Isolation of Sabin-like Polioviruses from Sewage in Poland.

As a complement to the active search for cases of acute flaccid paralysis, environmental sampling was conducted from January to December 2011, to test for any putative polio revertants and recombinants in sewage. A total of 165 environmental samples were obtained and analyzed for the presence of polioviruses by use of cell culture (L20B, RD and Caco-2) followed by neutralization and reverse-transcription polymerase chain reaction. Out of the 31 CPE positive samples, 26 contained one and 5 two different serotypes, yielding a total of 36 PVs. The microneutralization test revealed the presence of 7, 10 and 19 strains belonging to poliovirus serotype 1, 2 and 3, respectively. The genomic variability of 36 poliovirus strains was examined by the restriction fragment length polymorphism assay (RFLP). By combined analyses of two distant, polymorphic segments of the viral genome, one situated in the capsid protein VP1 coding region and the other in the 3D-polymerase coding region, we screened for the putative poliovirus revertants and recombinants. All detected PVs were classified as vaccine strains on the basis of RFLP-VP1 test. None of wild-type PVs or vaccine derived polioviruses were detected. RFLP assay also revealed the presence of 11 recombinants in 3D-polymerase coding region. Nine isolates appeared to be S3/S2, one S3/S1 and S1/S2 recombinant in analyzed 3Dpol region. This study revealed, through environmental monitoring, the introduction of SL PVs into the population associated with the routine use of OPV in Poland before the April 2016. Our findings demonstrate the usefulness of environmental surveillance in the overall polio eradication program.

Detection of Polioviruses in Sewage Using Cell Culture and Molecular Methods.

The work presented here demonstrates the utility of a two-step algorithm for environmental poliovirus surveillance based on: preselection of sewage samples tested for the presence of enteroviral genetic material-RT-PCR assay and detection of infectious viruses by cell culture technique (L20B for polioviruses and RD for polio and other non-polio enteroviruses). RD and L20B cell lines were tested to determine their sensitivity for isolation of viruses from environmental samples (sewage). Finally, we wanted to determine if sewage concentration affects the results obtained for RT-PCR and cell cultures.

Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy.

Ovarian cancer remains the most lethal gynecologic malignancy owing to late detection, intrinsic and acquired chemoresistance, and remarkable heterogeneity. Here, we explored approaches to inhibit metastatic growth of murine and human ovarian tumor variants resistant to paclitaxel and carboplatin by oncolytic vaccinia virus expressing a CXCR4 antagonist to target the CXCL12 chemokine/CXCR4 receptor signaling axis alone or in combination with doxorubicin. The resistant variants exhibited augmented expression of the hyaluronan receptor CD44 and CXCR4 along with elevated Akt and ERK1/2 activation and displayed an increased susceptibility to viral infection compared with the parental counterparts. The infected cultures were more sensitive to doxorubicin-mediated killing both in vitro and in tumor-challenged mice. Mechanistically, the combination treatment increased apoptosis and phagocytosis of tumor material by dendritic cells associated with induction of antitumor immunity. Targeting syngeneic tumors with this regimen increased intratumoral infiltration of antitumor CD8+ T cells. This was further enhanced by reducing the immunosuppressive network by the virally-delivered CXCR4 antagonist, which augmented antitumor immune responses and led to tumor-free survival. Our results define novel strategies for treatment of drug-resistant ovarian cancer that increase immunogenic cell death and reverse the immunosuppressive tumor microenvironment, culminating in antitumor immune responses that control metastatic tumor growth.

Seroprevalence of measles-specific antibodies in the group predominantly affected by measles in 2006-2009 in Poland.

INTRODUCTION: Irrespective of the high vaccination coverage against measles, sporadic measles outbreaks still occur in Poland. In 2006-2009, a slight increase in the number of measles cases was observed. Of these cases, people born in 1976-198 9 were predominantly affected. AIM: The aim of the study was to evaluate the immunity to measles in the aforesaid age group. MATERIAL AND METHODS: The serum samples were selected from the serum bank in which material collected from the general population living in 5 provinces in Poland is stored. These samples were collected from patients hospitalized due to emergencies in 2010-2011. The antibody titre against measles was determined in each serum sample by ELISA test (Genzyme Virotech). Linear regression models using log-transformed antibody titres were used to compare the values. RESULTS: The serum samples collected from 483 persons, including 111 females and 372 males were tested. All patients had antibody titres exceeding 0.5 IU/ml. The antibody titre was statistically significantly associated with the vaccination coverage in each age group in particular province. CONCLUSIONS: The results of this study suggest good immunity to measles in the general population in Poland. The disparities between randomly selected provinces demonstrated a relation with the coverage rates as well as the differences in measles incidence which is observed recently between these provinces.

The utility of Caco-2 cells in isolation of enteroviruses from environmental and clinical material.

The work presented here demonstrates the utility of Caco-2 cells in the isolation of enteroviruses (EVs) from environmental and clinical materials. Thirty-two samples of cerebrospinal fluid positive in Pan-entero RT-PCR were taken for EV strain isolation in cell culture. Out of the 32 samples analysed, 22 (68.75%) were positive for enteroviruses by isolation in Caco-2 cells, and 10 (31.25%) were positive by isolation in RD cells. High viral titre in clinical specimens resulted in rate increase for isolation in Caco-2 cells and RD cells (87.5% and 50%, respectively). Also, the probability of isolation of enteroviruses from sewage in Caco-2 cells was 20 times higher that in RD cells. We proved that Caco-2 cells were more effective than RD cells in enterovirus isolation, irrespective of the material used in the inoculation process.

Relationship between measles outbreaks based on genetic analysis of measles virus genomes detected in patients in Poland between 2006 and 2012.

This study describes the molecular characterization of 56 MeV strains obtained from 56 patients in Poland from 2006 to 2012. The C-terminal fragment of nucleoprotein gene was analysed. It has been found out during 2006 and 20012 MeV strains circulating in Poland belonged to genotypes D4, D5, D6 and B3. The D4 strains isolated in Poland were different from any other D4 strain circulating at the same time in Europe, whereas all other MeV strains isolated during 2007-2012 were related to strains from other countries. The present data suggest that after 2006 the MeV strains were imported.

Measles outbreak among Roma people in Wroclaw, Poland, 2012.

A measles outbreak that affected mainly the Roma ethnic group has been observed in Wroclaw, southwest Poland, in spring/summer 2012. There were 15 confirmed measles cases occurring among young Roma people aged from 0 to 16 years including a newborn infant, born by a mother who showed measles symptoms immediately after delivery. Measles virus transmission into the general Polish population was restricted to two contact cases. Initiation of the outbreak by MeV importation from Romania has been confirmed by detection of MeV variant "D4-Maramures" circulating in Romania from 2011 to 2012. The outbreak experience highlights once more the still existing prob- lem of immunity gaps in Roma groups moving throughout Europe with a high susceptibility among children and adolescents including young women of child-bearing age.

Non-influenza viruses in acute respiratory infections among young children. High prevalence of HMPV during the H1N1V.2009 pandemic in Poland.

UNLABELLED: In Poland the majority of hospitalized cases of pneumonia (annually more than 70000) were reported without determination of an aetiological agent (J18 of ICD-10), also because diagnosis of viral ARTI is limited to identification of influenza viruses or sometimes RSV. MATERIAL AND METHODS: For determination the contribution of non-influenza viruses in ARTI among children, 381 nasopharyngeal swabs from hospitalized in period X.2008-IV.2011y. children (aged 1 day - 5 y.o.) were tested for RSV, HMPV, HEV/HRV, HPIV 1-3, HAdV, HBoV. RESULTS: At least one viral agent was detected in 72.7% of patients. The most predominant was RSV infection (49%), followed by HEV/HRV (15.5%); HMPV (8.7%), Adenoviruses (7.4%), HPIVt.1-3 (5.8%) and HBoV (5.5%). Seven periods based on the median of examined children/month were determined: 3 with increased number of ARTI. RSV infections, diagnosed in all periods, were predominate in five periods, mainly in LRTI cases. In the 3th period - HMPV was predominant, in the 5th - HEV/HRV. It was found that clinical manifestation of HMPV infections varied depending on the period. CONCLUSIONS: Relatively high prevalence of HBoV or HMPV cases of ARTI, especially different clinical picture in some periods (ARTI without pneumonia or bronchiolitis), indicated necessary of more detailed molecular and epidemiological studies. Also our results indicate the need for improved diagnostic capabilities of virological tests in acute upper and lower respiratory tract infections in children.

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.

RSV respiratory infection in children under 5 y.o.--dynamics of the immune response Th1/Th2 and IgE.

INTRODUCTION: The imbalance of Th1/Th2 cytokine concentrations and increased level of IgE might be useful tool for prediction of severity of RSV infection among young children and possibility of sequels. The ratio of cytokines Th1/Th2 varied during the disease. THE AIM of our studies was the assessment of immunological response by dynamics of Th1 and Th2 cytokines and IgE in RSV infections. MATERIAL AND METHODS: Study was done on sera collected from 36 young children hospitalized because of RSV infection and from 16 children with other respiratory tract infection (HMPV, EV, HPIV1-3). Assaying of the serum levels of cytokine Th1 (IL-2, IFN-g, TNF), Th2 (IL-4, IL-6, IL-10) and concentration of IgE has been done. Paired sera (48 patients) were collected in the interval 4-14 days. Reference group consist of 18 children (< 6 months of life) hospitalised because other than respiratory diseases with negative results for viruses tested by PCR. RESULTS: Among children with respiratory infection the Th1/Th2 ratio was shifted towards Th2, level of IgE increased in comparison to the reference group. Changes in concentration of IL-6, IFN-g, IL-10 were related to RSV infection, mainly bronchitis and bronchiolitis, while the dynamic of TNF concentration was independent on aetiological agent. It was found that the risk factors (prematurity, artificial nutrition) correlated with RSV bronchitis and the levels of cytokines and IgE. Increased level of IL-6 and IL-10 were shown in prematures, and increased concentration of IgE--among artificial nourished children. The time of serum collection affected the level of cytokines and IgE and the effect was depended on the aetiological agent. In RSV infections was observed significant decrease with time of IL-6, IL-10 and IFN-gamma but not IgE (still significantly higher than in the reference group). While the significant decrease of IgE was determined only in other than RSV infections. CONCLUSIONS: Determined level of cytokines and IgE varied depending on the time of serum collection. Observed dynamics in paired sera and IgE might have prognostic value in disease and sequels of RSV infections. Prevention RSV infection in premature infants should be done in any possible way. Breastfeeding is one of the critical elements of prevention of RSV infection.

Sequence based typing and pre-absorption test in retrospective analysis of a pseudo-outbreak of Legionella infections differentiates true cases of legionellosis.

UNLABELLED: The aim of this study was elimination of false positive results obtained by the Chlamylege kit. Two serological kits (IgM ELISA L. pneumophila sgs1-7; ImmuView(TM) L. pneumophila sg1/sg3) and pre-absorption tests (with L. pneumophila sg1 and sg3 reference strains antigens) were used. 153 sera (79 patients) were examined. The high correlations were found between the results by both tests. Positive results by ELISA (sgs1-7) were found in 19/79 patients; by ImmuView(TM) (sg1+sg3) in 16/63. In 8 patients, the dynamics of the IgM in pairs of sera was high (ratio ≥2). In 5/8 of those patients seroconversion was determined. Selected pre-absorbed sera (15 pairs) were tested simultaneously by the same tests. In 8/15 pairs of sera, the reduction of IgM levels in pre-absorbed sera was higher than 10. The reduction of IgM differed in sg1 and sg3 tests. The probability of infections due to L. pneumophila sg3 (7 patients) and L. pneumophila sg1 (5 patients) was based on the results of pre-absorption tests. The correlation between ELISA and ImmuView(TM) tests of pre-absorbed sera was statistically significant (Po=0.0389). Moreover, genotyping of L. pneumophila (SBT) directly in the sera of selected 15 patients (high IgM reduction) was carried out. Completed 7 alleles profile (ST36) was determined in one patient. However, a second patient had the same profile of 5 alleles, and similar reactions in pre-absorption tests. At least 4 sources of infections were suggested on the base of genotyping and pre-absorption results. CONCLUSIONS: Positive results obtained by molecular techniques (eg.PCR) in the diagnosis of Legionella infections should be supplemented by other tests for confirmation of legionellosis. The sequence based typing carried out directly in clinical specimens seems to be a promising method.

Infections caused by RSV among children and adults during two epidemic seasons.

Respiratory Syncytial Virus (RSV) is one of the most common causes of lower respiratory tract infections in young children, immunocompromised patients (children and adults), patients with chronic respiratory diseases and elderly people. Reinfections occur throughout the life, but the severity of disease decreased with subsequent infection. The aim of this study was to analyze the frequency of RSV infections in two selected subpopulations: young children (below 5 y.) and adults with chronic respiratory diseases (25-87 y.). Nasopharyngeal swabs (334) collected from October 2008 to March 2010 were examined. The presence of RSV genome was determined by RT-PCR and the presence of RSV antigen by quick immunochromatographic test. Positive results of RT-PCR were found in 45.2% of all swabs: 48.6% samples in 2008; 41.5% in 2009; 50.8% in 2010. The highest frequency of RSV-positive samples was in fall-winter months, but differences in RSV epidemic seasons were found. In the first season (2008-2009) an increased number of RSV infections was observed from November 2008, but in the second season--from January 2010. Generally, the frequency of RSV-positive RT-PCR among children was 53%, among adults 25%. The highest difference was observed in the first three-month period of 2010. RT-PCR positive samples were found in 68.5% of children and 5.9% of adults. However, the RSV antigen was found in 44.4% of samples collected from adults in this period. Our results indicate that the contribution of RSV infections during epidemic season of respiratory tract infections in Poland was really high among children and adults.

Detection of hMPV antigen by EIA in clinical specimens.

UNLABELLED: Human Metapneumovirus (hMPV) is one of the latest discovered viruses. It has been classified to Paramyxoviridae family. It is the second viral etiological agent, after RSV, which causes respiratory tract infections (RTI) in children, especially children below 5 years old. It is estimated that 5-25% of RTI in children is due to hMPV. In adults hMPV reinfections are bounded to upper respiratory tract infections. The aim of the study was to establish usefulness of ELISA test in detecting hMPV antigen and to analyze hMPV infection in connection to clinical diagnosis. MATERIAL/METHODS: 273 nasopharyngeal swabs from children (189 swabs) and adults (84 swabs) with respiratory tract infections collected from 2008 to 2010 were examined. Due to similarity of hMPV and RSV viruses and overlapping of their epidemic season rapid immunochromatographic test for RSV antigen detection was also performed in case of 120 samples, hMPV antigen was detected in 24.5% of all swabs (n = 67): in 0.0% probes in 2008, 29.0% in 2009 and 36.8% in first quarter of 2010. The highest rate ofhMPV infection was detected from summer of 2009 till the end of March 2010 (VIII-IX 2009 - 62.5%, X-XII 2009 - 44.1% and I-III 2010 -36.8%). We analyzed respiratory tract diseases reported in patients with hMPV infection. Infection due to hMPV was found in 26.5% of children and 24.0% of adults with recognized pneumonia, respectively in 28.4 and 17.6% of patients with bronchitis. Bronchiolitis was diagnosed in two children with hMPV. RSV and hMPV coinfections were confirmed in 15 out of 120 examined probes. Cross reaction pattern was excluded thanks to ELISA hMPV antigen test which was performed with suspension of RSV and thanks to statistical analysis. Coinfections were confirmed in 8% of pneumonia, 11% of bronchitis and 24.2% of the rest concomitant diagnoses. CONCLUSIONS: We found hMPV infection as the significant agent ofpneumonia not only in children but also in adults. ELISA hMPV antigen test can be used in diagnosis of etiological agent of respiratory infections in children and adults and in coinfections as well.

[The influence of Namalwa cells on inhibition of HSV-1 replication]. / Wplyw komórek hodowli namalwa na replikacje HSV-1.

The main immunological elements that control infection with herpes simplex virus type 1 include interferon, NK cells, and specific T and B cells. The aim of this study was to determine the influence of Namalwa cells (B cell immortalized by EBV) which produce IFN alpha in large amount on inhibition of HSV-1 replication in CV-1 and MDBK cells. Inhibition of HSV-1 replication was measured by MTT assay. Addition Namalwa cells to CV-1 or MDBK cells infected HSV-1 inhibited virus replication. Degree of inhibition was connected with amount of added cells and with time of addition (before infection or after infection). The highest inhibition of HSV-1 replication showed Namalwa cells added one hour before infection (72% for CV-1 infected HSV-1 and 68% for MDBK infected HSV-1).

Seroprevalence of varicella-zoster virus in Polish population.

A varicella zoster virus (VZV) is the first herpesvirus for which a vaccine was developed. Since 1999, the varicella vaccine is licensed in Poland and recommended for use in adults without history of a varicella infection, and in children and young adults with remission of acute leukemia. While serological data is essential to assess the appropriate vaccination programme, we conducted the first in Poland serosurvey on a representative group of Polish population aged 1-19. Serum samples were selected from a serum bank collected in 1995-2004 with a catchment area of the all geographical regions of Poland. A total of 1300 serum samples collected over 9 years (1995-1996, 1998-2004) were selected using a stratified sampling design (stratification by age). Samples were selected, consisting of 100 samples for each 1-year band of age groups 0-9 years, and 40 samples for each 1-year band of age groups 10-19 years. IgG serum antibodies specific to VZV were detected using an indirect enzyme immunoassay and the antibody level was expressed in international units per millilitre (mIU/ml) and was refered to the international standard for VZV immunoglobulin of 50 IU. The overall seroprevalence estimate, adjusted for sampling design for the age group 1-19 was 76.6% (95% CI: 74.6%-78.7%). Seroprevalence correlated closely with age (p<0.0001) and among 18 and 19 year olds reached 95% and 98% respectively. No association was found between gender, rural/urban areas and geographical regions of Poland. For samples collected over the 5 year period (2000-2004), evidence of overall differences in seropositivity over these years was not observed. In Poland VZV vaccination is provided only for a limited group of high risk patients. The possible updates in the immunization program are discussed and the results of the presented study can contribute valuable information to base the vaccination policy decisions.

[Epidemiological surveillance of measles occurrence in Poland in 2006 by means of virological and molecular methods]. / Badania wirusologiczne i molekularne w nadzorze epidemiologicznym nad zachorowaniami na odre w polsce w 2006 roku.

The availability of sensitive cell line for isolation of measles virus from clinical samples and establishment RT-PCR and molecular sequencing metods have allowed for rapid genetic characterization of wild-type strains of measles virus. This sequence information makes it possible to identify the source of wild-type viruses and differentiate between native and reported cases. The aim of this study was evaluation of virus isolation in cell culture in National Laboratory in Poland (Department of Virology in NIH), presentation of RT-PCR and molecular genotyping results performed in Regional Reference Laboratory in Berlin (Robert Koch Institut).

Composition of inflammatory infiltrate and its correlation with HBV/HCV antigen expression.

AIM: To study the composition of liver inflammatory infiltrate in biopsy material from patients chronically infected with hepatotropic viruses and to evaluate the correlation of inflammatory infiltrate with hepatitis B virus (HBV) and hepatitis C virus (HCV) viral antigen expression in chronic B and C hepatitis. METHODS: The phenotype of inflammatory cells was evaluated by the EnVision system, using a panel of monoclonal antibodies. HBV and HCV antigens were detected with the use of monoclonal anti-HBs, polyclonal anti-HBc and anti-HCV antibodies, respectively. RESULTS: The cellular composition of liver inflammatory infiltrate was similar in the patients with B and C hepatitis: approximately 50%-60% of cells were T helper lymphocytes. Approximately 25% were T cytotoxic lymphocytes; B lymphocytes comprised 15% of inflammatory infiltrate; other cells, including NK, totalled 10%. Expression of HLA antigens paralleled inflammatory activity. Portal lymphadenoplasia was found more often in hepatitis C (54.5%) than in hepatitis B (30.6%). Expression of HBcAg was found more often in chronic B hepatitis of moderate or severe activity. Overall inflammatory activity in HBV-infected cases did not correlate with the intensity of HBsAg expression in hepatocytes. Inflammatory infiltrates accompanied the focal expression of HCV antigens. A direct correlation between antigen expression and inflammatory reaction in situ was noted more often in hepatitis C than B. CONCLUSION: Irrespective of the etiology and activity of hepatitis, components of the inflammatory infiltrate in liver were similar. Overall inflammatory activity did not correlate with the expression of HBsAg and HCVAg; HBcAg expression, however, accompanied chronic hepatitis B of moderate and severe activity.

[Epstein-Barr infections in children with acute leukaemia. Preliminary report]. / Zakazenie wirusem Epsteina-Barr u dzieci z ostrymi bialaczkami. Doniesienie wstepne.

INTRODUCTION: Epstein-Barr virus (EBV) infection is common all over the world. Primary EBV infection can lead to latent or chronic infection resulting in lymphoproliferative diseases. THE AIM: of this study was to evaluate the effectiveness of laboratory methods used to detect EBV and to monitor EBV infections in children with acute leukaemia. MATERIALS AND METHODS: we conducted the study on 30 children with acute leukaemia. For the present analysis we included 8 patients from whom samples were also taken three times. Studies were made on serum, blood and bone marrow. Specific IgM and IgG antibodies for viral capsid antigen (VCA), nuclear (EBN) and early antigen (EA) were tested by ELSA. The presence of DNA virus was estimated by nested PCR. The control group were 11 subjects, without chronic or neoplastic disease, undergoing routine laboratory tests in the Virology Department. RESULTS: on admission serologied tests indicated past EBV infection in 6 out of the 8 children. Primary infection was detected in 2 patients, in one of them on day 1 of the observation and on day 22 in the other. DNA EBV was found by nested PCR in only one of the 8 infected children. CONCLUSION: 1. Results of serological investigations indicated the type of EBV infection in our patients. 2. Confirmation of EBV virus active replication by nased PCR was obtained in only 1 out of 8 patients. 3. In order to assess the effectiveness of serological and molecular methods in evaluation of EBV infection type in children with acute leukaemia, it is necessary to investigate a larger group of patients and taken at least three times.

High genetic diversity of measles virus, World Health Organization European Region, 2005-2006.

During 2005-2006, nine measles virus (MV) genotypes were identified throughout the World Health Organization European Region. All major epidemics were associated with genotypes D4, D6, and B3. Other genotypes (B2, D5, D8, D9, G2, and H1) were only found in limited numbers of cases after importation from other continents. The genetic diversity of endemic D6 strains was low; genotypes C2 and D7, circulating in Europe until recent years, were no longer identified. The transmission chains of several indigenous MV strains may thus have been interrupted by enhanced vaccination. However, multiple importations from Africa and Asia and virus introduction into highly mobile and unvaccinated communities caused a massive spread of D4 and B3 strains throughout much of the region. Thus, despite the reduction of endemic MV circulation, importation of MV from other continents caused prolonged circulation and large outbreaks after their introduction into unvaccinated and highly mobile communities.

[Hantavirus specific IgG antibodies in population of zoologists and forest workers]. / Wystepowanie przeciwcial klasy IgG swoistych dla hantawirusów w populacji pracowników lesnych i zoologów.

Serological diagnostic tests were introduced for detection of immunoresponse against to etiological agents of current infectious diseases. Interpretation criteria for those tests should eliminate unspecific reactions, cross-reactivity with related viruses as well as traces amounts of immunological response against to the past infections. In the case of past hantaviruses infections for obtaining an adequate seroepidemiological data revalidation of this tests are required. In present studies Puumala IgG results obtained in sera from healthy population had been analysed by statistical method and interpretation criteria were recalculated. Panels of 86 sera from forest workers and 47 zoologists working with small mammals were evaluated for hantavirus specific IgG (Hantavirus Puumala ELISA test). Puumala specific antibodies were detected in 7 zoologist's sera and in 5 sera gave equivocal results. All sera collected from forest's workers were negative. Statistical analysis based on negative results in forest workers group suggests that the cut off of the ELISA test suitable for diagnosis of suspected nephropathia epidemica cases is too restrictive for seroepidemiological research of hantavirus in healthy population.