Wastewater Analysis Indicates that Genetically Diverse Astroviruses, Including Strains Belonging to Novel Clades MLB and VA, Are Circulating within Japanese Populations

Abstract

Human astroviruses (HAstVs) are a common etiological agent of infantile gastroenteritis. Recent studies revealed that novel astrovirus (AstV) strains of the MLB clade (MLB-AstVs) and VA clade (VA-AstVs), which are genetically distinct from the classic HAstVs, are circulating in the human population. In the present study, we quantified classic HAstVs as well as carried out a genetic analysis of classic and novel HAstVs in wastewater in Japan. The concentration of classic HAstVs in the influent water samples ranged from 10 4 to 10 5 copies per liter, and the amount removed by wastewater treatment was determined to be 2.4 ؎ 0.3 log 10 . Four types of classic HAstV strains (HAstV types 1, 2, 5, and 4/8) as well as novel AstV strains belonging to the MLB-2, VA-1, and VA-2 clades were identified using reverse transcription-PCR (RT-PCR) assays, including assays newly developed for the detection of strains of the MLB and VA clades, followed by cloning and nucleotide sequencing. Our results suggest that genetically diverse AstV strains are circulating among the human population in Japan. The newly developed (semi)nested RT-PCR assays for these novel AstV clades are useful to identify and characterize the novel AstVs in environmental waters.H uman astroviruses (HAstVs) are considered one of the most important causes of infantile gastroenteritis worldwide (1). The infections typically occur sporadically, and the cause of 2 to 10% of total viral gastroenteritis cases is attributed to HAstVs (1-6). Astroviruses (AstVs) possess nonenveloped, icosahedrally shaped virions containing approximately 6,800 nucleotides (nt) of single-stranded positive-sense RNA (7). It has been reported that the diameter of AstV particles shed in fecal specimens ranges from 28 to 31 nm, while the diameter of those grown in cell culture is 41 nm (8). Their viral genome consists of three open reading frames (ORFs), namely, ORF1a, ORF1b, and ORF2, encoding the serine protease, RNA-dependent RNA polymerase, and capsid proteins, respectively (7). HAstVs are antigenically or genetically classified into eight different types (HAstV type 1 [HAstV-1] to HAstV-8). Previous studies revealed that HAstV-1 is the most prevalent type among infected individuals and in the environment, followed by 5,[9][10][11][12][13].Because HAstV can be excreted in the feces of infected individuals at a high concentration (up to 10 15 particles/g) (14), examination of municipal wastewater samples could be an effective approach to understand the actual prevalence and epidemiology of these viruses (15, 16). In spite of their importance as enteric pathogens, the occurrence and other characteristics of HAstVs in water compared to those of other enteric viruses, such as noroviruses, rotaviruses, and adenoviruses, have not been well documented (17, 18). Due to the scarcity of knowledge, the fate of HAstVs in water environments and wastewater treatment processes is not well understood.Recent studies based on viral metagenomic analysis have identified some novel AstVs, namely, clade MLB ...

Continuous detection and genetic diversity of human rotavirus A in sewage in eastern China, 2013–2014

“…Environmental sewage surveillance has been successfully conducted to obtain molecular characterizations of human enteric viruses in some regions [ 19 – 21 ]. In this study, this novel surveillance method was performed to monitor the circulation of rotavirus in Jinan and Linyi of eastern China.…”

Rotavirus and Astrovirus

“…Recently, the emergence of MLB and VA HAstV genotypes have been also observed in sewage samples in Japan and Uruguay (Hata et al, 2015;Lizasoain et al, 2015). Sewage surveillance conducted in sewage samples from Uruguay also detected canine astrovirus clustering with strains detected previously in Italy (2008) and Brazil (2012) (Lizasoain et al, 2015b).…”

“…The virus was present in influent at concentrations ranging from 10 4 to 10 5 GC/L, in secondary treated wastewater (after activated sludge process) and chlorinated wastewater samples ranged from 10 2 to 10 3 GC/L and in effluent samples (after sand filtration) ranged from 10 1 to 10 2 GC/L. The highest HAstV reduction was observed after the activated sludge process and the mean reduction of the whole process was 2.4 log 10 (Hata et al, 2015). Similar results were observed by Morsy El-Senousy et al (2007) who studied the reduction of HAstV concentration in three different WWTP in Egypt.…”

Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

“…Additionally, as a microfluidic PCR technique has been applied for the detection and quantification of DNA viruses as well as RNA viruses ( Ishii et al, 2014 ), with suitable modifications, our MFnPCR–MiSeq method can also be applied to detect DNA viruses. Moreover, recent advances in DNA sequencing techniques have accelerated the accumulation of viral sequence data, which has led to the development of novel PCR primer sets ( Hata et al, 2015 ). These novel primer sets can be included in MFnPCR–MiSeq, along with the primers tested in the present study.…”

“…For example, NoV is composed of seven genogroups, and each genogroup is further subdivided into genotypes ( Vinjé, 2015 ). These RNA viruses circulate in aquatic environments and the human population ( Iwai et al, 2009 ; Sano et al, 2011 ; Hata et al, 2015 ; Miura et al, 2016 ; Pu et al, 2016 ). For instance, the viruses exist at high concentration in feces of patients and are released into sewage system ( Lee et al, 2007 ; Drexler et al, 2011 ).…”