Ingrid E Fakkert

MD, PhD Liesbeth Jansen

RN Kees Meijer

MD, PhD Theo Kok

MD, PhD Jan C Oosterwijk

Marian J E Mourits

Geertruida H Bock

Bsc

MD, PhD Marian Je Mourits

Prof. Dr Geertruida H De Bock
email: g.h.de.bock@epi.umcg.nl

Breast cancer screening in and mutation carriers after risk reducing salpingo-oophorectomy

Keywords: pBSO, RRSO, BRCA1, BRCA2, breast cancer screening, MRI

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Introduction

Breast cancer is the most common cancer in women and the life time risk of developing breast cancer for individual women in western countries is approximately 12-13% [START_REF]Borstkankerincidentie in vrouwen per 100 000[END_REF]. Women with a family history of breast and/or ovarian cancer can have substantially higher risks [START_REF] Claus | Autosomal dominant inheritance of early-onset breast cancer. Implications for risk prediction[END_REF]. For women with a proven BRCA1 or BRCA2 mutation, life time risk can reach up to 65-80% for breast cancer and 45% for ovarian cancer by the age of 70 [START_REF] Antoniou | Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies[END_REF][START_REF] Chen | Meta-analysis of BRCA1 and BRCA2 penetrance[END_REF]. In the Northern Netherlands we found a cumulative breast cancer risk of 71.4% (95% CI: 67%-82%) for BRCA1 mutation carriers and 87.5% (95% CI: 82%-93%) for BRCA2 mutation carriers by age of 70 [START_REF] Van Der Kolk | Penetrance of breast cancer, ovarian cancer and contralateral breast cancer in BRCA1 and BRCA2 families: high cancer incidence at older age[END_REF]. For ovarian cancer these risks were 58.9% (95% CI: 54%-64%) and 34.5% (95% CI: 25%-44%) respectively [START_REF] Van Der Kolk | Penetrance of breast cancer, ovarian cancer and contralateral breast cancer in BRCA1 and BRCA2 families: high cancer incidence at older age[END_REF].

Female mutation carriers are counselled on different risk reducing strategies, e.g. screening or prophylactic surgery. Prophylactic mastectomy strongly reduces the breast cancer risk, with about 90% at the age of 70 when conducted at a mean age of 38 [START_REF] Rebbeck | Bilateral prophylactic mastectomy reduces breast cancer risk in BRCA1 and BRCA2 mutation carriers: the PROSE Study Group[END_REF][START_REF] Heemskerk-Gerritsen | Prophylactic mastectomy in BRCA1/2 mutation carriers and women at risk of hereditary breast cancer: long-term experiences at the Rotterdam Family Cancer Clinic[END_REF]. Intensive breast cancer screening in mutation carriers consists of annual MRI, mammography and clinical breast examination (CBE) and can reach a sensitivity of more than 90% in finding early stage breast cancer in mutation carriers [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Warner | Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination[END_REF][START_REF] Leach | Screening with magnetic resonance imaging and mammography of a UK population at high familial risk of breast cancer: a prospective multicentre cohort study (MARIBS)[END_REF][START_REF] Kuhl | Mammography, breast ultrasound, and magnetic resonance imaging for surveillance of women at high familial risk for breast cancer[END_REF][START_REF] Rijnsburger | BRCA1-Associated breast cancers present differently from BRCA2-associated and familial cases: long-term follow-up of the Dutch MRISC study[END_REF]. For ovarian cancer it was recently shown that current screening protocols are ineffective in detecting early stage ovarian cancer in mutation carriers [START_REF] Van De Velde | Time to stop ovarian cancer screening in BRCA1/2 mutation carriers?[END_REF][START_REF] Hermsen | No efficacy of annual gynaecological screening in BRCA1/2 mutation carriers; an observational follow-up study[END_REF] and the only effective strategy to prevent ovarian cancer death is a risk reducing salpingo-oophorectomy (RRSO), which is eventually chosen by approximately 70% of these women [START_REF] Meijers-Heijboer | Use of genetic testing and prophylactic mastectomy and oophorectomy in women with breast or ovarian cancer from families with a BRCA1 or BRCA2 mutation[END_REF][START_REF] Metcalfe | International variation in rates of uptake of preventive options in BRCA1 and BRCA2 mutation carriers[END_REF].

When this procedure is performed before the age of 50 years, the risk of breast cancer is also reduced, with up to 50%, depending on the timing of RRSO [START_REF] Rebbeck | Meta-analysis of risk reduction estimates associated with riskreducing salpingo-oophorectomy in BRCA1 or BRCA2 mutation carriers[END_REF]. Nevertheless, Breast cancer screening before the age of 50 is indicated for women with a breast cancer risk that is 3 times higher than the population risk [START_REF]Richtlijn mammacarcinoom 2008[END_REF][START_REF](NICE) guideline CG41 Familial Breast Cancer[END_REF] BRCA1 and BRCA2 mutation carriers continue to receive the same three modality breast screening program after RRSO as they had before.

Intensive breast cancer screening combining MRI, mammography and CBE was proven to be effective in women at high risk for breast cancer [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Warner | Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination[END_REF][START_REF] Leach | Screening with magnetic resonance imaging and mammography of a UK population at high familial risk of breast cancer: a prospective multicentre cohort study (MARIBS)[END_REF][START_REF] Kuhl | Mammography, breast ultrasound, and magnetic resonance imaging for surveillance of women at high familial risk for breast cancer[END_REF]. One of the reasons that MRI was introduced in the screening program is that mammography is less effective in younger premenopausal women with higher breast density, as is the case in BRCA1/2 mutation carriers. We hypothesize that the breast cancer screening protocol might need adaptation in women after RRSO, because of the lower breast cancer incidence and the lower breast density after surgical menopause. The aim of this study is therefore to determine the effectiveness of breast cancer screening after RRSO in BRCA1/2 mutation carriers.

Materials and methods

Study population

To determine the effectiveness of breast cancer screening in BRCA1/2 mutation carriers after RRSO, a retrospective cohort study was performed in BRCA1 or BRCA2 mutation carriers who underwent a RRSO. All women who were younger than 52 years of age at RRSO (which is the mean age at menopause the Netherlands) were included. Results from subsequent breast cancer screening visits at the family cancer clinic from the University Medical Centre Groningen between January 1995 and the end of September 2009 were analyzed.

Women needed to have at least 1 breast eligible for screening. Women with uni-or bilateral breast conserving therapy were included. The use of hormonal replacement therapy (HRT) was recorded and this was not an exclusion criterion. Women in whom ovarian cancer was diagnosed during RRSO were not included in the study.

Surveillance protocol

In our family cancer clinic, RRSO was initially an option from the age of 35-40 years in BRCA1 mutation carriers and from the age of 40-45 years in BRCA2 mutation carriers. As of 2007, counseling has moved towards a specific advice to have RRSO around the age of 40, because ovarian screening appeared not effective in reducing ovarian cancer death [START_REF] Van De Velde | Time to stop ovarian cancer screening in BRCA1/2 mutation carriers?[END_REF][START_REF] Hermsen | No efficacy of annual gynaecological screening in BRCA1/2 mutation carriers; an observational follow-up study[END_REF]. However, the actual timing of this decision depends on many personal circumstances such as previous breast cancer, marital status, previous or planned pregnancies and mental acceptance of this definitive procedure. The operative procedure is a daycare surgical procedure, performed by laparoscopy [START_REF] Kenkhuis | Short-term surgical outcome and safety of risk reducing salpingo-oophorectomy in BRCA1/2 mutation carriers[END_REF].

As of 1995, annual clinical breast examination (CBE) by oncologic surgeons and nurse practitioners was performed together with annual mammography from the age of 25. From 1999, annual MRI was added to the protocol in a part of our population that participated in the MRISC study [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Rijnsburger | BRCA1-Associated breast cancers present differently from BRCA2-associated and familial cases: long-term follow-up of the Dutch MRISC study[END_REF], After 2005 it is advised to screen BRCA1/2 mutation carriers with annual CBE and MRI from age 25 till 60, to which mammography is added from age 30 onwards [START_REF]Richtlijn mammacarcinoom 2008[END_REF][START_REF](NICE) guideline CG41 Familial Breast Cancer[END_REF]. At our centre, MRI and mammography imaging is ideally performed alternating, i.e. with a six months interval.

Variables and endpoints

Information on date of birth, mutation status and personal oncologic history, date of RRSO and HRT use were collected for all women. For previous breast cancers, we collected information about age at diagnosis and therapy. We reviewed every visit were CBE, mammography and/or MRI was performed. For all these visits, the reason for visiting (screening or interval visit due to signs or symptoms), the performed diagnostic procedures and the outcomes of these investigations were collected. The result of a CBE consult is considered possibly malignant in case of referral for further investigation. The results of mammography and MRI screening are scored in a standardized way, according to the Breast Imaging Reporting and Data System (BI-RADS) classification. BI-RADS categories vary between 0 to V and BI-RADS category III or higher are considered abnormal and indicate further analysis by ultrasound, MRI and/or tissue sampling [START_REF] Tardivon | Breast imaging and reporting data system (BIRADS): magnetic resonance imaging[END_REF][START_REF] Liberman | Breast imaging reporting and data system (BI-RADS)[END_REF][START_REF] Obenauer | Applications and literature review of the BI-RADS classification[END_REF].

When additional diagnostic procedures were performed, the type of procedure and the findings were noted.

Findings could be "no malignancy" (when no malignancy was found at the additional diagnostic procedures), "suspect for malignancy" (when more additional investigation was necessary) and "malignancy" (as confirmed by biopsy). For each new breast cancer, tumour size in millimeters and the presence or absence of lymph node metastases in the (sentinel) axillary lymph nodes was noted.

Data collection

All relevant data were retrieved from the patients" hospital file. Physician"s letters, pathology reports and imaging reports were used to collect detailed information about above mentioned variables and endpoints as defined below. Data from all consecutive screening visits after RRSO were entered into an SPSS database.

Protection of the patient"s identity was guaranteed by a patient identification number. Those numbers were only retraceable to an individual woman when entered in the hospital database, which is only accessible for medical staff from the hospital, having a personal account. Complying with Dutch law, no further Institutional Review Board approval was needed.

Statistical analysis

Duration of follow-up was calculated for each woman. Follow-up time was calculated from the date of RRSO to the following endpoints: date of prophylactic mastectomy, date of ablation or date of last screening visit. Newly detected malignancies were defined as prevalent, screen-detected or interval cancers. Women were assigned to 2 groups, based on their mutation (BRCA1 or BRCA2). Characteristics of women are presented with median and range when they are continuous variables. For the other variables numbers are given and percentage for each mutation group. Differences between groups were calculated with the Kruskall Wallis test for continuous variables and χ 2 tests for the percentages within mutation groups. To compare the 3 different screening modalities, we calculated the sensitivity, specificity, PPV and NPV of each screening modality. For these 4 outcomes, 95% confidence interval (95% CI) was calculated with the Wilson score interval method. For MRI and mammography, calculations were made for BI-RADS III and IV as cut off point for a positive (suspect) test.

Statistic analyses were performed using the software of SPSS 16.0 for Windows and the VassarStats website [START_REF] Lowry | Clinical calculator 1: from an observed sampleestimates of population prevalence, sensitivity, specificity, predictive values, and likelihood ratios[END_REF].

Results

Population characteristics

Of all the mutation carriers who were screened at the UMCG after RRSO during the study period, 139 women met our inclusion criteria; 88 (63.3%) BRCA1 mutation carriers and 51 (36.7%) BRCA2 mutation carriers (Table 1). BRCA1 mutation carriers had a median age at RRSO of 40.5 years (range 30-51 years) and BRCA2 mutation carriers had a median age of 42 years (range 33-51 years) Of all women, 18% had a history of breast cancer, BRCA1 mutation carriers more often than BRCA2 mutation carriers (22.7% and 9.8% respectively, p=0.081). Of all women, BRCA1 mutation carriers reported significantly more often breast cancer before and at the age of 45 years than BRCA2 mutation carriers. (80.0% and 40.0% respectively, p=0.034).

Screening visits and events during follow-up

The total follow-up in this study was 5064 months (422 women years). The median follow-up was 24.0 months per woman (range 2-236 months). Women were censored because of preventive mastectomy (n=32, 23.0%), breast cancer (n=2, 1.4%), referral to another hospital (n=7, 5.0%) or loss to follow-up (n=10, 7.2%), the other women were still in screening after the end of our study (Table 2). In the women for whom screening ended after detection of breast cancer, one underwent mastectomy and one did not undergo further screening after being diagnosed with metastases after breast cancer in history.

Malignancies after RRSO

In a total of 1,146 hospital visits, 1,191 screening modalities (CBE, MRI or mammography) were performed in 422 women years. During the total screening period, 14 new breast cancers were detected: 12 new tumors and two prevalent tumors, both within 6 months after RRSO. The median time between RRSO and detection of a new tumor was 16.5 months. Three patients with a new breast cancer had a history of breast cancer before RRSO. All of the three were contra lateral new breast cancers, there were no local recurrences. In 6 of the 10 invasive cancers the lymph nodes were positive. One of the 2 interval cancers was node positive. Of the 14 women who were diagnosed with breast cancer after RRSO, 5 used HRT. One of these breast cancers was an interval tumor.

Performance of the screening methods

The performance of the 3 screening modalities is summarized in table 3. The overall sensitivity of the breast cancer screening program is 85.7%. Two tumors were interval cancers No tumors were found by CBE (Table 3). This gives CBE a sensitivity of 0%, a specificity of 97.7%, a PPV of 0% and a NPV of 98.1. In 9 women with breast cancer after RRSO, mammography screening was conducted and 5 of the 9 cancers were identified by mammography. This gives mammography a sensitivity of 55.6%, a specificity of 98.4%, a PPV of 50.0% and a NPV of 98.8. In 10 women with breast cancer after RRSO MRI screening was conducted, 6 of these cancers were identified by MRI. This gives MRI a sensitivity of 60.0%, a specificity of 95.9%, a PPV of 35.3% and a NPV of 98.5%.

Of the 2 interval tumors, the first, (20 mm, node positive), was detected 5 months after screening CBE. Mammography screening had not yet been performed. This was in 1999 and MRI was not yet part of the screening protocol at that time. Five years after diagnosis, this patient died of metastatic disease after a relapse in the mastectomy scar. The second interval cancer (11 mm, node negative) was detected by the patient herself 2 weeks after a negative CBE screening. MRI had not been conducted and the last mammography was 17 months before the new tumor was found.

Screening visits and additional investigation

Of the 138 women who attended screening after RRSO, 127 women had at least one screening CBE, 117 women had at least one screening mammography and 109 women had at least one screening MRI (Table 4). The total number of screening investigations was highest for CBE (583), and lowest for MRI (278). CBE generated the least additional diagnostic procedures 3% of the screening visits led to additional research. For MRI 21% of the screening visits led to additional research and for mammography, 14% of the screening visits led to additional research. In the study period, a total of 27 invasive diagnostic procedures (15 fine needle aspirations and 12 biopsies) were conducted, also most after MRI. Most additional diagnostic procedures were performed by using ultrasound (64 times).

While no new cancers where found with CBE, CBE was carried out as additional investigation 11 times. Of the tumors found during the screening period, 5 were palpable when CBE was carried out after suspect findings at mammography or MRI. In 3 of these cases, the women had had a negative CBE screening in the month before the suspect findings.

When screening consisted of MRI and mammography, 43 screening visits were needed to find 1 new breast cancer. When CBE was added to the screening, 85 screening visits were needed to find 1 new breast cancer.

Discussion

In this group of 139 BRCA1/2 mutation carriers who had had RRSO at a premenopausal age and were under surveillance at our Family Cancer Clinic, 14 breast cancers were detected in 14 women during 442 women years. There were 2 prevalent, 10 screen detected and 2 interval breast cancers. The program sensitivity was 85.7% and the specificity 97.5%. For the individual screening modalities, the sensitivity for CBE was 0% (95% CI: 0% -32.1%), for mammography 55.6% (95% CI: 22.7% -79.9%), and for MRI 60.0% (95% CI: 27.4% -86.3%). Of all these cancers, 70% was high grade (III) and 60% of the tumors were node positive.

The program sensitivity of screening with MRI, mammography and CBE was 85.7% (95% CI: 56.2% -97.6%).

Earlier studies on screening effectiveness in women at high risk of breast cancer found sensitivities varying from 91.1% to 97.7% [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Warner | Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination[END_REF][START_REF] Leach | Screening with magnetic resonance imaging and mammography of a UK population at high familial risk of breast cancer: a prospective multicentre cohort study (MARIBS)[END_REF][START_REF] Kuhl | Mammography, breast ultrasound, and magnetic resonance imaging for surveillance of women at high familial risk for breast cancer[END_REF], which seems to be higher. Only one study on the long-term effectiveness of the MRISC study found a total program sensitivity of 86.6% [START_REF] Rijnsburger | BRCA1-Associated breast cancers present differently from BRCA2-associated and familial cases: long-term follow-up of the Dutch MRISC study[END_REF]. The study population in these previous studies was more diverse, consisting of pre-and postmenopausal women. Also, their studies contained mutation carriers, but also women at high risk for breast cancer without mutation. In our study, all women were postmenopausal after premenopausal RRSO and they were all mutation carriers. These factors may have contributed to the differences in effectiveness.

Both Kriege et [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF]. The amount of women analyzed in both the MRISC study and our study is probably small. A main factor that might have contributed to the lower sensitivity of our surveillance program is that we evaluated the program sensitivity of the screening over a period of 14 years (1995 -2009) during which screening guidelines changed several times. MRI was offered from 1999 to a subset of women as part of the MRISC study, and as of 2005 it is offered to all women at high risk. One interval cancer was found in 1999.

Excluding this tumor from the analysis, would increase the program sensitivity of our screening from 84.6% to 91.7%, which is in the range that can be expected. Another consideration is that the four series mentioned [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Warner | Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination[END_REF][START_REF] Leach | Screening with magnetic resonance imaging and mammography of a UK population at high familial risk of breast cancer: a prospective multicentre cohort study (MARIBS)[END_REF][START_REF] Kuhl | Mammography, breast ultrasound, and magnetic resonance imaging for surveillance of women at high familial risk for breast cancer[END_REF] are prospective studies with a strict screening protocol in which every woman receives MRI, mammography and CBE at regular intervals. In daily practice, many factors may influence the regularity of the screening visits.

Planning MRI, mammography and CBE on the first visit is sometimes difficult because of agenda and capacity issues, women do not always attend appointments, or there may be contra indications for MRI scanning. It is reasonable to assume that the second interval cancer is related to "protocol deviances". This interval cancer was detected by the patient herself, 2 weeks after a negative CBE screening, and her last mammography was 17 months before the new tumor was found. Though this woman was in screening from 2005 to 2007, she was not screened by MRI.

For the separate screening modalities, the sensitivity of 0% (95% CI: 0 % -32.1%) for CBE found in our study was comparable to sensitivities of 9.1-17.8% found in other studies [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Warner | Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination[END_REF]. In general, CBE is considered to have no additional benefit over mammography, whereas it does increase the false positive rate [START_REF] Chiarelli | The contribution of clinical breast examination to the accuracy of breast screening[END_REF][START_REF] Obdeijn | Assessment of falsenegative cases of breast MR imaging in women with a familial or genetic predisposition[END_REF]. When CBE was excluded from program sensitivity analyses, sensitivity did not change. This would mean exclusion of 538 CBE screenings visits in which no new tumors were detected. Three of the new tumors found by imaging were palpable at diagnosis (CBE was conducted as additional research), while CBE screening within the month before diagnosis was negative. Also, CBE has proven to be useful in detecting locoregional recurrences and contralateral breast cancer in an early stage in women with breast cancer in history (eg. after mastectomy, breast conserving therapy and radiotherapy) [START_REF] Lu | The value of routine physical examination in the follow up of women with a history of early breast cancer[END_REF]. This indicates that CBE should not be eliminated from our post-RRSO screening protocol, especially in women with previous breast cancer.

The sensitivity of 55.6% (95% CI: 22.7% -79.9%) for mammography was not significantly different from sensitivities of 32%-40% reported in previous studies. [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Warner | Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination[END_REF][START_REF] Leach | Screening with magnetic resonance imaging and mammography of a UK population at high familial risk of breast cancer: a prospective multicentre cohort study (MARIBS)[END_REF][START_REF] Kuhl | Mammography, breast ultrasound, and magnetic resonance imaging for surveillance of women at high familial risk for breast cancer[END_REF]. Long-term follow-up in the MRISC study revealed a sensitivity for mammography of 25% in BRCA1 mutation carriers. [START_REF] Rijnsburger | BRCA1-Associated breast cancers present differently from BRCA2-associated and familial cases: long-term follow-up of the Dutch MRISC study[END_REF] This is much lower than the sensitivity found in our study, while the majority of tumours we found, were also in BRCA1 mutation carriers. Due to the lower breast density after RRSO, we expected that the sensitivity of mammography in our population would be higher than in other high risk screening studies. The sensitivity of mammography we found was indeed higher than previously reported though the difference was not significant. This may be due to small numbers, or to the fact that about 47% of the women used HRT after RRSO, which is known to preserve breast density in postmenopausal women [28].

The sensitivity of 60.0% (95% CI: 27% -86%) for MRI in our study was lower (though not significant) than the sensitivities for MRI reported in previous studies, ranging from 71.1%-77.0% [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Warner | Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination[END_REF][START_REF] Leach | Screening with magnetic resonance imaging and mammography of a UK population at high familial risk of breast cancer: a prospective multicentre cohort study (MARIBS)[END_REF] and significantly lower than the sensitivity of 90.7% for MRI found by Kuhl et al [START_REF] Kuhl | Mammography, breast ultrasound, and magnetic resonance imaging for surveillance of women at high familial risk for breast cancer[END_REF]. It is reasonable to suppose that the effectiveness of MRI screening in daily practice is less than the performance of MRI screening in prospective trials. At our centre, MRI software, protocols and scanners evolved in the past years. It is likely that MRI sensitivity will improve in the future along with experience and further improvement of MRI technology. Another issue is that, 2 of the 4 tumors that where false negative on MRI were full DCIS. Most tumors missed on MRI in the MRISC study were also DCIS, which indicates that MRI is significantly less reliable in detecting DCIS [START_REF] Obdeijn | Assessment of falsenegative cases of breast MR imaging in women with a familial or genetic predisposition[END_REF]. This is illustrated by the fact that for invasive tumors only, MRI sensitivity in our study is 75%, which lowers to 60.0% (95% CI: 27.4% -86.3%) when DCIS are included. Rijnsburger et al. found an MRI sensitivity of 66.7% for BRCA1 mutation carriers and 69.2% for BRCA2 mutation carriers, which was significantly higher than the sensitivity of 25% they found for mammography. In addition, they showed that MRI enabled the detection of breast tumors in a more favorable stage than mammography. They suggest that MRI-screening should be considered biannually in certain categories [START_REF] Rijnsburger | BRCA1-Associated breast cancers present differently from BRCA2-associated and familial cases: long-term follow-up of the Dutch MRISC study[END_REF].

In our study, tumor size was also comparable to other studies, with 83.3% of the tumors after RRSO being smaller than 20 mm, compared to percentages ranging from 75% -94% in other studies [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Warner | Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination[END_REF][START_REF] Leach | Screening with magnetic resonance imaging and mammography of a UK population at high familial risk of breast cancer: a prospective multicentre cohort study (MARIBS)[END_REF][START_REF] Kuhl | Mammography, breast ultrasound, and magnetic resonance imaging for surveillance of women at high familial risk for breast cancer[END_REF]. However, 60% of the tumors in our study were node positive, while others found percentages of 15.4-36.0%, [START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Warner | Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination[END_REF][START_REF] Leach | Screening with magnetic resonance imaging and mammography of a UK population at high familial risk of breast cancer: a prospective multicentre cohort study (MARIBS)[END_REF][START_REF] Kuhl | Mammography, breast ultrasound, and magnetic resonance imaging for surveillance of women at high familial risk for breast cancer[END_REF][START_REF] Rijnsburger | BRCA1-Associated breast cancers present differently from BRCA2-associated and familial cases: long-term follow-up of the Dutch MRISC study[END_REF]. These findings need further evaluation. This is the first study on the effectiveness of breast cancer screening with CBE, mammography and MRI, in BRCA1/2 mutation carriers who had undergone RRSO at premenopausal age, conducted in daily practice and not in the context of a clinical trial.

It is an interesting finding that MRI sensitivity in this study was slightly lower than MRI sensitivity in earlier trials. The question is if this difference is caused by protocol deviances in general practices our by effects generated by RRSO. Also, mammography sensitivity seems to be higher after RRSO than before. This might be caused by lower breast density after RRSO, but this should be evaluated during long term follow-up. We used the number of new tumors detected by screening to evaluate the effectiveness of the surveillance program. Eventually, the purpose of screening is to increase survival after breast cancer. For evaluating the effect

  al. and Rijnberger et al. present results of the MRISC study. Of the 2275 participants in these studies, 148 were from our family cancer clinic.(12) Kriege et al. noted that 19% of the mutation carriers had RRSO in history

Table 1 : Characteristics of the women at baseline (N=139)

 1

		BRCA1 (N=88)	BRCA2 (N=51)	Total (N=139)	Statistics
	Age at RRSO in
	years
	Median(Range) 40.5 (30-51)	42.0 (33-51)	41.0 (30-51)	P = 0.059
	Previous breast
	cancer
	No	77.3% (68/88)	90.2% (46/51)	82.0% (114/139)	P = 0.081
	Yes unilateral	15.9% (14/88)	9.8% (5/51)	13.7% (19/139)
	≤45 years	85.7% (12/14)	40.0% (2/5)	73.7% (14/19)	P = 0.034*
	Yes bilateral	6.8% (6/88)	-	4.3% (6/139)
	≤45 years	100% (6/6)	-	100% (6/6)
	Age at diagnosis
	first breast cancer
	in years (N=25)
	Median	40.0 (30-49)	46.0 (34 -50)	41.0 (30-50)	P = 0.143
	(Range)
	Breast cancer
	therapy**
	BCT unilateral	40.0% (8/20)	80.0% (4/5)	48.0% (12/25)	P = 0.427
	Mast unilateral	40.0% (8/20)	20.0% (1/5)	36.0% (9/25)
	BCT bilateral	15.0% (3/20)	-	12.0% (3/25)
	BCT and Mast	5.0% (1/20)	-	4.0% (1/25)
	HRT use #
	No	51.2% (41/80)	56.2% (27/48)	53.1% (68/128)	P = 0.583
	Yes	48.8% (39/80)	43.8% (21/48)	46.9% (60/128)

* P < 0.05 is significant. Mann-Whitney U test for continuous variables, Pearsons" χ 2 test for proportions. ** BCT: Breast Conserving Therapy, Mast: Mastectomy, if noted as BCT and Mast, 1 breast is treated with BCT and 1 with Mast.

# Hormonal replacement therapy after RRSO.

Table 2 : Follow-up

 2

	BRCA1 (N=88)	BRCA2 (N=51) Total (N=139)
	Follow-up in months
	Total
	Median (range)

Table 3 : Performance of the screening tests

 3

		Cancer present	Cancer absent	Total
	CBE
	11 cancers (2 interval), 9 invasive, 2 DCIS
	Positive	-	13	13
	Negative	11	559	570
	Total	11	572	583
	Sensitivity: 0% (95% CI: 0% -32%)	PPV: 0% (95% CI: 0% -28%)
	Specificity: 97.7% (95% CI: 96% -99%)	NPV: 98,1% (95% CI: 97% -99%)
	Mammography BI-RADS ≥III
	9 cancers (no interval), 7 invasive, 2 DCIS
	Positive	5	5	10
	Negative	4	316	320
	Total	9	321	330
	Sensitivity: 55.6% (95% CI: 23% -85%)	PPV: 50.0% (95% CI: 20% -80%)
	Specificity: 98.4% (95% CI: 96% -99%)	NPV: 98.8% (95% CI: 97% -100%)
	MRI BI-RADS ≥III
	10 cancers (no interval), 7 invasive, 3 DCIS,
	Positive	6	11	17
	Negative	4	257	261
	Total	10	267	278
	Sensitivity: 60.0% (95% CI: 27% -86%)	PPV: 35.3% (95% CI: 15% -61%)
	Specificity: 95.9% (95% CI: 93% -98%)	NPV: 98.5% (95% CI: 96% -100%)
	Total screening
	14 cancers (2 interval), 11 invasive, 3 DCIS
	Positive	12	28	40
	Negative	2	1149	1151
	Total	14	1177	1191
	Sensitivity: 85.7% (95% CI: 56% -98%)	PPV: 30.0% (95% CI: 17% -47%)
	Specificity: 97.6% (95% CI: 97% -98%)	NPV: 99.8% (95% CI: 99% -100%)

Table 4 : Screening visits and additional research

 4

	Screening	Visits	CBE	Mammo	MRI	Ultra	FNA*	Biopsy	Total
	modality			graphy		sound
	CBE (N=127)	583	4	2	-	7	2	2	17
	Mammography	330	2	3	2	31	3	4	45
	(N=117)
	MRI (N=109)	278	5	4	5	27	10	6	57
	Total	1191	11	9	7	64	15	12	118
	*FNA: Fine Needle Aspiration

of survival, long term follow-up is needed. We continue to collect data of this study population in a prospective database.

To conclude, the sensitivity of three-modality breast cancer screening in BRCA1/2 mutation carriers after RRSO is lower than breast cancer screening before RRSO. It is the lower sensitivity of MRI that contributed largely to the lower sensitivity of the total screening program, while mammography sensitivity increases after RRSO. In our post RRSO population, CBE did not contribute to tumor detection. Our findings suggest that intensive breast cancer screening with both MRI and mammography should be continued in BRCA1/2 mutation carriers, after RRSO.


V Pliaka

Z Kyriakopoulou

D Tsakogiannis

I G A Ruether

C Gartzonika

S Levidiotou-Stefanou

A Krikelis

P Markoulatos
email: markoulatos@bio.uth.gr

Prof P Markoulatos

Correlation of mutations and recombination with growth kinetics of Poliovirus vaccine strains

Keywords: OPV isolates, mutations, recombination, temperature sensitivity, growth kinetic analysis

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and in rare cases may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight OPV isolates (two non-recombinants and six recombinants) as well as of Sabin vaccine strains was evaluated using two different assays, Reproductive Capacity at different Temperatures test (RCT test) and one-step growth curve test in Hep2 cells at two different temperatures (37 o C and 40 o C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All the recombinant OPV isolates showed a thermoresistant phenotype in RCT test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37 o C and 40 o C in one-step growth curve test. All the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light in the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

Introduction

Poliovirus, the causal agent of poliomyelitis, is a member of the enterovirus genus of the Picornaviridae family and exists as three immunologically defined serotypes (P1, P2 and P3). The poliovirus is a nonenveloped virus composed of a 7.5-Kb positive sense single-stranded polyadenylated RNA genome surrounded by an icosahedral capsid [START_REF] Wimmer | Genetics of poliovirus[END_REF]. The capsid is made of 60 protomers, each containing a single copy of the four structural capsid proteins (VP1 to VP4). The viral RNA contains a long open reading frame flanked by 5' and 3' noncoding regions (5'-NCR and 3'-NCR, respectively) involved in viral replication and translation. The predicted secondary structure of the 5'-NCR defines six domains (numbered from I to VI) corresponding to highly conserved structures formed by stems and loops [START_REF] Pilipenko | Conserved structural domains in the 5'-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence[END_REF][START_REF] Skinner | New model for the secondary structure of the 5' non-coding RNA of poliovirus is supported by biochemical and genetic data that also show that RNA secondary structure is important in neurovirulence[END_REF]. Domains II, IV, and V and part of domain VI contribute to the internal ribosomal entry site which allows cap-independent initiation of translation of the viral genome [START_REF] Pelletier | Capindependent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region[END_REF].

Structural and nonstructural viral proteins are coded by a single polypeptide precursor which is cleaved by viral proteases.

Since 1960, poliomyelitis has been effectively controlled by the use of inactivated or live attenuated vaccines. The Sabin live oral poliovirus vaccine (OPV) is constituted of attenuated strains of each of the three serotypes (Sabin 1, 2, and 3) which have been selected by numerous passages of wildtype strains in monkey tissues in vivo and in vitro [START_REF] Sabin | History of Sabin attenuated poliovirus oral live vaccine strains[END_REF]. The Sabin strains are temperature sensitive (ts) and the ts phenotype correlates with the attenuated phenotype [START_REF] Christodoulou | Mapping of mutations associated with neurovirulence in monkeys infected with Sabin 1 poliovirus revertants selected at high temperature[END_REF][START_REF] Macadam | Reversion of the attenuated and temperature-sensitive phenotypes of the Sabin type 3 strain of poliovirus in vaccinees[END_REF][START_REF] Macadam | The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity[END_REF]. The molecular determinants of attenuation and temperature sensitivity have been intensively studied to elucidate the mechanisms involved and to improve the safety of Sabin vaccine strains. The genome of Sabin 1 differs from that of its parental virulent Mahoney virus by 54 point mutations.

Molecular determinants of the attenuation and temperature sensitivity of Sabin 1 have been found in the 5'-NCR and 3'-NCR of the genome, as well as in the regions encoding VP1, VP3 and VP4 capsid proteins and the RNA polymerase (3D) [START_REF] Bouchard | Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine[END_REF][START_REF] Georgescu | Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus[END_REF][START_REF] Kawamura | Determinants in the 5' noncoding region of poliovirus Sabin 1 RNA that influence the attenuation phenotype[END_REF][START_REF] Omata | Genetic analysis of the attenuation phenotype of poliovirus type 1[END_REF][START_REF] Tardy-Panit | A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice[END_REF][START_REF] Otelea | Genomic modifications in naturally occurring neurovirulent revertants of Sabin 1 polioviruses[END_REF][START_REF] Ogra | Effect of prior immunity on the shedding of virulent revertant virus in feces after oral immunization with live attenuated poliovirus vaccines[END_REF]. For Sabin types 2 and 3, the vaccine strains differ from their progenitors by 23 and 11 mutations, respectively, and there is evidence in both cases that attenuation is determined by just 2 or 3 of these mutations. One substitution in the 5'-NCR of the genome and another that changes an amino acid residue in a capsid protein (VP1 in Sabin 2 and VP3 in Sabin 3) are the major determinants of attenuation and temperature sensitivity [START_REF] Macadam | Reversion of the attenuated and temperature-sensitive phenotypes of the Sabin type 3 strain of poliovirus in vaccinees[END_REF][START_REF] Macadam | The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity[END_REF][START_REF] Ogra | Effect of prior immunity on the shedding of virulent revertant virus in feces after oral immunization with live attenuated poliovirus vaccines[END_REF][START_REF] Minor | The temperature sensitivity of the Sabin type 3 vaccine strain of poliovirus: molecular and structural effects of a mutation in the capsid protein VP3[END_REF][START_REF] Westrop | Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine[END_REF][START_REF] Guest | Molecular mechanisms of attenuation of the Sabin strain of poliovirus type 3[END_REF][START_REF] Gutiérrez | Attenuating mutations in the poliovirus 5' untranslated region alter its interaction with polypyrimidine tract-binding protein[END_REF][20][START_REF] Gnanashanmugam | Shedding and reversion of oral polio vaccine type 3 in Mexican vaccinees: comparison of mutant analysis by PCR and enzyme cleavage to a real-time PCR assay[END_REF][START_REF] Martinez | Shedding of 21 sabin poliovirus Type 3 containing the nucleotide 472 uracil-to-cytosine point mutation after administration of oral poliovirus vaccine[END_REF].

Undoubtedly, the use of OPV in mass vaccinations has resulted in drastic reductions in the number of poliomyelitis cases due to infections with wild-type polioviruses from an estimated 350,000 cases in over 125 endemic countries in 1988 to just 1,310 cases in four countries in 2007. However, in rare cases (1 case per 750,000 primary vaccinees), OPV strains have been implicated in vaccine-associated paralytic poliomyelitis (VAPP) [START_REF] Centers For | Poliomyelitis prevention in the United States: introduction of a sequential vaccination schedule of inactivated poliovirus vaccine followed by oral poliovirus vaccine. Recommendations of the Advisory Committee on Immunization Practices (ACIP)[END_REF]. The underlying cause of VAPP is the genetic instability of OPV strains. During multiplication in the human intestine, mutations at specific sites of the genome are associated with the attenuated and temperature sensitive phenotype of Sabin strains and recombination between Sabin strains may result in the loss of the attenuated phenotype of OPV strains and the acquisition of traits characteristic of wild polioviruses, such as increased neurovirulence and loss of temperature sensitivity [START_REF] Furione | Polioviruses with natural recombinant genomes isolated from vaccineassociated paralytic poliomyelitis[END_REF][START_REF] Georgescu | Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis[END_REF][START_REF] Cherkasova | Spread of vaccine-derived poliovirus from a paralytic case in an immunodeficient child: an insight into the natural evolution of oral polio vaccine[END_REF]. Moreover, changes are frequently observed in antigenic properties of OPV derivatives representing a selection of viral variants that are less prone to be neutralized by human antibodies [START_REF] Yakovenko | Evolution of the Sabin vaccine into pathogenic derivatives without appreciable changes in antigenic properties: need for improvement of current poliovirus surveillance[END_REF].

The aim of the present study was to correlate the reverted phenotypic traits of OPV isolates such as temperature sensitivity and growth kinetics with mutations and recombination events on the viral genome.

Materials and methods

Virus isolation

Virus isolation was performed from faecal samples during 1978-1985 from VAPP patients and healthy vaccinees in Hep-2 cells (Table 1) and the isolates were re-cultured for the purpose of the present study in Hep-2 cells. Initial isolation at that time occurred at the Hellenic Pasteur Institute and identification/serotyping was done by sero-neutralization with rabbit polyclonal antibodies, (National Institute for Public Health and Environment, RIVM, The Netherlands), according to enclosed instructions. To avoid viral mixtures serial ten-fold dilutions were performed; the last dilution presenting a complete cytopathic effect (CPE) was passaged once again in Hep-2 cells, and the serotype was retested as described above. Vaccine strains (Sabin types 1, 2, 3) used in this study were provided by the World Health Organization (WHO).

Genotyping and identification of recombinant OPV isolates

Primer pair UG52/UC53 was used for the identification of the vaccine origin and for the genotyping of the isolates in the 5'-NCR genomic region [START_REF] Georgopoulou | Improved genotyping vaccine and wild-type poliovirus strains by restriction fragment length polymorphism analysis: clinical diagnostic implications[END_REF].

Moreover, the genotype of isolates in VP1 capsid protein was determined.

The primer pairs used for complete VP1 gene sequencing were UG1/222 [START_REF] Balanant | The natural genomic variability of poliovirus analyzed by a restriction fragment length polymorphism assay[END_REF][START_REF] Mulders | A Sabin vaccine-derived field isolate of poliovirus type 1 displaying aberrant phenotypic and genetic features, including a deletion in antigenic site 1[END_REF] for the first half of the VP1 gene and serotype-specific primer pairs S 1 37/S 1 688, S 2 30/S 2 688 and S 3 26/S 3 651 for Sabin 1, Sabin 2 and Sabin 3 isolates, respectively, for the second half of the VP1 gene [START_REF] Dedepsidis | Detection of unusual mutation within the VP1 region of different re-isolates of poliovirus Sabin vaccine[END_REF].

The identification of recombination sites of isolates EP9, IF, ID, EP12, EP16, EP23 and the sequencing from 2C-3D genomic region have been previously published by our group [START_REF] Karakasiliotis | Site analysis of recombinant and mutant poliovirus isolates of Sabin origin from patients and from vaccinees[END_REF][START_REF] Paximadi | Genomic analysis of recombinant sabin clinical isolates[END_REF].

Rct marker test

Reproductive capacities at different temperatures (Rct marker) were evaluated by Rct test. The Rct value is defined as the difference between the log 10 virus titer of a viral stock measured at the optimal temperature (37 o C) and that of the supraoptimal temperature (40 o C). Briefly, each virus stock was decimally diluted through 10 -7 . A 0.1-ml of medium containing 4 X 10 4 Hep-2 cells was added into each well of two 96-well plastic plates. A 0.1-ml volume of each virus dilution was inoculated into four wells of each of the two plates.

One plate was incubated at 37 o C and the other at 40 o C. After 5 days of incubation at the appropriate temperatures, the titers were determined by an endpoint micromethod [START_REF] Nakano | Parameters for differentiating vaccine-derived and wild poliovirus strains[END_REF] and were expressed in log 10 50% tissue culture infective dose units (TCID 50 ) per ml. Viruses were considered thermosensitive (ts) if the RCT value (between 37 o C and 40 o C) was greater or equal to 2.00 and thermo resistant (not-ts) when the RCT value was inferior to 2.00.

One-step growth curve experiment

One-step growth curve experiments were performed in Hep-2 cells at 37 and 40 o C, with a multiplicity of infection of 10 as determined by titration of virus stocks on Hep-2 cells, as has been previously described [START_REF] Malnou | Poliovirus internal ribosome entry segment structure alterations that specifically affect function in neuronal cells: molecular genetic analysis[END_REF]. Briefly, 100 μl of Hep-2 cells (10 4 cells/100 μl) were added into each well of twelve 96well plastic plates. After the cells were attached to the plates, the isolates and the reference Sabin vaccine strains (Sabin 1, Sabin 2 and Sabin 3) were inoculated each into five replica wells of 96-well plastic plates. After the plates were rocked for 2 hours at 37 o C, the cells were thoroughly washed two times each with 300 μl of Eagle MEM to remove unbound virus. Then, 100 μl of Eagle MEM was added into each well and the plates were incubated at 37 o C or 40 o C for 0, 1, 2, 4, 8 and 16 hours. The plates were subjected to three consecutive freeze-thaw cycles and the viral titers of the supernatants were determined by the TCID 50 assay on Hep-2 cells at 37 o C for each of the above mentioned hours post-infection. The titration was repeated five times for each of the post-infection hours.

Computational analysis

ClustalW was used for alignment of the sequences (nucleotide and amino acid) of 5'-NCR, VP1, 2C, 3A, 3B, 3C and 3D genomic regions of all virus isolates with those of the reference strains cited in GenBank (Sabin type 1: AY184219, Sabin type 2: AY184220 and Sabin type 3: AY184221). Amino acid sequences of VP1, 2C, 3A, 3B, 3C and 3D genomic regions of all virus isolates were obtained by using Gene-Runner V 3.05.

Results

Genotypes and recombination types of OPV isolates

The 5'-NCR and VP1 genotypes of isolates were identified. One strain was genotyped as Sabin 1, four strains as Sabin 2 and three strains as Sabin 3 (Table 1). Among the eight isolates, two (II and 729) were identified as nonrecombinant and six (EP9, IF, ID, EP12, EP16 and EP23) were identified as recombinant in one site. The recombination type was S2/S1 for four isolates (EP9, IF, ID, EP12) and the recombination site was located in the 3D region for isolates IF and EP12, in the 3C region for isolate ID and in the 3A region for isolate EP9. Isolates EP16 and EP23 were S3/S2 and S3/S1 recombinant respectively and the recombination site was located in the 2C region (Table 1).

Temperature sensitivity of OPV isolates

The Rct marker assay is based on a comparison of the temperaturesensitive multiplication of poliovirus Sabin strains and the non-temperaturesensitive multiplication of virulent strains.

The non-recombinant Sabin-1 isolate II showed a partial reversion to non-ts phenotype with a Rct value of 2.0 units. Three of recombinant Sabin-2 isolates (EP9, IF, ID: S2/S1) showed a non-ts phenotype with Rct values ranging from 0.0 to 0.75 units while one (EP12: S2/S1) showed partial reversion to non-ts phenotype with Rct value of 1.75 units. Both recombinant Sabin-3 isolates, (EP16: S3/S2) and (EP23: S3/S1), showed partial reversion to non-ts phenotype with Rct values of 1.0 and 1.5 units, respectively. The nonrecombinant Sabin-3 isolate 729 showed a ts phenotype with Rct value of 3.5 units (Table 2). The Sabin vaccine strains (Sabin1, Sabin2, Sabin3) exhibited, as expected, Rct values greater to 2.00 units.

One-step growth curve experiment

To determine whether the ts phenotype, as determined by Rct assay, was indicative of growth defects, one-step growth curve experiments were carried out for each isolate. We compared the growth rates and virus yields at The non-recombinant Sabin-1 isolate II replicated with kinetics similar to that of its progenitor Sabin 1 vaccine strain showing that the viral yield rises after 2 hours post-infection at 37 o C (Fig. 1) while at 40 o C its growth was blocked as was also the case for Sabin 1 vaccine strain. However, the isolate II showed 1.0 log 10 unit lower viral yield than Sabin 1 vaccine strain at the initial stages of the infectious life cycle (0-2 hours post-infection) resulting in a difference of 2.5 log 10 units at 4 hours post infection.

The recombinant Sabin-2 isolates EP9, EP12 and ID displayed similar growth kinetics to that of their progenitor Sabin 2 vaccine strain (Fig. 2). More specifically, they showed the same or lower viral yield than Sabin 2 vaccine strain during the infectious life cycle at both 37 and 40 o C. The recombinant Sabin-2 isolate IF also displayed similar growth kinetics to that of its progenitor Sabin 2 vaccine strain (Fig. 2). However, it showed higher viral yield than Sabin 2 vaccine strain at 4 and 8 h post-infection at 37 o C. Moreover, it showed 1.0-1.5 log 10 units higher viral yield than Sabin 2 vaccine strain at 4, 8 and 16 h post-infection at 40 o C. Both recombinant Sabin-3 isolates EP16 and EP23 showed different growth kinetics from that of their progenitor Sabin 3 vaccine strain (Fig. 3).

Isolates EP16 and EP23 showed 2.0 and 1.7 log 10 units, respectively, higher viral yield than the Sabin 3 vaccine strain at 0 and 1 hour post-infection.

Moreover, the viral yield of isolates EP16 and EP23 rises after 1 hour postinfection at 37 o C while that of Sabin 3 vaccine rises after 2 hours postinfection. The isolates EP16 and EP23 replicated efficiently at both 37 and 40 o C. More specifically, the isolates EP16 and EP23 showed 2.0-3.5 and 1.0-2.0 log 10 units, respectively, higher viral yield than Sabin 3 vaccine strain at both 37 and 40 o C during the infectious life cycle. The non-recombinant Sabin-3 isolate 729 replicated with kinetics similar to that of Sabin 3 vaccine strain at both 37 and 40 o C (Fig. 3).

Nucleotide and amino acid sequence analysis

In an attempt to correlate the phenotypic markers of the isolates with some molecular markers, we investigated the nucleotide and amino acid positions in the genomes of the isolates that have been involved in the attenuated and thermo sensitive phenotype of Sabin vaccine strains.

The nucleotide sequences of 5'-NCR, VP1, 2C, 3A, 3B, 3C and 3D genomic regions were investigated in order to establish their respective predicted amino acid sequences. Tables 2 and3 show the sequence differences with respect to Sabin vaccine strains. Mutations that result in loss of the attenuated and thermo sensitive phenotype of Sabin vaccine strains have been identified in the genomes of all isolates.

In non-recombinant Sabin-1 isolate II, the mutation U525C was identified (Table 2). The suppressive mutation U525C restores the stability of a base pair between nt 480 and 525 (AU in Mahoney, GU in Sabin 1, and GC in isolate II) in domain V of 5'-NCR and has been correlated with reversion to neurovirulence [START_REF] Christodoulou | Mapping of mutations associated with neurovirulence in monkeys infected with Sabin 1 poliovirus revertants selected at high temperature[END_REF][START_REF] Rezapkin | Microevolution of Sabin 1 strain in vitro and genetic stability of oral poliovirus vaccine[END_REF]. The mutation C583G is situated in a double stranded region of domain VI of IRES and leads to a G-G mismatch. In VP1 coding region, the reversion at nt 2795 (A→G [Thr-106-Ala]), a well-known determinant of attenuation [START_REF] Christodoulou | Mapping of mutations associated with neurovirulence in monkeys infected with Sabin 1 poliovirus revertants selected at high temperature[END_REF][START_REF] Bouchard | Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine[END_REF] was also identified. Amino acid residue 106 is located at the exterior of the capsid, specifically in VP1 BC loop, and is involved in hydrophobic interactions with residue in position 90 [START_REF] Cherkasova | Long-term circulation of vaccine-derived poliovirus that causes paralytic disease[END_REF]. Moreover, substitutions Ala-96-Thr and Lys-99-Asn were identified in isolate II. Both of them are highly exposed in the exterior of the capsid and are located in N-AgI site as well as in VP1 BC loop, which is participating in the formation of the north rim of the canyon and is involved in the binding of receptor CD155 to the virus [START_REF] Minor | Antigenic structure of polioviruses of serotypes 1, 2 and 3[END_REF][START_REF] Harber | Canyon rim residues, including antigenic determinants, modulate serotype-specific binding of polioviruses to mutants of the poliovirus receptor[END_REF][START_REF] He | Interaction of the poliovirus receptor with poliovirus[END_REF]. Amino acid substitution Glu-168-Gly is exposed at the outer surface of the virion and is located in EF loop which is implicated in hydrophobic interactions with receptor CD155 [START_REF] Harber | Canyon rim residues, including antigenic determinants, modulate serotype-specific binding of polioviruses to mutants of the poliovirus receptor[END_REF][START_REF] He | Interaction of the poliovirus receptor with poliovirus[END_REF]. Amino acid substitution Gln-220-Arg is situated in the exterior of the virion, in N-AgIIa site [START_REF] Minor | Antigenic structure of polioviruses of serotypes 1, 2 and 3[END_REF].

All Sabin-2 isolates displayed the mutation A481G in 5'-NCR (Table 2). This substitution is a reversion to the wild-type genotype and has been associated with increased neurovirulence and thermo resistance [START_REF] Macadam | The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity[END_REF][START_REF] Georgescu | Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus[END_REF][START_REF] Muzychenko | Coupled mutations in the 5'untranslated region of the Sabin poliovirus strains during in vivo passages: structural and functional implications[END_REF][START_REF] Macadam | Genetic basis of attenuation of the Sabin type 2 vaccine strain of poliovirus in primates[END_REF][START_REF] Minor | Genetic basis of attenuation of the Sabin oral poliovirus vaccines[END_REF][START_REF] Guillot | Point mutations involved in the attenuation/neurovirulence alternation in type 1 and 2 oral polio vaccine strains detected by site-specific polymerase chain reaction[END_REF][START_REF] Georgescu | High diversity of poliovirus strains isolated from the central nervous system from patients with vaccineassociated paralytic poliomyelitis[END_REF], probably by preventing a new base pair to form between residues 481 and 511, which would weaken the structure of 5'-NCR. Additionally, the mutation U398C was identified in two isolates (EP9 and IF) and the mutation U437C was identified in one isolate (EP9). These substitutions have also some effect in attenuation and thermo sensitivity of Sabin 2 vaccine strain even though minor [START_REF] Friedrich | Genomic modifications in Sabin vaccine strains isolated from vaccination-associated cases, healthy contacts and healthy vaccinees[END_REF]. Both of these mutations are situated in a double stranded region of domain IV of IRES and in particular they convert a base pair GU into GC. It is noteworthy the presence of mutation C584G in three Sabin-2 isolates (EP9, IF and EP12) which leads to a G-G mismatch. In VP1 coding region, amino acid substitutions in residue 143 were identified in two isolates (EP9 and EP12). Residue 143 is exposed on the external surface of the virion in the DE loop and the substitution of the attenuating Ile by Val, Thr or Asn has been correlated with increased neurovirulence [START_REF] Macadam | The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity[END_REF][START_REF] Georgescu | Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus[END_REF][START_REF] Muzychenko | Coupled mutations in the 5'untranslated region of the Sabin poliovirus strains during in vivo passages: structural and functional implications[END_REF][START_REF] Macadam | Genetic basis of attenuation of the Sabin type 2 vaccine strain of poliovirus in primates[END_REF][START_REF] Georgescu | High diversity of poliovirus strains isolated from the central nervous system from patients with vaccineassociated paralytic poliomyelitis[END_REF][START_REF] Equestre | Identification of a consistent pattern of mutations in neurovirulent variants derived from the sabin vaccine strain of poliovirus type 2[END_REF]. In isolate EP12, the substitution Phe-136-Cys was also identified. The residue 136 is situated in the hydrocarbon-binding pocket of VP1 interacting with a sphingosine molecule.

All Sabin-3 isolates displayed the mutation U472C in 5'-NCR (Table 2). This substitution is a reversion to the wild-type genotype and has been associated with increased neurovirulence and thermo resistance [START_REF] Macadam | Reversion of the attenuated and temperature-sensitive phenotypes of the Sabin type 3 strain of poliovirus in vaccinees[END_REF][START_REF] Westrop | Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine[END_REF][START_REF] Monica | A mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans[END_REF][START_REF] Monica | Differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line SH-SY5Y[END_REF][START_REF] Chumakov | Correlation between amount of virus with altered nucleotide sequence and the monkey test for acceptability of oral poliovirus vaccine[END_REF]. The nt 472 and 537 pair in the 5'-NCR in a predicted secondary structure.

The attenuating U at nt 472 of 5'-NCR of Sabin 3 vaccine strain resulted in the alteration of a C-G base pair (nt 472-537) found in the neurovirulent wild strain P3/Leon/37 to a U-G base pair found in Sabin 3 vaccine strain, suggesting that the attenuating phenotype is partially associated with a weakening of a base pairing in a highly conserved structure. It is noteworthy the presence of mutation C586G in all three Sabin-3 isolates. This substitution is situated in a double stranded region of domain VI of IRES and leads to a G-G mismatch as the mutations C583G in Sabin-1 isolate (II) and C584G in three Sabin-2 isolates (EP9, IF, EP12). These mutations may confer a selective advantage in OPV isolates as they have been also observed in isolates of previous studies [START_REF] Pliaka | Use of mutational pattern in 5'-NCR and VP1 regions of polioviruses for molecular diagnosis[END_REF][START_REF] Pliaka | Growth kinetic analysis of bi-recombinant poliovirus vaccine strains[END_REF]. In VP1 coding region, all three Sabin-3 isolates displayed the reversion at nt 2493 (C→U [Thr-6-Ile]) which is a determinant of attenuation [START_REF] Weeks-Levy | Identification and characterization of a new base substitution in the vaccine strain of Sabin 3 poliovirus[END_REF][START_REF] Tatem | A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating[END_REF][START_REF] Mento | Significance of a newly identified attenuating mutation in Sabin 3 oral poliovirus vaccine[END_REF]. Residue 6 is situated at the N-terminus of VP1 in the interior of the capsid and the substitution of the attenuating Thr by Ile probably strengthens the hydrophobic interactions between the VP1 amino terminus and endosomal cell membranes during virus cell entry [START_REF] Tatem | A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating[END_REF][START_REF] Fricks | Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding[END_REF]. In isolate EP23, the substitution Ile-258-Val, partially exposed at the outer surface of the virion and near to N-AgI, was also identified.

In the 2C-3D non-structural genomic region of the isolates, nucleotide and amino acid substitutions were also identified (Table 3). The reversion at nt 6203 (C→U [His-73-Tyr]) of 3D polymerase coding region has been correlated with the attenuated and thermo sensitive phenotype of Sabin 1 vaccine strain [START_REF] Christodoulou | Mapping of mutations associated with neurovirulence in monkeys infected with Sabin 1 poliovirus revertants selected at high temperature[END_REF][START_REF] Georgescu | Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus[END_REF][START_REF] Tardy-Panit | A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice[END_REF][START_REF] Georgescu | Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis[END_REF][START_REF] Martin | Use of type 1/type 2 chimeric polioviruses to study determinants of poliovirus type 1 neurovirulence in a mouse model[END_REF][START_REF] Mcgoldrick | Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus[END_REF]. The C6203U mutation was identified in two recombinant Sabin-2 isolates (EP9: S2/S1 and ID: S2/S1) in a Sabin 1 background but was not identified in the recombinant isolate EP23 (S3/S1) which has also a Sabin 1 background in 3D region. The 2C coding region is known to contain a conserved stem-loop structure (nt 4443-4504 in Poliovirus type 2) serving as an essential cis-acting replicative element (CRE) involved in the uridylylation of VPg [START_REF] Paul | Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg[END_REF][START_REF] Rieder | Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation[END_REF]. The substitution of either of the three A of a conserved AAACA sequence (in Poliovirus type 1 and type 3) in the loop of the CRE reduces both VPg uridylylation and virus yield [START_REF] Rieder | Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation[END_REF]. The substitution G4473A in isolate EP9 (S2/S1), converts the conserved AAGCA sequence of Sabin 2 vaccine strain to the conserved AAACA sequence of Sabin 1 vaccine strain. It has been proposed that the presence of G at nt 4473 is implicated in the attenuated phenotype of Sabin 2 vaccine strain [START_REF] Rieder | Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation[END_REF].

Discussion

In this study, eight OPV isolates which were isolated from healthy vaccinees and VAPP cases during the time period 1978-1985, were investigated. More specifically, we determined the phenotypic traits such as thermo sensitivity and growth kinetics of each isolate and attempted to correlate them with genomic modifications.

The Sabin vaccine strains differ from their progenitors in biological properties other than neurovirulence, some of which have been used as in vitro markers to monitor the quality of new batches of vaccine prior to monkey neurovirulence assays. Temperature sensitivity and plaque size have been used as in vitro phenotypic markers in previous studies and were showed to correlate with the neurovirulence of poliovirus [6-10, 13, 25, 45, 58, 61].

MAPREC test which is based on the presence of mutations at known major determinants of attenuation has also been correlated with the neurovirulence of poliovirus [START_REF] Horie | Isolation of vaccine-derived type 1 polioviruses displaying similar properties to virulent wild strain Mahoney from sewage in Japan[END_REF]. One-step growth curve experiment was also used as an indication of viral multiplication ability that might correlate with viral virulence [START_REF] Bouchard | Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine[END_REF][START_REF] Gutiérrez | Attenuating mutations in the poliovirus 5' untranslated region alter its interaction with polypyrimidine tract-binding protein[END_REF][START_REF] Cello | Growth phenotypes and biosafety profiles in poliovirus-receptor transgenic mice of recombinant oncolytic polio/human rhinoviruses[END_REF][START_REF] Malnou | Effects of vaccine strain mutations in domain V of the internal ribosome entry segment compared in the wild type poliovirus type 1 context[END_REF][START_REF] Jesus | Mutation of a single conserved nucleotide between the cloverleaf and internal ribosome entry site attenuates poliovirus neurovirulence[END_REF].

In the present study, the growth phenotype of eight OPV isolates (two nonrecombinants and six recombinants) as well as of Sabin vaccine strains in Hep2 cells at two different temperatures (37 o C and 40 o C) was evaluated using two different assays, RCT test and one-step growth curve analysis. In nonrecombinant Sabin-1 isolate (II) the presence of mutations at known determinants of attenuation and thermo sensitivity or in other positions of 5'-NCR and VP1 regions involved in virus replication, does not favor the reversion of its growth phenotype at both 37 o C and 40 o C. It is noteworthy the lower virus titre (1 log 10 unit) of isolate II than Sabin 1 vaccine strain at the first stages of the infectious life cycle (0 and 1 h post-infection). This initial difference leads to a lower virus titre of isolate II than Sabin 1 vaccine strain throughout the infectious life cycle and may originate from the different adsorption and/or penetration properties of isolate II in comparison to that of Sabin 1 vaccine strain. However, isolate II showed partial reversion to non-ts phenotype (Rct value=2.0). The presence of many other determinants of thermo sensitivity (nt 935, 1944, 2438 and 2741) in the 5' end of the genome of Sabin 1 vaccine strain makes difficult the reversion of Sabin-1 isolates to a non-ts phenotype [START_REF] Bouchard | Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine[END_REF]. All Sabin-2 isolates were S2/S1 recombinants. Isolates EP9, IF and ID showed a non-ts phenotype while isolate EP12 showed partial reversion to non-ts phenotype as it was proved by RCT test values. The different phenotypes of Sabin-2 isolates may correlate with the presence of mutations at nt 398 (isolates EP9 and IF) and at nt 6203 (isolates EP9 and ID). The presence of mutations at nt 481, 584 and 2909 in isolate EP12 seems to have minor contribution to the reversion to a non-ts phenotype. In the one-step growth curve test, all Sabin-2 isolates displayed similar growth kinetics to Sabin 2 vaccine strain. However, isolates EP9, ID and EP12 displayed lower virus titre (≤1 log 10 unit) than Sabin 2 vaccine strain at the first stages of the infectious life cycle (0 and 1 h post-infection) as in the case of Sabin-1 isolate II. Moreover, isolate IF showed higher viral yield than Sabin 2 vaccine strain at 4, 8 and 16 h post-infection at 40 o C. The discrepancy of the results of the two assays concerning the reversion of thermosensitivity may correlate with the fact that they show the viral multiplication for different time periods.

The recombinant Sabin-3 isolates EP16 (S3/S2) and EP23 (S3/S1) reverted partial to non-ts phenotype while the non-recombinant Sabin-3 isolate 729 displayed a ts phenotype as it seems from Rct test value. Isolates EP16 and EP23 replicated efficiently at both 37 and 40 o C in one-step growth curve test. More specifically, isolates EP16 and EP23 showed 2.0 and 1.7 log 10 units, respectively, higher viral yield than the Sabin 3 vaccine strain at 0 and 1 hour post-infection. The presence of specific mutations (U472C, C586G in 5'-NCR and C2493U in VP1 coding region) and the recombination events may lead to different properties of isolates in comparison to Sabin 3 vaccine strain. These different properties may lead to the higher viral yield of isolates EP16 and EP23 at the initial stages of the infectious life cycle and to the earlier raise of virus titre than the Sabin 3 vaccine strain. The nonrecombinant isolate 729 replicated with kinetics similar to that of Sabin 3 vaccine strain at both 37 and 40 o C. However, it showed higher viral yield (<1 log 10 unit) than Sabin 3 vaccine strain at 0 and 1 h post-infection as isolates EP16 and EP23. The reversion at nt 2493 may be responsible for the higher viral yield of all three Sabin-3 isolates than the Sabin 3 vaccine strain at the initial stages of the infectious life cycle. Isolates EP16 and EP23 showed mutations at the same determinants of attenuation and thermosensitivity (nt 472 of 5'-NCR and 2493 of VP1) as the isolate 729. Consequently, the different phenotypes of isolates EP16 and EP23 in comparison with that of 729 are probably associated with the recombination event. This would be in disagreement with a previous report by Macadam et al. 1989 [7] showing that similar type 3 recombinant viruses were slightly more ts than their nonrecombinant counterparts containing the same reverted mutations.

Consequently, in non-recombinant Sabin-1 isolate the presence of mutations at known determinants of attenuation and thermo sensitivity does not affect its growth phenotype. Sabin 1 vaccine strain is genetically more stable than Sabin 2 and Sabin 3 vaccine strains and this fact may explain why Sabin 3 and Sabin 2 are more frequently isolated from VAPP cases than are Sabin 1 (only about 12% of cases) [START_REF] Strebel | Epidemiology of poliomyelitis in the United States one decade after the last reported case of indigenous wild virus-associated disease[END_REF][START_REF] Strebel | Paralytic poliomyelitis in Romania, 1984-1992. Evidence for a high risk of vaccine-associated disease and reintroduction of wild-virus infection[END_REF]. Moreover, the presence of S1/SX (SX: S2 or S3) recombinants is extremely rare. The sequences of Sabin type 1 origin are regularly found in SX/S1 recombinant strains [START_REF] Macadam | Reversion of the attenuated and temperature-sensitive phenotypes of the Sabin type 3 strain of poliovirus in vaccinees[END_REF][START_REF] Georgescu | High diversity of poliovirus strains isolated from the central nervous system from patients with vaccineassociated paralytic poliomyelitis[END_REF][START_REF] Cuervo | Genomic features of intertypic recombinant sabin poliovirus strains excreted by primary vaccinees[END_REF][START_REF] Driesel | Vaccine-associated cases of poliomyelitis over a 30 year period in East Germany[END_REF][START_REF] Gavrilin | Evolution of circulating wild poliovirus and of vaccine-derived poliovirus in an immunodeficient patient: a unifying model[END_REF][START_REF] Savolainen-Kopra | Comparison of poliovirus recombinants: accumulation of point mutations provides further advantages[END_REF]. The above may correlate with the increased fitness of SX/S1 recombinants than that of the S1/SX recombinants. The determinants of attenuation and thermo sensitivity of Sabin 1 vaccine strain are scattered along the whole genome but most of them are situated in the 5'-NCR and capsid region [START_REF] Bouchard | Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine[END_REF]. Sabin 1 vaccine strain may exchange the 5' end of its genome with that of Sabin 2 or Sabin 3 vaccine strains in order to get rid of these fitness-decreasing mutations.

In Sabin-2 isolates the presence of mutations at nt 398 of 5'-NCR and 6203 of 3D coding region favours considerably the reversion to non-ts phenotype and may lead to the reversion towards neurovirulence. The mutation at nt 398 of 5'-NCR has been frequently observed in Sabin-2 isolates from VAPP cases [START_REF] Macadam | The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity[END_REF][START_REF] Muzychenko | Coupled mutations in the 5'untranslated region of the Sabin poliovirus strains during in vivo passages: structural and functional implications[END_REF][START_REF] Friedrich | Genomic characterization of type 2 polioviruses isolated from vaccineassociated cases in Brazil[END_REF]. The mutation at nt 6203 of 3D coding region has been also observed in Sabin-1 isolates from VAPP cases [START_REF] Otelea | Genomic modifications in naturally occurring neurovirulent revertants of Sabin 1 polioviruses[END_REF][START_REF] Furione | Polioviruses with natural recombinant genomes isolated from vaccineassociated paralytic poliomyelitis[END_REF][START_REF] Georgescu | High diversity of poliovirus strains isolated from the central nervous system from patients with vaccineassociated paralytic poliomyelitis[END_REF].

In Sabin-3 isolates, the recombination of Sabin 3 vaccine strain with Sabin 1 or Sabin 2 and the acquisition of genome structure S3/S1 or S3/S2 favour the growth phenotype and the reversion towards neurovirulence. This observation is in accordance with the fact that S3/SX recombinants have been frequently isolated from VAPP cases [START_REF] Georgopoulou | Improved genotyping vaccine and wild-type poliovirus strains by restriction fragment length polymorphism analysis: clinical diagnostic implications[END_REF][START_REF] Karakasiliotis | Site analysis of recombinant and mutant poliovirus isolates of Sabin origin from patients and from vaccinees[END_REF][START_REF] Paximadi | Genomic analysis of recombinant sabin clinical isolates[END_REF][START_REF] Cuervo | Genomic features of intertypic recombinant sabin poliovirus strains excreted by primary vaccinees[END_REF][START_REF] Blomqvist | Characterization of a recombinant type 3/type 2 poliovirus isolated from a healthy vaccinee and containing a chimeric capsid protein VP1[END_REF][START_REF] Martín | Isolation of an intertypic poliovirus capsid recombinant from a child with vaccineassociated paralytic poliomyelitis[END_REF][START_REF] Paximadi | Vaccine derived bi-and multi-recombinant Sabin strains[END_REF].

Thus, both mutations and recombination could affect the phenotype traits of Sabin derivatives and may lead to neurovirulent viral strains. Moreover, the recombination allows either the congregation of reversed determinants of attenuation and thermo sensitivity in the viral genome or the elimination of some determinants of attenuation and thermo sensitivity from the viral genome.

In conclusion, both Rct assay and one-step growth curve analysis are effective markers for the estimation of neurovirulence of Sabin derivatives.

Moreover the use of phenotypic markers such as the one-step growth curve analysis and the Rct assay along with the genomic analysis may shed additional light in the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

  37 o C and 40 o C of each isolate with that of the corresponding genotype of Sabin vaccine strain (Sabin 1, Sabin 2 or Sabin 3) in one-step growth experiments in Hep-2 cells.

Fig. 1 . 2 Fig. 2 .Fig. 3 .

 1223 Fig.1. One-step growth curve analysis of Sabin-1 isolate II in comparison with

Table 1

 1 Data for the OPV isolates.

	Isolate	5'-NCR	Source	Recombination	Crossover	Accession numbers	Reference
		and VP1		type	point	5'NCR/VP1
		genotype				/2C-3'NCR
	II	S1	Cord	nonrecombinant	-	EU598476/FJ609769/-	[32, 52]
			tumor
	EP9	S2	Healthy	S2/S1	5314-5318/S2 a	EU598464/EU598478	[33]
			vaccinee			/AY736178
	IF	S2	Healthy	S2/S1	6247-6281/S2	FJ609757/FJ609772	[32]
			vaccinee			/AY297764 (2C),
						AY297760 (3D)
	ID	S2	VAPP	S2/S1	5521-5526/S2	FJ609758/FJ609773	[32]
						/EU715814
	EP12	S2	Healthy	S2/S1	6337-6362/S2	EU598465/EU598479	[33]
			vaccinee			/AY736179
	EP16	S3	Healthy	S3/S2	4892-4914/S3	EU598466/FJ609775	[33]
			vaccinee			/AY736180
	EP23	S3	VAPP	S3/S1	4880-4887/S3	EU598468/EU598480	[33]
						/AY736181
	729	S3	Healthy	nonrecombinant	-	EU598477/FJ609778/-	[52]
			vaccinee
		a Numbering according to Sabin 2 (AY184220) or Sabin 3 (AY184221) vaccine
		strains.

Acknowledgements

The work was supported by research grants of the Postgraduate Program "Applications of Molecular Biology-Genetics. Diagnostic Biomarkers", code 3817, of the University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology.

Table 2

Location of mutations in 5'-NCR and VP1 sequences of OPV isolates and comparison with the corresponding nucleotide and amino acid positions in Sabin vaccine strains. Numbering according to vaccine strains Sabin 1(AY184219), Sabin 2 (AY184220) and Sabin 3 (AY184221). Mutations identified in more than one isolates are underlined. Reproductive capacity values of OPV isolates and Sabin vaccine strains at different temperatures (Rct marker) are also indicated. Location of mutations in 2C-3D genomic region of isolates EP9, IF, ID, EP12, EP16, EP23 and comparison with the corresponding nucleotide and amino acid positions in Sabin vaccine strains. The genomic region from 2C-3D of no recombinant isolates II and 729 was not sequenced. Numbering according to vaccine strains Sabin 1 (AY184219), Sabin 2 (AY184220) and Sabin 3 (AY184221). Mutations identified in more than one isolates are underlined.

Isolate Recombination type

Mutations Δ logRct 40 5'-NCR VP1

Isolate Region Origin