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Virus Research 133 (2008) 74–87

A review of studies on animal reservoirs of the SARS coronavirus

Zhengli Shi, Zhihong Hu ∗
State Key Laboratory of Virology and Joint-Lab of Invertebrate Virology, Wuhan Institute of Virology,
Chinese Academy of Sciences, Wuhan 430071, PR China
Available online 23 April 2007

Abstract
In this review, we summarize the researches on animal reservoirs of the SARS coronavirus (SARS-CoV). Masked palm civets were suspected
as the origin of the SARS outbreak in 2003 and was confirmed as the direct origin of SARS cases with mild symptom in 2004. Sequence analysis
of the SARS-CoV-like virus in masked palm civets indicated that they were highly homologous to human SARS-CoV with nt identity over 99.6%,
indicating the virus has not been circulating in the population of masked palm civets for a very long time. Alignment of 10 complete viral genome
sequences from masked palm civets with those of human SARS-CoVs revealed 26 conserved single-nucleotide variations (SNVs) in the viruses
from masked palm civets. These conserved SNVs were gradually lost from the genomes of viruses isolated from the early phase to late phase
human patients of the 2003 SARS epidemic. In 2005, horseshoe bats were identified as the natural reservoir of a group of coronaviruses that are
distantly related to SARS-CoV. The genome sequences of bat SARS-like coronavirus had about 88–92% nt identity with that of the SARS-CoV.
The prevalence of antibodies and viral RNA in different bat species and the characteristics of the bat SARS-like coronavirus were elucidated. Apart
from masked palm civets and bats, 29 other animal species had been tested for the SARS-CoV, and the results are summarized in this paper.
© 2007 Elsevier B.V. All rights reserved.

Keywords: SARS-CoV; Animal reservoirs; Bats; Masked palm civets

1. Introduction that the animal traders had significantly higher prevalence of

IgG antibodies against the SARS-CoV than vegetable traders
Severe acute respiratory syndrome (SARS), a disease that first or control groups (Xu et al., 2004a,b; CDC, 2003). These data
occurred in November 2002 in Guangdong Province of China pointed to the fact that animals being sold in markets as a likely
(Xu et al., 2004a,b), has spread to 28 regions around the world source of the virus. Therefore, after the outbreak of SARS, many
in 2003 causing 774 deaths in 8096 infected individuals (WHO, scientists concentrated their efforts on investigating the animal
2004). A novel coronavirus, SARS-CoV, was identified as the origin of SARS-CoV.
etiological agent of the disease (Drosten et al., 2003; Ksiazek et A breakthrough came in May of 2003 when viruses related
al., 2003; Peiris et al., 2003) and phylogenetic analysis indicated to SARS were identified in animals. A team led by Yi Guan
that the virus was different from previously known coronaviruses (Hong Kong University) and colleagues from the Center for
(Marra et al., 2003; Rota et al., 2003). Although the identi- Disease Control (CDC) Shenzhen, studied 25 individuals from
fication of the etiological agent and molecular studies on the 8 different species including 6 masked palm civets (Paguma
SARS-CoV proceeded rather rapidly, the puzzle on the origin larvata), 1 raccoon dog (Nyctereutes procyonoides), 2 Chinese
of the disease was still not fully resolved. From the very begin- ferret-badgers (Melogale moschata), 3 hog-badgers (Arctonyx
ning, data suggested that SARS was originated from animals. collaris), 3 beavers (Castor fiber), 4 domestic cats (Felis
For examples: (1) Animal food handlers who handle, kill, or catus), 3 Chinese hares (Lepus sinensis) and 2 Chinese muntjac
butcher “exotic” animals for food were over represented among (Muntiacus reevesi) gathered in Dongmen Market, Shenzhen,
early-onset SARS cases (Xu et al., 2004a,b; Zhong et al., 2003). Guangdong Province (Guan et al., 2003). Nasal and faecal swabs
(2) Serological studies of traders from several selected animal from the animals were tested for SARS-CoV nucleic acid by
markets in Guangzhou, Guangdong Province in 2003 indicated using reverse transcription-polymerase chain reaction (RT-PCR)
on the N gene of the human SARS-CoV, and all the swaps
were inoculated into FRhK-4 cells for virus isolation. All of
∗ Corresponding author. Tel.: +86 27 87197180; fax: +86 27 87197180. the six masked palm civets and one raccoon dog were positive
E-mail addresses: zlshi@wh.iov.cn, huzh@wh.iov.cn (Z. Hu). for SARS-CoV by either RT-PCR and/or virus isolation. Sera

0168-1702/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.virusres.2007.03.012
 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87 75

from five animals including three masked palm civets, a rac- higher than 99.6%, implying that SARS-CoV-like viruses might
coon dog and a Chinese ferret badger, had neutralizing antibody have recently entered into the masked palm civet population. (2)
against a SARS-CoV isolated from a masked palm civet (SZ16). Although masked palm civets in markets had a high prevalence
The neutralizing antibody titer was ≥640, 40, 40 for the three for SARS-CoV during the epidemic (Guan et al., 2003; Kan et
masked palm civets, ≥640 for the raccoon dog and 160 for the al., 2005; Tu et al., 2004), investigations on farmed masked palm
Chinese ferret badger (Guan et al., 2003). It is interesting to civets were often negative for the SARS-CoV (Kan et al., 2005;
note that one of the masked palm civet, which was tested posi- Tu et al., 2004) with only a few exceptions (Hu et al., 2005; Tu
tive for SARS-CoV nucleic acid both in nasal and faecal swabs et al., 2004). (3) An investigation of 21 wild masked palm civets
was negative for neutralizing antibodies against its own SZ16 trapped in Hong Kong indicated that those animals were free
virus. It implied that this animal was either newly infected or of SARS-CoV-like viruses suggesting the virus is not widely
it had developed mechanisms to avoid generating neutralizing circulating in wild masked palm civets (Poon et al., 2005).
antibodies. This study raised the initial alert that masked palm For a number of reasons, attention turned to bats as a likely
civets and other wildlife animals may play a role in the transmis- reservoir of the SARS-CoV. Bats have long been known as an
sion of SARS-CoV and led to a temporary ban on the hunting, important reservoir for many zoonotic viruses including rabies
sale, transportation and export of all wild animals in Guangdong virus, Hendra virus, Nipha virus, Ebola virus and St. Louis
Province. encephalitis viruses (Halpin et al., 2000; Leroy et al., 2005;
Later in 2003, the role of masked palm civets and other Mackenzie et al., 2001; Mackenzie and Field, 2004). An early
animals was questioned and a debate ensued on whether these study by Poon et al. (2005) on 81 bats belonging to 12 different
animals are actually the origin of the SARS-CoV. A rather species identified a novel bat coronavirus (Bat-CoV) from three
convincing evidence implicating masked palm civets came in different bat species of the same genus, Miniopterus magnater,
approximately half a year later in the winter of 2003–2004. Miniopterus pusillus, and Miniopterus schreibersi. However, the
Between 16 December 2003 and 8 January 2004, a total of Bat-CoV belonged to group 1 coronavirus and was not closely
four patients were identified as SARS cases with mild flu-like related to SARS-CoV since its RNA fragments shared only
syndromes and no secondary transmission (Liang et al., 2004). 41–62% nucleotide sequence identities to SARS-CoV (Poon
Epidemiological information collected by the Guangdong CDC et al., 2005). This initial failure to identify a SARS-CoV-like
and Guangzhou CDC indicated that three of the patients had virus in bats was soon compensated by the successful detection,
direct or indirect contact to masked palm civets. The second by two other independent groups, of several SARS-CoV-like
patient was a waitress in a local restaurant (TDLR), where live viruses in different species of horseshoe bats (Lau et al., 2005;
masked palm civets were kept for exotic dishes, and she often Li et al., 2005b).
passed by or stood in close proximity of the animal cages (Wang This review summarizes the research aimed at investigating
et al., 2005b). The fourth patient dined in the same restaurant the origin in masked palm civets, bats, as well as other ani-
and his dining table was within 5 m of civet cages (Wang et mals. We tried to cover investigations on ecology, epidemiology,
al., 2005b). The third patient dined in a neighboring restaurant as well as molecular virology. For readers interested in more
(SJR), which shared the kitchen with TDLR (Song et al., detailed information on interaction of SARS-CoV spike protein
2005). The masked palm civets kept in TDLR restaurant were with receptors of different species in relation to animal origin of
reported to have been originally purchased from the Xinyuan SARS-CoV, we highly recommend the recent review paper by
Live Animal Market (Wang et al., 2005b). Three research teams Li et al. (2006).
published their results on the SARS-CoV-like viruses in masked
palm civets from the TDLR restaurant and from the Xinyuan 2. Masked palm civets and SARS-CoV
Live Animal Market as well as the four patients (Kan et al.,
2005; Song et al., 2005; Wang et al., 2005b). Almost identical 2.1. Masked palm civets
sequences of the SARS-CoV spike genes were identified in
samples from some of the patients and from masked palm civets Masked palm civets (P. larvata) belong to Carnivora, Viver-
in either the restaurant or the Xinyuan Live Animal Market (Kan ridae. In China, they are distributed mainly south of Yangzhi
et al., 2005; Song et al., 2005; Wang et al., 2005b). Based on River, but are also found in other parts such as HeBei, Shaanxi
virological and epidemiological findings, Guangdong provin- and Shanxi Provinces and south of Tibet (Zhang, 1999; Gao et
cial officials took an aggressive action on 5 January 2004 to al., 1987). There are nine subspecises of P. larvata in China,
destroy any animals that might harbor the SARS-CoV in farms including P. l. larvata, P. l. taivana, P. l. reevesi, P. l. hainana,
and in animal food markets. Quarantine on all civets reared for P. l. intrudens, P. l. chichingensis, P. l. neglecta, P. l. nigriceps,
human consumption in farms all over China was imposed. Since and P. l. lanigera (Gao et al., 1987). The animals are mainly
then, no additional community-acquired SARS cases have been arboreal, nocturnal and largely solitary. They live in mountain
reported. and hill forests taking shelter in hollows of trees. It was reported
Although masked palm civets were identified as direct ori- that they live in a family based group of 2–15 individuals (Zhang
gin of at least some of the SARS cases, the following data et al., 1991b). They eat mostly fruits but also supplement their
indicated that it might not necessarily be the natural animal diet with rodents, birds, insects and roots.
reservoir of the SARS-CoV: (1) The so far sequenced SARS- Farming of masked palm civets in China started in late 1950s
CoVs from masked palm civets harbored a nucleic acid identity mainly for fur (Liu, 1959), but breeding became popular in the
76 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87

Table 1
Summary of investigations of SARS-CoV in masked palm civets from markets, farms and wilda
Market/ Samples Anitibody Nucleic acid/virus Reference
farm/wild
Location (Province) Sample date Methods Prevalence Method Prevalence

Market Shenzheng (Guangdong) May, 2003 VNT 3/4 (75%) RT-PCR, isolation 6/6 (100%) Guan et al. (2003)
Market Xinyuan (Guangdong) May, 2003 ND ND RT-PCR 2/7 (29%) Tu et al. (2004)
Market Xinyuan (Guangdong) January, 2004 ND ND RT-PCR 91/91 (100%) Kan et al. (2005)
Market Xinyuan (Guangdong) January, 2004 VNT, IFA, WB 14/18 (78%) RT-PCR 0/18 Tu et al. (2004)
Market Guangzhou (Guangdong) November, 2004 ND ND Real-time RT-PCR 0/12 Wang et al.
(2005a,b)
Market Shenzheng (Guangdong) December, 2004 ND ND Real-time RT-PCR 0/12 Wang et al.
(2005a,b)
Farm Luoning (Henan) June, 2003 VNT, IFA 0/17 ND ND Tu et al. (2004)
Farm Changsha (Hunan) June, 2003 VNT, IFA 0/30 ND ND Tu et al. (2004)
Farm Qianguan (Guangdong) January, 2004 VNT, IFA 0/9 RT-PCR 0/9 Tu et al. (2004)
Farm Shanguan (Guangdong) January, 2004 VNT, IFA 0/10 RT-PCR 0/10 Tu et al., 2004
Farm Shanwei (Guangdong) January, 2004 VNT, IFA, WB 4/10 (40%) RT-PCR 0/10 Tu et al. (2004)
Farm Zhuhai (Guangdong) January, 2004 VNT, IFA 0/10 RT-PCR 0/10 Tu et al. (2004)
Farm Guangzhou (Guangdong) May, 2003 ND ND RT-PCR 2/9 (22%) Tu et al. (2004)
Farm (Jiangxi) May, 2003 ND ND RT-PCR 0/15 Tu et al. (2004)
25 Farms (12 Provinces) January–September, ND ND RT-PCR 0/1107 Kan et al. (2005)
2004
Farm (Hubei) April, 2004 ND ND Real-time RT-PCR 7/7 (100%) Hu et al. (2005)
Wild Hong Kong 2003–2004 VNT 0/21 RT-PCR 0/21 Poon et al. (2005)
a ND, not done; VNT, virus neutralization test; IFA, immunofluorescent antibody test; WB, Western blot test.

late 1980s with increasing demands for them as an exotic food. 2.2. SARS-CoV-like viruses in masked palm civets
It was reported that in 2003 about 40,000 masked palm civets
were raised in 660 farms all over China (Jiang et al., 2003). The When SARS-CoV-like viruses were first identified in masked
farms started either from capture and breeding of local wild ani- palm civets from a Shenzhen market (Guan et al., 2003), several
mals or from breeding stocks brought in from other farms. The investigators concentrated their efforts on exploring the preva-
feed for the farmed animals varied depending on the particu- lence of these viruses in this animal (Table 1).
lar farm but usually contained grains such as maize, beans, rice Tu et al. (2004) examined 56 masked palm civets including
and animal feed such as powdered products from fish or bones 38 from four farms in different regions of Guangdong Province
and often supplemented with fresh meat and/or parts from other and 18 from the Xinyuan Live Animal Market in Guangzhou,
animals (Zhou, 1994; Shao and Li, 1997). By studying body Guangdong Province in January 2004. They found anti-SARS-
temperature and weight, food intake, shedding and living habits CoV antibodies in 78% of the market animals (14 out of 18),
of 87 wild masked palm civets in farms, Zhang et al. (1991a) while only one out of the four farms contained animals with
found that masked palm civets in Shanxi Province hibernated anti-SARS-CoV antibodies with a prevalence of 40% (4 out of
slightly from December to February for about 75–85 days. A 10). SARS-CoV antibody levels in farmed animals were lower
similar study showed that the masked palm civets in Hunan than those from the market. Intestinal tissues collected from the
Province (south of Yangzhi River) also hibernated slightly 56 civets were tested by RT-PCR and none were positive. Anal-
from December to February as their body temperature dropped ysis of 47 serum samples collected in June 2003 from two farms
3–4 ◦ C and other physiological indexes decreased significantly in Henan and Hunan Provinces were negative by a neutraliza-
compared with those during normal period (Kang et al., tion test and an immunofluoresence antibody assay. The authors
1997). also mentioned previously unpublished data on SARS-CoV in
Masked palm civets are naturally timid. Farm breeding the Xinyuan Live Animal Market in Guangzhou, in Guangdong
seemed to have been successful and only limited number dis- Centre for Rescue and Care of Wildlife Animals, and in a civet
eases have been reported. Viral enteritis caused by a parvovirus farm in Jiangxi Province. These data indicated a high percentage
was one of the common diseases in masked palm civets. An of infected civets in one particular market at a specific time, while
inactivated vaccine against this virus was developed to immu- no indication of civet infection was seen on most farms during
nize the animals (Duan et al., 2001). An investigation of four the same period. The authors suggested that the high preva-
apparently healthy masked palm civets in Taiwan has shown lence in market civets might be associated with trading activities
that they had antibodies against feline parvovirus (FPV) but not under conditions of overcrowding and mixing of various animal
to feline herpesvirus type 1 or to feline calicivirus (Ikeda et al., species (Tu et al., 2004).
1999). Moreover, the titer of neutralizing antibodies against FPV Kan et al. (2005) investigated 1107 palm civets from 25 farms
ranged from 160 to >5120 (Ikeda et al., 1999). Bacterial and par- in 12 Provinces from January to September of 2004 in China.
asitic diseases have also been reported for masked palm civets All of the 1107 civets sampled tested negative for the virus by
(Yang et al., 2000; Zhao, 2003). RT-PCR. While all of 91 animals from the Xinyuan Live Animal
 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87 77

Market in January of 2004 were positive by RT-PCR (Kan et al., patients but all were lost in the late phase patients. At least one
2005). The authors suggested that the palm civets were infected of the mutations, aa 487 in the spike protein, was shown to be
at the market by other palm civets or by other animals harboring important in the adaptation of SARS-CoV to human cells (Li
the virus rather than at the farm (Kan et al., 2005). In contrast, by et al., 2005c; Qu et al., 2005). Analysis of the structures of the
using a real-time RT-PCR assay, Hu et al. (2005) reported seven spike protein and its cellular receptor angiotensin-converting
out of seven masked palm civets in a farm in Hubei Province enzyme 2 (ACE2) (Li et al., 2003) indicated that aa 487 of spike
were positive, although the copy numbers of the nucleic acid of is important for spike-ACE2 interaction and a mutation from
most of the samples were quite low. Ser487 (civet) to Thr487 appeared to enhance human-to-human
Wang et al. (2005a) examined the prevalence of the virus transmission of the SARS-CoV (Li et al., 2005c). Residues 344
in civets present in a Guangdong market in late 2004. Twelve and 360 of the spike protein are distant from the binding inter-
and 10 masked palm civets were sampled from markets from face in the spike-ACE2 complex (Li et al., 2005a), and these
Guangzhou and Shenzhen, respectively and none of them tested two mutations do not affect binding affinity or infectivity of
positive by real-time RT-PCR. Tests on 21 wild civets captured the SARS-CoV (Li et al., 2005c; Qu et al., 2005). Whether the
in Hong Kong did not reveal an infection by SARS-CoV-like rest of the aa residues play important roles during cross species
virus (Poon et al., 2005). transmission or human-to-human transmission shall need further
Taken all together, the above data indicate that one cannot investigations.
derive an unequivocal conclusion at the present time. It seems, All the so far identified masked palm civet SARS-CoV
however, that masked palm civets are not widely infected in the genomes except PC4-227 contain a 29-nt sequence which is
wild or when raised in farms. Therefore, investigation of larger missing in the epidemic strain of SARS-CoV. It is a 27-nt
number of masked palm civets in a wider distribution is needed sequence in PC4-227 lacking the last two nts of the 29-nt. The
in the future. 29-nt sequence was first identified in SZ3 and SZ16 (Guan et
al., 2003) and was also found in some early epidemic human
2.3. Viral sequence analysis from masked palm civets samples such as GD01, GZ02, HSZ-Bb, HSZ-Bc, HSZ-Cb, and
HSZ-Cc (Chinese SARS Molecular Epidemiology Consortium,
To date, the genomes of 10 viruses from civets have been 2004). The 29-nt was suggested as a marker to distinguish the
completely sequenced, including 2 (SZ3 and SZ16) from Shen- strain that originated from masked palm civet and the later epi-
zhen market in 2003 (Guan et al., 2003), 4 (civet010, civet007, demic strains (Guan et al., 2003; Chinese SARS Molecular
civet020, PC4-13) from TDLR restaurant in 2004 (Wang et al., Epidemiology Consortium, 2004). The 29-nt fuses ORF8a and
2005b; Song et al., 2005) and 4 (PC4-136, PC4-227, A022, ORF8b into a single ORF. A recent study by Yount et al. (2005)
B039) from the Xinyuan Live Animal Market in 2004 (Song using recombinant virus has shown that the 29-nt had little if any
et al., 2005; Kan et al., 2005). When the genome sequences impact on in vitro growth or RNA synthesis. It was predicted
were compared with 92 human SARS-CoVs (Song et al., 2005), that the occurrences of deletions in ORF8a/8b region might be
a total of 212 single-nucleotide variation (SNV) sites with mul- related to the RNA secondary structure of this region (Chinese
tiple occurrences were identified, among which 209 sites were SARS Molecular Epidemiology Consortium, 2004). Neverthe-
in the protein coding regions (data not shown). Among the 209 less, this 29-nt deletion reflected an important evolutionary event
SNV sites, 73 have synonymous amino acid (aa) mutations and of SARS-CoV-like viruses in their adaptation from masked palm
136 have nonsynonymous aa mutations. There was only one site civets to humans. Interestingly, the bat SARS-CoV-like viruses
with multiple base substitutions: the original nucleotide at posi- also harbor a 29-nt sequence and encode a single ORF8, although
tion 28,590 (Tor2) is C, while Civet020 and A022 have T instead, the similarity of the ORF8 is very low to that of SARS-CoV-like
and B039 has the nucleotide G. All these mutations resulted in viruses in masked palm civets (see below).
synonymous aa substitutions.
One hundred and twenty-nine SNV sites contain variations 2.4. Evolutionary perspectives
in viral sequences from masked palm civets, among which 26
SNVs were conserved in the civet viruses (Table 2). Twenty of The rate of evolution of a virus in a certain host could
the SNVs cause nonsynonymous aa substitution, including seven sometimes infer whether the host is a natural animal reservoir
in ORF1ab (aa 1121, 1663, 2746, 2971, 3047, 3072, 5767), eight of the virus. Several studies have been conducted to estimate
in the spike protein (aa 77, 244, 344, 360, 487, 665, 778, 1163), the substitution rate and to identify the most recent common
three in ORF3a (aa 7, 81, 121), one in M protein (aa 5) and one in ancestor of the SARS-CoV. By analyzing 10 genome sequences
N protein (aa 17) (Table 2). It is interesting to see that all but one of the 2003 Chinese patient samples and their epidemiological
of the 26 SNVs from masked palm civets were also conserved in information, it was determined that the synonymous substitution
sequences from the 2004 patients with mild symptoms (Table 2). rate of SARS-CoV genome to be 8.26 × 10−6 (±2.16 × 10−6 )
Based on epidemiological and molecular data, the patients of the nucleotides per site per day and the date for the most recent
2003 epidemic were classified as early, middle and late phases common ancestor was estimated to range from early June
(Chinese SARS Molecular Epidemiology Consortium, 2004). to the end of December 2002 (Chinese SARS Molecular
Many of the 26 SNVs from masked palm civets were conserved Epidemiology Consortium, 2004). Zhao et al. (2004) analyzed
in the genomes from early human cases in the 2003 epidemic. 11 genomic sequences from different parts of the world during
Some remained unchanged in the genomes of the middle phase 2003 SARS epidemic and found that the mutation rate to be in
78 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87

Table 2
SNVs of SASR-CoVs from masked palm civets and human patientsa

a MPC, masked palm civets; HP, human patients; 03, year 2003; 04, year 2004; E, M and L stands for early, middle and late phase of 2003 epidemic (according to

the Chinese SARS Molecular Epidemiology Consortium, 2004).

the order of 0.80–2.38 × 10−3 nucleotide substitution per site substitutions per site per day, with the rates for synonymous
per year (2.2–6.7 × 10−6 nucleotide substitutions per site per and non-synonymous mutations at 2.5 × 10−6 and 3.2 × 10−6
day) and the most recent common ancestor of the sequences nucleotide substitutions per site per day, respectively (Vega
was inferred to be present as early as the spring of 2002. et al., 2004). All the above studies used human samples
Analysis on the Singapore isolates from the 2003 epidemic with available clinical data from the 2003 outbreak and the
revealed that the mutation rate to be 5.7 × 10−6 nucleotide substitution rates of SARS-CoV genome obtained from the
 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87 79

various studies were in good agreement with each other. In pendent research teams (Lau et al., 2005; Li et al., 2005b). Both
comparison to other coronaviruses, the estimated substitution teams reported their findings of coronaviruses, which are highly
rate of SARS-CoV is lower than that in mouse hepatitis virus similar to SARS-CoV in several horseshoe bat species of the
(Chen and Baric, 1995) and similar to that in the transmissible genus Rhinolophus within the family Rhinolophidae.
gastroenteritis virus (Enjuanes et al., 1992), but higher than that
in the infectious bronchitis virus (Cavanagh et al., 1998). 3.1. Prevalence of antiviral antibodies and viral nucleic
Song et al. (2005) used the concatenated five major coding acids in bats
sequences (ORF1ab, S, E, M, and N) of SARS-CoV genomes to
estimate the neutral mutation rate. The total length of the con- The prevalence of anti-SARS-CoV nucleocapsid antibodies
catenated sequence accounted for 91.25% of the whole genome. was examined in bats in the Provinces of Hubei, Guangdong
By taking GZ02 as an outgroup, the number of synonymous sub- Guangxi and Tianjing (Li et al., 2005b) and in the Hong Kong
stitutions per synonymous site, Ks , was calculated for two 2003 Special Administrative Region (Lau et al., 2005) (Table 3). A
masked palm civet SARS-CoVs (SZ16 and SZ3), three 2004 rather high prevalence of antibodies was observed in the lesser
masked palm civet SARS-CoVs (PC4-136, PC4-227, PC4-13), horseshoe bat Rhinolophus pussilus (2/6), the great-eared horse-
and two 2004 human patient sequences (GZ03-01 and GZ03- shoe bat Rhinolophus macrotis (5/7) and the Pearson’s horseshoe
02). Based on these Ks values, the neutral mutation rate among bat Rhinolophus pearsoni (13/46) by a sandwich ELISA method
this group of SARS-CoVs was estimated to be 8.00 × 10−6 (Li et al., 2005b). It was also seen in the Chinese horseshoe bat
nucleotides per site per day. This suggested that SARS-CoV Rhinolophus sinicus (12/18 by Western blots, 31/37 positive by
evolves at a relatively constant neutral rate both in humans and EIA with titer ≥1:400, and 8/19 by a neutralization assay for
in masked palm civet (Song et al., 2005). human SARS-CoV with titer ≥1:20) (Lau et al., 2005). Antivi-
The spike genes of late human patient isolates were under- ral antisera from bats did not neutralize human SARS-CoV (Li
going negative selection during 2003 SARS outbreak (Hu et et al., 2005b). A very low percentage of positive samples were
al., 2003). However, when spike sequences from masked palm detected in the fruit bat Rousettus leschenaultia (1.2%). No anti-
civets were used as an outgroup to estimate the Ka /Ks (the ratios bodies were detected in other bat species sampled, including the
of the rates of nonsynonymous to synonymous changes) of the lesser dog-faced fruit bat Cynopterus sphinx, the mouse-eared
human isolates, the value was significantly over 1, indicating bats Myotis ricketti, Myotis altarium and Myotis chinensis, the
that the spike gene was under positive selection pressure when noctutule bats Nyctalus plancyi and N. noctula, the great round
transmitted from civets to human (Yeh et al., 2004). Phyloge- leaf bat Hipposideros armiger and the intermediate horseshoe
netic analysis showed that the viruses from masked palm civets Rhinolophus affinus (Table 3).
collected in 2003 were different from those of 2004 indicating Both teams subsequently confirmed by RT-PCR with SARS
the viral transmission from animal-to-human occurred indepen- specific primers the presence of SARS-CoV-like virus in some
dently in these two instances (Song et al., 2005). feacal swabs. A relatively low prevalence of viruses was detected
in Rhinolophus ferrumequinum (1/8), R. macrotis (1/8) and R.
2.5. Isolation of SARS-CoV-like virus from masked palm pearsoni (3/30), compared with a high prevalence in R. sinicus
civets (23/59) (Table 3). The authors pointed out that bats with neutral-
izing antibodies had a lower viral load in their anal swabs (Lau
Isolation of virus is an important step for identifying the et al., 2005). The quantitative RT-PCR was more sensitive than
viral host. So far, propagation in cell culture of SARS-CoV-like routine RT-PCR method. No positive samples were found in 219
viruses from masked palm civets seems to be difficult. Initially, respiratory swabs collected in these studies (Table 3). Unfortu-
it was reported that SZ16 replicated in FRhK-4 cells (Guan et al., nately, both teams failed in their attempt to isolate the viruses.
2003), but no further reports have been published. Wang et al.
(2005b) reported that SARS-CoV-like virus from a rectal swab 3.2. The sequences of the SARS-CoV-like viruses from bats
specimen of a masked palm civet replicated in FRhK-4 cells
cultured but not in Vero E6 cells. Similarly, viruses from the The complete genome sequences of three viruses from
four mild symptom patients of 2004 could not be propagated in the R. sinicus bat were determined by direct sequencing of
cell lines (Liang et al., 2005). Because it has been reported that the RT-PCR products. The three genomes, termed HKU3-1,
certain aa residues are important for the viral spike protein to HKU3-2 and HKU3-3 were 29,709 bp in size (Lau et al., 2005).
interact with its specific receptor ACE2 (Li et al., 2005a,c, 2006; HUK3-1 and HUK3-3 are identical and HKU3-1 and HKU3-2
Qu et al., 2005), selecting or engineering a cell line capable of have only 18 nucleotide difference, indicating that these
supporting the interaction of receptor and S proteins of SARS- virus isolates belong to the same genotype. Three full-length
CoV-like viruses may be essential to overcome the obstacles in genomes of bat SL-CoV were obtained from R. pearsoni (Rp3),
isolation of live viruses from civets and other animal sources. R. ferrumequinum (Rf1) and R. macortis (Rm1) by Li et al.
(2005b) and Ren et al. (2006). All full genomes have similar
3. Bats and SARS-CoV gene organization to the SARS-CoV and contained a 5 leader
sequence followed by non-structural proteins ORF1ab and the
Bats were recently identified as reservoirs of the SARS-like structural proteins S, E, M and N, and 3 polyA tail. There are
coronavirus (SL-CoV) or SARS-CoV-like virus by two inde- some SARS specific accessory proteins termed ORF3a, ORF6,
80 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87

Table 3
Detection of prevalence of antibodies and RNAs of SARS-CoV-like viruses in bats and genome identity between bat SL-CoVs and SARS-CoV
Sampling Bat species Antibody test RT-PCRa Genome size (nt) Identity with
location positive/total (%) positive/total (%) SARS-CoV (%)

Rousettus leschenaulti 1/142 (1.4%)b 0/165

Guangxi Rhinolophus pearsoni 13/46 (28.3%)b 3/30 (10%) 29,736 92
Rhinolophus pussilus 2/6 (33.3%)b 0/6
Rousettus leschenaulti 0/42b 0/45
Guangdong
Cynopterus sphinx 0/17b 0/27
Tianjin Myotis ricketti ND 0/21
R. pussilus ND 0/15
Rhinolophus ferrumequinum 0/4b 1/8 (12.5%) 29,743 87
Rhinolophus macrotis 5/7 (71%)b 1/8 (12.5%) 29,749 87
Hubei
Nyctalus plancyi 0/1b 0/1
Miniopterus schreibersi 0/1b 0/1
Myotis altarium 0/1b 0/1
Hipposideros armiger ND 0/12
Miniopterus magnater ND 0/23
Miniopterus pusillus ND 0/24
Myotis chinensis ND 0/3
Hong Kong
M. ricketti ND 0/2
Nyctalus noctula ND 0/2
Rhinolophus affinus ND 0/2
Rhinolophus sinicus 12/18 (67%)c 23/59 (39%) 29,709 88

Modified according to Lau et al. (2005) and Li et al. (2005b). ND, not done.
a Sandwich ELISA based on SARS-CoV N protein (Li et al., 2005b).
b Western blot with recombinant N protein of bat SL-CoV (Lau et al., 2005).
c Faecal swabs were used for RT-PCR.

ORF7a, ORF7b, ORF8, ORF9b and 9c between the S and lates. Except for the putative S1, ORF3, ORF8 and ORF9b and
N genes (the nomenclature of genes and ORFs followed the c, most of bat SL-CoVs gene products shared an aa identity
nomenclature used for other coronaviruses) (Spaan et al., 2005) of 90–100% with that of human SARS-CoVs or civet SARS-
(Fig. 1). All gene products of SARS-CoV and bat SL-CoV CoV-like viruses. The most conserved proteins were ORF1b
were similar in size except that bat SL-CoV HKU3-1 and Rf1 (98–99% identity), E (98–100% identity), M (97–98% iden-
had a shorter ORF1a (4376 and 4377 aa respectively). tity) and N (97–96% identity). The less conserved proteins
The genome sequences of SL-CoVs isolated from horseshoe were of ORF1a (94–96% identity), putative S2 (92–96% iden-
bat shared a sequence identity of 89–90%. Similar sequence tity), ORF6 (92–93% identity), ORF7a (91–95% identity), 7b
identity, 87–92%, exists between bat and human or civet iso- (90–93% identity), 9b (81–90% identity), 9c (80–91% iden-

Fig. 1. The genome organization of SARS-CoV in human and masked palm civet, and SL-CoV in bat. The black arrows indicate the conserved genes of the family
Coronaviridae; the gray arrows indicate SARS-CoV unique genes; the red indicate the most variable regions in SARS-CoV and SL-CoV. The bar scale may not
represent the real size of the genes.
 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87 81

tity) and ORF3a (81–86% identity). Moreover, the transcription 2005b). Others have suggested that these viruses form group 2b
regulatory sequences (TRS) and the stem-loop II motif (s2m) which are distinct from other group 2 coronavirus (group 2a)
similar to SARS-CoV were identified in bat SL-CoVs. Phyloge- within the genus Coronavirus (Lau et al., 2005).
netic analyses based on full sequences of bat SL-CoV revealed Nucleotide variations are present throughout all bat SL-CoV
that bat SL-CoVs are closely related to SARS-CoVs in humans genomes when compared with SARS-CoV. The most variable
and civets and distantly related to other coronaviruses in group regions in bat SL-CoVs relative to SARS-CoVs are in the non-
2 (Fig. 2). Some authors suggested that these groups of viruses structural protein nsp3, the spike protein (particularly in S1
constitute a SARS coronavirus cluster, which are assembled into domain, Fig. 3), ORF3 and ORF8. Most of deletion and insertion
group 2 coronavirus within the genus Coronavirus (Li et al., sites are present in nsp3, S and ORF8 (Fig. 1, Fig. 3), which result

Fig. 2. Phylogenetic trees based on aa sequence of ORF1b, nucleotide sequence of S, N and ORF8 gene. Sequence used for analysis in this study are as follows
(GenBank accession number given in parenthesis): Tor2 (NC-004718), human isolate from late phase of the 2002/3 oubreaks; GD01 (AY278489), human isolate from
early phase of the 2002/3 outbreaks; SZ3 (AY304486), masked palm civet isolate from 2003; PC4-227 (AY613950), masked palm civet isolate from 2004; HKU3-1
(DQ022305), isolate from Rhinolophus sinicus; Rp3 (DQ071615), isolate from Rhinolophus pearsoni; Rf1 partial sequence (DQ71611, DQ159956), isolate from
Rhinolophus ferrumequinum; Rm1 partial sequence (DQ71612, DQ159957), isolate from Rhinolophus macrotis. The phylogenetic trees were constructed using the
Neighbor-Joining algorithm in the MEGA3.1 software with a bootstrap of 1000 replicates. Other coronavirus sequences used in this study: HCoV-229E (AF304460),
HCoV-OC43 (AY391777), PEDV (AF353511), MHV (AY700211), IBV (AY851295), and BCoV (AF391542) and TGEV (NC 002306). Genetic variation scales
are indicated for each tree and different genetic scales are used for different trees.
82 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87

Fig. 3. Comparison of the receptor binding motif of S protein of bat SL-CoV with that of SARS-CoV. Alignment between the receptor binding motif of S protein
(RBM) of SARS-CoV Tor2, SZ3 (masked palm civet) and the corresponding regions of bat SL-CoVs S proteins is shown. The displayed RBM region represents aa
424–495 of the Tor2 spike protein sequence.

in a low similarity with SARS-CoV in the S protein (76–78%) 4.1.1. Raccoon dogs
and in ORF8 (35% for Rp3, HKU3-1, HKU3-2 and Rm1, 80% Raccoon dogs (N. procyonoides), belong to Carnivora,
for Rf1). The 29-nt region, which is present only in SARS- Canidae. There are three main subspecises in China: N. p. procy-
CoV from masked palm civets and in the early phase human onoides, N. p. ussuriensis, and N. p. orestes. N. p. procyonoides
patients, also existed in all bat SL-CoVs and resulted in a fused is naturally distributed in the east and the central-south parts
ORF (ORF8). The low similarity in ORF8 between SARS-CoV of China, including Fujian, Jiangxi, Hubei, Hunan, Jiangsu,
from human/civet and bat SL-CoV is also seen between bat SL- Zhejiang, Anhui, Guangxi and Guangdong Provinces. N. p.
CoV Rf1 and other three isolates. Analysis of ORF8 indicated ussuriensis is naturally distributed in the northeast part of China
that Rf1 was more closely related to SARS-CoV than to other including Heilongjiang, Liaoning and Jinlin. While N. p. orestes
bat SL-CoV isolates. The high mutated region in ORF8 fur- is naturally distributed in the southwest part of China including
ther demonstrates that this locus is prone to deletions (Chiu Yunnan, Guizhou and Sichuan Provinces (Gao et al., 1987). Nat-
et al., 2005; Lau et al., 2005; Li et al., 2005b; Song et al., urally, raccoon dogs are omnivores and change their food accord-
2005). ing to the seasonal food availability. Rodents, frogs, lizards,
The key difference between bat SL-CoVs and SARS-CoVs invertebrates, birds and their eggs are often eaten, specially in
in humans and masked palm civets is seen in the spike protein, the early summer. Plants and mushrooms are eaten, while berries
which determines host range and tissue tropism in coronaviruses and fruits are favored in late summer and autumn. Raccoon dogs
(Holmes, 2001). The putative S2 domain of all SL-CoVs has a are nocturnal and they hibernate in winter. They are strictly
high identity with that of SARS-CoVs (96%). All sites that are monogamous and live in pairs (Kauhala and Saeki, 2004).
critical for SARS virus fusion to cell membranes are highly Farming of the raccoon dogs started in 1950s in the northeast
conserved in SL-CoVs putative S2 domain, suggesting that part of China for fur. The local wild raccoon dogs (N. p. ussurien-
SL-CoVs possess the same fusion mechanism to host cells as sis) were domesticated successfully in early 1960s in Jilin and
SARS-CoVs. However, the putative S1 domain of SL-CoVs has Heilongjiang Provinces (Tong, 1990). In 1988, the farmed rac-
low similarity to that of SARS-CoVs, especially in N-terminus coon dogs were over 300 million in China (Tong, 1990). Unlike
and the receptor-binding domain (RBD, 318-510 aa of Tor2 S1 the farmed masked palm civets which were mainly for food and
domain) (Wong et al., 2004). One insertion of 6 aa residues and almost disappear after 2004 in China, the farmed raccoon dogs
three deletion sites are present in the S1 domain of bat SL-CoVs, are mainly for fur and are still in very large abundance, specially
among which two deletion sites (5 and 12–13 aa, respectively) in northeast China.
are present in RBD particularly in the receptor binding motif Two independent surveys have shown that raccoon dogs from
(RBM, 424–494 of Tor2 S1 domain) (Li et al., 2005a) (Fig. 3). live animal markets harbored SARS-CoV at a very high preva-
These differences may imply that bat SL-CoVs may use a vastly lence (100%) (Guan et al., 2003; Kan et al., 2005). In May 2003,
diverse ACE2 or even a different molecule as the entry receptor. Guan et al. isolated a SARS-CoV-like virus from the feacal sam-
ple of a raccoon dog (SZ13) from the Dongmen animal Market,
4. Are there other animal reservoirs of SARS-CoV? in Shenzhen. The neutralization antibody titer of the serum of
the raccoon dog was ≥640 against the SARS-CoV-like virus of a
4.1. Other animal species that have been investigated for masked palm civet (SZ16) (Guan et al., 2003). In January 2004,
SARS-CoV Kan et al. investigated 15 raccoon dogs from the Xinyuan ani-
mal market, Guangzhou. Of the 15 raccoon dogs, by real-time
Apart from masked palm civets and bats, 29 other animal and nested RT-PCR, 12 were tested positive with both throat
species from 13 families, 8 animal orders and 2 classes that and rectal swabs, while 3 tested positive with throat swabs only
have been tested for SARS-CoV (Table 4). Among them, seven (Kan et al., 2005). So far there are three spike sequences of rac-
species were shown to harbor the virus under certain circum- coon dogs, that of SZ13 (Guan et al., 2003), A030G (Kan et al.,
stances. 2005), and A031G (Kan et al., 2005). All of them, however, were
 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87 83

Table 4
Summary of investigations of SARS-CoV in other animal speciesa
Order Family Species Common name Methods Prevalence Reference

Primates Cercopithecidae Macaca mulatta Rhesus macaque RT-PCR 0/20 Lau et al. (2005)
Antibody 0/20 Chen et al. (2005)
Canis familiaris Dog
Real-time RT-PCR 0/5 Wang et al. (2005a,b)
Canidae RT-PCR, virus isolation 1/1 Guan et al. (2003)
Nyctereutes procyonoides Raccoon dog
RT-PCR 15/15 Kan et al. (2005)
Vulpes vulpes Red fox Real-time RT-PCR 3/5 Wang et al. (2005a,b)
Melogale moschata Chinese ferret-badger Antibody 1/2 Guan et al. (2003)
Carnivora Mustelidae Arctonyx collaris Hog-badger RT-PCT, antibody 0/3 Guan et al. (2003)
Mustela vison Mink Real-time RT-PCR 0/1 Wang et al. (2005a,b)
RT-PCT, antibody 0/4 Guan et al. (2003)
Real-time RT-PCR 4/20 Wang et al. (2005a,b)
Felidae Felis catus Domestic cat Real-time RT-PCR 0/13 Wang et al. (2005a,b)
Real-time RT-PCR 0/3 Wang et al. (2005a,b)
Antibody 0/11 Chen et al. (2005)
Sus scrofa domestica Pig Antibody, RT-PCR 2/108 Chen et al. (2005)
Suidae
S. scrofa Wild boar Real-time RT-PCR 1/19 Wang et al. (2005a,b)
RT-PCT, antibody 0/2 Guan et al. (2003)
Artiodactyla Cervidae Muntiacus reevesi Chinese muntjac
Antibody 0/9 Wang et al. (2005a,b)
Bos tarurs Cattle Real-time RT-PCR 0/60 Chen et al. (2005)
Bovidae
Capra hircas Goat Real-time RT-PCR 0/3 Wang et al. (2005a,b)
Real-time RT-PCR 0/6 Wang et al. (2005a,b)
Oryctolagus cuniculus Rabbit
Lagomorpha Leporidae Real-time RT-PCR 0/5 Wang et al. (2005a,b)
Lepus sinensis Chinese hare RT-PCT, antibody 0/3 Guan et al. (2003)
Castoridae Castor fiber Beaver RT-PCT, antibody 0/3 Guan et al. (2003)
Niviventer fulvescens Chestnut spiny rat RT-PCR 0/12 Lau et al. (2005)
Rattus rattus flavipectus Buff-bellied rat RT-PCR 0/4 Lau et al. (2005)
Rodentia
Muridae Rattus sikkimensis Sikkim rat RT-PCR 0/44 Lau et al. (2005)
Real-time RT-PCR 1/6 Wang et al. (2005a,b)
Rattus losea Lesser rice field rat
Real-time RT-PCR 0/16 Wang et al. (2005a,b)
Anas domestica Duck Antibody 0/30 Chen et al. (2005)
Real-time RT-PCR 0/13 Wang et al. (2005a,b)
Anas platyhynchos Spotbill duck
Anseriformes Anatidae Real-time RT-PCR 0/9 Wang et al. (2005a,b)
Real-time RT-PCR 1/10 Wang et al. (2005a,b)
Anser anser Greylag goose
Real-time RT-PCR 0/14 Wang et al. (2005a,b)
Gallus domestiaus Chicken Antibody 0/11 Chen et al. (2005)
Gallus gallus Red jungle fowl Real-time RT-PCR 0/46 Wang et al. (2005a,b)
Real-time RT-PCR 0/31 Wang et al. (2005a,b)
Galliformes Phasianidae Francolinus pintadeanus Chinese francolin
Real-time RT-PCR 0/2 Wang et al. (2005a,b)
Phasianus colchicus Common pheasant Real-time RT-PCR 0/8 Wang et al. (2005a,b)
Pavo muticus Green peafowl Real-time RT-PCR 0/2 Wang et al. (2005a,b)
Columbiformes Columbidae Columba livia Pigeons Real-time RT-PCR 0/6 Wang et al. (2005a,b)
a This table does not include masked palm civets and bats.

found to be identical to certain sequences found in masked palm also harbor the virus (Wang et al., 2005a). Real-time RT-PCR
civets. For example, the sequence of SZ13 is identical to that of revealed that 4 out of 20 cats, 3 out of 5 red foxes, and 1 out of
SZ16 (Guan et al., 2003), and A030G and A031G are identical to 6 Lesser rice field rats were positive for SARS-CoV. The N and
A022G (Kan et al., 2005). It remains unclear whether the raccoon P genes sequences were determined and shown to be identical
dogs were infected by the virus from the masked palm civets or to those of the human SARS-CoVs. After culling of animals
the other way around. It is also possible that both animals were and disinfection of the market place, follow up surveillance was
infected by the virus from a hitherto unidentified host. Unlike conducted 2 weeks later and only one greylag goose out of 119
the situation with masked palm civets, very limited research has animals tested positive for SARS-CoV. At this time cats did not
been conducted to survey wild or farmed raccoon dog. test positive for the virus (Wang et al., 2005a). Also, the same
team reported that in 2004, one wild boar out of 102 animals
4.1.2. Other investigated animals tested positive (Wang et al., 2005a) (Table 4). Since the envi-
A survey conducted on 5 January 2004 in a Guangzhou mar- ronment of the animal market was heavily contaminated with
ket before the mass culling of masked palm civets demonstrated SARS-CoV-like viruses during that period (Kan et al., 2005), it
that red foxes, domestic cats, and Lesser rice field rats may remains unknown whether those animals that tested positive by
84 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87

PCR were truly susceptible hosts or simply mechanical carriers of the SARS-CoV in masked palm civets will continue to play
of SARS-CoV genetic materials. In this regard, it is worth to note an important role in the prevention of potential future SARS
that under experimental conditions, goose was not susceptible outbreaks.
to SARS-CoV infection (Swayne et al., 2004). Bats, the only flying mammals, constitute about 20% of the
In another study carried out in a village close to Beijing (Chen mammalian diversity (Kunz and Fenton, 2003). They are dis-
et al., 2005), 2 of 108 pigs sampled were positive for the SARS- tributed widely in the world and most of them are gregarious.
CoV by antibody analysis and by RT-PCR during a survey which Because of unknown mechanisms, they appear to be natural
also included 60 cattle, 20 dogs, 11 cats, 11 chickens and 30 reservoirs of several new and re-emerging viruses, such as Hen-
ducks. Based on the sequence and available epidemiological dra virus, Nipah virus, Menangle virus, lyssavirus and Ebola
data, the authors suggested that the pigs were infected from feed virus, etc. over the last 20 years (Leroy et al., 2005; Mackenzie
materials contaminated by human SARS patients and were not et al., 2001). Horseshoe bats have been found to be a natural
a natural host of the SARS-CoV. reservoir of the SARS-CoV-like virus. The genetic diversity of
It needs to be pointed out that there were no standard screen- coronaviruses found in bats highlighted our poor understand-
ing regimens being used for detecting SARS-CoVs in the open ing of viruses in wild animals. Moreover, bats that harbor the
markets. During the SARS epidemic and shortly after, many viruses rarely display clinical signs or overt symptoms of pathol-
teams were involved into the animal reservoir investigation, and ogy. Even though there was little direct evidence to indicate that
different teams used their own sampling and screening meth- viruses in bats can cause the severe disease in other domestic
ods. The culling circumstances also added the complexity of the animals and in humans, there is an increased possibility of virus
sampling. Therefore, some of the open market data need to be variants crossing the species barrier and causing outbreaks in
further proven. humans as people come into closer contact with wild animals.
It is therefore essential to study the viral pathology in the host
4.2. Experimental infection of animals and to enhance our knowledge and understanding of reservoir
host distribution, animal-to-animal and human-to-animal inter-
Experimental data have indicated that SARS-CoV might actions. It is also interesting to find out why bats are such a
infect a wide-range of hosts, including masked palm civets (Wu natural reservoir for so many viruses. It is intriguing to spec-
et al., 2005), monkeys (Fouchier et al., 2003; McAuliffe et al., ulate if this is related to the bats’ immune system and/or to
2004; Qin et al., 2005; Rowe et al., 2004), cats and ferrets their ecology. During the search for the natural reservoir of the
(Martina et al., 2003), mice (Glass et al., 2004; Roberts et al., SARS-CoV, the investigators also found several new types of
2005a; Subbarao et al., 2004; Wentworth et al., 2004), pigs and coronavirus which are closely related to group 1 and group 2
chickens (Weingartl et al., 2004), guinea pigs (Liang et al., 2005), coronaviruses, further indicating that bats can harbour a much
and Golden Syrian Hamsters (Roberts et al., 2005b). However, wider spectrum of coronaviruses (Lau et al., 2005; Poon et al.,
as most experimental tests were performed to find a suitable 2005). It is not known whether the coronaviruses in bats would
animal model for vaccine evaluation, not much is known about cause a zoonotic disease in humans or in other animals. All these
transmission of the virus from the inoculated animals. Pigs and uncertainties underscore the need for further investigations to
chickens, for example, may support SARS-CoV replication to a understand the ecology and pathogenicity of these viruses.
very limited degree but are not likely to play a role as an amplify- So far the sequence data showed that the average genome
ing host (Weingartl et al., 2004). A study by Swayne et al. (2004), identity of the SARS-CoV-like virus from horseshoe bat to the
showed that by intratracheally injecting SARS-CoV into chick- SARS-CoV is about 87–92%. As it was pointed out by Li et al.
ens, turkeys, geese, ducks, and quail, failed to cause disease or (2006) that substantial genetic changes in the spike protein of bat
replicate with all the avian species, suggesting domestic poultry SL-CoV are likely to be necessary for the virus to infect humans.
are unlikely the reservoir of the SARS-CoV. Further investigations might reveal viruses with higher identity
to SARS-CoV in bats or with lower identity in masked palm
5. Conclusions civets. However, it is likely that in the emerging path of SARS-
CoV, there are still other species missing between horseshoe
Evidence so far indicated that the origin of the 2003 SARS bats and masked palm civets. One way of revealing possible
epidemic and the 2004 mild SARS cases was from masked palm links and suspects is to look at the ecological circles of both
civets (Guan et al., 2003; Kan et al., 2005; Song et al., 2005; bats and masked palm civets. Alternatively, constant survey of
Wang et al., 2005b). It is still uncertain whether the masked wild animal species for SARS-CoV-like viruses should provide
palm civets were infected during trading in animal market or further information on animal reservoirs.
were naturally infected in the wild or under farmed conditions.
The very high nt sequence identity among the masked palm Acknowledgements
civet SARS-CoV-like viruses (>99.6%) clearly suggested that
the virus has not resided in the masked palm civet population The authors thank the supports for the European Com-
for a very long time. Nevertheless, it is quite clear that masked mission DISSECT (no. SP22-CT-2004-511060) and EPISARS
palm civets are highly susceptible to the SARS-CoV (Wu et (no. SP22-CT-2004-511603) contracts, the Programme de
al., 2005; Kan et al., 2005) where the virus can be amplified to Recherche en Réseaux Franco-Chinois (Épidémie du SRAS: de
very high titers (Kan et al., 2005). Therefore, detailed surveys l’émergence au contrôle), and MOST projects (2003CB514118,
 Z. Shi, Z. Hu / Virus Research 133 (2008) 74–87 85

2005CB523004, and a special grant for “Animal Reservoir of Hu, W., Bai, B., Hu, Z., Chen, Z., An, X., Tang, L., Yang, J., Wang, H., Wang,
SARS-CoV”). We would like to thank Dr. Basil M. Arif for H., 2005. Development and evaluation of a multitarget real-time Taqman
scientific editing. reverse transcription-PCR assay for detection of the severe acute respiratory
syndrome-associated coronavirus and surveillance for an apparently related
coronavirus found in masked palm civets. J. Clin. Microbiol. 43, 2041–2046.
Ikeda, Y., Miyazawa, T., Nakamura, K., Naito, R., Inoshima, Y., Tung, K.C.,
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