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Dispatch
SARS-associated Coronavirus
Transmitted from Human to Pig
Weijun Chen,*†1 Minghua Yan,‡1
Ling Yang,*1 Boliang Ding,‡ Bo He,† Yingzhen
Wang,‡ Xiuli Liu,‡ Chenhui Liu,* Hui Zhu,‡ Bo You,† Shengyong Huang,†
Jiangguo Zhang,* Feng Mu,*† Zhao Xiang,*§ Xiaoli Feng,* Jie Wen,*† Jianqiu
Fang,*†Jun Yu,* Huanming Yang,* and Jian Wang*†
*Chinese Academy of Sciences, Beijing, China; †Beijing BGI-GBI Biotech
Co., Ltd, Beijing, China; ‡Tianjin Institute of Animal Husbandry and Veterinary
Science, Tianjin, China; and §BGI Hangzhou Bio-Environment Technology
Co., Ltd, Hangzhou, China
Suggested
citation for this article
Severe acute respiratory
syndrome–associated coronavirus (SARS-CoV) was isolated from a pig during
a survey for possible routes of viral transmission after a SARS epidemic.
Sequence and epidemiology analyses suggested that the pig was infected
by a SARS-CoV of human origin.
Severe acute respiratory syndrome (SARS) was first identified in Guangdong
Province, China, in November 2002 (1). A novel coronavirus,
SARS-CoV, was identified as the pathogen; several possible origins of
the coronavirus were suggested from wild animal reservoirs, such as Himalayan
palm civets and raccoon dogs (2–8). The virus infects
many other wild and domesticated animals, such as Mustela furo,
Felis domesticus, and Nyctereutes procyonoides (9,10),
but infection of domesticated pigs has not been previously reported.
The Study
We surveyed 6 major domestic animal species that are in close contact
with humans and could be infected by SARS-CoV if transmission were possible.
The survey was conducted in a suburban area and its extended farming villages,
Xiqing County of Tianjin, China, where a SARS outbreak occurred in late
spring of 2003. Animal samples, blood and fecal swab specimens, for antibody
and RNA detection were collected from the sites and transported on ice
to a biosafety level 3 laboratory within 24 hours. We used 2 types of
assays for the initial viral screen, immunologic assays to identify antibodies
and reverse transcription–polymerase chain reaction (RT-PCR) to detect
the viral genome. The immunoassays were carried out by the double-antigen
sandwich method with a recombinant N protein and a partial S protein of
SARS-CoV, and results were confirmed by Western blot (11).
RT-PCR with virus-specific primers was used to detect viral genome RNA,
which was extracted from blood samples with a QIAamp RNA Blood Mini Kit
(Qiagen, Hilden, Germany) and from fecal swabs with Trizol Reagent (Invitrogen,
Carlsbad, CA, USA). Total RNA was then reverse transcribed with random
hexamers, and cDNA was amplified with a nested PCR method (12).
We also isolated viruses from Vero E6 cultures, performed a cross-neutralization
test, and sequenced the viral genome (13).
Of 242 animals surveyed, we identified 2 antibody-positive samples from
2 pigs; test results for the other 240 animals were negative (Table
1 and Figure 1). Of 93 blood specimens and
15 fecal swabs on which we performed RT-PCR, 1 of the same 2 pigs tested
positive. We subsequently obtained 2 viral isolates from its blood and
fecal samples, designated TJB and TJF, respectively. We also performed
follow-up studies for 4 weeks on the infected pig until its blood tested
negative with our RT-PCR assay. The animal later died giving birth. We
also tested serum samples from the swineherd on the farm and a few persons
who may have had contact with the swineherd. All were negative by the
tests that we conducted.
Using a viral isolate, TJF, we conducted cross-neutralization experiments
with antisera and an early viral isolate, BJ01 (8), to
prove their equivalent virulence (Table 2). We then
sequenced TJF completely (GenBank accession no. AY654624) and compared
its sequence to that of BJ01. Eighteen nucleotide (nt) substitutions are
between the TJF and BJ01 sequences, and 4 of them are nonsynonymous over
the entire length (29,708 bp). Two pieces of evidence strongly suggested
a human origin for the TJF strain. First, it is only distantly related
to SZ16, which was isolated from Himalayan palm civets of southern China,
in which 64 substitutions over a length of 29,731 bp were found, 3.6 times
more than were identified between TJF and BJ01. Second, a sequence signature
(a 29-nt insertion [246 nt upstream of the N gene, from residues 27869
to 27897]) found only in an early isolate, GD01 (from Guangdong Province),
but absent from all the SARS-CoV isolates so far discovered, is also absent
in the TJF sequence (14). This sequence has been found
in all coronavirus isolates of animal origin except from the pig identified
in this study. Therefore, direct viral transmission of SARS-CoV from a
human host to the pig bearing TJF is most likely. To further elucidate
our point, we constructed a phylogenetic tree based on S-gene sequences;
it shows that TJF is more closely related to human SARS-CoV isolates than
to animal coronaviruses (Figure 2).
Conclusions
We have shown that human SARS-CoV can infect domesticated mammals, in
particular, the pig. The direct source of SARS-CoV transmission to the
identified infected pigs was most likely virus-contaminated animal feed
because the farm where the infected pig was identified is rather remote,
>1 km from the nearest village. The only person routinely in close
contact with the animals is the swineherd, whose serum samples were negative
for SARS-CoV on all tests. Swineherds in rural areas often obtain leftovers
from restaurants in the cities for use as hogwash (without thoroughly
fermenting it). Thus, even if no direct evidence for human-to-swine SARS-CoV
transmission exists, a strong warning should be issued to prevent such
a practice, or regulatory procedures should be instituted to block this
route of disease propagation (15). Whether or not other
domesticated (such as dogs and cats) and wild animals that are common
in and around human settlements can easily contract and pass on SARS-CoV
remains to be seen in future studies. Intensive surveillance and investigations
on animals, especially during and after an outbreak of SARS, will lead
to a better understanding and ability to control this disease's natural
animal reservoirs and to prevent interspecies transmission events.
This study was supported
by grants from Chinese Academy of Sciences and Tianjin Institute of
Animal Husbandry and Veterinary Science.
Mr. Chen is a PhD
candidate who works at the Beijing Genomics Institute, Chinese Academy
of Sciences, Beijing, China. His primary research interests are molecular
virology and infectious disease genomics.
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| Table
1. Animal surveys for antibodies to SARS-CoV and viral RNA* |
|
|
Animals
|
Antibodies in serum samples
|
RT-PCR
|
Virus isolation
|
|
|
|
|
ELISA
|
WB
|
Blood
|
Fecal swab
|
Blood
|
Fecal swab
|
|
|
|
|
|
|
|
N
|
P
|
N
|
P
|
N
|
P
|
N
|
P
|
N
|
P
|
N
|
P
|
|
|
Pigs
|
108
|
2
|
5
|
2
|
14
|
1
|
14
|
1
|
6
|
1
|
6
|
1
|
|
Cattle
|
60
|
0
|
0
|
0
|
22
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
|
Dogs
|
20
|
0
|
0
|
0
|
14
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
|
Cats
|
11
|
0
|
0
|
0
|
11
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
|
Chickens
|
11
|
0
|
0
|
0
|
11
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
|
Ducks
|
30
|
0
|
0
|
0
|
20
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
|
| *Numbers in table refer
to number of animals tested with the assays. SARS-CoV, severe acute
respiratory syndrome–associated coronavirus; RT-PCR, reverse transcriptase–polymerase
chain reaction; ELISA, enzyme-linked immunosorbent assay; WB, Western
blot; N, negative; P, positive. |
| Table
2. Cross-neutralization tests for severe acute respiratory syndrome
(SARS)–associated coronavirus* |
|
|
Virus strains
|
Sera (ND50)
|
|
|
S1
|
S2
|
S3
|
H1
|
H2
|
H3
|
|
|
TJF
|
1:160
|
1:640
|
<1:10
|
1:1,280
|
1:640–1,280
|
<1:10
|
|
BJ01
|
1:160–320
|
1:640
|
<1:10
|
1:640–1,280
|
1:320–640
|
<1:10
|
|
| *S1 and S2 were sera from
swine that were positive by enzyme-linked immunosorbent assay (ELISA).
H1 and H2 were sera from SARS patients. H3 and S3 were controls from
sera of a normal human and an ELISA-negative pig, respectively. ND50,
50% neutralization dose. |
1W. Chen, M. Yan, and L. Yang contributed
equally to this article.
Suggested citation
for this article:
Chen W, Yan M, Yang
L, Ding B, He B, Wang Y, et al. SARS-associated coronavirus transmitted
from human to pig. Emerg Infect Dis [serial on the Internet]. 2005 Mar
[date cited]. Available from http://www.cdc.gov/ncidod/EID/vol11no03/04-0824.htm
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