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Environmental surveillance for human astrovirus in Shandong Province, China in 2013.

Human astroviruses (HAstVs) are one of the leading viral agents of acute gastroenteritis. However, there is limited information on HAstVs in China. Here, we describe the molecular characterization of HAstVs in Shandong, China via sewage surveillance. A total of 23 sewage samples were collected from sewage treatment plants in the cities of Jinan and Linyi in 2013. After concentration via adsorption-elution method, 9 samples (39.1%) were positive by reverse transcription PCR (RT-PCR) for the presence of the 719-nt HAstV nucleotide sequence. Genetic cloning and sequencing were performed on positive PCR products, and 26 HAstV sequences were obtained. Phylogenetic analysis on these sequences revealed 4 genotypes (HAstV-1, -2, -4 and -5), with HAstV-1 and -5 as the most common genotypes in Jinan and Linyi, respectively. Homologous comparison revealed Shandong sequences had relatively less genetic divergence among themselves than with foreign sequences. This study represents the first effort to investigate the genotypes and molecular epidemiology of HAstVs via sewage surveillance in China. The high detection rate in this study reflects that HAstVs circulated at a relatively high frequency in the local population, and demonstrates that environmental surveillance is an effective method in investigating circulating HAstVs.


Results
HAstVs in raw sewage. By using the virus concentration and RT-PCR, a total of 23 raw sewage samples were collected during the 12month surveillance period in 2013. Of these, HAstVs were detected in 9 samples (39.1%, 9/23), 4 (33.3%, 4/12) in Jinan and 5 (45.5%, 5/ 11) in Linyi, respectively. HAstV sequences and positive sewage specimens were illustrated in table 1. In Jinan, the positive samples were mostly in winter (October, November, December and January). However, in Linyi, the positive samples did not show an apparent seasonal distribution (detected in April, May, June, October and November).
Homologous comparison and phylogenetic analysis. Homologous comparison and phylogenetic analysis were carried out among all 26 environmental sequences and 26 reference sequences of all eight HAstV genotypes. Table 2 shows the alignment results of nucleotide and amino acid in acquired genotypes. Phylogenetic analysis revealed that Shandong HAstV-1 sequences in this study can be divided into two clusters comprising 13 and 1 sequences, respectively (Fig 1). Fourteen environmental HAstV-1 sequences displayed 92.0-100% nucleotide identity to each other. HAstV-1 sequences in Jinan had 98.3-100% nucleotide identity, and the HAstV-1 sequences in Linyi had 92.0-100% nucleotide identity among themselves. In the major cluster, the environmental sequences had 97.4-100% nucleotide identity to each other, and 97.9-99.9% identity to the Shanghai HAstV-1 strain (FJ375759). In the minor cluster, the sequence LY45-2 was closely related to the Oxford strain (L23513) and a Hungarian strain (HQ398856), with 96.8% and 98.1% nucleotide identity, respectively. When compared with strains identified in other regions of China, including Beijing (FJ755403 and FJ755404), the Inner Mongolia (HM1200877) and Liaoning Province (KF211475), all the HAstV-1 strains in two clusters displayed lower nucleotide identity (91.5 to 95.9%) to them. Moreover, no closer relationship was observed when compared with strains from other countries, such as Germany (AY720892), Korea (JN887820), and Italy (JX087965), with 91.7-92.3%, 92-92.4% and 91.6-92.2% nucleotide identity, respectively.
All HAstV-5 strains in this study were segregated into one cluster, and the nucleotide identity among themselves was 99.3 to 100%. These sequences showed a close relationship with strains isolated in Liaoning Province of China (JQ403108) and Hungary (KF157967), with 98.3 to 98.7% and 98.1 to 98.3% identity, respectively. However, great genetic divergence was observed in comparison with HAstV-5 strains isolated in Beijing (AB037274) and Brazil (DQ028633), with 4.1-4.5% and 4.5-4.9% genetic difference, respectively.
The HAstV-2 strain isolated in this study had a high similarity of 98% with a strain isolated in Italy (JX087964), and the HAstV-4 strain displayed 93.7% and 94.4% identities to the reference strains from Brazil (DQ070852) and Germany (AY720891), respectively.

Discussion
HAstVs are one of the important viral agents of diarrhea. However, no specialized surveillance system on these viruses has been established in China. In this study, based on the well established enterovirus environmental surveillance system in Shandong Province, we investigated the prevalence and genetic characteristics of HAstVs in 2013.
As a result, 23 raw sewage samples were collected and examined for HAstVs, and 26 HAstV sequences were obtained, with a high HAstV-positive rate of 39.1%. This positive rate was in accordance with other reported detected frequencies. For example, in a previous report in Hungary, HAstVs were detected in 43% of raw sewage samples 19 . A detection of enteric viruses in Germany environmental waters, depending on the samples sites, 24-42% of the samples from the sewage plant influent contained HAstVs 33 . These high positive rates of HAstVs in sewage reflect the high prevalence and circulation of HAstVs in population, and the inhabitants are faced with a higher health risk.
Four genotypes were recognized in this study, in which HAstV-1 accounted for the largest proportion (53.8%), suggesting that HAstV-1 is the main prevalent genotype in the cities of Jinan and Linyi, consistent with studies in most countries 5,12,34,35 , including China 11 . In Jinan, only HAstV-1 was identified, however, the absence of other genotypes does not mean that only HAstV-1 was prevalent in Jinan, because the sample size was limited in this study. In Linyi, HAstV-1, -2, -4 and -5 were detected, indicating the simultaneous circulation of different HAstV genotypes in this region and the circulating genotype varies in different geographic location. In addition, the HAstV-1 and -5 were the most prevalent genotypes in Linyi, and the HAstV-5 isolates (accounting for 50%) even outnumbered the HAstV-1 isolates (accounting for 40%), indicating that HAstV-5 may be more frequent than HAstV-1 in Linyi. This result is different from many studies mentioned above 5,11,12,34,35 . The small sample set may be a credible reason, however, the dominant genotypes can vary in different geographical areas, and the isolates from a study conducted in Houston and Mexico City were all HAstV-5 after typed by RT-PCR 36 . Therefore, in spite of HAstV-1 being the most common genotype worldwide, HAstV-5 may be the dominant genotype in some areas. Further investigation should be conducted to demonstrate whether HAstV-5 is the most frequent genotype in Linyi.
HAstV-2 and -4 were detected at a low rate (both 5%). The emergence of HAstV-2 and -4 suggests the need to monitor the minor HAstV genotypes besides the overarching dominant genotypes. HAstV-6 to -8 are rare all over the world 5 . In this study, HAstV-3, -6, -7 and -8 were not detected, and it might be explained that these genotypes were not prevalent in our sampling sites. However, HAstV-3 and -6 has been identified in the cities of Beijing, Wuhan and Jiangmen of China and other countries 11,[37][38][39][40] . Furthermore, HAstV-3 was reported to be able to induce more severe gastroenteritis and shed more virus 41 . HAstV-8 also has been examined in Spain, the South Africa and Australia 4,5,27 . Therefore, more attention should be paid to the circulating possibility of these less frequent genotypes.
HAstV prevalence is known to be seasonal, and more HAstV infection was observed in winter and spring in temperate regions 3 . In this study, the seasonal distribution was observed in Jinan with most positive samples detected in winter, and no positive sample was detected in summer, which supports the epidemiology reports 5,7 . This phenomenon may be due to the reason that the viral stability increases in the lowered temperature 42 . However, the HAstVs detected in Linyi did not follow a seasonal pattern. Moreover, a study from Australia has reported that HAstVs could display an unusual biennial winter peak 4 , and the distinct winter peak may be not observed in each year. But, owning the biggest small commodity wholesale market in Northern China, Linyi is a city of frequent population flow. This is quite different from the situation in Jinan. This may partially explain the difference in detection between the two cities. Considering the limit of investigation period in this study, further study should be performed to testify whether the HAstV infection in Linyi follow this seasonal distribution.
According to the homologous comparison and phylogenetic analysis, the environmental HAstV-1 isolates can be divided into 2 clusters. The major viral cluster contain the sequences from Jinan and Linyi, indicating that one HAstV-1 transmission strain circulated in the two cities concurrently and the occurrence of frequent inter-city spread during the survey period. The HAstV-1 strains in Linyi belonged to 2 clusters, suggesting the presence of two HAstV-1 transmission links. Additionally, the strain found in Shanghai of China (FJ375759) had a close relationship (97.9-99.9% identity) with the major viral strain cluster. Therefore, it may be inferred that the strains in the major viral cluster may be transmitted from Shanghai, and these strains, along with the circulating strain in Shanghai should have the same ancestor. Another less transmission strain in Linyi was similar to a Hungarian strain (HQ398856, 98.1% identity). Though HAstV-1 has been identified in some regions in China 37,38,43 , no close relationship was observed when compared with other Chinese reference strains (Beijing, Liaoning and the Inner Mongolia).
The HAstV-5 strains in this study displayed a high identity to each other and were located in one cluster, indicating the circulating of one transmission link. A Hungarian HAstV-5 strain (KF157967) showed a high similarity with these strains, with addition of the relationship of HAstV-1 strain in Linyi and Hungary as is mentioned above, moreover, in 2010, a HAstV-gastroenteritis outbreak occurred in Hungary 44 . On this account, the HAstVs surveillance in Linyi should be strengthen to avoid the outbreak of HAstVgastroenteritis. In addition, the HAstV-5 isolate in Liaoning Province of China displayed 98.3 to 98.7% identity with our isolates, so the common origin of HAstV-5 strains in Linyi, Liaoning Province and Hungary is probable. No appropriate HAstV-2 and -4 reference strains in China were found. By comparing them with foreign strains, the two minor genotypes keep close relationship with Italian and Brazilian strain, respectively.
Environmental surveillance has been proved to be an alternate tool to obtain the genetic diversity of enteric viruses in urban population [19][20][21]27 . Data from sewage can provide an overview on the viral molecular epidemiology. In this study, a one-year environmental surveillance on HAstVs was conducted successfully, and the genetic characteristics of HAstVs in Jinan and Linyi was observed, which offers another convincing evidence on the benefit of environmental surveillance. And long-term surveillance of circulating viruses should be conducted in the inhabitants to control and prevent virus infection. The monitoring time and genotypes obtained are limited in this investigation, and due to the lack of clinical data, we did not compare the environmental isolates with the clinical isolates circulating, however, this study provides the only surveillance data on HAstVs via sewage surveillance.
In conclusion, this study proves the presence of HAstVs in raw sewage in Jinan and Linyi of China. The high detection rate of HAstVs suggests that HAstVs circulated frequently in the population. HAstV-1 and -5 are the most frequent genotypes in Jinan and Linyi, respectively. In the absence of clinical data on gastroenteritis viruses, environmental surveillance can be a sensitive approach to assess the presence of virus and characterize the viral genetic evolutionary relationships.

Methods
Sampling. The permission for each sampling location was issued by Shandong Provincial Environmental Protection Department. From January to December in 2013, sewage samples were collected monthly in the inlet collector canal of the municipal sewage treatment plants, Jinan Everbright Water and Linyi Shouchuang Water, by using grab sampling procedure. The samples were transferred to the laboratory, stored at a cold temperature of 4uC, and processed within 24 h.
Concentration. Sewage samples were concentrated by adsorption-elution method as described previously 20 . Briefly, the raw sewage was centrifuged at 3000 3 g for 30 min at 4uC. MgCl 2 was added to the 800 ml of the supernatant to a final concentration of 0.05 M, and the pH value was adjusted to 3.5 by hydrochloric acid. Then the supernatant was filtered through a mixed cellulose ester membrane filter (pore size of 0.45 mm, ADVANTEC, Tokyo, Japan). The membranes were eluted with 10 ml 3% beef extract solution (pH value, 9.0) by ultrasonication for 5 min. The eluted solution was centrifuged at 3000 3 g for 30 min, and the supernatant was filtered through a 0.2 mm filter (PALL, Ann Arbor, USA).
RNA extracted and reverse transcription-PCR. Viral genomic RNA was extracted from 140 ml of concentrated sewage using QIAamp viral RNA mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's recommended procedure. Reverse transcription-PCR (RT-PCR) was carried out to amplify 719-nt HAstV ORF2 sequence (nt position 4235 to 4959 according to Oxford strain) by using Access RT-PCR System (Promega, USA), with primers pairs Precap1 (59 -GGACTGCAAAGCAGCTTCGTG -39) and 82b (59 -GTGAGCCACCAGCCAT CCCT -39), which can amplify all classic serotypes of human astrovirus 45 .
Cloning and sequencing. The RT-PCR products were analyzed by electrophoresis with 1% agarose gels. The positive products were gel-purified using a QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). The purified products were ligated into the pGEMH-T Easy vector (Promega, USA) by TA cloning. The ligation products were transformed into competent Escherichia coli JM109 cells using the heat shock method. After blue and white screening, ten positive recombinant clones were selected for each transformation. The plasmid was extracted and sequenced with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosys-tems, Foster City, CA). Sequences were analyzed by an ABI 3130 genetic analyzer (Applied Biosystems). Molecular typing was performed by using BLAST.