key: cord-0791810-ughtuxpu
authors: Chu, Daniel K. W.; Chin, Alex W. H.; Smith, Gavin J.; Chan, Kwok-Hung; Guan, Yi; Peiris, J. S. Malik; Poon, Leo L. M.
title: Detection of novel astroviruses in urban brown rats and previously known astroviruses in humans
date: 2010-10-03
journal: J Gen Virol
DOI: 10.1099/vir.0.022764-0
sha: 49952f26e28224dcb6345f7bd9abcbc1b1dc32d8
doc_id: 791810
cord_uid: ughtuxpu

Several novel astroviruses have been recently discovered in humans and in other animals. Here, we report results from our surveillance of astroviruses in human and rodent faecal samples in Hong Kong. Classical human astroviruses (n=9) and a human MLB1 astrovirus were detected in human faecal samples (n=622). Novel astroviruses were detected from 1.6 % of the faecal samples of urban brown rat (Rattus norvegicus) (n=441), indicating the prevalence of astrovirus infection in rats might be much lower than that recently observed in bats. These rat astroviruses were phylogenetically related to recently discovered human astroviruses MLB1 and MLB2, suggesting that the MLB viruses and these novel rat astroviruses may share a common ancestor.

The family Astroviridae comprises non-enveloped, positivesense, single-stranded RNA viruses. The members of the family are classified into mamastroviruses and avastroviruses and are known to infect mammalian and avian species, respectively (Mendez & Arias, 2007) . In human, the classical human astroviruses are genetically closely related and can be classified into eight serotypes (HAstV1-8). In addition, several genetically distinct human astroviruses (e.g. MLB1) have been recently identified in stool samples from patients suffering from gastroenteritis (Finkbeiner et al., 2008a (Finkbeiner et al., , 2009a Kapoor et al., 2009) . Although astrovirus is one of the major causative agents for gastroenteritis, there is relatively little information on the ecology and evolution of these viruses. The recent discoveries of genetically diverse astroviruses in bats (Chu et al., 2008b; Zhu et al., 2009) and other animals (Atkins et al., 2009; Rivera et al., 2010) highlight the genetic diversity of astroviruses in nature and suggest that there might be many more novel astroviruses circulating in peridomestic and wild animals.

Bats represent the second largest group of mammals (comprises 20 % of all mammals). The remarkably high detection rate of astroviruses in bats prompted us to perform a similar surveillance study on rodents, which is the largest group of mammals accounting for 40 % of known mammalian species. As we were primarily interested in viruses that might be at the animal-human interface, we specifically targeted rodent species that have a long history of living in close proximity to human populations (Sullivan, 2003) . Here, we report the discovery of rat astroviruses (RAstVs) from urban rats. In addition, we also report our recent surveillance of astroviruses in humans.

A total of 622 human stool samples collected from patients with diarrhoea admitted to the Queen Mary Hospital in Hong Kong during the years 2004 and 2005, and a total of 441 rat faecal samples collected from Rattus norvegicus (brown rat, n5371) and Rattus rattus (black rat, n570) captured from urban areas in Hong Kong during the years 2007 and 2008 were resuspended in 1 ml of viral transport medium and tested as described before (Chu et al., 2008b) . RNA was extracted using QIAamp virus RNA mini kit (Qiagen) following the protocol recommended by the manufacturer. Hemi-nested RT-PCR assay with degenerated primers designed to detect the RNA-dependent RNA polymerase (RdRp) gene of all known human and other mammalian astroviruses (Chu et al., 2008b) were used for the screening of astroviruses in these samples. All PCR amplicons obtained were subsequently analysed by sequencing.

Ten (1.6 %) of 622 samples from patients with diarrhoea were positive for astroviruses. Direct sequencing of the RT-PCR products revealed that nine (1.4 %) of these were classical human astroviruses and one (0.2 %) was MLB1 astrovirus. Though sequence identity suggests that the primers employed in our PCR screening test should detect other recently discovered human astroviruses MLB2, HAstV-VA1, VA2, VA3 (Finkbeiner et al., 2009a, c) and HMO astroviruses (Kapoor et al., 2009) , none of these viruses was detected from our human samples. To determine the sequence variation between MLB1 found in Hong Kong and the viruses previously detected from other continents, the complete genome sequence of MLB1 virus identified in this study (MLB1 HK05) was deduced. Primers used for determining the MLB1 HK05 genome were designed based on other MLB1 virus genomes previously reported in GenBank (available upon request). The MLB1 HK05 genome was found to be similar to other reported MLB1 genomes detected in Australia (Finkbeiner et al., 2008b) and USA (Finkbeiner et al., 2009b) (Fig. 1a) , with overall nucleotide sequence identities of 95.2 and 91.8 %, respectively. The complete genome sequence of MLB1 HK05 can be accessed through GenBank (accession no. HM450380).

The screening by the RT-PCR assay of astrovirus in rats revealed that six (1.6 %) of 371 faecal samples from R. norvegicus and none of the samples from R. rattus were positive. The positive specimens were collected in 2007 (n54) or 2008 (n52) and were collected from different geographical locations in Hong Kong.

Phylogenetic analysis on the partial RdRp sequences of these rat astroviruses revealed a group of previously unknown astroviruses. The rat astrovirus sequences deduced in this study were found to have a remarkable Novel astroviruses in rats degree of sequence variation (82-93 % for the RdRP gene) and they formed two genetically closely related, but distinct, clusters in a phylogenetic analysis (represented by RS118 and RS126 in Fig. 1a) . Samples RS118 and RS126 were selected for further sequence analyses. Primers used in determining the sequences of these viruses are summarized in Supplementary Table S1 (available in JGV Online). All nucleotide sequences of the RAstVs generated in this study can be accessed through GenBank (accession nos HM450381-HM450386). Attempts in culturing these viruses in BHK, CaCo-2, Vero and HEK 293 cells with or without trypsin (1-10 mg ml 21 ) and attempts in amplifying the 59 end of these RAstVs (predicted length:~1500 nt) by using consensus primers deduced from other AstVs sequences were unsuccessful.

Excluding the poly(A) tail, the partial genome sequences of RS118 (4918 nt) and RS126 (4853 nt) were constructed by aligning multiple overlapping PCR fragments. Each of these assembled sequences represented the 39 two-third of the corresponding viral genome, which includes partial ORF1a, complete ORF1b, complete ORF2 and 39 UTR regions. Serine protease gene and RdRp gene can be identified in ORF1a and ORF1b, respectively, from both RAstV sequences. A heptameric slippery sequence (AAAAAAC) can be identified at the putative ORF1a/1b junction of each of these RAstVs. Similar to the MLB1 virus (Finkbeiner et al., 2008b) , a stem-loop II-like motif (s2m), which can be commonly found in the 39 UTR of many astroviruses, could not be found in the RAstVs.

Pair-wise amino acid identities of different ORFs between astroviruses indicated that these rat viruses are genetically distinct from other previously known astroviruses (Table  1) . Between the two representative RAstVs, sequence identities of ORF1a (partial), ORF1b and ORF2 regions were 79.4, 91.6 and 70.9 %, respectively (Table 1) . These representative rat viruses are phylogenetically distinct from each other and have a sister-clade relationship, which is supported by high posterior probability values in all ORFs (Fig. 1a) . Sequence analysis of these and other astroviruses by using Recombination Detection Program 2 (Martin et al., 2005) and Simplot (Lole et al., 1999) did not reveal strong evidence of recombination events in the rat viral genomes. Overall, our results suggested that these are two groups of astroviruses circulating in R. norvegicus in Hong Kong.

Interestingly, these RAstVs are shown to be phylogenetically closely related to the human MLB1 and MLB2 astroviruses (hereinafter MLB viruses) with strong statistical support (Fig. 1a, ORF1b and ORF2 ). It should be noted that this sister-clade relationship does not have a strong statistical support in the ORF1a sequence analyses (Fig. 1a) . However, the lack of resolving power in the ORF1a analysis is very probably due to the lack of other viral sequences available for analysis.

We further selected ORF1b gene, which is the most conserved gene in astrovirus genomes, for a dating analysis using BEAST (Drummond & Rambaut, 2007) . Complete ORF1b nucleotide sequences of selected mamastroviruses were aligned at the amino acid level. Dating analysis was done using the uncorrelated relaxed clock model, with rates at each branch being allowed to vary independently from a lognormal distribution. Markov chain Monte Carlo sample chains were run for 60 000 000 generations with sampling every 3000 generations under GTR nucleotide substitution model and allowing gamma-rate heterogeneity for all datasets. As indicated in Fig. 1(b) , RAstVs and MLB viruses formed a unique clade which is supported by a posterior probability of 0.992. This strongly indicated that RAstVs and MLB viruses might share a common ancestor virus. The mean date of the most recent common ancestor (MRCA) virus was estimated to be AD 1054 (95 % highest posterior densities: AD 837-1827). In addition, the aligned ORF1b sequences were subjected to a d N /d S analysis by GA-Branch method incorporated in Datamonkey (http://www. datamonkey.org/) (Pond & Frost, 2005) . This analysis failed to find an accelerated positive selection for the RAstVs or for the MLB viruses (not shown), suggesting that these RAstVs and MLB viruses have been circulating in and adapted to rats and humans, respectively, over a long period of time.

Phylogenetic analyses on ORF1b strongly indicated that the newly found rat astroviruses are genetically related to human astrovirus MLB viruses. R. norvegicus and R. rattus are two of the most commonly found rodent species in human settlements and are long known as major reservoirs for some zoonotic diseases (Morse, 1995) . Our results suggest that the human MLB viruses and the rat astroviruses shared a common ancestor. However, with the limited sequencing data available at present, questions concerning the evolutionary history of these viruses cannot be answered in greater detail. Further surveillance in rodent species might help to improve the preciseness of the MRCA dating and better explain the origins of the rat astroviruses and MLB viruses.

We previously reported the detection of genetically highly diverse astroviruses and coronaviruses in bats (Chu et al., 2008a, b; Poon et al., 2005; Zhu et al., 2009) . The prevalence of these novel RNA viruses in bat faecal samples was found be extremely high (10-50 %) (Chu et al., 2008a, b; Poon et al., 2005; Zhu et al., 2009) . By contrast, less than 2 % of the studied rodent faecal samples were found to be positive for astrovirus. We also screened about 60 % of these rodent samples (n5403) for coronavirus, but none of them was positive in the assay (data not shown). These findings suggest that bats are unusual in their propensity to harbour astroviruses and coronaviruses and may have a more prominent role in shaping the evolution of these groups of viruses. However, as rats are more likely to have close and repeated contact to human populations, further systematic surveillance of novel viruses in rodent populations in different geographical locations should be encouraged.

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We would like to acknowledge the support from National Institutes of Health (NIAID contract HHSN266200700005C), Seed Funding Programme for Basic Research (HKU 200711159027, L. L. M. P.) and EMPERIE (grant no. EU FP7 223498). We thank Vijaykrishna Dhanasekaran for facilitating the data analysis and Joyce Choi for her technical support.