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

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Abstracts / Journal of Clinical Virology 82S (2016) S1–S142 S91
ues. Differences between the groups were analyzed for statistical
significance using T-test.
A significantly higher expression of mRNA levels of XRCC1, DNA
PkI and FasR was detected in HIV infected individuals than in HD
(XRCC1: CT naïve = 13.7; CT INI-cART = 15.2 and CT HD = −5.5;
p < 0.05. DNA Pk1: CT naïve = 9.7; CT INI-cART = 14.3 and CT
HD = −2.2; p < 0.05; FasR: CT naïve = 14.5; CT INI-cART = 12.3
and CT HD = −0.4; p < 0.05).
No significant differences in expression of DNA Pk II, Lig III
and Parp-1 mRNA levels between treatment naïve patients, ART
containing INI treated patients and healthy donors were detected
(DNA Pk II: CT naïve = 6.7; CT INI-cART = 5.3; CT HD = 11.14;
Lig III : CT naïve = 8.1; CT INI-cART = 8.44; CT HD = 13.08;
Parp-1: CT naïve = 4.6; CT INI-cART = 5.6; CT HD 8.1; p > 0.05).
The expression levels of some DNA damage genes (XRCC1, FasR
and DNA PKI) are higher in HIV+ patients than in healthy donors. No
difference of DNA PK II, Parp-1 and Lig III alpha mRNA expression
levels were observed between HIV+ patients and HD. Interest-
ingly, no significant difference between naïve and INI-cART treated
patients was observed. This data suggests that a cellular damage
persist despite suppression of viral replication.
Abstract no: 305
Presentation at ESCV 2016: Poster 141
Molecular studies on HSV: Replication rate,
infection capacity and progeny
A. Azevedo ∗, A. Nunes, C. Roque, I. Costa,
J.P. Gomes, S. Lopo
Department of Infectious Diseases, National Institute
of Health-Lisbon, Portugal
Introduction: In the last years genital herpes has emerged as
one of the most prevalent sexually transmitted infections. Herpes
simplex virus (HSV) is the most common cause of genital ulcer dis-
ease, with infections caused by both sub-types HSV-1 and HSV-2.
A better understanding of the virus replication cycle is relevant to
the pathogenesis of human diseases and is essential for the devel-
opment of antiviral chemotherapy.
Objectives: We aimed to shed some light on the HSV-1 andHSV-
2 infectious cycle, namely their capacity of infection, replication
rate and progeny, in three distinct cell lines (Vero, Vero E6 and
HeLa229). We also aimed to evaluate whether the concentration of
virus has any influence on the degree of the infection.
Methodology: Preliminary assays were performed in order
to understand which cellular concentration, viral load, nutrients’
availability and inoculation modus operandi (centrifugation versus
agitation) best mimic the HSV infection. Confluent cell monolayers
were infected with two HSV-2 and two HSV-1 at MOIs of 1:10, 1:1,
10:1 and 100:1. Inoculations were performed in parallel in two 24-
well plates, one for quantitative real-time PCR (kPCR) and one for
immunofluorescence assays, whichwere incubated for 30 h at 37 ◦ C
and 5% CO2. At different times-points of infection (6, 12, 18, 24 and
30 h p.i.), the wells were scratched for kPCR and the slides were
stained with monoclonal antibodies. For kPCR assays, appropriate
standard curves were generated by serial diluting plasmids cloned
with HSV-1 and HSV-2 single copy genes.
Results and conclusions: Preliminary assays showed that,
regardless of the viral load, it takes approximately 23 h for the virus
to complete the infectious cycle taking into account that no replica-
tion is observed after this time point. Considering the comparison
between the two inoculation procedures (centrifugation versusagi-
tation), we only observed relevant differences for lower viral loads,
with centrifugation yielding more viral progeny. More specific data
regarding both the HSV-1 and HSV-2 replication capacity for differ-
ent MOIs are currently under evaluation.
Abstract no: 318
Presentation at ESCV 2016: Poster 142
Genetic diversity and drug resistance profiles of
human immunodeficiency virus type 1 (HIV-1)
strains infecting pregnant women in the
Greater Lisbon
C. Simões ∗, E. Pádua , A. Mendes , A. Esteves ,
R. Parreira iedade
1 GHTM, Grupo de Virologia, Unidade de
Microbiologia Médica, Instituto de Higiene e
Medicina Tropical, Universidade Nova de Lisboa,
Lisbon, Portugal
2 Laboratório Nacional de Referência IST – VIH e
Hepatites B e C, Departamento de Doenc¸ as
Infecciosas, Instituto Nacional de Saúde Dr. Ricardo
Jorge, Lisbon, Portugal
According to the UNAIDS (The Joint United Nations Programme
on HIV/AIDS), human immunodeficiency virus (HIV) infected 36.9
million people at the end of 2014, of whom 2.6 million were chil-
dren under 15 years of age. Vertical transmission is the main cause
of infection in children and while the associated risk has decreased
dramatically with the introduction of highly active antiretroviral
therapy (HAART), this transmission continues to occur. Although
it has been the subject of significant progress in recent years,
the access of pregnant women to therapy still remains difficult
in certain regions. Moreover, in some cases, the presence of viral
mutations associated with resistance is responsible for the failure
of the implemented prophylactic regimens and may consequently
lead to transmission of resistant viruses to newborns.
This study included a group of 34 multiparous women infected
with HIV-1, fromwhom a sample of peripheral blood was collected
within two days after delivery, between 1999 and 2008. The great
majority of the women had followed therapeutic regimens for pre-
vention of vertical transmission of HIV-1 during pregnancy. The
proviral DNA of 70 samples analyzed was extracted and purified
from peripheral blood mononuclear cells, being the amplification
of the protease coding region carried out by double nested PCR.
After nucleotide sequencing, the genetic characterization of the
viral strains bymanual phylogenetic analysis was performed, along
with the characterization of resistance-associated mutations, as
well as other genetic polymorphisms, using the HIVdb program
(available at ).
The study revealed a high genetic diversity of HIV-1 within
this population, with predominance of G (47.8%), C (14.9%), and B
(11.9%) subtypes, and also a high prevalence of unique recombinant
forms (16.4%). Non-B subtypes were responsible for the infection
in all women of African origin, and the B subtype was only found
in Portuguese women. Additionally, African women were the only
infectedwith subtype C. Considering themutations associatedwith
resistance to protease inhibitors (PIs), two major mutations (D30N
and M46I) and seven minor mutations (L10I, L10V, L33F, G48E,
A71T, A71V, and T74S) were identified, in 19 of the sequences
studied. Of these, 16 were classified as non-B subtypes, but no
statistically significant association was found. Furthermore, most
of these mutations were detected in women whose prophylac-
tic regimens included PIs, which may have led to their selection.
The remaining genetic polymorphisms, not associated with anti-