L20B Cell Research Articles

Enteroviruses as a possible cause of hypertension, dilated cardiomyopathy (DCM) and hypertensive heart failure (HHF) in South western Nigeria

Human enteroviruses have long been associated with various diseases of man resulting into a wide range of acute symptoms involving the cardiac and skeletal muscles, central nervous system, pancreas, skin and mucous membranes. To assess the role of enteroviruses in the etiology of hypertension, DCM and HHF. We obtained stool specimens from 70 subjects comprising 65 patients and 5 controls and isolation was carried out on RD, L20B, HEp-2C and Vero cell lines and identified by neutralization with standard antisera (RIVM). Thirty-six enteroviruses were isolated and identified to be Coxsackieviruses-B5, A9, Echoviruses 1, 6, 7, 9, 11, 12, 22, 30 and Poliovirus type 1 and 3. Three most frequently occurring enterovirus serotypes which constitute 60.0% of the 30 NPEV typed and 50.0% of all the isolates were Echoviruses, Coxsackie-B5-virus and Coxsackievirus-A9. Echoviruses constituted 50.0% of all the serotypes while Coxsackieviruses-B5 and A9 accounts for the 27.8 % and 5.6% respectively. Enteroviral isolation rate was higher in age groups 51 years and above. The percentage of study subjects who had Coxsackie-B5-viruses and echoviruses was significantly (P<0.05) higher in cases of hypertension, HHF and DCM than in control subjects. Coxackie-B5-virus, Echovirus-6 and Echovirus-11 were found in both study locations. The findings of this study showed that Enteroviruses may likely be involved in the etiology of hypertension, DCM and HHF. Further studies would therefore be necessary for the prevention and control of these diseases.

Environmental surveillance of poliovirus and non-polio enterovirus in urban sewage in Dakar, Senegal (2007-2013).

IntroductionGlobal poliomyelitis eradication initiative relies on (i) laboratory based surveillance of acute flaccid surveillance (AFP) to monitor the circulation of wild poliovirus in a population, and (ii) vaccination to prevent its diffusion. However, as poliovirus can survive in the environment namely in sewage, environmental surveillance (ES) is of growing importance as the eradication target is close. This study aimed to assess polioviruses and non polio enteroviruses circulation in sewage drains covering a significant population of Dakar.MethodsFrom April 2007 to May 2013, 271 specimens of raw sewage were collected using the grab method in 6 neighborhoods of Dakar. Samples were processed to extract and concentrate viruses using polyethylene glycol and Dextran (two-phase separation method). Isolation of enteroviruses was attempted in RD, L20B and Hep2 cell lines. Polioviruses were identified by RT-PCR and Elisa. Non Polio Enteroviruses (NPEVs) were identified by RT-PCR and microneutralisation tests.ResultsPolioviruses and NPEVs were respectively detected in 34,3% and 42,8% sewage samples. No wild poliovirus neither circulating vaccine-derived Poliovirus (cVDPV) was detected. Neutralization assays have identified 49 non polio enteroviruses that were subsequently classified in 13 serotypes belonging to HEV-A (22, 4%), HEV-B (12, 24%), HEV-C (26, 53%) and HEV-D (6, 12%) species.ConclusionThis study is the first documentation of enteroviruses environmental detection in Senegal. It shows the usefulness of environmental surveillance for indirect monitoring of the circulation and distribution of enteroviruses in the community.

Detection of Enteroviruses in Water Samples from Yopougon, Côte d’Ivoire by Cell Culture and Polymerase Chain Reaction

The objective of this study was to compare sensitivities of enterovirus isolation from wastewater in different cell lines as well as to compare the sensitivity and specificity of isolation in cell culture with direct detection by reverse transcription polymerase chain reaction (RT-PCR). Sixty-eight samples of wastewaters were collected between September 2008 and January 2009 in Yopougon, Abidjan. Enteroviruses were concentrated according to World Health Organization recommendations. Viruses were inoculated into various cell lines while direct RT-PCR was performed on water concentrates. The buffalo green monkey kidney cell line was the most sensitive with 58.8% of viral isolation. This was followed by the rhabdomyosarcoma cell line with sensitivity of 51.6%, with human epidermoid carcinoma cell line showing sensitivity of 50% and fibroblastic cells derived from transgenic mice LTK-1 (L20B) cell showing 23.50% sensitivity. However, a lower specificity of 2.9% was observed with the L20B cell line. 44.1% of the samples were positive by direct RT-PCR detection while 51.47% samples were positive by using RT-PCR on infected cell cultures. No difference in percentage positivity was observed using RT-PCR on infected tissue culture isolates or using RT-PCR directly on wastewater samples.

ISOLATION SOME ACTIVE MATERIALS IN THE IRAQI FRUITS AND SEEDS OF CITRULLUS COLOCYNTHIS L. AND STUDY OF THEIR EFFECTS ON AQUEOUS AND ALCOHOLIC EXTRACTS OF SOME PATHOLOGICAL BACTRIA AND CANCER CELLS TYPE L20B

The aim of this research is to detect and identify the functional chemical materials in the fruits and seeds of citrulluscolocynthisL..The results show that the fruits contain Tannins , phlobatannins , Saponins , flavonoids , glycosides , terepens , phenols , resins , carbohydrates , alkaloids , and iridoiglycosids . while the seeds contain tannins , saponins , flavonoids , terepens , phenols , resin , amino acids , carbohydrates , alkaloids , and iridoidglycosides .The percentage of isolated saponins , tannins , and volatile oils in fruits 10.92% , 17.6% , and 2.04% whereas their percentage in seeds are 3.77% , 7.39% , and 18.7% respectively .The concentrations of isolated materials , aqueous extracts and alcoholic extracts (1 , 5 , 12 , 25 , 50 mg/ml )have been studied as a biological activities against E.coli and staph.aureus the results show the highest inhibition diameter is 18mm against E,coli using 12mg /ml while against staph.aureus is 20mm using 25mg/ml concentration .The aqueous and alcoholic extracts of fruits and seeds gave a good inhibition towards the growth of L20B cell line and the highest growth inhibition is 67.7% .

Reemergence of Recombinant Vaccine–derived Polioviruses in Healthy Children, Madagascar

Reemergence of Recombinant Vaccine–derived Polioviruses in Healthy Children, Madagascar

Electron microscope detection of an endogenous infection of retrovirus-like particles in L20B cells

L20B cells are a cell line commonly used for the isolation of poliovirus. The current study indicates that L20B cells are chronically infected with a retrovirus-like particle that replicates in the cytoplasm and buds through the plasma membrane. The findings indicate that care is needed in the use of L20B cells for certain virus isolation studies and emphasize the importance of electron microscope studies as an adjunct to the development of diagnostic virology protocols.

In Vitro CYTOTOXIC STUDY FOR PARTIAL PURE LIGNAN EXTRACT FROM FLAX SEED (Linum usitatissirnurn) ON RHABDOMYOSARRCOMA (RD) AND MURINE MAMMARY ADENOCARCINOMA (MMA) CELL LINES

The lignan (Secoisolaricinesinal diglucoside, SDG), was obtained from flaxseed ( Linum usitatissirnurn) appear to act as both anti-estrogens and weak estrogens. The anti-estrogenic potential of lignans has been postulated to protect against hormone sensitive cancers such as breast, prostate and colon cancer. The aim of this study was to examine the cytotoxic effect of partial pure lignan on some cell lines including, the Murine Mammary Adenocarcinoma, Rhabdomyosarcoma RD, L20B and normal cell line (Rat Embryo Fibroblast REF). Four concentrations of partial pure SDG which obtained by using alcohol hydrolysis dioxin / ethanol flaxseed extraction and liquid/liquid partition, have been prepared 5, 10, 15, 20 μM with different of exposure periods 24, 48, 72 h. These parameters have been applied on all cancer and normal cell line. The highest percentage of inhibition appears in 5, 10,15 μM at 24 h (60.11, 49.99, 50.28 %) respectively on RD cell line when treated with partial pure. and the with same concentrations this percentage decreases without any significant difference. In case of L20B cell line in 72 h, the inhibitory effect of partial pure at 5,10 μM, 57.67 and 52.11 % respectively. While the cytotoxicity of partial pure SDG on ANM3 cell line appeared in 24h at 15um which was 64.88%. The lignans are growth inhibitors of colon tumor cells and suggest act through mechanisms other than anti-estrogenic activity.

The Detection of Enteroviruses in Sewage Using Caco-2 Cells

The work presented here demonstrates the utility of Caco-2 cells to detect enteroviruses in sewage. Viruses were concentrated by beef extract elution and organic flocculation prior to analysis by cell culture assays and RT-PCR. Enteroviruses were detected in all sewage samples, but only one sample was positive solely in RT-PCR assay. We proved that Caco-2 cells were more effective than RD and L20B cells in enterovirus isolation, depending on procedures used in the inoculation process.

Sebaran Geografi Non Polio Enterovirus (NPEV) dari Kasus Layu Akut di Bagian Barat Indonesia 2007-2010

Acute Flaccid Paralysis (AFP) surveillance which comprises cases and laboratory investigation has been established by WHO in order to achieve the goal of global eradication of polio. NIHRD is one of the national reference polio laboratories in Indonesia that conduct diagnostic test for AFP case specimens from the Western part of Indonesia. AFP cases can be caused by poliovirus and non polio viruses, such as enteroviruses. This recent study investigate the serotype of Non Polio Entero Viruses (NPEV) from AFP cases in 2007-2010 and its geographical distribution. This recent study used biological archieve from AFP cases in 2007-2010. APF specimens was cultured in cell line (RD/L20B) in BSL-2 laboratory. Positive isolates from RD cell lin in negative in L20B cell line in were tested by neutralization test to determine serotype of NPEV. Demography data were also collected from case investigation form of the patients and geographical data was analyzed using GIS arc View 3.3. From a total 2525 AFP cases, 6,8% are NPEV. NPEV cases were mostly found in North Sumater province (20,4%), There is variation fo the number of NPEV cases found each year in 16 province that sen AFP specimens to NIHRD. NPEV cases is commonly found in age group 1-5 years old, (54,3% in 2007, 86,7% in 2008, 62,2% in 2009 and 66,3% in 2010). Serotyping using WHO standard antisera showed that Echovirus is the common serotype found in 2007-2010. However, there were unidentified or untypeable NPEV isolates that need further investigation. NPEV is one of the etiologies that cause Acute Flaccid Paralysis (AFP) in Western part of Indonesia. Further detection of NPEV serotype using molecular tehcnology is needed to reveal other etiologies of AFP. Key words : Acute Flaccid Paralysis, Non Polio Enterovirus, Serotype Abstrak Surveilans lumpuh layu akut atau Acute Flaccid Paralysis (AFP) yang terdiri dari investigasi kasus dan pemeriksaan laboratorium telah ditetapkan oleh WHO sebagai bagian program pemberantasan polio global. Laboratium Virologi Badan Litbangkes adalah salah satu laboratium rujukan Nasional penyakit polio di Indonesia yang melakukan tes diagnostik untuk spesimen kasus AFP dari Indonesia bagian Barat. Kasus AFP dapat disebabkan oleh virus polio dan virus non polio seperti enterovirus. Tujuan penelitian ini adalah untuk menyelidiki serotipee Non Polio Entero Virus (NPEV) dari kasus AFP pada tahun 2007-2010 dan distribusi geografisnya. Studi ini menggunakan spesimen tersimpan dari kasus AFP pada tahun 2007-2010. Spesimen AFP dikultur dalam lini sel (RD/L20B) di laboratorium BSL-2. Isolat positif dari lini sel RD dan negatif pada lini sel L20B diuji netralisasi untuk menentukan serotipe NPEV. Data denografi juga dikumpulkan dari bentuk kasus investigasi dari pasien sedangkan, data geografis dianalisis menggunakan GIS View 3.3. Dari total 2625 kasus AFP, ditemukan 6,8% kasus NPEV. Kasus NPEV terbanyak ditemukan di provinsi Sumatera Utara (20,4%). Terdapat variasi jumlah kasus NPEV yang ditemukan setiap tahunnya pada 16 provinsi yang mengirim spesimen AFP ke Laboratium Badan Litbangkes. Kasus NPEV umunya ditemukan dalam kelompok usia 1-5 tahun, (54,3% pada tahun 2007, 86,7% pada tahun 2008, 62,2% pada tahun 2009, dan 66,3% pada tahun 2010). Serotyping menggunakan antiserum standar WHO menunjukkan bahwa Echovirus adalah serotipe yang paling banyak ditemukan pada spesimen AFP yang dikumpulkan pada tahun 2007-2010. Namun, terdapat isolat NPEV tidak dikenal atau untypeable yang perlu penyelidikan lebih lanjut. Echovirus merupakan salah satu etiologi yang menyebabkan Acute Flaccid Paralysis (AFP) di Indonesia bagian Barat. Deteksi lebih lanjut serotipe NPEV menggunakan teknologi molekuler diperlukan untuk mengungkapkan etiologi lain dari AFP. Kata Kunci : Acute Flaccid Paralysis, Non Polio Enterovirus,Serotype

Comparative study of molecular and antigenic characterization for intratypic differentiation (ITD) of poliovirus strains

This study was designed to compare the sensitivity of a Sabin vaccine strain-specific PCR assay and an enzyme-linked immunosorbent assay with polyclonal cross-absorbed antisera (PAb-E) for intratypic differentiation (ITD) of polioviruses (PVs). These were used for the definitive characterization of the strains. Poliovirus strains isolated in L20B and RD cell lines were subjected to both PCR and ELISA. Both PCR and ELISA identified 3 (13.6%) out of 22 isolates, respectively as poliovirus Sabin 1. PCR identified 4 (18.2%) out of 22 isolates as poliovirus Sabin 2 and ELISA identified 2 (9.1%) out of 22 isolates as poliovirus Sabin 2. None of the two assay identified poliovirus Sabin 3. Both PCR and ELISA identified 12 (54.5%) out of 22 isolates, respectively as wild poliovirus (WPV) 1. None of the assays identified any of the isolates as WPV 2 and 3. Only PCR assay was able to identify the mixture of two poliovirus Sabin serotypes (a mixture of Sabin 1 and 2) and two mixtures of poliovirus Sabin 2 and 3. In this study, only ELISA was able to identified two invalid results. Invalid results observed in this study are of important practical implication to the emergence of vaccine-derived poliovirus. This may have epidemic potential. Hence, the two ITD assays are of paramount importance for identification of PVs. It is therefore recommended in line with WHO guideline that at least two methods be used for the ITD of poliovirus isolates, and each method should be based on a different principle (i.e., antigenic and genetic properties).

Identification of human parechovirus genotype, HPeV-12, in a paralytic child with diarrhea

BackgroundNew genotypes of human parechoviruses have been readily identified after improvement of diverse diagnostic tools. We hereby report the detection of a new genotype, HPeV 12, from a child presented with diarrhea and paralysis. ObjectivesThe genetic variability of human parechoviruses has recently expanded defining 16 genotypes however data available covers only 11 genotypes. The present study was designed to determine the genetic characterization of human parechovirus identified in a child with gastroenteritis and acute flaccid paralysis (AFP). Study designStool samples are referred to Virology Department, NIH-Pakistan for the routine detection of enteroviruses and polioviruses through cell culture and RT-PCR. Five of isolates showing cytopathic effect on L20B cell line but negative for poliovirus were further explored for human parechovirus using multiple cell lines and RT-PCR. ResultsHuman Coxsackie A virus type 2, 3, 6 and 20 were found in four samples whereas the fifth sample contained human parechovirus genotype 12. Efficient growth of human parechovirus was found on L20B cells while Vero and LLC-MK2 cells showed no apparent cytopathic effect. ConclusionsThis study describes the detection of a new human parechovirus genotype (HPeV-12) in a paralytic child with diarrhea. Human parechoviruses are now considered as potential pathogens that may cause a number of serious clinical complications especially in infants and young children. These findings emphasize to conduct large scale epidemiological surveys in the country to understand their association with clinical diseases especially gastroenteritis, respiratory and neurological disorders.

Evaluating the Inhibitory Activity of Apigenin Extracted from Salvia officinalis leaves on the Growth of L20B Cancer Cell Line

The study aimed to evaluating the inhibitory activity of apigenin extracted from Salvia officinalis leaves on the growth of L20B cancer cell in vitro, and through two incubation periods; 48 and 72 hours. Accordingly, eight concentrations (1.56, 3.13, 6.25, 12.5, 25.0, 50.0, 100.0 and 200.0 micromol) of apigenin and similar concentrations of vitamin C and carbon tetrachloride (CCl4) were tested. The apigenin revealed its significant inhibitory potentials against the growth of L20B cell line, especially at the low concentrations (1.56, 3.13 and 6.25 micromol) and at 72 incubation period in comparison with vitamin C and CCl4.

Evaluating the Inhibitory Activity of Apigenin Extracted from Salvia officinalis leaves on the Growth of L20B Cancer Cell Line

The study aimed to evaluating the inhibitory activity of apigenin extracted from Salvia officinalis leaves on the growth of L20B cancer cell in vitro, and through two incubation periods; 48 and 72 hours. Accordingly, eight concentrations (1.56, 3.13, 6.25, 12.5, 25.0, 50.0, 100.0 and 200.0 micromol) of apigenin and similar concentrations of vitamin C and carbon tetrachloride (CCl4) were tested. The apigenin revealed its significant inhibitory potentials against the growth of L20B cell line, especially at the low concentrations (1.56, 3.13 and 6.25 micromol) and at 72 incubation period in comparison with vitamin C and CCl4.

Feasibility of Quantitative Environmental Surveillance in Poliovirus Eradication Strategies

The progress of the Global Polio Eradication Initiative is monitored by acute flaccid paralysis (AFP) surveillance supplemented with environmental surveillance in selected areas. To assess the sensitivity of environmental surveillance, stools from (re)vaccinated elderly persons with a low seroprevalence and from wastewater were concurrently collected and analyzed in the Netherlands over a prolonged period of time. A total number of 228 healthy individuals with different levels of immunity were challenged with monovalent oral polio vaccine serotype 1 or 3. Poliovirus concentrations were determined by the titration of fecal suspensions on poliovirus-sensitive L20B cells and of sewage concentrates by L20B monolayer plaque assay. Almost half of the individuals (45%) shed poliovirus on day 3 after challenge, which peaked (57%) on day 8 with an average poliovirus excretion of 1.3 × 10(5) TCID(50) per g of feces and gradually decreased to less than 5% on day 42. The virus concentrations in sewage peaked on days 6 to 8 at approximately 100 PFU per liter, remained high until day 14, and subsequently decreased to less than 10 PFU per liter on day 29. The estimated poliovirus concentration in sewage approximated the measured initial virus excretion in feces, within 1 log(10) variation, resulting in a sensitivity of detection of 100 infected but mostly asymptomatic individuals in tens of thousands of individuals. An additional second peak observed in sewage may indicate secondary transmission missed by enterovirus or AFP surveillance in patients. This enables the detection of circulating poliovirus by environmental surveillance, supporting its feasibility as an early warning system.

Synergism between the compound Dichloroflavan and Chalcone Against poliovirus in vitro

Flavone originally founded in some herbs and plants such as beans, tomato and grapefruit. Dichloraflavan (DCF) and the compound chalcone relatively non-toxic, we find in this study that the toxicity of the two compounds to RD and L20B cells ranging between (16-32)µg/ml when they used alone. The minimal inhibitory concentration (MIC) of chalcone was 0.06 µg/ml while the MIC of DCF was 0.03 µg/ml to inhibit (100TCID50) of poliovirus in RD cells and L20B cells. Binary combination of these two compounds showed synergistic activity. We used the Therapeutic Index (TI) to determine the safety of the compound the TI which is the ratio between the toxic concentrations of the drug over the MIC. If the TI one or less means that the compound not advise to use due to the side effect while if the TI more than one compound may be used for treatment, And the fractional inhibitory concentration (FIC) index to determined the synergy between the two compounds MIC of drug A in combination MIC of drug B in combination FIC index = + MIC of drug A alone MIC of drug B alone The interpretation of the index is as follows: &lt;0.5; significant synergism; 0.5-0.9; suggestive of synergism; =1; effects are additive; 1.1-1.9 partial antagonism; .2 antagonisms.

Détection d’entérovirus dans les eaux usées urbaines à Yopougon, Abidjan

AimThe aim of this study was to determine prevalence of enteroviruses in raw municipal wastewater and specify serotypes circulating. MethodsAfter virus concentration by WHO recommend method, aqueous phase of 68 samples was inoculate on BGM, RD, L20B and Hep2 cells lines. Typing after titration was performed by microplate neutralization test with specific horse immune serum against EV (kit RIVM/WHO). NPEV strains were confirmed by RT-PCR test in 5′NC genomic region. Antigenic and molecular tests of Polioviruses intratypic differentiation were performed to determine vaccine or wild origin of Poliovirus strains. ResultsViral isolation obtained 43/68 (65.15 %) positives samples. Among these 35/43 (81. 39%) were EV and 8/43 (19.04 %) non EV strain. Among EVs, 34/35 (97.14 %) were NEPV and 2/35 (5.71 %) vaccinal Poliovirus strains type 2 (Sabin-like). NEPV typing gave 77 % untypeable strains by conventionally method. The average virus concentration estimated in the raw sewage is 4.28×105 PFU/L. ConclusionVarious contacts between people and these waters expose them to health risks.

Estandarización de un método de concentración y detección de virus entéricos en aguas de consumo

Enteric viruses have been implicated in acute diarrheal disease, food-borne disease, hepatitis A and meningitis outbreaks, in which water was the vehicle of transmission. A concentration method was standardized for the detection of enteric viruses in drinking water. Twenty liters of water were concentrated to 6 ml by filtration and tangential ultrafiltration. Viral solutions of 20 L each were prepared at 1, 10, 50 and 100 TCID50 of Sabin poliovirus type 1 as positive controls. Viral particles were recovered by tissue culture and detected by conventional polymerase chain reaction (PCR), according to the international standards recommended by the Enterovirus Laboratory at the Centers for Disease Control and Prevention, Atlanta, GA. All positive controls showed cytopathic effect on L20B and RD cells and were amplified by conventional PCR directly from samples. Negative controls did not show any amplification or viral cytopathic effect. Tangential ultrafiltration for concentrating viruses proved to be a fast, efficient recovery and reproducible. It has the advantage of allowing the detection (at the 1 TCID50 level) and identification of viruses by RT-PCR and the demonstration of viral infectivity by tissue culture.

Public health approach after detection of an iVDPV case in Argentina

Background: Argentine has been polio free since 1984 and has a sustained and active surveillance of the acute flaccid paralysis (AFP) that involves an epidemiology and laboratory approach, reaching all the PAHO indicators. In this context we describe the emergence of a case of AFP due to an VDPV in a 15-months old boy with polyclonal agammaglobulinemia. Methods: The case was notified to the National Program, and a stool sample and a throat swab were sent to the Regional Reference Center for Polio Diagnosis. Samples were inoculated in Rd and L20B cells following the new algorithm recommended by WHO. In less than 2 days a virus was isolated in both samples which were characterized as a polio type 1. They were sequenced in the VP1 region, 5́NCR. As a result we found a 3,7% (stool sample) and 3,5% (throat swab) nucleotide difference in VP1 compared with the Sabin strain. These results confirm the presence of an iVDPV1. The sequences of 5́NCR showed the 480 nucleotide change, proving the reversion of the sabin strain to the infectivity. These findings were sent to the National Program in less than 15 days. Results: After the detection of AFP notification was sent to the field, Epidemiology actions were made as in all cases. In the investigation three different locations were established as the child residence. All of them were visited and vaccination of all children under 18 years old was done. A national and international alert were sent Active community surveillance was made and contact and environmental samples were collected and sent to the Regional Lab. In none of them the iVDPV 1 was detected. Four serial samples from the case were taken each month, in all of them the same iVDPV1 was isolated. Conclusion: The country has sustained the surveillance of AFP through 22 years based on the collaborative work between the laboratory and the epidemiologists. No other cases appeared although the vaccine coverage in one of the district was very low. As consequence of this finding a national vaccination campaign was made. Although poliomyelitis is a threat to the region Argentine is ready to face it. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive

Isolation of Sabin-Like Polioviruses from Wastewater in a Country Using Inactivated Polio Vaccine

From 2001 to 2004, Switzerland switched from routine vaccination with oral polio vaccine (OPV) to inactivated polio vaccine (IPV), using both vaccines in the intervening period. Since IPV is less effective at inducing mucosal immunity than OPV, this change might allow imported poliovirus to circulate undetected more easily in an increasingly IPV-immunized population. Environmental monitoring is a recognized tool for identifying polioviruses in a community. To look for evidence of poliovirus circulation following cessation of OPV use, two sewage treatment plants located in the Zurich area were sampled from 2004 to 2006. Following virus isolation using either RD or L20B cells, enteroviruses and polioviruses were identified by reverse transcription-PCR. A total of 20 out of 174 wastewater samples were positive for 62 Sabin-like isolates. One isolate from each poliovirus-positive sample was analyzed in more detail. Sequencing the complete viral protein 1 (VP1) capsid coding region, as well as intratypic differentiation (ITD), identified 3 Sabin type 1, 13 Sabin type 2, and 4 Sabin type 3 strains. One serotype 1 strain showed a discordant result in the ITD. Three-quarters of the strains showed mutations within the 5' untranslated region and VP1, known to be associated with reversion to virulence. Moreover, three strains showed heterotypic recombination (S2/S1 and S3/S2/S3). The low number of synonymous mutations and the partial temperature sensitivity are not consistent with extended circulation of these Sabin virus strains. Nevertheless, the continuous introduction of polioviruses into the community emphasizes the necessity for uninterrupted child vaccination to maintain high herd immunity.

Phytochemical and biological studies on Acanthus hirsutus Boiss

Acanthus L. is a genus of 30 species of flowering plants in the family Acanthaceae and represented by five species in Turkish flora [1]. Some of the Acanthus species are used as a herbal remedy for various ailments such as cough, chest pain, snake bite, paralysis, asthma, stomach upset, urinary disorders and rheumatic pains in different countries [2, 3]. The titled plant, Acanthus hirsutus Boiss. is endemic for Turkey and used as expectorant, for wound healing and against constipation in Anatolia [4]. There is no phytochemical or biological record in the literature for this species up to now. After serial chromatographies of the methanol extract of the plant, three lignan glucosides (-)-syringaresinol-di-O-β-D-glucopyranoside, (-)-medioresinol-di-O- β -D-glucopyranoside, (-)-pinoresinol-4'-O- β -D-glucopyranoside), two benzoxazinoids (4-hydroxy-2H-1,4-benzoxazin-3(4H)-one, (2R)-2-O- β -D-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one), four phenylethanoid glycosides (acteoside, martynoside, leucosceptoside A, hattushoside) and two phenylpropanoid glucosides (sinapyl aldehyde-4-O- β -D-glucopyranoside, sinapyl alcohol-4-O- β -D-glucopyranoside) were isolated. Aqueous A. hirsutus extract (IC50=201–700µg/ml) and its isolated compounds (IC50=11.17–262.42µg/ml) showed significant cytotoxic activity in vitro against Hep-2, RD and L20B cell lines in MTT assay. Their radical scavenging activities were also found against 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical (IC50=86.30µg/ml for the extract, IC50=10–200µg/ml for the compouds.