Between 2013 and 2016, 225 rodents (Rattus norvegicus and Rattus flavipectus) and 196 shrews (Suncus murinus) were trapped by mouse traps, using fried foods as bait, in Shenzhen city, Guangdong province, southern China. Samples of various tissues (liver, lung, and intestine) were collected. Rodents and shrews were first identified based on morphology; the species of animals from which virus-positive samples had been collected were further confirmed by sequencing of the mitochondrial cytochrome b gene (Ge et al., 2013). The tissue samples were reserved at –80 °C until nucleic acid isolation.
Tissue samples were suspended at 10% w/v in 1 mL of phosphate-buffered saline (pH 7.4), and centrifuged at 12,000 × g for 10 minutes at 4 °C (Eppendorf, Hamburg, Germany). Total viral nucleic acids from liver, lung, and intestine samples were extracted from 200 μL of tissue suspension separately, using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Nucleic acids were aliquoted with elution buffer and stored at –80 °C until subsequent use.
First, we mixed RNA extracted from liver, lung, and intestine samples from each animal. RT-PCR assays were conducted for the distinct viruses, using the One-Step RT-PCR Kit (Invitrogen, Karlsruhe, Germany); sequences were amplified to perform phylogenetic analyses. Primer combinations targeting specific virus are listed in Table 1.
Virus Primer Sequence (5′–3′)a Region Amplicon size (bp) Reference Mammarenavirus Arena-F1 AYNGGNACNCCRTTNGC L gene First round: 938 Li et al., 2015 Arena-R1 TCHTAYAARGARCARGTDGGDGG Arena-F2 GGNACYTCHTCHCCCCANAC Second round: 611 Arena-R2 AGYAARTGGGGNCCNAYKATG Hantavirus HAN-L-F1 ATGTAYGTBAGTGCWGATGC L gene First round: 453 Klempa et al., 2006 HAN-L-R1 AACCADTCWGTYCCRTCATC HAN-L-F2 TGCWGATGCHACIAARTGGTC Second round: 385 HAN-L-R2 GCRTCRTCWGARTGRTGDGCAA HEV DE-F4228 ACYTTYTGTGCYYTITTTGGTCCITGGTT RdRp gene First round: 371 Drexler et al., 2012 DE-R4598 CCGGGTTCRCCIGAGTGTTTCTTCCA DE-R4565 GCCATGTTCCAGAYGGTGTTCCA Second round: 338 Note: aR: G/A; Y: C/T; S: G/C; W: A/T; M: A/C; K: G/T; H: A/C/T; N: A/T/C/G; I: inosine.
Table 1. Sequences of primers used for RT-PCR and sequencing
For mammarenaviruses, samples were screened by nested RT-PCR with primers amplifying the L segment as described previously (Li et al., 2015). For hantaviruses, samples were screened by RT-PCR using consensus primers based on the conserved domain of the L fragment (Klempa et al., 2006). For HEV, a hemi-nested RT-PCR targeting the viral RNA dependent RNA polymerase (RdRp)gene was performed, as described previously (Drexler et al., 2012).
RT-PCR products were first examined by agarose gel electrophoresis. Bands of the expected length were purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), and then cloned into the vector pGEM-T Easy (Promega, Madison, WI, USA). The inserts of plasmids were sequenced with primers M13F and M13R (Invitrogen, Karlsruhe, Germany), using a 3100 Sequencer (ABI, Waltham, MA, USA). Preliminary sequence data were analyzed using Geneious (Version 10.0.2, Biomatters Limited, Auckland, New Zealand) (Wang et al., 2017). The derived sequences were submitted to the GenBank database under the subsequent accession numbers: mammarenavirus (KY659332− KY659346), hantaviruses (KY659347−KY659375), and HEV (KY659376−KY659382). Sequence similarity comparisons were implemented using the BLASTn search engine (https://blast.ncbi.nlm.nih.gov). Sequence alignment with other representative viral sequences was performed using MAFFT (Katoh et al., 2002). The phylogenetic trees were built using the neighbor-joining algorithm with bootstrap values supported by 1,000 replications in MEGA version 7 (Kumar et al., 2016).
In total, 421 rodents and shrews were captured between 2013 and 2016 in 10 regions of Shenzhen city: Guangming, Longhua, Bao’an, Nanshan, Futian, Luohu, Longgang, Pingshan, Yantian, and Dapeng districts. Tissue samples harvested from these animals were examined by RT-PCR for the presence of mammarenavirus, hantavirus, and HEV. As illustrated in Table 2, the detection rates for these viruses varied: mammarenavirus infection rates in Rattus norvegicus and Suncus murinus were 6.70% and 0.51%, respectively, with an overall positive rate of 3.56% (15 of 421 animals). Regarding hantavirus, the infection rates in Rattus norvegicus and Suncus murinus were 13.40% and 0.51%, respectively, with an overall positive rate of 6.89% (29 of 421 animals); therefore, hantaviruses were the most prevalent viruses in this study. HEV was detected in 7/209 Rattus norvegicus samples (2.87%) and 1/196 Suncus murinus samples (0.51%), with an overall positive rate of 1.66% (7 of 421 animals). However, R. flavipectus was negative for all three viruses. Positive samples for the different viruses were derived from 8 of the 10 districts.
Virus Species Total R. norvegicus R. flavipectus S. murinus Mammarenavius a14/209 (6.70) 0/16 (0.00) 1/196 (0.51) 15/421 (3.56) Hantavirus 28/209 (13.40) 0/16 (0.00) 1/196 (0.51) 29/421 (6.89) HEV 6/209 (2.87) 0/16 (0.00) 1/196 (0.51) 7/421 (1.66) Note: apositive samples/tested samples (%)
Table 2. Detection rates of mammarenavius, hantavirus and HEV by RT-PCR in small mammals derived from Shenzhen city, China
A 589-bp sequence of the L fragment of mammarenavirus, from 15 samples from 14 rodents and 1 shrew, was amplified and sequenced. The 15 partial L fragment sequences had 93.8%–100% nucleotide sequence identity. Similarity comparisons reflected the highest nucleotide sequence similarity of these strains from Shenzhen city with the Cardamones virus isolate C617 from Cambodia (90.2%), followed by the Wenzhou virus isolate Rn-366 from eastern China (89.5%). Comparison of these strains with Lassa virus (LASV) revealed a lower nucleotide sequence similarity of up to 74.5%. A phylogenetic tree was built from the amplified sequences together with representative mammarenavirus strains (Figure 1). As shown in the tree, 15 strains clustered within mammarenaviruses from the Old World, consisting of Africa, Europe, and Asia, which is clearly separated from the New World comprising the Americas.
Figure 1. Phylogenetic relationships for 15 rodent and shrew mammarenavirus strains derived from Shenzhen city, China. The tree, which was based on a 589-bp sequence of the L fragment of the mammarenavirus, was built using a neighbor-joining algorithm implemented in Mega version 7. Bootstrap values (> 70%) are shown at specific nodes. Scale bars reflect the number of nucleotide substitutions per site. The tree is divided between the Old World and New World mammarenaviruses. The strain designations are reflected at each branch. The newly determined Shenzhen rodent and shrew strains are indicated via circles and triangles, respectively.
A 359-bp sequence of the L fragment of hantavirus was amplified and sequenced from 29 samples collected from 28 rodents and 1 shrew. The 28 partial L fragment sequences from rodents had 96.5% to 100% nucleotide sequence similarity; these strains reflected the highest nucleotide sequence identity with the Seoul virus isolate 2012–289 from Fujian province, eastern China (99.7%). A shrew strain had the highest nucleotide sequence similarity with the Thottapalayam virus strain VRC-66412 from shrews in India (88.9%), followed by the Imjin virus strain Cl 04-55 from Crocidura lasiura in South Korea (70.6%); these viruses are highly divergent from human strains. A phylogenetic tree was constructed using representative hantavirus strains (Figure 2). In this tree, 28 rodent strains clustered together with the human Seoul virus, and a shrew strain clustered with the shrew Thottapalayam virus.
Figure 2. Phylogenetic relationships for 29 rodent and shrew hantavirus strains derived from Shenzhen city, China. The tree, which was based on a 359-bp sequence of the L fragment of the hantavirus, was built using a neighbor-joining algorithm implemented in Mega version 7. Bootstrap values (> 70%) are shown at specific nodes. Scale bars reflect the number of nucleotide substitutions per site. Seoul virus and Shrew virus are indicated on the right. The strain designations are indicated at each branch. The newly determined Shenzhen rodent and shrew strains are indicated via circles and triangles, respectively.
A 280-bp fragment of the ORF1 of HEV, from 7 samples from 6 rodents and 1 shrew, was amplified and se-quenced. The 7 partial ORF1 fragment sequences had 77.7%–99.7% nucleotide sequence identity. Among them, 4 HEV strains from 3 rodents and 1 shrew had the highest nucleotide sequence similarity of 84.7% with Hepatitis E virus rat/R63/DEU/2009 from rodents in Germany; the other 3 rodent HEV strains had the highest identity of 84.9% with Hepatitis E virus isolate Vietnam-105 from rats in Vietnam. A phylogenetic tree was constructed using representative HEV strains (Figure 3). According to the tree, all 8 strains from Shenzhen city belonged to species Orthohepevirus C (strains from rat, shrew, greater bandicoot, ferret, and mink). Of note, 3 rodent and 1 shrew HEV strains clustered together with rat HEV strains from Asia, whereas the other 3 rodent HEV strains clustered with rat strains from Europe.
Figure 3. Phylogenetic relationships for 7 rodent and shrew HEV strains derived from Shenzhen city, China. The tree, which was based on a 280-bp sequence of the RdRp gene of HEV, was constructed using a neighbor-joining algorithm implemented in Mega version 7. Bootstrap values (> 70%) are indicated at specific nodes. Scale bars indicate the number of nucleotide substitutions per site. The strain designations are reflected at each branch. The newly determined Shenzhen rodent and shrew strains are indicated via circles and triangles, respectively
Detection and characterization of three zoonotic viruses in wild rodents and shrews from Shenzhen city, China
- Received Date: 10 March 2017
- Accepted Date: 27 May 2017
- Published Date: 17 July 2017
Abstract: Diverse species of rodents and shrews,which are abundant worldwide,harbor a variety of viruses; some of these are closely related to human viruses and possess zoonotic potential.Previously studies have demonstrated that the mammarenavirus and hantavirus carried by rodents or shrews could cause diseases in human population.To determine the distribution of zoonotic viruses in Shenzhen city,the major city in southern China with a high population density,we analyzed 225 rodents (Rattus norvegicus and Rattus flavipectus) and 196 shrews (Suncus murinus) from urban and rural districts for the presence of mammarenavirus,hantavirus,and hepatitis E virus (HEV) by RT-PCR targeting the conserved regions.The infection rates for mammarenavirus,hantaviruses, and HEV in rodents and shrews were 3.56%,6.89%,and 1.66%,respectively.Partial genome fragment analysis indicated that mammarenavirus and hantavirus strains had more than 90% and 99% nucleic acid identity with Cardamones virus and Seoul virus,respectively,which cause diseases in humans.Although the present HEV strains identified are typically found worldwide, phylogenetic analysis demonstrated a divergence of 16%.To our knowledge,the present work is the first report of the prevalence of mammarenavirus,hantaviruses,and rat HEV strains in rodents and shrews from Shenzhen city,China.Our findings highlight the zoonotic potential of rodent-and shrew-borne mammarenavirus and hantavirus,and the biodiversity of rat HEV isolates in Shenzhen city.The present work suggests that utilization of good hygiene habits is important to minimize the risk of zoonosis.