Continuous detection of norovirus and astrovirus in wastewater in a coastal city of China in 2014–2016

Norovirus (NoV) and human astrovirus (HAstV) are important causative agents of acute gastroenteritis in children and adults. They are comprised of multiple genotypes and attention should be paid to genotype changes or emergence of new genetic variants. To study the prevalence and diversity of NoV GI, GII, and HAstV circulating in eastern China, we conducted a three-year environmental surveillance in a coastal city of Yantai. Thirty-six sewage samples were collected, processed, and examined for the presence of viral genomes by PCR. The results showed that NoV GI, GII, and HAstV were detected in all 36 samples. Six NoV GI genotypes, 11 NoV GII genotypes, and 5 HAstV serotypes were identified; GI.6, GII.17, and HAstV-5 were the most prevalent types, respectively. Persistent existence of NoV GII.17 Kawasaki 308 variant was observed during whole study period. Phylogenetic analysis reflected multiple transmission lineages in local population for both viruses. Our results reflect continuous presence of enteric viruses in sewage, improve our understanding on their molecular epidemiology, and demonstrate surveillance on sewage is an effective approach in understanding the local circulation of enteric viruses.     

Evidence of the co‐circulation of enteric viruses in sewage and in the population of Greater Cairo

Aims: To characterize major enteric viruses (enterovirus, rotavirus, norovirus, astrovirus and adenovirus) in the sewage of Greater Cairo and to compare the results with clinical data collected during the same period. Methods and Results: Seventy‐two sewage samples from two waste water treatment plants were collected from April 2006 through February 2007. Enteroviruses, noroviruses (NoVs) and rotaviruses (RVs) were detected by RT‐PCR in 22%, 18% and 8·3% of the samples, respectively. No adenovirus and astrovirus was detected. G2P[8], G9P[8], G1P[8], G2P[4] and rare G12 RV isolates were detected in the environment as well as a bovine RV. The environmental NoV strains mostly belonged to genogroup I (84%). Rotaviruses and some of the NoVs were similar to those found in the clinical samples at the same time. Conclusions: The comparison of environmental and clinical data suggests that similar RV and NoV isolates were circulating in the environment and in the population during the same period. Significance and Impact of the Study: Few studies have investigated the prevalence and the epidemiology of RVs and NoVs in Cairo. This work is the first to establish a correlation between viral gastroenteritis and the concomitant presence of enteric viruses in the environment for Greater Cairo where combined environmental and clinical surveys should help to prevent infections caused by these major pathogens.     

Virus movement in soil columns flooded with secondary sewage effluent.

Secondary sewage effluent containing about 3 X 10(4) plaque-forming units of polio virus type 1 (LSc) per ml was passed through columns 250 cm in length packed with calcareous sand from an area in the Salt River bed used for ground-water recharge of secondary sewage effluent. Viruses were not detected in 1-ml samples extracted from the columns below the 160-cm level. However, viruses were detected in 5 of 43 100-ml samples of the column drainage water. Most of the viruses were adsorbed in the top 5 cm of soil. Virus removal was not affected by the infiltration rate, which varied between 15 and 55 cm/day. Flooding a column continuosly for 27 days with the sewage water virus mixture did not saturate the top few centimeters of soil with viruses and did not seem to affect virus movement. Flooding with deionized water caused virus desorption from the soil and increased their movement through the columns. Adding CaCl2 to the deionized water prevented most of the virus desorption. Adding a pulse of deionized water followed by sewage water started a virus front moving through the columns, but the viruses were readsorbed and none was detected in outflow samples. Drying the soil for 1 day between applying the virus and flooding with deionized water greatly reduced desorption, and drying for 5 days prevented desorption. Large reductions (99.99% or more) of virus would be expected after passage of secondary sewage effluent through 250 cm of the calcareous sand similar to that used in our laboratory columns unless heavy rains fell within 1 day after the application of sewage stopped. Such virus movement could be minimized by the proper management of flooding and drying cycles.     

Virus movement in soil columns flooded with secondary sewage effluent.

Secondary sewage effluent containing about 3 X 10(4) plaque-forming units of polio virus type 1 (LSc) per ml was passed through columns 250 cm in length packed with calcareous sand from an area in the Salt River bed used for ground-water recharge of secondary sewage effluent. Viruses were not detected in 1-ml samples extracted from the columns below the 160-cm level. However, viruses were detected in 5 of 43 100-ml samples of the column drainage water. Most of the viruses were adsorbed in the top 5 cm of soil. Virus removal was not affected by the infiltration rate, which varied between 15 and 55 cm/day. Flooding a column continuosly for 27 days with the sewage water virus mixture did not saturate the top few centimeters of soil with viruses and did not seem to affect virus movement. Flooding with deionized water caused virus desorption from the soil and increased their movement through the columns. Adding CaCl2 to the deionized water prevented most of the virus desorption. Adding a pulse of deionized water followed by sewage water started a virus front moving through the columns, but the viruses were readsorbed and none was detected in outflow samples. Drying the soil for 1 day between applying the virus and flooding with deionized water greatly reduced desorption, and drying for 5 days prevented desorption. Large reductions (99.99% or more) of virus would be expected after passage of secondary sewage effluent through 250 cm of the calcareous sand similar to that used in our laboratory columns unless heavy rains fell within 1 day after the application of sewage stopped. Such virus movement could be minimized by the proper management of flooding and drying cycles.     

我国河南与北京腹泻患儿中的星状病毒感染

人星状病毒（ＨＡｓｔＶ）是ＲＮＡ病毒的新家族,从我国河南和北京两地区的急性腹泻患儿便样中鉴定了７株星状病毒,经电镜观察病毒颗粒直径约为２８ｎｍ,表面呈五角或六角星状形态,用ＲＴ－ＰＣＲ检定为阳性,比较ＰＣＲ扩增产物的核苷酸序列构建的序列同源性树表明,１９９０年在河南流行的星状病毒主要为ＨＡｓｔＶ－１,另有一株ＨＡｓｔＶ－５,１９９６年北京流行株为ＨＡｓｔＶ－５。对ＨＡｓｔＶ患儿的临床症状进行了讨论     

Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks

Most persons infected with enterically transmitted viruses shed large amounts of virus in feces for days or weeks, both before and after onset of symptoms. Therefore, viruses causing gastroenteritis may be detected in wastewater, even if only a few persons are infected. In this study, the presence of eight pathogenic viruses (norovirus, astrovirus, rotavirus, adenovirus, Aichi virus, parechovirus, hepatitis A virus [HAV], and hepatitis E virus) was investigated in sewage to explore whether their identification could be used as an early warning of outbreaks. Samples of the untreated sewage were collected in proportion to flow at Ryaverket, Gothenburg, Sweden. Daily samples collected during every second week between January and May 2013 were pooled and analyzed for detection of viruses by concentration through adsorption to milk proteins and PCR. The largest amount of noroviruses was detected in sewage 2 to 3 weeks before most patients were diagnosed with this infection in Gothenburg. The other viruses were detected at lower levels. HAV was detected between weeks 5 and 13, and partial sequencing of the structural VP1protein identified three different strains. Two strains were involved in an ongoing outbreak in Scandinavia and were also identified in samples from patients with acute hepatitis A in Gothenburg during spring of 2013. The third strain was unique and was not detected in any patient sample. The method used may thus be a tool to detect incipient outbreaks of these viruses and provide early warning before the causative pathogens have been recognized in health care.     

Detection and molecular characterization of enteric viruses in environmental samples in Monastir,Tunisia between January 2003 and April 2007

Aims: A prospective study was performed to characterize the main human enteric viruses able to persist in sewage samples and in shellfish tissues, and to establish the correlation between environmental strains and viral infantile diarrhoea observed in the same area during the same period. Methods and Results: A total of 250 sewage (raw and treated) and 60 shellfish samples were collected between January 2003 and April 2007 in Monastir region, Tunisia. Group A rotavirus (RVA) was detected in 80 (32%) sewage samples, norovirus (NoV) in 11 (4·4%) and enteric adenovirus (AdV) in 1 (0·4%). Among 60 shellfish samples collected near sewage effluents, one was contaminated by NoV (1·6%). Conclusion: Our data represent the first documentation in Tunisia, combining gastroenteritis viruses circulating in the environment and in clinical isolates. We observed a correlation between environmental strains and those found in children suffering from gastroenteritis during the same period study. This suggests the existence of a relationship between water contamination and paediatric diarrhoea. Significance and Impact of the Study: Our results address the potential health risks associated with transmission of human enteric viruses through water‐related environmental routes. The research findings will aid in elucidating the molecular epidemiology and circulation of enteric viruses in Tunisia and in Africa, where data are rare.     

Prevalence of nptII and Tn5 in kanamycin-resistant bacteria from different environments

Abstract Kanamycin (Km)-resistant bacterial populations in different soil, river water, sewage and pig manure slurry samples were enumerated and their prevalence in the total populations determined. About 350 Km-resistant Gram-negative colonies grown in the presence of kanamycin were identified using a rapid presumptive identification scheme. They were then screened for the presence of Tn5 and npt II sequences using hybridization of cells in dot blots, of Southern-blotted genomic DNA extracts and of PCR amplification products. Colonies reacting positively with a 2.7 kb probe of the central region of Tn5, or with a 925 bp npt II specific probe were primarily obtained from sewage samples, whereas fewer were obtained from pig manure slurry, river water and soil. However, in soil samples bacteria containing Tn5 or npt II were not found. Transposon Tn5 carrying the npt II gene could be unequivocally demonstrated in 3 isolates from sewage, identified as Aeromonas spp. (2x) and Escherichia coli . Hin dIII digests of chromosomal DNA obtained from these strains were cloned and shown to confer Km resistance to a sensitive E. coli strain. Further, various strains revealed the presence of npt II homologous sequences in a non-Tn5 background. The occurence of Tn5 and npt II in the samples was also assessed via PCR analysis of total community DNA extracts obtained from the aforementioned environmental samples. Evidence for the occurence of npt II was obtained for sewage, pig manure slurry, for 2 (out of 3) river water (Avon, Rhine) and 3 (out of 6) soil (Flevo silt loam, Westmaas silt loam, Ahlum rhizosphere) samples. Tn5 was not detectable via PCR in any of these environmental DNA extracts but it was found in Ede loamy sand and Flevo silt loam samples taken from a field microplot 2 and 4 weeks after release of a Tn5-containing genetically modified organism.     

Genetic characterization of Human astrovirus infection in Wuhan, People's Republic of China, 2007-2008

Human astrovirus (HAstV) was an important cause of viral gastroenteritis in infants in Wuhan city based on our previous study. The aim of the study was to investigate the nature of HAstV infection in Wuhan, People's Republic of China, especially in adults. Stool specimens were collected from 361 children and 301 adults with diarrhea from July 2007 to June 2008 and were tested for HAstV RNA by RT-PCR. The 348-bp PCR product of positive samples was further sequenced and analyzed for multiple sequence alignment and phylogenetic tree. HAstV RNA was detected in 2.33% (7/301) adults, which was significantly lower than that in children (13.57%, 49/361). HAstV-positive patients were either older than 50 years of age or younger than 3. Genetic analysis showed that the HAstV strain in adults was the same as that in children in 2007-2008. Contrarily, HAstV strains prevalent in 2007-2008 showed genetic characteristics different from those in 2004-2005 and belonged to two new groups of HAstV-1b. Thus, our data characterized HAstV infection in Wuhan 2007-2008, suggesting that HAstV infection also played an important role in adults in Wuhan, especial in patients of >50 years, and should be included for routine diagnosis in the population with diarrheal illness.     

Environmental surveillance and molecular characterization of human enteric viruses in tropical urban wastewaters

Aims: To study the prevalence and genotypes of waterborne pathogenic viruses in urban wastewaters in the tropical region. Methods and Results: Viruses in wastewaters collected at three water reclamation plants in Singapore were studied by molecular methods. Over a 6‐month sampling period, adenoviruses, astroviruses and both norovirus genogroups I (GI) and II (GII) were detected in 100% of the sewage and secondary effluent. Enteroviruses and hepatitis A viruses (HAV) were found in 94 and 78% of sewage, and 89 and 28% of secondary effluent, respectively. By using quantitative real‐time PCR, estimated concentrations of astrovirus in the sewage were 1–2 orders of magnitude higher than those for adenovirus, noroviruses GI and GII. Genotyping of environmental isolates revealed multiple genotypes of GI and GII noroviruses. Coxsackieviruses A, astrovirus type 1 and adenovirus type 41 were prevalent. Norovirus GII/4 and coxsackievirus A24 isolates in wastewaters were closely related to respective outbreak strains isolated previously in Singapore. Conclusions: This study showed the widespread occurrence of all tested enteric virus groups in urban wastewaters. Genetic diversity of astroviruses, enteroviruses and noroviruses in the tropical region was observed. Significance and Impact of the Study: The high prevalence and great genetic diversity of human enteric viruses in urban wastewaters strongly supports the need of further comprehensive studies for evaluating the public health risk associated with viral pathogens in water environments.     

Identification of Human Astrovirus Genome-Linked Protein (VPg) Essential for Virus Infectivity

Viral genome-linked proteins (VPgs) have been identified in several single-stranded positive-sense RNA virus families. The presence of such protein in the family Astroviridae has not been fully elucidated, although a putative VPg coding region in open reading frame 1a (ORF1a) of astrovirus with high amino acid sequence similarity to the VPg coding region of Caliciviridae has been previously identified. In this work we present several experimental findings that show that human astrovirus (HAstV) RNA encodes a VPg essential for viral infectivity: (i) RNase treatment of RNA purified from astrovirus-infected cells results in a single protein of 13 to 15 kDa, compatible with the predicted astrovirus VPg size; (ii) the antibody used to detect this 13- to 15-kDa protein is specifically directed against a region that includes the putative VPg coding region; (iii) the 13- to 15-kDa protein detected has been partially sequenced and the sequence obtained is contained in the computationally predicted VPg; (iv) the protein resulting from this putative VPg coding region is a highly disordered protein, resembling the VPg of sobemo-, calici- and potyviruses; (v) proteolytic treatment of the genomic RNA leads to loss of infectivity; and (vi) mutagenesis of Tyr-693 included in the putative VPg protein is lethal for HAstV replication, which strongly supports its functional role in the covalent link with the viral RNA.