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Hu et al. Emerging Microbes & Infections (2018) 7:154 Emerging Microbes & Infections
DOI 10.1038/s41426-018-0155-5 www.nature.com/emi
ARTICLE Open Access
Genomic characterization and infectivity of
a novel SARS-like coronavirus in Chinese
bats
Dan Hu
1,2
, Changqiang Zhu
2
,LeleAi
2
,TingHe
2
,YiWang
3
, Fuqiang Ye
2
,LuYang
2
,ChenxiDing
2
, Xuhui Zhu
2
,
Ruicheng Lv
2
,JinZhu
2
, Bachar Hassan
4
, Youjun Feng
5
, Weilong Tan
2
and Changjun Wang
1,2
Abstract
SARS coronavirus (SARS-CoV), the causative agent of the large SARS outbreak in 2003, originated in bats. Many SARS-
like coronaviruses (SL-CoVs) have been detected in bats, particularly those that reside in China, Europe, and Africa. To
further understand the evolutionary relationship between SARS-CoV and its reservoirs, 334 bats were collected from
Zhoushan city, Zhejiang province, China, between 2015 and 2017. PCR amplification of the conserved coronaviral
protein RdRp detected coronaviruses in 26.65% of bats belonging to this region, and this number was influenced by
seasonal changes. Full genomic analyses of the two new SL-CoVs from Zhoushan (ZXC21 and ZC45) showed that their
genomes were 29,732 nucleotides (nt) and 29,802 nt in length, respectively, with 13 open reading frames (ORFs). These
results revealed 81% shared nucleotide identity with human/civet SARS CoVs, which was more distant than that
observed previously for bat SL-CoVs in China. Importantly, using pathogenic tests, we found that the virus can
reproduce and cause disease in suckling rats, and further studies showed that the virus-like particles can be observed
in the brains of suckling rats by electron microscopy. Thus, this study increased our understanding of the genetic
diversity of the SL-CoVs carried by bats and also provided a new perspective to study the possibility of cross-species
transmission of SL-CoVs using suckling rats as an animal model.
Introduction
Coronaviruses (CoVs) are a family of RNA viruses
belonging to the Coronaviridae family and the
Coronavirinae subfamily and are the largest group of
positive-sense single-stranded RNA viruses. From an
academic perspective, CoV can be divided into four
genera, namely Alphacoronaviruses,Betacoronaviruses,
Gammacoronaviruses, and Deltacoronaviruses. The
alphacoronaviruses and betacoronaviruses are usually
found in mammals, while the gammacoronaviruses and
deltacoronaviruses are mainly associated with birds
1,2
.
SARS-CoV is the causative agent of the severe acute
respiratory syndrome (SARS) outbreak that occurred in
2002–2003. This SARS outbreak was the first human
pandemic to break out since the beginning of the 21st
century, and it resulted in nearly 8000 cases of infection
and 800 deaths worldwide
3,4
. SARS-CoV belongs to the
Betacoronavirus genus, and its genomic sequence exhibits
low levels of similarity with the previously identified
human CoVs-OC43 and 229E. Thus, we hypothesized
that SARS-CoV underwent a long and independent evo-
lutionary process. The SARS-CoV genome usually
encodes four structural proteins: the spike protein (S),
envelope protein (E), membrane protein (M), and
nucleocapsid protein (N). Among them, the S protein is a
trimeric, cell-surface glycoprotein that consists of two
subunits (S1 and S2), whereas the S1 subunit is respon-
sible for receptor binding. Variations in the S protein, to a
© The Author(s) 2018
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Correspondence: Youjun Feng (fengyj@zju.edu.cn) or Weilong Tan
(njcdc@163.com) or Changjun Wang (science2008@hotmail.com)
1
Department of Epidemiology, College of Preventive Medicine, Third Military
Medical University, Chongqing 400038, China
2
Department of Epidemiology, Research Institute for Medicine of Nanjing
Command, Nanjing 210002, China
Full list of author information is available at the end of the article.
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large extent, are responsible for the tissue tropism and
host ranges of different CoVs
5,6
.
The origin of SARS-CoV has always been a focus of
research. Palm civets were initially considered the natural
reservoir of SARS-CoV due to the isolation of several
strains of SARS-CoV from palm civets that were traded in
the wet markets of the Guangdong province of China in
2003
7
. However, subsequent studies showed that the virus
was detected only in palm civets of market origin that
were tested prior to culling, but not in those tested later;
palm civets captured from the wild also tested negative for
the virus. This finding suggested that palm civets served
only as an intermediate reservoir and are therefore not a
natural reservoir for SARS-CoV
8,9
. Recently, bats have
captured our attention due to their ability to act as natural
reservoirs for a wide variety of viruses, including many
important zoonotic viruses that are associated with sev-
eral severe forms of emerging infectious diseases, such as
Ebola virus, Nipah virus, Hendra virus, and Marburg
virus
10,11
. In 2005, teams from Hong Kong and Mainland
China almost simultaneously discovered the presence of
SL-CoVs in wild Chinese horseshoe bats (Rhinolophus
sinicus) from China. These findings suggested that the
bats were the natural hosts of SARS-CoV
12
. Notably,
during longitudinal surveillance of the Rhinolophus sini-
cus colony in the Yunnan Province of China over the past
few years, a Chinese research team successfully isolated a
live SL-CoV sample from Vero E6 cells that were incu-
bated in the bat feces in 2013
13
. The isolated virus showed
more than 95% genome sequence identity with human
and civet SARS-CoVs. Further studies on these indicated
that the SL-CoV from bats may directly infect humans
and does not require an intermediate host. SL-CoV,
similar to SARS-CoVs, possesses the ability to infiltrate
cells using its S protein to combine with angiotensin-
converting enzyme 2 (ACE2) receptors
14
. This observa-
tion indicated that SARS-CoV originated from Chinese
horseshoe bats and that SL-CoV isolated from bats
therefore poses a potential threat to humans.
In recent years, many novel SL-CoVs have been
identified in a variety of bat species throughout the
world, including Asia, Europe, Africa, and America.
Most SL-CoVs were discovered in rhinolophids from
China, Slovenia, Bulgaria, and Italy
15–17
,whilenovel
beta-coronaviruses related to SARS-CoV have been
detected in Hipposideros and Chaerophon species from
Kenya and Nigeria
18,19
. However, analysis of the RNA-
dependent RNA polymerase (RdRp) amino acid
sequence showed that the genomic sequences of these
bat SL-CoV samples obtained from different parts of the
world shared 80–90% identity among themselves and
exhibited 87–92% identity with the SARS-CoVs
extracted from human or civet sources
20,21
.These
findings indicated that SARS-CoV likely evolved in bats
over longer periods of time. Previous research con-
ducted by our group revealed that bats found in
Southeast China have high carrying capacities for
SL-CoVs
22
. After conducting an epidemiological survey
on the bats carrying CoVs, two novel SL-CoVs were
identified in the Rhinolophus pusillus specimens from
Zhoushan city, Zhejiang Province, China; subsequently,
a rat infection model was utilized to assess the cross-
species transmission potential of the viruses.
Results
Sampling
Between 2015 and 2017, 334 bats were sampled from
Zhoushan, China. These bats belonged to the species
Rhinolophus pusillus as determined by the sequences of
the mitochondrial cytochrome bgene in their muscle
tissues
23
. All 334 bat samples were screened for CoV RNA
using a pan-coronavirus reverse transcription (RT)-PCR
assay. The overall prevalence of the virus was 26.65% (89/
334, bats; Table 1). Additionally, a higher prevalence was
observed in samples collected in July (66.7% in 2015) than
in those collected in October (21% in 2016) or February
(13% in 2017). A phylogenetic tree was constructed
according to the 440-bp RdRp partial sequences, and the
positive samples were classified into Alphacoronaviruses
and Betacoronaviruses. As shown in Fig. S1, 89 amplicons
were grouped into five clades with 66–100% nucleotide
identities between them, and they shared 94–100%
Table 1 Summary of the bat-CoVs detection in bats from the Zhejiang province of China
Time Locus Sample number Bat species CoV positive
July, 15 Dinghai, Zhoushan city (ZXC) 45 Rhinolophus sinicus 66.7% (30/45)
January, 16 Dinghai, Zhoushan city (Z2) 120 Rhinolophus sinicus 25% (30/120)
October, 16 Daishan, Zhoushan city (DXC) 84 Rhinolophus sinicus 21% (18/84)
February, 17 Dinghai, Zhoushan city (ZC) 85 Rhinolophus sinicus 13% (11/85)
Total 334 26.65 (89/334)
Hu et al. Emerging Microbes & Infections (2018) 7:154 Page 2 of 10
identities with the viruses that were extracted from Hong
Kong, Guangdong, and Hainan in China as well as those
from Spain.
Full genomic sequence comparison and recombination
analyses
To further explore the evolution of SL-CoV from
Zhoushan, two complete genomic sequences of the
representative bat-derived CoVs were generated by
sequencing several overlapping amplicons. Specifically,
sequences were generated from the following samples:
SL-CoV ZXC21 (MG772934) bat that was extracted from
a sample procured in July 2015, and SL-CoV ZC45
(MG772933) bat that was extracted from a sample pro-
cured in February 2017. The full genomes of ZXC21 and
ZC45 consisted of 29,732 nt and 29,802 nt, respectively.
The genomic organization in both cases was similar to
that of the most well-known bat-SL-CoVs. Using the RDP
program, the potential recombinant events between
ZXC21, ZC45 and other representative strains of 13
human/civet and bat SARS-like CoVs were initially pre-
dicted. The results did not identify any potential recom-
bination events. The genomic sequence similarity among
the five bat-SL-CoVs and the SARS-CoV SZ3 strain was
examined by Simplot analysis (Fig. 1). The results showed
that the genomes had 38.9% GC content and had 13 open
reading frames (ORFs) similar to the HKU3-1 strain. The
two new bat SL-CoVs shared 97% genomic sequence
identity among themselves. The overall nucleotide
sequence identity of these two genomes with civet SARS-
CoV (SZ3 strain) was 81%, which was lower than the
previously reported observations associated with bat SL-
CoVs collected from China (88–92%). From homology
analyses of different ORFs, ORF8 fragments showed the
lowest homology with the reported SL-CoV homology
data
24
, presenting a shared identity of only 60% with its
closest relatives.
The S protein is responsible for the entry of the virus
and is functionally divided into two domains, S1 and S2.
The bat SL-CoV Rs3367 is the most closely related virus
to the human SARS-CoV and has 89.9% amino acid
sequence identity to the SARS-CoV with respect to the
whole spike protein. Comparatively speaking, the S pro-
teins of ZXC21 and ZC45 identified in this study were
slightly more different than their counterpart in SARS-
CoV, which showed 77% identity at the amino acid level.
Phylogenetic analyses based on the S protein suggested
that the S proteins of ZXC21 and ZC45 represented a
separate clade related to the lineage B CoVs (Fig. 2b). The
highest amino acid sequence identity shared with the
Rs806 strain was only 83%. Like other bat-SL-CoVs, the
S1 domain of the bat SARS-like CoVs exhibited a very low
nucleotide similarity with SARS CoV, and there are sev-
eral key deletions and mutations in most of the variable
regions within the receptor-binding domain (RBD)
(Fig. 2a).
Rat infection and virus detection test
Despite the failed isolation of the infectious virus from
PCR-positive samples in Vero E6 cells, we attempted to
isolate the virus from suckling rats by infecting them with
tissue samples that were positive for the coronavirus.
After 15 days, pathological analysis showed that the tis-
sues and organs of the infected rats exhibited varying
degrees of inflammation, and the inflammatory reaction
in the brain tissues was most evident. Of the ten suckling
rats, four showed clinical symptoms, including drowsi-
ness, slow action, and mental depression. The new suck-
ling rats infected with the diseased brain tissue still had
irregular onset, whereas five of the 11 suckling rats in one
nest had clinical symptoms. Numerous apoptotic neurons
were seen in the focal areas of the brain tissue, and the
chromatin in the nuclei was condensed and unclear. The
lung tissues were well structured, but the alveolar cavities
were partly fused together and showed clear signs of mild
emphysema. Intestinal tissue analysis showed a loss in the
structure of the intestinal mucosa; the mucous mem-
branes were thin, the crypts were shallow, the intrinsic
glands were reduced, and the stroma showed a dispersed
inflammatory infiltrate (Fig. 3). Subsequently, the viral
load of different tissues was detected by quantitative PCR,
and the viral loads of the lung tissues remained the
highest, showing approximately 10
4
viral genome copies
per 1 μl of tissue suspension (data not shown).
Suspected viral particles were observed in the nuclei of
denatured neurons in the brain tissues of the rats using
transmission electron microscopy (TEM). These viral
particles presented the typical coronavirus morphology
and were approximately 100 nm in size with apparent
surface spikes (Fig. 4). Simultaneously, various viral RT-
PCR tests were conducted on the tissues to detect viral
particles. The tissues were tested for the presence of viral
particles associated with a wide variety of viruses, such as
CoVs, henipaviruses, respiroviruses, avulaviruses, rubula-
viruses, and the influenza-A virus of the Orthomyxovir-
idae family, using previously published methods
25,26
. The
test results revealed that the tissues were positive only for
CoV.
Analysis of the N protein antigen and western blotting
Similar to other CoVs, the nucleocapsid protein is one
of the core components of the SARS-CoV. The N protein
is one of the most predominantly expressed proteins
during the early stages of SARS-CoV infection and has
been an attractive diagnostic tool due to the initiation of
strong immune response against it. Evolutionary analyses
have shown that the homology between the N protein
and its counterparts in the well-known SARS-CoV
Hu et al. Emerging Microbes & Infections (2018) 7:154 Page 3 of 10
and bat SL-CoV ranged from 89 to 91%. The antigenic
analysis was based on the amino acid sequence of the N
protein (Fig. 5), and the results suggested that the two
alternative antigenic peptides, including KHD2016288-1:
KDKKKKADELQALPQ and KHD2016288-2:QQQG
QTVTKKSAAEA, were selected for peptide synthesis
To further characterize the antigenic reactivity of the
virus in infected murine tissues with ZC45-specificanti-
bodies compared to that of ZC45, polyclonal antibodies
against the polypeptides (KHD2016288-1:KDKKKKA-
DELQALPQ) derived from ZC45 N proteins were gener-
ated and then subjected to western blotting analysis (Fig. 6).
The anti-polypeptides were derived from the ZC45 N
protein antibodies from six different sources of N pro-
teins (50 kDa), including the intestinal tissues, brain
tissues and lung tissues of infected rats.The results indicated
that the polypeptide antigen was synthesized correctly,
and the polyclonal antibodies produced against this
Fig. 1 A gene map of the two novel SL-CoVs and the recombination analysis of novel SL-CoVs with other SL- CoVs. Similarity plots were
conducted with SARS CoV SZ3 as the query and bat SL-CoVs, including Rs3367, Longquan-140, and HKU3-1, as potential parental sequences. The
analysis was performed using the Kimura model, with a window size of 2000 base pairs and a step size of 200 base pairs
Hu et al. Emerging Microbes & Infections (2018) 7:154 Page 4 of 10
polypeptide could react with the N proteins of the bat SL-
CoV. The polyclonal antibodies reacted specifically with
the infected rat tissues, but not with the rat tissues
derived from the control specimens. These results
indicated that the virus can circulate and proliferate in
infected rats.
Fig. 2 Characterization of S1 domains of the SARS CoV and SL-CoVs. a Amino acid sequence comparison of the S1 subunit. The receptor-
binding domain (aa 318–510) of SARS-CoV. bA phylogenetic analysis of the entire S1 amino acid sequences based on the neighbor-joining method.
The SARS-CoV-GD01, BJ302, and GZ02 strains were isolated from patients of the SARS outbreak in 2003. The SARS-CoV SZ3 was identified from civets
in 2003. Other bat-SL-CoVs were identified from bats in China.The sequences of SL-CoVs in this study are marked as filled triangles
Fig. 3 Light microscopy observations of rat tissues infected with bat-SL-CoVs: Sectioned brain, intestine, lung and liver tissues were sampled
from rats infected with bat-SL-CoV ZC45
Hu et al. Emerging Microbes & Infections (2018) 7:154 Page 5 of 10
Discussion
Since the first report on the origin of SL-CoVs from bats
in 2005, CoVs have been found in ten different bat species
within six families from more than ten countries,
including China, Africa, and Europe
21,27
. Our 2-year
longitudinal surveillance of bats in Zhoushan indicated
that all 334 bats that were collected belonged to the
species Rhinolophus sinicus, suggesting that it was the
dominant bat species found in our study and has been
shown to be the natural reservoir of SARS-CoV. Nested
PCR amplification of the conserved region of RdRp
showed that the CoV carrying rate associated with this
species of bat was much higher than that reported pre-
viously
28,29
. At the same time, the summer carrying rate
was higher than that associated with the other seasons
due to the influence of seasonal distribution. In this
region, there were two clades of Alphacoronaviruses and
three clades of Betacoronaviruses identified, indicating
that a wide variety of CoVs circulate in the bats of the
Zhoushan area, and these CoVs were the most widely
transmitted in the bat colonies found in this region.
To explore the possibility of CoV transmission from
bats in this area, two full-length samples of bat-SL-CoVs
were procured from the viral-infected bats. These two bat
SL-CoVs were obtained from the same location but dur-
ing different seasons; a genomic sequence identity of
88–99% was presented among them, indicating that the
bats are the natural reservoirs of these SL-CoVs and that
these SL-CoVs can circulate within single colonies.
Meanwhile, there was a great difference between the two
viruses described in this study and the viruses described in
earlier studies, especially with respect to the hypervaria-
bility of the S1 domain
30,31
. It was noted that the gene
Fig. 4 Transmission electron micrographs of infected rat brain tissues. a, b CoV-like particles are considered SL-CoVs ZC45 in different locations
of the infected rat brain tissues
Fig. 5 Prediction of the antigenicity of the bat SL-CoV N protein.
aThe predicted antigenicity for the N protein. bAmino acid sequence
of the N protein. The high antigenicity portion is indicated in the red
circle. The two synthesized polypeptides are indicated in red
Fig. 6 Detection of N protein expression in infected rat tissues by
western blotting. Proteins from the following tissues were analyzed:
rat brain from the control specimen (lane 1), intestinal tissue from bat
ZC45 (lane 2), intestinal tissue from the infected rat (lane 3,6), lung
tissue from the infected rat (lane 4,7), and brain tissue from the
infected rat (lane 5,8)
Hu et al. Emerging Microbes & Infections (2018) 7:154 Page 6 of 10
encoding the S protein showed a high degree of variability.
The S protein is responsible for viral entry and is func-
tionally divided into two domains, namely, S1 and S2. The
S1 domain is involved in receptor binding, while the S2
domain is involved in cellular membrane fusion. The S1
domain can be functionally subdivided into two domains,
an N-terminal domain (S1-NTD) and a C-terminal
domain (S1-CTD), and both can bind to host receptors,
hence functioning as RBDs
32
. ZXC21 and ZC45 showed
huge diversities with the previously reported CoVs of bats
associated with the S1 region, and the highest level of
shared identity was only 83%. An attempt was made to
perform a recombination analysis during the course of
this study. In our study, no potential recombination
events could be identified. This could be because the two
strains originated from an unsampled SL-CoV lineage
residing in a bat species that is phylogenetically closer to
ZXC21 and ZC45 than all other known bat SL-CoV
samples. Then, we used simplot to analyze the sequence
similarity of five bat-SL-CoVs and the SARS-CoV SZ3.
The Longquan-140 strain is the most homologous to
ZC45 and ZXC21, the Rs3367 is the closest strain of bat
origin to the human pathogenic SARS coronavirus, and
SZ3 is the representative strain of civetorigin.
In this study, a suckling rat model was initially used to
study the possibility of the proliferation of bat-derived
CoVs in other animals. Previously, only one report had
shown promising results associated with the isolation of
live SL-CoVs from the fecal samples of bats with Vero E6
cells
13
. The live SL-CoV cultured in Vero E6 cells pre-
sented a typical CoV morphology and has the ability to
use ACE2 from humans, civets, and Chinese horseshoe
bats for cell entry
33
. An attempt to isolate the virus with
Vero E6 cells was unsuccessful, which was likely due to a
low viral load or a lack of compatibility with Vero E6 cells.
This study found that the SL-CoVs derived from bats
could replicate successfully in suckling rats, and patho-
logical examination showed the occurrence of inflamma-
tory reactions in the examined organs of the suckling rats.
This result indicated that the virus can proliferate in rats
and has the potential of cross-species transmission. When
CoV particles procured from the infected brain tissues of
the rats were studied by electron microscopy, the mor-
phology of the particles was found to be identical to the
typical coronavirus particles, as described in previous
studies
34
. However, the typical spikes could not be
visualized by electron microscopy. This observation can
be partially explained by the hypothesis that the S1 and S2
domains of the S protein (which are not well-connected)
were easily detached from the virion using excessive
freeze-thawing or ultracentrifugation
6
. Thus, there was a
loss of S1 domains, which likely occurred during the
preparation of the samples for electron microscopy.
Meanwhile, the infected rat tissues could react with the
polyclonal antibodies associated with the ZC45 N protein,
according to the results from the western blotting assay,
indicating that the virus can circulate in rats. Despite the
negative western blotting results in the intestinal tissues of
rat and the positive results of western blotting in the brain
and lung tissues, we considered that these differences may
be caused by different viral loads in different tissues.
In conclusion, based on the early detection of a high
carrying rate for SL-CoVs, which originated from the
bats in Zhoushan, China, this study involved continuous
surveillance of the SL-CoVs that originated from the
bats of this region. Diverse bat SL-CoVs were identified
in this region, and the SL-CoVs in this region remained
stable and could be transmitted to each other. Although
there were several differences between the SARS-CoVs
and the bat-SL-CoVs procured from this region based
on the two-full-length samples obtained in this study,
especially pertaining to the S protein region, this strain
could still cause infection in neonatal rats. This obser-
vation highlights the possibility of cross-species trans-
mission of these viruses. These findings strongly suggest
the need for continued surveillance of viruses originat-
ing from wild animals andpromote further research to
study the possibility of cross-species transmission of
these viruses.
Materials and methods
Ethics statement
The procedures for sampling of bats were reviewed and
approved by the Administrative Committee on Animal
Welfare of the Institute of Zhejiang CDC Veterinary
(Laboratory Animal Care and Use Committee Author-
ization). All live bats were maintained and handled
according to the Principles and Guidelines for Laboratory
Animal Medicine (2006), Ministry of Science and Tech-
nology, China. All animal experiments were approved by
the Ethics Committee of the Research Institute for Med-
icine, Nanjing Command. All methods were performed in
accordance with the relevant guidelines and regulations
(Approval number: 2015011).
Sampling
Overall, 334 adult bats were captured live at the
mountain cave with mist nets at four separate times from
July 2015 to February 2017 in Zhoushan city (including
Dinghai and Daishan), Zhejiang Province, China. All bats
appeared healthy and had no obvious clinical signs at
capture. After completion of collection from each sample
site, all bats were immediately dissected, and bat details
are shown in Table 1. Each sample (approximately 1 g of
intestinal tissues) was immediately transferred into viral
transport medium (Earle’s balanced salt solution, 0.2%
sodium bicarbonate, 0.5% bovine serum albumin, 18 g/l
amikacin, 200 g/l vancomycin, 160 U/l nystatin), stored in
Hu et al. Emerging Microbes & Infections (2018) 7:154 Page 7 of 10
liquid nitrogen prior to transportation to the laboratory,
and ultimately stored at −80 °C.
RNA extraction and RT-PCR screening
All specimens were pooled and subjected to nested
RT-PCR analysis as reported in the previous study
22
.
Briefly, each intestinal sample (approximately 0.1 g) was
homogenized in a glass grinder with ten volumes of SM
buffer(50mMTris,10mMMgSO4,0.1MNaCl,pH
7.5). The homogenate was centrifuged at 12,000 g for
10 min at 4 °C, but only the supernatant was used. The
supernatant of each sample was passed through 0.22 μm
Pellicon II filters (Millipore, Billerica, MA) to filter out
the ruptured tissues, bacteria, and other impurities. The
viral RNA was extracted with a Viral RNA Mini Kit
(Qiagen, Hilden, Germany) according to the manu-
facturer’s recommendations. RNA was eluted in 35 μl
RNase-free H
2
O and stored at −80 °C. Reverse tran-
scription was carried out using the first cDNA synthesis
kit (TaKaRa, Dalian, China) according to the manu-
facturer’s protocol with double-distilled water (ddH
2
O)
as a negative control. All samples were amplified by a
nested PCR that targeted a 440-nt fragment in the gene
RdRp of all known alpha and betacoronaviruses
35,36
.For
the first round PCR, the 20 μl reaction mix contained 18
μl of PCR reaction solution (Takara), 10 pmol of each
primer and 1 μl of the DNA template. The amplification
was performed under the following conditions: 94 °C for
3 min; 40 cycles at 94 °C for 30 s, 52 °C for 30 s and 72 °C
for 1 min for 40 cycles of in-house reaction; and exten-
sion at 72 °C for 10 min. For the second round PCR, the
20 μl reaction mix contained 18 μl of PCR reaction buf-
fer, 10 pmol of each primer, and 1 μl product of the first
round PCR. The amplification was performed under
the following conditions: 94 °C for 3 min followed by
30 cycles consisting of 94 °C for 30 s, 52 °C for 30 s, 72 °C
for 30 s, and a final extension of 72 °C for 10 min with
ddH
2
O as a negative control. Positive PCR products were
sequenced in both directions by an ABI 3730 DNA
Analyzer (Invitrogen, Beijing, China).
Sequencing of full-length genomes
To obtain the full genomic sequences of ZXC21 and
ZC45, 19 degenerated PCR primer pairs were designed by
multiple alignment of available SARS-CoV and bat SL-
CoV sequences deposited in GenBank, targeting almost
the full length of the genome. Primer sequences are
available upon request. Sequences of 5′and 3′genomic
ends were obtained by 5′and 3′RACE (Takara), respec-
tively. PCR products with expected size were gel-purified
and directly subjected to sequencing. The sequences of
overlapping genomic fragments were assembled to obtain
the full-length genome sequences, with each overlapping
sequence longer than 600 bp.
Phylogenetic analysis of amplicons
All 440-bp-long amplicons were aligned with their
closest phylogenetic neighbors in GenBank using Clus-
talW v.2.0. Representatives of different species in the
genera of Alphacoronavirus and Betacoronavirus as well
as some unapproved species were included in the align-
ment. Phylogenetic trees based on nucleotide sequences
were constructed using the neighbor-joining method
using MEGA v.7 with the Maximum Composite Like-
lihood model and a bootstrap value of 1000
37
.
The aligned full sequences were initially scanned for
recombination events using the Recombination Detection
Program (RDP)
38
. The potential recombination events
between ZXC21, ZC45, Rs3367 (KC881006), Longquan-
140 (KF294457.1), and HKU3-1 (DQ022305.2), as sug-
gested by RDP with strong Pvalues (<10
−20
), were
investigated further by similarity plot and bootscan ana-
lyses using SimPlot v.3.5.1
39
.
Suckling rat infecting assay
To test the pathogenicity of the ZC45 agent, infection
experiments were performed in suckling rats. 3-day-old
suckling BALB/c rats (SLAC, China) were intracerebrally
inoculated with 20 μl of volume grinding supernatant of
ZC45 intestinal tissue. Animal housing care and all animal
experiments were performed in a biosafety level 3 (BSL-3)
facility and were approved by the local ethics committee.
After 14 days, the brain, lungs, intestine, and liver tissues
from infected rats were selected to prepare pathological
sections. Briefly, the tissues were fixed in 10% (vol/vol)
neutral-buffered formalin. After routine tissue processing,
including dehydration by graded alcohol solutions,
washing, and incubation in paraffin, 4 µm thick sections
were cut and stained with hematoxylin and eosin (H&E).
Approximately 2 h later, the prepared tissue sections were
imaged using optical microscopy (Olympus, Japan).
TEM was utilized to obtain more detailed pathological
information responsible for the major symptoms. The
tissue samples were fixed in 2.5% (vol/vol) dialdehyde for
2 h, postfixed in 1% (vol/vol) osmium tetroxide for 1 h,
dehydrated in graded ethanol, and embedded in Epon-812
epoxy resin. Then, 70 nm ultrathin sections were pro-
duced and quickly stained in aqueous uranyl acetate and
Reynolds’lead citrate. Finally, the generated tissue sec-
tions were examined using a JEM-1200 TEM (Jeol Ltd.
Tokyo, Japan).
Quantitative RT-PCR was performed using tissue sus-
pensions of rats positive for SL-CoV by RT-PCR. cDNA
was amplified in SYBR Green I fluorescence reactions
(Roche) using specific primers (5′-TGTGACA
GAGCCATGCCTAA-3′and 5′-ATCTTATTACCAT
CAGTTGAAAGA-3′)
12
. A plasmid with the target
sequence for generating the standard curve was used. At
the end of the assay, PCR products (280-bp fragment of
Hu et al. Emerging Microbes & Infections (2018) 7:154 Page 8 of 10
pol) were subjected to melting curve analysis (65–95 °C,
0.1 °C/s) to confirm the specificity of the assay.
Preparation of rabbit antiserum against two peptides
To obtain the polyclonal antibody of bat SL-CoV ZC45
N protein, two partial peptides with 15-amino acid resi-
dues of N protein were synthesized (Sangon Biotech,
Shanghai, China) after a homology search according to
the bioinformatics analysis and prediction of signal pep-
tide (SignalIP-4.1), hydrophilicity and antigenicity of N
protein. New Zealand White rabbits (2–2.3 kg) were
injected subcutaneously using 0.6 mg of two peptides in
1 ml phosphate-buffered saline (PBS) emulsified with 1 ml
Freund’s complete adjuvant (Sigma). Animals were
boosted twice by the same route at 2-week intervals with
approximately 0.3 mg of two peptides in 1 ml of PBS
emulsified with 1 ml of Freund’s incomplete adjuvant
(Sigma). One week after the last booster immunization,
blood samples were collected, and sera were isolated for
biological activity assays. The antibody titer was tested by
indirect enzyme-linked immunosorbent assay. Pre-
immune rabbit serum was collected before the first
injection.
Determination of virus infectivity by western blotting
assay
Western blotting was performed to characterize the
antigenic reactivity of infected rat tissue with N protein
antibody of bat SL-CoV-ZC45. Infected intestine, lung
and brain tissue samples were homogenized and lysed in
RIPA buffer supplemented with proteinase inhibitors.
Equal amounts of proteins (40 μg) were loaded and
separated on 8% SDS-PAGE (sodium dodecyl sulfate-
polyacrylamide gel electrophoresis) gel. Following elec-
trophoresis, the proteins were transferred onto a PVDF
(polyvinylidene difluoride) membrane, blocked with 5%
(w/v) milk, and incubated with primary and secondary
antibodies. Blots were developed and detected by
enhanced chemiluminescence (GE Healthcare, Little
Chalfont, UK). Rat tissues from the control specimens and
intestinal tissues from bat ZC45 were used as negative and
positive controls, respectively.
Nucleotide sequence accession numbers
All amplicon sequences and the full genomes of ZXC21
and ZC45 generated in this study have been deposited in
GenBank under accession numbers MG772844 through
MG772934.
Acknowledgements
This study was supported by National Major Infectious Diseases
(2017ZX10303401-007), National Natural Science Foundation of China
(U1602223), Army Logistics Scientific Research Projects (BWS14C051), Jiangsu
Province Science and Technology Support Program Project (BE2017620),
National Postdoctoral Special Aid (2016T91011), and Jiangsu Postdoctoral Fund
(1501147C).
Author details
1
Department of Epidemiology, College of Preventive Medicine, Third Military
Medical University, Chongqing 400038, China.
2
Department of Epidemiology,
Research Institute for Medicine of Nanjing Command, Nanjing 210002, China.
3
Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu Province 214064, P.R.
China.
4
Stony Brook University, Stony Brook 11794, USA.
5
Department of
Pathogen Biology & Microbiology and Department of General Intensive Care
Unit of the Second Affiliated Hospital, Zhejiang University School of Medicine,
Hangzhou, Zhejiang 310058, China
Conflicts of interest
All authors declare that they have no conflicts of interest.
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies this paper at (https://doi.org/
10.1038/s41426-018-0155-5).
Received: 10 April 2018 Revised: 9 July 2018 Accepted: 2 August 2018
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