whether HTLV-1 RNA can be detected in plasma;2) if detectable, whether this has comparable diagnostic and prognostic value to the current assays, which quantify HTLV-1 proviralDNA in PBMCs; finally, 3) to investigate whether viral load was quantifiable also in plasma, as in PBMC. .

Methods

Plasma samples from 65 patients, whose HTLV-1 infection had been confirmed by Western Blot, were analysed. HTLV-1 proviral load in PBMCs for each sample time point was measured routinely as described previously [Demontis et al. 2013]. Samples were categorized according to the donor’s HTLV-1-related clinical state:27 asymptomatic carriers, 17 patients with HAM/TSP, 14 patients with ATLL, two patients co-infected with HIV, two with polymyositis, two with neurological symptoms not HAM and one with strongyloidiasis (TableI). Plasma samples from 13 HTLV-1 uninfected subjects were used as negative controls. Seventeen samples (four from AC, seven from patients with HAM/TSP and six from patients with ATLL) were repeated to assess the reproducibility of the method. From six patients (two AC, two patients with HAM/TSP and two with ATLL) a second time point,at least two years late, was analysed (sample pair had a proviral load within the range of intra-patient variability)

RNA Extraction and Reverse Transcription

Samples were thawed at room temperature and 1ml plasma was aliquoted. Plasma was centrifuged at 20,000g, 4°C, for one hour. The upper 860µl of plasma were removed and the remaining 140µl were processed using spin column protocol of the QIAMP Viral RNA MiniKit (Qiagen, Germany) according to the manufacturer’s instructions. The resulting RNA was eluted in 60µl of Elution Buffer and stored at -80°C until used. RNA was reverse transcribed (RT) using the QuantiTect RNA Reverse Transcription Kit (Qiagen, Germany) according to the manufacturer’s instructions. All apparatus and work surfaces were treated with Ambion DNA zap and RNase zap prior to conducting reverse transcription. A Geneamp 9700 PCR system was used for incubation at 42°C for 15 minutes followed by 95°C for 3min. The protocol also includes addition of 2µl gDNAwipeout per 12µl RNA to prevent carryover of DNA contaminants. To exclude the presence of genomic DNA from RNA preparation, a minus reverse-transcriptase control was included in RT-PCR experiments. All resulting 20µl cDNA samples were purified with the QIAquick PCR Purification Kit (Qiagen, Germany) and suspended in 50µl elution buffer for storage at -80°C.

HTLV-1 viral RNA detection.

All the cDNA samples were subjected to HTLV-1 detection byquantitative PCRin duplicate [Demontis et al 2013] and by highly sensitive semi-quantitative nested PCR in quadruplicate [Tosswill et al. 1998]. Primers published previously,SK43IF(5’-CGGATACCCAGTCTACGTGT-3’)and SK44IR (5’-GAGCCGATAACGCGTCCATCG-3’)[Kwok et al. 1988], were used to amplify a 159-bp fragment of DNA from the tax region of HTLV-1 cDNA.The lower detection limit was three copies of HTLV cDNA per milliliter of plasma.

To confirm the nature of the PCR products four of these were sequenced by cycle sequencing using an ABI 3100 GeneticAnalyser according to the manufacturer’sinstruction. The sequences were then edited and analysed and compared to the HTLV-1 tax reference sequence, using the programmeSequencher (Sequencer, GeneCodes Corporation (URL:

Validation of RT efficiency

To ensure the reliability of the RNA extraction and cDNA generation, serial dilutions of cDNA obtained from a plasma sample of a patient with untreated HTLV/HIV co-infection and with aknown HIV viral load with a commercially used assay (1300 copies/ml, COBAS TaqMan HIV-1 assay) was amplified by semi-nested PCR using HIV integrase specific primers (SID1: 5’-AAGACAGCAGTACAAATGGCAGT-3”, SID2: 5’- TACTGCCCCTTCACCTTTCCA-3’ and SID3: 5’- CAATTTTAAAAGAAAAGGGGGGATT-3) to generate a 198bp product.In the first round the reaction volume of 25µl (composed of 2.5µl 10x buffer, 1.5µl MgCl2 (25mM), 0.5µl Primers, SID1 and SID2, 0.5µl dNTPs (10mM), 0.1µl Taq Polymerase (5U/µl), 14.9µl Nuclease free water and 5µl of sample cDNA) was subjected to the following thermo cycling conditions: 94°C for 10mins, 25 cycles of 94°C for 30s, 55°C for 30s, 72°C for 30s and a single cycle of 72°C for 7mins.

The second round of PCR was conducted immediately after the first round using a reaction mix of 25µl composed of 2.5µl 10x buffer, 1.5µl MgCl2 (25mM), 0.5µl Primers, SID2 and SID3, 0.5µl dNTPs (10mM), 0.1µl Taq Polymerase (5U/µl), 18.9µl Nuclease free water and 1µl of first round product. Amplification conditions were: 94°C for 10mins, 39 cycles of 94°C for 30s, 55°C for 30s, 72°C for 30s and a single cycle of 72°C for 7mins. Amplification products were separated on a 2% agarose gel at 100V for 40minutes employing SYBR Green (Invitrogen).

Results

Using the HTLV-1 specific nested PCR, products of the correct size (159bp) were detected in the first plasma sample of18/65 (28%) patients.

The frequency of detection of HTLV-1 viral RNA did not differ by clinical status.

The selected amplicons were confirmed to be HTLV-1 by sequencing.Using Poisson distribution the HTLV-1 cDNA copy was 3-13 cDNA copies generated from 1 ml of plasma.

All no template controls, minus reverse-transcriptase controls and HTLV-1 uninfected plasma controls were negative by quantitative PCR and nested PCR.HTLV RNA was not detected in any sample from the patients by quantitative PCR. HIV integrase RNA was detected in the stored plasma of the HIV-1/HTLV-1 co-infected patient at a concentration of 560 RNA copies/ml compared to the 1300 RNA copies/ml obtained with the commercial assay.

Due to the very low concentration of cDNA, to test the variability of the method and how consistently results could be reproduced,the same samples of 16 patients (12 initially negative and four initially positive) were assayed again. A concordant result was obtained in 12/16 (75%) whilst 4/16 (25%) were discordant: two initially negative were positive and two initially positive were negative.This degree of discordancy is consistent with the target sequence being present at the limit of detection. A plasma sample from a different time point was also assayed for six patients. Two were repeatedly negative and four were discordant:negative to positive at the Lowest Detection Limit (threecDNAs copies/ml) for two patients and positive to negative fortwo patients.

Plasma HTLV RNA was detectable in similar proportions of symptomatic patients (grouped together) and asymptomatic carriers (p=0.41 by Fisher’s exacrt test).

The proviral load values in PBMC were lower in subjects who were positive for HTLV in the plasma (10.8±21.9, mean±SD) compared to those who were negative (14.6±21.4); however this difference was not significant (p=0.46 by analysis of variance, figure I).

Discussion

Although whole blood, cellular blood products and tissue can transmit HTLV-1 it has long been recognized that plasma from patients infected with HTLV-1 is not infectious. Retrospective studies have never conclusively reported HTLV-1 transmission to recipients of cell free blood products from infected donors[Sullivan et al. 1991; Manns et al. 1992; Kleinman et al. 1993; Blejer et al. 1995; Namen-Lopes et al. 2009].

The apparent inability of plasma to transmit HTLV-1 could be explained by one, or a combination of two theories. Either the plasma of patients infected with HTLV-1 does not contain HTLV-1 virions, or only in quantities insufficient for transmission, or such plasma contains virions but they are not infectious. In support of the second theory Fan et al. demonstrated that only a very small minority of cell-free virions produced by HTLV-1 infected cells or continuous producer cell lines such as MT-2, are infectious (in vitro) [Fan et al. 1992]. However, the first hypothesis is widely believed, but data are sparse with the single paper addressing this reporting the detection of HTLV-1 RNA in the plasma of 8% of HTLV-1 infected persons, who were either asymptomatic carriers or patients with HAM/TSP[Cabral et al. 2012].

In this study the frequency at which HTLV-1 RNA can be detected in patient plasma as well as the relationship of plasma HTLV-1 RNA to disease including patients with ATLL, who have the highest HTLV-1 proviral burden, has been explored. HTLV-1 viral RNA was detected by a highly sensitive and specific nested RT-PCR in 28% of the subjects, being AC, patients with HAM/TSP, or ATLLbut only at or just above the threshold of detection by nPCR and below that of qPCR.Using this semi-quantitative method 3 – 13 HTLV-1 cDNA copies were found per 1ml plasma which, assuming 25% RT efficiency,equates to 12 – 56 HTLV-1 RNA copies per ml plasma. This low level of HTLV-1 RNA was confirmed by repeated testing of the same or subsequent samples indicating that at such a low copy number sampling contributes significantly to the frequency of detection. The presence of such a small number of HTLV-1 RNA copies is likely to be the prime reason why plasma from HTLV-1 infected patients does not transmitthis infection.

High proviral load is known to be a risk factor for the development of ATLL[Okayama et al. 2004] but this did not correlate with detectability of HTLV-1 in plasma as patients with and without malignant disease manifestations had comparable HTLV-1 plasma viraemia.The potential of HTLV-1 plasma viraemia as a marker of disease progression is thus extremely limited. Molecular detection of HTLV-1 RNA in plasma cannot be used to screen for HTLV-1 infection and does not correlate with the presence of HTLV-1 associated disease.Since all patients in the study had chronic infection the possibility of HTLV-1 RNA in plasma during acute infection cannot be discounted.

These data support the importance of the virological synapse and the role of cell-to-cell transmission in infectious spread of HTLV-1 in vivo.Although not examined these data also suggest that cell-to-cell spread is important in both mother-to-child transmission and sexual transmission of HTLV-1 but this needs to be confirmed.

Sources of Funding

Internal. GPT is supported by the Imperial Comprehensive Biomedical Research Centre

Competing interests

The authors declare that they have no competing interests.

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