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

137 / 152

Abstracts / Journal of Clinical Virology 82S (2016) S1–S142 S133
persistent primary infection or a reactivation, with a lot of different
clinical symptoms.
It is well known, that the favourite location of EBV is repre-
sented by B-cells on one hand, but also by epithelial and particularly
oropharyngeal cells, on the other.
So we developed an immunotherapy administered by sublin-
gual way, as it is already the case in allergy treatment, allowing a
close and immediate contact between the virus and the molecular
complex for its neutralization.
Besides clinical observation two biological parameters allow us
to measure the impact of our treatment – the evolution of the viral
serology on one side, – the extent of the virus load on the other
side.
In both cases we observe very often an improvement of one
and/or the other.
Abstract no: 310
Presentation at ESCV 2016: Poster 227
Evaluation of the antiviral activity of an
aqueous extract from Solidago virgaurea
against Herpes simplex virus type 2
A.R. Mendes ∗, S. Lopo , L. Ascensão , P.L. Fale ,
M.L. Serralheiro F. Caeir
1 Centro de Estudos do Ambiente e do Mar Lisboa,
Faculdade de Ciencias, Universidade de Lisboa,
1749-016 Lisboa, Portugal
2 Department of Infectious Diseases, National
Institute of Health-Lisbon, Portugal
3 Centro de Quimica e Bioquimica, Faculdade de
Ciencias, Universidade de Lisboa, Campo Grande,
1749-016 Lisboa, Portugal
Herpes simplex virus type 2 (HSV-2) is widely distributed
through the human population, infecting more than 500 million
people globally lthough typically causing mild diseases this
virus may be responsible for severe infections, mainly in immuno-
compromised patients. Currently, there is a number of systemic
antiviral agents against herpesvirus, the most commonly used
being acyclovir and related drugs. However, long term treatments
with these drugs may result in the development of resistance,
especially in immunocompromised patients, which leads to a con-
tinuous search for new and better therapeutic alternatives
According to the World Health Organization plants are the best
sources for obtaining a wide variety of drugs So, in the last
decades many pharmacological and chemical studies have focused
on medicinal plants to the discovery of new natural antiviral com-
pounds.
In the current study the anti-herpetic action of an aqueous
extract, obtained by decoction from stems/leaves of Solidago vir-
gaurea L. (Asteraceae) was evaluated. Experiments were made in
Vero E6 cell cultures infected with HSV-2 and treated with the
plant extract at different non-cytotoxic concentrations. Infected
non-treated cells were used as controls. Extract cytotoxicity against
Vero E6 cells was assessed by the MTT test. The virucidal effect of
the extract, evaluated by comparison of the titers of virus parti-
cles incubated in contact or in the absence of the extract, proved
null. Anti-herpetic activity was investigated by two types of exper-
iments: (1) treatment of infected cells during virus production
revealed a mean yield reduction of 94% relatively to non-treated
and an IC50 of 35.1 g/mL; (2) treatment of infected cells dur-
ing virus titration shown a slighter inhibition, but significant size
differences between virus plaques formed in treated and control
conditions (smaller in treatment conditions). To a further evalu-
ation of the mechanisms that mediate the inhibitory effect of the
extract, a kinetic of the first 7 h of infectionwas performedwith and
without treatment, to assess possible differences in viral DNA syn-
thesis. DNA samples from infected cells were subjected to PCR with
primers that target the viral DNA polymerase gene and PCR prod-
ucts were visualized in agarose gels. Preliminary results showed
the expected amplicon both in treated and non-treated conditions.
Amplification of viral DNA appears to start after 4 h of infection but,
during the period assayed, only increases under the non-treated
conditions. This result is consistent with the low inhibition induced
by the extract when it is added later than 4 h post-infection. Our
results suggest that S. virgaurea aqueous extract inhibits HSV-2
replication cycle, if added in the early phase of the infection, possi-
bly by interfering with the viral DNA synthesis.
Acknowlegements: Thanks are due for the financial support
to CESAM (UID/AMB/50017), to Centro de Química e Bioquimica
(UID/MULTI/00612/2013) and to FCT/MEC through national funds
and the co-funding by the FEDER, within the PT2020 Partnership
Agreement and Compete 2020.
Reference
[1] K.J. Looker, et al., Bull. World Health Organ. 86 (2008) 805–812.
[2] J. Piret, G. Boivin, Antimicrob. Agents Chemother. 55 (2011) 459–472.
[3] J.C.O. Sardi, et al., J. Med. Microbiol. 62 (2013) 10–24.
Abstract no: 88
Presentation at ESCV 2016: Poster 228
Ex vivo expansion of human cytomegalovirus
specific T cells for adoptive transfer therapy of
patients after hematopoietic stem cell
transplantation – Optimization of cultivation
conditions
Sarka Nemeckova ∗, Jitka Krystofova, Jan Musil,
Petr Hainz
Department of Immunology, Institute of Hematology
and Blood Transfusion, Prague, Czech Republic
Ex vivo expanded virus specific memory donor T cells can be
used in allogeneic hematopoietic stem cell transplantation recipi-
ents for prevention of severe disease caused by infections such as
HCMV, EBV, ADV, BKV, etc. Phenotype and functional properties
of T cell lines are influenced by cultivation conditions. We com-
pared properties of HCMV specific T cells expanded in RPMI 1640
or Cellgro medium with or without addition of 5% human serum
(HS), cell growth factors IL4, IL7, IL21 and HCMV derived peptide
pools. Expanded cells were characterized by flow cytometry. Func-
tional HCMV T cell response specific for viral antigens pp65, IE1,
US3, UL55 was measured by ELISPOT IFN-gamma.
Cultivation in RPMI 1640, 5% human AB serum (CTLmedium) for
13 days gave highest T cell expansion rate (4×) and favoured CD4+
T cells against CD8+ T cells. Supplementation with high concentra-
tion of IL4 (1666U/ml) increased expansion rate and IFN-gamma
response in comparisonwith 1000 lower concentration. The expan-
sion rate was slower in serum free Cellgro medium (expansion by
1.8× at day 15) but the latter favoured CD8+ Tcells against CD4+
Tcells. Cultivation in the CellGro medium resulted in high yields of
Streptamer HLA B07-RPHERNGFTVL (pp65) positive CD8+ T cells
which expanded 120× in comparison with day 0 whereas in CTL
medium they expanded 30× only. Phenotype of HCMV Strep+ T
cells resembled the phenotype of effector memory T cells (CCR7−,