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

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Abstracts / Journal of Clinical Virology 82S (2016) S1–S142 S131
analysis of primer and probe regions from positive clinical materi-
als, selection and validation of new, updated primers and probes.
Results: Analysis of sequences obtained from GenBank and
clinical materials showed that primer and probe sequences for
detection of influenza (Flu) A virus, parainfluenza virus (PIV) 1,
human rhinovirus (HRV) and human coronaviruses (HCoV) 229E,
NL63, and HKU-1 still showed a 100% match to the circulating virus
genomes. However, primer and/or probe sequences for detection
of Flu B, RSV, PIV2, PIV3, PIV4, human metapneumovirus (HMPV),
and HCoV OC43 required some adjustment. The RSV assay, that
detects both RSV-A and RSV-B, consisted of two sense primers,
one antisense primer and two probes. All sequences obtained from
RSV positive clinical isolates from 2013 contained a mismatch to
both probe sequences. This mismatch was also observed in two
sequences from GenBank (both from The Netherlands, 2012) but
not in other L gene sequences fromGenBank. Validation of adapted
RSV probes was ongoing at the moment that a RSV RT-PCR neg-
ative sample resulted in a RSV positive culture. The cultured RSV
strain was negative in the diagnostic RT-PCR, but positive in the
updated RSV RT-PCR that was validated at that moment. However,
the relative fluorescent unit (RFU) signal of the RSV strainwas lower
than that of positive control material and sequence analysis of the
strain showed amismatchwith the newprobe. This additional mis-
match was not observed in sequences obtained from GenBank, but
in January and February 2014 several clinical samples tested in our
setting showed RSV signals with low RFU and turned out to have
the samemismatch. Therefore, a newupdate of the RSV RT-PCRwas
started. Another target of the RT-PCR was considered, but align-
ment of over 100 whole genomes of RSV showed that the current
target region in the L gene remained the target of choice. The two
relatively short taqman probes were replaced by a longer taqman
probe that should be able to better tolerate mismatches. The new
RSV assay will be used next RSV season.
Conclusion: Due to the highmutation frequency in RNA viruses,
regular update of RT-PCR assays ismandatory for reliablemolecular
detection of respiratory viruses.
Reference
[1] Templeton et al., Rapid and sensitive method using multiplex real-time PCR for
diagnosis of infections by influenza A and influenza B viruses, respiratory
syncytial virus, and parainfluenza viruses 1, 2, 3, and 4.
Abstract n◦: 84
Presentation at ESCV 2016: Poster 223
Genetic characterization of human respiratory
syncytial virus (hRSV) infecting children in
France during two winter seasons
F. Boulouard ∗, A. Luthon , J. Hamel ,
J. Brouard , V. Gajdos , A. Vabret ,
J. Dina
1 CHU de Caen, Department of Virology, Caen
F-14000, France
2 Université Caen Normandie, Medical School, Caen
F-14000, France
3 National Reference Center for Measles and
Respiratory Paramyxovirus, France
4 CHU de Caen, Department of Pediatrics, Caen
F-14000, France
5 Hôpitaux de Paris (APHP), Department of
Pediatrics, Hôpital Antoine Béclère and Université
Paris 11, Clamart, France
Background: Worldwide, the human respiratory virus (hRSV)
genetic characterization takes a significant place and highlights the
importance tomonitor the circulation of different genotypes and/or
the emergence of new variants. These can affect the susceptibility
to the current or future treatments of hRSV infection. There is no
information to date regarding the molecular epidemiology of hRSV
in France. The aimof this studywas to investigate the genetic diver-
sity of group A and B hRSV isolates, obtained from children under
1 year old, during two recent consecutive epidemic periods.
Material and methods: Nasopharyngeal swabs or aspirates
obtained from children included in a study who evaluate the
efficacy of the use of salt solution 3% in the management of non-
complicated bronchiolitis, the “GUERANDE” study, were analyzed.
The samples were collected in hospital centers in France who
participate to the study during two winter seasons, 2012–2013
and 2013–2014. Viral ARN was extracted using Qiasymphony DSP
Virus/Pathogen Mini kit® . All samples were tested by a real time
RT-PCR for the detection and group typing of the hRSV A/B. The
amplification and sequencing of the second variable region (HRV-2)
of the G gene were performed using One-step RT-PCR kit (Qia-
gen, Hilden, Germany) and specific primers and protocols. The
sequences obtained and reference sequences for different geno-
types were analyzed with BioEdit® software and phylogenetic tree
were constructed by the neighbor-joining method in MEGA7 soft-
ware.
Results: A total of 719 samples were included in the
“GUERANDE” study. These samples were collected from children
under 1 year old consulting for a non-complicated bronchi-
olitis in 24 hospital centers distributed in 12 different French
regions. The hRSV group typing identified 375(52.16%) hRSV-A,
247(34.35%) hRSV-B, as well as 14(1.95%) hRSV-A/B co-detections
and 83(11.54%) were negatives for hRSV detection. The ampli-
fication and sequencing of the HRV-2 G gene were successfully
undertaken for 228(60%) of the 375 hRSV-A and 215(87%) of the
247 hRSV-B.
The analyzed sequences of hRSV-A fell within different clusters
genotypes, corresponding to ON1 in the majority of cases, but also
NA1 and GA2. The ON1 identified sequences were closely related to
GA2. The sequences that had been sampled in different epidemics
dose not formed distinct clusters.
The phylogenetic analysis of hRSV-B sequences allows the iden-
tification of 3 genotypes, BA-9, BA-10 and BA-C. A distinct BA-9
cluster was observed for the sequences sampled in Toulouse.
This cluster was confirmed by different phylogenetic analysis. The