Selective Growth Suppressive Effect of Pravastatin on Senescent Human Lung Fibroblasts.

Various chemical reagents containing inhibitors of mitochondrial activity, antioxidants, nuclear factor-kappa B (NF-kB) inhibitor, mammalian target of rapamycin (mTOR) inhibitor and other clinical therapeutics were screened in order to identify those that selectively decrease the viability of senescent human lung fibroblasts. Cell viability was measured using the CCK-8 assay. The results showed that pravastatin, a drug for hyperlipidemia, decreased the viability of senescent cells but not non-senescent cells. The effect of pravastatin on senescent cells is thought to be due to the inhibition of cell proliferation, rather than cell death. The effect of pravastatin was further investigated using the glucose metabolism assay, which showed that glucose consumption was inhibited both in non-senescent and senescent cells and intracellular nicotinamide adenine dinucleotide (NAD) was decreased in senescent cells. Changes to the mRNA expression levels of senescence-associated genes in response to pravastatin treatment were quantified by real-time-qPCR. There were no significant changes in the relative mRNA expression levels of IL-1ß, p16, p21, and p53 in pravastatin-treated non-senescent cells, whereas the expression of IL-1ß and p16 were increased by pravastatin only in senescent cells. The results of this study suggest that pravastatin does not induce senolysis, but rather selectively inhibits the proliferation of senescent cells and that cellular senescence is enhanced by decreasing intracellular NAD and promoting IL-1ß production.

Occurrence of noroviruses in recycled water and sewage sludge: emergence of recombinant norovirus strains.

AIMS: To investigate the presence of noroviruses in recycled water and sewage sludge obtained from a wastewater treatment plant in Bangkok Metropolitan Region, Thailand. METHODS AND RESULTS: Twenty-seven recycled water and twenty-three sewage sludge samples were tested for the presence of norovirus genogroup (G)I and GII using RT-nested PCR. Molecular characterization of noroviruses was undertaken by DNA sequencing and phylogenetic analysis. The level of the RNA genome of the noroviruses was determined using quantitative real-time RT-PCR. Noroviruses were detected in 44·4% of recycled water samples and 73·9% of sewage sludge samples. Norovirus GI.2 and GII.4 were identified in recycled water samples at levels of 2·19 × 101 and 3·26 × 104 RNA copies per litre. Six different genotypes of GI (GI.1, GI.2, GI.5a, GI.5b, GI.6b and GI.7) and GII.17 were identified in sewage sludge samples at levels ranging from 1·99 × 101 -1·43 × 105 RNA copies per gram wet weight. Four recombinant norovirus strains were detected in sewage sludge samples, namely GII.P16-GII.2, GII.P16-GII.4, GII.P16-GII.13 and GII.P21-GII.13. CONCLUSIONS: These findings provide evidence that noroviruses may be spread to the community and environment via the use of recycled water for plant areas, and sewage sludge for land application. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study demonstrating recombinant norovirus strains in sewage sludge samples. The presence of noroviruses in recycled water and sewage sludge contributes to a health risk of environmental exposure.

Immune responses against norovirus GII.4 virus-like particles in mice.

Norovirus (NoV) is a causative agent of gastroenteritis in children and adults worldwide. To evaluate the safety and effectiveness of a NoV vaccine candidate, 100 µg of GII.4 NoV-like particles (VLPs) was challenged orally (oral and intrabuccal administration) and by subcutaneous injection without adjuvant in mice. The subcutaneous injection induced IgG in sera, but not IgA in feces. The oral delivery method induced IgA in both sera and feces, but not IgG in sera. However, challenging by the intrabuccal administration induced IgG in sera and IgA in both sera and feces, especially by 2-dose immunization. The peak of specific immune responses by the intrabuccal administration was detected later than that of the oral delivery method. Heterologous immune responses against other genotypes were also recognized. NoV-specific IFN-γ was detected after the intrabuccal administration. These findings indicated that the administration of NoV VLPs by intrabuccal administration could induce the best immune responses against NoV in mice.

Clinical and epidemiological characteristics of acute respiratory virus infections in Vietnamese children.

Information about viral acute respiratory infections (ARIs) is essential for prevention, diagnosis and treatment, but it is limited in tropical developing countries. This study described the clinical and epidemiological characteristics of ARIs in children hospitalized in Vietnam. Nasopharyngeal samples were collected from children with ARIs at Ho Chi Minh City Children's Hospital 2 between April 2010 and May 2011 in order to detect respiratory viruses by polymerase chain reaction. Viruses were found in 64% of 1082 patients, with 12% being co-infections. The leading detected viruses were human rhinovirus (HRV; 30%), respiratory syncytial virus (RSV; 23·8%), and human bocavirus (HBoV; 7·2%). HRV was detected all year round, while RSV epidemics occurred mainly in the rainy season. Influenza A (FluA) was found in both seasons. The other viruses were predominant in the dry season. HRV was identified in children of all age groups. RSV, parainfluenza virus (PIV) 1, PIV3 and HBoV, and FluA were detected predominantly in children aged 24 months, respectively. Significant associations were found between PIV1 with croup (P < 0·005) and RSV with bronchiolitis (P < 0·005). HBoV and HRV were associated with hypoxia (P < 0·05) and RSV with retraction (P < 0·05). HRV, RSV, and HBoV were detected most frequently and they may increase the severity of ARIs in children.

Human rhinovirus infections in hospitalized children: clinical, epidemiological and virological features.

Molecular epidemiology and clinical impact of human rhinovirus (HRV) are not well documented in tropical regions. This study compared the clinical characteristics of HRV to other common viral infections and investigated the molecular epidemiology of HRV in hospitalized children with acute respiratory infections (ARIs) in Vietnam. From April 2010 to May 2011, 1082 nasopharyngeal swabs were screened for respiratory viruses by PCR. VP4/VP2 sequences of HRV were further characterized. HRV was the most commonly detected virus (30%), in which 70% were diagnosed as either pneumonia or bronchiolitis. Children with single HRV infections presented with significantly higher rate of hypoxia than those infected with respiratory syncytial virus or parainfluenza virus (PIV)-3 (12·4% vs. 3·8% and 0%, respectively, P < 0·05), higher rate of chest retraction than PIV-1 (57·3% vs. 34·5%, P = 0·028), higher rate of wheezing than influenza A (63·2% vs. 42·3%, P = 0·038). HRV-C did not differ to HRV-A clinically. The genetic diversity and changes of types over time were observed and may explain the year-round circulation of HRV. One novel HRV-A type was discovered which circulated locally for several years. In conclusion, HRV showed high genetic diversity and was associated with significant morbidity and severe ARIs in hospitalized children.

Evaluation of immunochromatography tests for detection of novel GII.17 norovirus in stool samples.

A novel GII.17 norovirus has emerged as a major cause of epidemic and endemic acute gastroenteritis in several countries in Asia. We used a small panel of stool samples in which GII.17 virus had been quantified by real-time RT-PCR to evaluate four commercially available norovirus immunochromatography (IC) kits. At least 10(8) copies/mL of GII.17 virus were required by each IC kit for a positive result, which is 1,000-fold more than that reported for these assays for GII.4 viruses.

Prevalence, seasonality, and peak age of infection of enteric adenoviruses in Japan, 1995-2009.

A total of 7185 faecal specimens collected from infants and children with gastroenteritis in seven different regions of Japan during 1995-2009 were examined for adenovirus by polymerase chain reaction. Adenovirus was detected in 568 (7.9%) patients. The adenovirus activity peak was in winter and spring seasons (December-March) during the study period in the Japanese paediatric population. During the last 15 years, adenovirus 41 was the most predominant strain in Japan.

Seasonal pattern and genotype distribution of sapovirus infection in Japan, 2003-2009.

Sapovirus, a member of the family Caliciviridae, is one of the major causative agents of viral gastroenteritis affecting all age groups. A total of 3232 faecal specimens collected from infants and children with gastroenteritis in five different regions of Japan during 2003-2009 were examined for sapovirus by reverse transcription-polymerase chain reaction. Sapoviruses were detected in 131 (4·05%) patients with the peak observed mainly in the cold season (November-March) in Japan during 2003-2009. During the last 6 years, sapovirus GI/1 was the predominant strain in Japan followed by GIV, GII/3, GII/6, GII/2, GII/12 and GI, respectively.

Genetic characterization of group C rotavirus isolated from a child hospitalized with acute gastroenteritis in Chiang Mai, Thailand.

During an epidemiological survey of human rotavirus infection in Chiang Mai, Thailand, from 2002 to 2004, in which 263 stool specimens tested, one isolate of group C rotavirus was detected from a two-year-old child admitted to hospital with acute gastroenteritis. The human group C rotavirus, named CMH004/03, was characterized further by molecular analyses of its VP4, VP6, and VP7 gene segments as well as determination of RNA pattern by polyacrylamide gel electrophoresis (PAGE). Molecular characterization of VP4, VP6, and VP7 genes by sequence analyses showed high levels of sequence identities with those of human group C rotavirus reference strains isolated worldwide at 95.2% to 99.4% on nucleotide and 97.5% to 100% on amino acid levels. In contrast, the CMH004/03 strain exhibited far lesser nucleotide and amino acid sequence identities at 67.7% to 84.1% and 68.7% to 91.3%, respectively, when compared with those of porcine and bovine group C rotaviruses. Phylogenetic analyses of VP4, VP6, and VP7 genes clearly confirmed that the CMH004/03 strain clustered in a monophyletic branch with other human group C rotavirus reference strains and distantly related to the clusters of animal group C rotavirus strains. In addition, the RNA electrophoretic migration pattern of CMH004/03 showed a typical pattern (4-3-2-2) of group C rotavirus. To our knowledge, this study is the second report of group C rotavirus infection in pediatric patients in Thailand after it was reported for the first time about two decades ago.

Use of sequence analysis of the VP4 gene to classify recent Vietnamese rotavirus isolates.

Twenty-eight strains of P(8), four of P(4) and one of P(19) rotavirus, isolated in Ho Chi Minh City, Vietnam, during 2002-2003, were investigated by sequence analysis of the VP4 gene. Seven of the 28 P(8) rotavirus VP4 sequences clustered in the P(8)-3 lineage, or the rare, so-called OP354-like lineage. Amino-acid sequence comparison revealed that Vietnamese P(8)-3 rotaviruses were generally very similar to Malawian strains, including the prototype OP354 strain. The numerical severity scores of diarrhoeal disease caused by the Vietnamese P(8)-3 rotaviruses were statistically higher than those of diarrhoeal disease caused by rotaviruses in the more common P(8)-2 lineage. Sequence and phylogenetic analysis of the VP4 gene of a Vietnamese G9P(19) rotavirus isolate showed a high degree of homology with the cognate genes of other human and porcine rotaviruses, including the prototype 4F strain.

Perinatal bisphenol A affects the behavior and SRC-1 expression of male pups but does not influence on the thyroid hormone receptors and its responsive gene.

Bisphenol A (BPA) has been shown to interfere with thyroid hormone receptors (THRs) and to influence the expression of THR-responsive elements in vivo and in vitro, while some studies reported hyperactivity induced by BPA treatment. In the present study, our purpose was to investigate the effect of BPA exposure on behavioral alteration and its mechanism of action, especially focusing on the thyroid hormone pathway. Significant sexual difference on behaviors was observed in perinatal BPA exposure, as manifested by hyperactivity and impaired spatial learning/memory in male pups after matured. Dams treated with 0.1mg/l BPA showed transient hypothyroidism, while male pups were found to exhibit a transient hyperthyroidism followed by hypothyroidism. Furthermore, significant up-regulated expression levels of mRNA and protein of SRC-1 in the hippocampus were observed in male pups by 0.1mg/l BPA treatment. However the expression of THRalpha/beta and RC3/neurogranin were not affected by BPA treatment. These results indicate that perinatal BPA exposure at a very low level may influence thyroid function and then consequently affects brain development, but at the same time, suggest that thyroid hormone receptor may not be a direct target of BPA action, but instead, another factor may be involved in this action.

Emergence of rare sapovirus genotype among infants and children with acute gastroenteritis in Japan.

A total of 1,154 fecal specimens from infants and children with acute gastroenteritis in five cities in Japan (Maizuru, Tokyo, Sapporo, Saga, and Osaka), collected from July 2003 to June 2005, were tested for the presence of diarrheal viruses by reverse transcriptase multiplex PCR. Overall, 469 of 1,154 (40.6%) were positive for diarrheal viruses, of which 49 (10.4%) were positive for sapovirus. The peak of sapovirus infection shifted from April-June in 2003-2004 to October-December in 2004-2005. The observations show that maximum sapovirus prevalence can occur during warmer seasons. Sapovirus was subjected to molecular genetic analysis by sequencing. The results indicated that sapovirus genogroup I was a dominant group (100%). Sapovirus strains detected in this study were further classified into four genotypes (GI/1, GI/4, GI/6, and GI/8). Of these, sapovirus GI/1 was the most predominant, followed by sapovirus GI/6; these accounted for 93% (13 of 14) and 7% (1 of 14), respectively, in 2003-2004. However, it was noteworthy that sapovirus GI/6 suddenly emerged to become the leading genotype, accounting for 77% (27 of 35) of isolates in 2004-2005. This is believed to be the first report of the changing distribution of sapovirus genotypes and of the emergence of the rare sapovirus GI/6.

Genomic characterization of HBV genotype F in Bolivia: genotype F subgenotypes correlate with geographic distribution and T(1858) variant.

Hepatitis B virus (HBV) strains were classified into eight genotypes from A to H. Genotype F, an indigenous genotype in Central and South America, has been classified into subgenotypes. An in-depth phylogenetic analysis was performed using two full-length Bolivian HBV sequences and other genotype F strains from the database. A novel nomenclature of subgenotypes of genotype F was proposed, in which Bolivia strains belonged to subgenotype F4. This subgenotype had both Leu(45) and Ile(110) in the S gene, and linked to the T(1858) in the precore. This novel nomenclature demonstrated the relation between variability of the HBV genome and the restricted geographical distribution of the virus in some parts of Central and South America.

Changing distribution of group A rotavirus G-types and genetic analysis of G9 circulating in Japan.

A total of 1,797 fecal specimens from infants and children with acute gastroenteritis in Japan from July 2000 to June 2003 were tested for group A rotavirus by ELISA, RT-PCR, RNA-PAGE and latex agglutination methods. Of these, 439 were found to be positive for group A rotavirus and this presented 24.4%. In 2000-2001, G1 was the most prevalent (45.5%) followed by G2 (32.5%), G3 (12.3%), G9 (5.9%) and G4 (2.6%). However, G2 was found predominant with 40% in the following year (2001-2002). Interestingly, G9 had a rapid increase of infection up to 17.8%. In 2002-2003, G3 dominated over other G-types with 34%. Another interesting feature of the study was the demonstration of great genetic diversity among G9 strains in Japan. Worth of note was the first prevalence pattern of rotavirus G-types with an increase of G2, G3 as well as G9 and a decrease of G1 during the 20 year-survey of rotavirus infection in Japan.

Emergency medical services in Islamabad, Pakistan: a public-private partnership.

OBJECTIVES: The objective of this study was to evaluate the emergency medical services (EMS) based in Islamabad, Pakistan. Rescue-15 is an innovative EMS collaboration project between the police, the private sector and the community. METHODS: Data from Rescue-15 were used for systems analysis. The institutional set-up, private-public partnership, client satisfaction and sustainability issues were examined. The access and efficiency of EMS were assessed in terms of ambulance response time. RESULTS: Primarily, systems analysis showed community participation to explain the project's strength. Since its establishment, the project has been meeting its own recurrent expenditures without levying an extra burden on the Government. Sustainability issues such as amendments to legislation have been addressed at departmental and governmental levels. Data analysis showed that rescue time is, on average, 10.4 min (SD=2.6 min). A client survey also demonstrated user satisfaction and increased confidence in the service. CONCLUSIONS: This EMS programme exemplifies the potential of public-private partnership involving the police and the private sector in project implementation and management in a developing country with scarce resources. This initiative to involve the public and the private sector may provide a model for implementation of such services in other resource-poor developing countries, which may in turn facilitate realistic solutions for better prehospital care in developing countries.

Emergency obstetric care in Pakistan: potential for reduced maternal mortality through improved basic EmOC facilities, services, and access.

OBJECTIVE: To ascertain and compare compliance with UN emergency obstetric care (EmOC) recommendations by public health care centers in Pakistan's Punjab and Northwest Frontier Province (NWFP) provinces. METHOD: Cross-sectional data were collected from July through September 2003 using UN process indicators. From each province, 30% of districts (n=19); were randomly selected; all public health facilities providing EmOC services (n=170) were included. RESULTS: The study found that out of 170 facilities only 22 were providing basic and 37 comprehensive EmOC services in the areas studied. Only 5.7% of births occurred in EmOC health facilities. Met need was 9% and 0.5% of women gave birth by cesarean section. The case fatality rate was a low 0.7%, probably due to poor record keeping. Access and several indicators were better in NWFP than in Punjab. CONCLUSION: Almost all indicators were below UN recommendations. Health policy makers and planners must take immediate, appropriate measures at district and hospital levels to reduce maternal mortality.

Existence of multiple outbreaks of viral gastroenteritis among infants in a day care center in Japan.

A total of 921 fecal specimens collected from 44 infants in a day care center (DCC) in Tokyo, Japan during June 1999 to July 2000 were tested for the presence of rotavirus, norovirus, sapovirus, astrovirus and adenovirus by reverse-transcription-multiplex polymerase chain reaction (RT-multiplex PCR) and sequence analysis. Of 88 fecal specimens from infants with acute gastroenteritis, 51.1% (45) were found to be positive for diarrheal viruses. Astrovirus was the most prevalent (15.9%, 14 of 88), followed by norovirus GII (14.8%, 13 of 88), adenovirus (12.5%, 11 of 88), and sapovirus (2.3%, 2 of 88). Viral mixed infection accounted for 5.7% (5 of 88). Interestingly, 230 of 833 (27.6%) fecal specimens collected from asymptomatic infants were also infected with diarrheal viruses. Of these, astrovirus, norovirus GII, adenovirus and sapovirus were identified in 53, 46, 96 and 22 fecal specimens (23%, 20%, 41.7%, and 9.6%, respectively). Moreover, 13 of 833 (1.6%) normal specimens showed mixed viral infections. Surprisingly, no rotavirus (known as the most common causative agent of acute gastroenteritis in DCCs) was detected in those subjects. Another interesting feature was the demonstration of five separate outbreaks of acute gastroenteritis identified in a single DCC. Outbreak A was associated with both astrovirus serotype 1 and norovirus GII/3 (known as Toronto virus cluster); Outbreak B with adenovirus 12; Outbreak C with norovirus GII/4 (Lordsdale virus cluster); Outbreak D with sapovirus GIV and Outbreak E with astrovirus serotype 1. To our knowledge, this is the first proof of multiple outbreaks of viral gastroenteritis in Japanese infants in a single DCC. Our results confirm the presence as well as the importance of these viruses and warn of the threat they pose.

Characterization of polyclonal antibodies raised against sapovirus genogroup five virus-like particles.

Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains were recently divided into five genogroups (GI to GV). We characterized novel polyclonal antibodies raised against SaV GV virus-like particles (VLPs) by Western blot analysis, and both antibody and antigen enzyme-linked immunosorbent assays (ELISAs). Our results have indicated SaV GI and GV VLPs were antigenically distinct by Western blotting and ELISAs. These reagents may be useful for genogroup specific detection of SaV.

Etiologic agents of acute gastroenteritis among Japanese infants and children: virus diversity and genetic analysis of sapovirus.

A total of 371 fecal specimens from infants and children with acute gastroenteritis in Maizuru, Tokyo and Osaka, Japan from July 2002 to June 2003 were tested for the presence of diarrheal viruses by reverse transcription-polymerase chain reaction (RT-PCR), reverse passive hemagglutination (PRHA), RNA-polyacrylamide gel electrophoresis (PAGE), latex agglutination and sequence analysis methods. Among diarrheal viruses detected, group A rotavirus was the most prevalent (42.2%) followed by norovirus (28.9%), group C rotavirus (8.4%), sapovirus (6.7%), adenovirus (5.3%) and astrovirus (0.9%), respectively. There was the high rate (7.6%) of viral mixed infections. Sapovirus was classified into 6 genotypes (GI/1, GI/4, GI/5, GI/6 and GII/1 and one novel tentatively called GII/5). It is noteworthy that genogroup II sapovirus can be classified into 5 genotypes. Our findings confirmed the presence of many diarrheal viruses co-circulating among Japanese infants and children and showed the great genetic diversity among sapoviruses.