Journal of Clinical Virology - Volume 82 - Supplement 1 - September 2016

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Abstracts / Journal of Clinical Virology 82S (2016) S1–S142 S13
due to lack of pre-existing immunity. Conversely, non-protective
immunity from previous exposure to circulating GII.4 subtype
strains may reduce the duration of shedding.
Abstract no: 73
Presentation at ESCV 2016: Oral 23
Molecular characterization of long-term
shedding of respiratory syncytial viruses
isolated from consecutive samples collected in
haematological patients
Julia Tabatabai ∗, Alexander Thielen ,
Nicola Lehners rtin Daeumer
Paul Schnitzler
1 Department of Infectious Diseases, University
Hospital Heidelberg, Germany
2 Institute of Immunology and Genetics
Kaiserslautern, Germany
3 Department of Internal Medicine V, University
Hospital Heidelberg, Germany
Objective: Respiratory syncytial virus (RSV) is a frequent cause
of upper respiratory tract infections, but can be associated with
severe and prolonged infection in immunocompromised patients.
The objective of this study was to investigate the genetic variability
of RSV in haematological patients with prolonged RSV infection.
Methods: To inform for infection control measures during the
winter seasons, haematological patients at the University Hospital
Heidelberg are routinely screened for respiratory viruses on admis-
sion to a ward or presentation in the outpatient department. In
RSV positive patients, screening was continued on a regular basis
until three consecutive samples were tested negative. Nasopha-
ryngeal swabs (NPS) were screened for influenza, parainfluenza
and RSV using rtPCR during the winter seasons 2011-2013. In
patients with prolonged RSV infection (defined as viral shedding
≥28 days), Sanger sequencing of the second hypervariable region
of the RSV G gene was performed in consecutive samples. Further,
ultra-deep sequencing (UDS) by next-generation-sequencing was
performed in representative samples (three each with the shortest
and longest viral shedding) to identify further RSV variants. Readily
available medical records were retrospectively reviewed to obtain
basic characteristics, clinical and laboratory data.
Results: In total 16 patients with prolonged RSV A infection
were analysed (median shedding 79.5 days, 81.2% male). Phylo-
genetic analysis identified RSV strains as genotype NA1 (2011/12)
or ON1 (2012/13). Almost all patients showed identical sequences
of the G gene at the beginning compared to the end of the shed-
ding period (n = 14/16). However, in two patients with particularly
long viral shedding (333 and 142 days) Sanger sequencing revealed
the presence of mutations leading to premature stop codons. The
G protein was thereby shortened by between 60 and 71 amino
acids. In addition, UDS revealed RSV strain variants (67%) with
premature stop codons at different positions in a patient with the
second longest viral shedding period (155 days). All three patients
with premature stop codons received intravenous immunoglobu-
lins. Interestingly, UDS revealed also a loss of the 72nt duplication
with no further changes in strain variants of the ON1 genotype in
5 of 6 patients.
Conclusion: Long-term shedding of RSV in haematological
patients showed only minor changes of RSV strains and is thereby
not likely caused by re-infections. However, long shedding periods
and lack of immune selective pressure in the immunocompromised
host seems to allow the virus to strip a part of the C-terminus of the
G glycoprotein. To the best of our knowledge, the loss of the char-
acteristic 72nt duplication in ON1 variant strains is here described
for the first time.
Abstract no: 92
Presentation at ESCV 2016: Oral 24
Plasma Torquetenovirus (TTV) DNA load as a
surrogate marker of reconstitution of
CMV-specific immunity in the allogeneic stem
cell transplantation setting
Estela Giménez ∗, Eliseo Albert , Carlos Solano ,
Lisa Macera niele Focosi vid Navarro
1 Microbiology Service, Hospital Clínico
Universitario, Valencia, Instituto de Investigación
INCLIVA, Spain
2 Hematology Service, Hospital Clínico Universitario,
Valencia, Instituto de Investigación INCLIVA, Spain
3 Virology Unit, Pisa University Hospital, Italy
4 North-Western Tuscany Blood Bank, Pisa
University Hospital, Italy
5 Microbiology Service, Hospital Clínico
Universitario, Valencia, Instituto de Investigación
INCLIVA, Department of Microbiology, School of
Medicine, University of Valencia, Spain
Background:Torquetenoviruses (TTV) are small circular ssDNA
viruses classified within the family Anelloviridae. TTV DNA can be
detected in plasma of most individuals at variable levels. Trans-
plant recipients are particularly prone to carry high TTV burdens in
blood. The dynamics of TTV viremia in allogeneic stem cell trans-
plant recipients (Allo-SCT) were previously shown to correlatewith
that of graft reconstitution, suggesting that TTV replicatesmainly in
hematopoietic cells. In this context, we investigated whether mon-
itoring of TTV DNAemia early after transplant permits to infer the
level of CMV-specific T-cell reconstitution following Allo-SCT.
Material and methods: The patients included (n = 23) in
the study underwent Allo-SCT at the University Clinic Hospi-
tal of Valencia between 2013 and 2014. CMV DNAemia was
monitored weekly by a RealTime PCR (Abbott Molecular, Des
Plaines, IL). The quantification of TTV DNA was performed by
a real-time Taqman assay, using serial dilutions of standards
TTV (from 102 to 107). The PCR targeted a segment of the
untranslated region of the viral genome: forward primer 5 -
GTGCCGIAGGTGAGTTTA-3 , reverse primer 5 -AGCCCGGCCAGTCC-
3 and probe 5 -TCAAGGGGCAATTCGGGCT-3 . The detection of TTV
vas carried out in plasma samples obtained at baseline and on days
21, 30 and 60 after transplant. Enumeration of CMV pp65 and IE-
1-specific gamma interferon-producing CD8+ and CD4+ T cells was
performed on day 30 after transplantation by flow cytometry for
intracellular cytokine staining.
Results: TTV DNA was detected in 22 out of 23 patients (95.7%).
At baseline (median day −7, range −8 to +7) TTV DNA load val-
ues ranged between 1.59 log10 to 7.97 log10 (median 3.08 log10).
Baseline median TTV DNA levels were comparable regardless of the
conditioning regimen used. Overall, median TTV DNA levels were
not related to the immunossupressive regimen used for graft vs.
host disease prophylaxis. A total of 14 (60.9%) patients developed
an episode of CMV infection during the first 100 days after trans-
plantation (median 31 days, range 5–61). Plasma TTV DNA levels at
baseline and on day 21 were comparable in patients with or with-
out subsequent CMV DNAemia (P = 0.74). In general there was an
increase in the TTV load over time, which paralleled that of total