Published online ahead of print on 11/06/2008 as DOI 10.1099/vir.0.2008/001529-0.
An infectious cDNA clone of a highly pathogenic porcine reproductive and
respiratory syndrome virus variant associated with porcine high fever syndrome
Jian Lv,1,2 Jianwu Zhang,2 Zhi Sun,2 Weiquan Liu1 and Shishan Yuan2
China Agricultural University, College of Biological Sciences, Beijing 100094, PR China
Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, The Key Laboratory of Animal Parasitology,
Chinese Ministry of Agriculture, Shanghai 200232, PR China
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Since May 2006, a so-called ‘porcine high fever syndrome’ (PHFS) has spread all
over China. The arterivirus porcine reproductive and respiratory syndrome virus
(PRRSV) was believed to be the main causative agent, although the involvement of
other pathogens was not formally excluded. The genome of a representative
Chinese PRRSV strain, named JX143, was sequenced and used to develop
infectious cDNA clones, pJX143 and pJX143M, with the latter containing an
engineered MluI site that served as a genetic marker. In various virological assays,
the rescued viruses, vJX143 and vJX143M, were indistinguishable from their
parental virus. Animal experiments showed that these recombinant viruses retained
the high pathogenicity and induced the typical clinical symptoms observed during
PHFS outbreaks. This is the first report describing infectious cDNA clones of this
highly pathogenic PRRSV. Our results unambiguously fulfil Koch’s postulates and
define highly pathogenic PRRSV as the aetiological agent of PHFS in China.
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In the last two years, an epidemic of what is locally called ‘porcine high fever syndrome’ (PHFS)
has spread over the Chinese swine industry, resulting in the culling of an estimated 20 million pigs
annually. Infected animals suffer from laboured breath, pyrexia, lethargy, anorexia and reproductive
failure. Furthermore, the mortality rate of infected piglets is alarmingly high. Following extensive
epidemiological investigations, PRRSV was suspected to be the causative agent of the PHFS
epidemic (Tian et al., 2007). However, it remained to be further investigated whether PRRSV is the
sole aetiologic agent of the disease, since other pathogens like classical swine fever virus (CSFV)
and type 2 porcine circovirus (PCV2) were occasionally isolated from PHFS cases (Li et al., 2007;
Ning et al., 2006). In this study, we have therefore characterized a PRRSV strain isolated from a
typical PHFS case and have investigated its possible role in the PHFS outbreak.
The PRRSV strain used in this study, named PRRSV JX143 strain, was isolated from the serum of a
dying piglet displaying the clinical signs of PHFS. The third virus passage (P3) from MA-104 cells
was used in the present study. Specific primer pairs (Supplementary Table S1, available with the
online version of this paper) were used for RT-PCR amplification and sequence analysis of the
PRRSV JX143 genome. Primers were based on genomic sequences (GenBank accession nos:
AF184212; DQ176021) of the prototypic type II PRRSV. Viral RNA purification, RT-PCR, and
nucleotide sequencing were carried out as previously described (Yuan & Wei, 2008) and the full-
length genome sequence was assembled with the Lasergene package (DNASTAR). The genomic
RNA of PRRSV JX143 turned out to be 15320 nt in length, excluding the poly(A) tail (GenBank
accession no. EU708726). As summarized in Supplementary Table S2, the JX143 nucleotide
sequence shared an overall identity of 61.6% and 89.5%, respectively, with the LV strain (Type I,
GenBank accession no. M96262) and VR-2332 strain (Type II, GenBank accession no. DQ176021)
and was 99.3% identical to the JXA1 sequence (GenBank accession no. EF112445), another
Chinese PRRSV strain isolated during the PHFS outbreak (Tian et al., 2007). Compared to VR-
2332 (Nelsen et al., 1999), the PHFS-related PRRSV isolates contained a single nucleotide deletion
in both the 5′ UTR and the 3′ UTR. Moreover, these PRRSV isolates shared the same consecutive
deletions of 1 and 29 amino acids in their nsp2-coding region, which is the most variable part of the
PRRSV genome (Fang et al., 2004; Gao et al., 2004; Han et al., 2007; Ropp et al., 2004).
Curiously, the nsp2 deletion partially overlaps with that in the genome of PRRSV MN184, an
atypical high pathogenicity (HP) PRRSV strain from North America, thus promoting the hypothesis
that such a deletion may be related to the increased virulence of these viruses (Han et al., 2007).
Tian et al. (2007) first demonstrated that this PRRSV variant caused almost 100% mortality in 35-
day-old pigs and 57% mortality in 77-day-old growing pigs. Based on these results, Tian and co-
workers proposed that the causative agent of PHFS was a HP PRRSV variant. Other groups also
confirmed the HP nature of PRRSV isolated from typical PHFS cases by using cell-culture-
amplified viruses for challenge studies in animals (An et al., 2007; Tong et al., 2007), although it
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remained to be proven that the virus inoculum was free of other pathogens. In fact, it has been
debated whether other agents or unknown novel pathogens may cause or contribute to PHFS.
Therefore, the role of the prevalent HP PRRSV strain in the PHFS outbreak remained to be
characterized in more detail.
Reverse genetic systems are a powerful tool for the molecular dissection of arteriviruses and
infectious cDNA clones have been developed for equine arteritis virus and traditional PRRSV
strains (Balasuriya et al., 1999, 2007; Meulenberg et al., 1998; van Dinten et al., 1997; Wang et al.,
2008). In this study, we set out to construct infectious cDNA clones of HP PRRSV to further clarify
the role of this virus as the major aetiological agent of PHFS. The strategy we adopted is shown in
Supplementary Fig. S1 and was described previously (Yuan & Wei, 2008). Briefly, cDNA
fragments covering the entire JX143 genome were cloned and assembled into full-length cDNA
clone pJX143, in which the viral sequence was placed downstream of a T7 promoter. The viral
genome sequence was determined and deposited in GenBank (accession no. EF488048). Compared
to the parental genome sequence (EU708726), a total of 34 nucleotide variations were identified
(Supplementary Table S3). Among these, two substitutions, C328T and C6267T, were attributed to
the quasispecies nature of the PRRSV genome, whereas the remaining 32 nucleotide differences
might be PCR artefacts. Among these mutations, 15 nucleotide changes were translationally non-
silent, almost all of which were located in ORF1 except for nt 12309 (Asn→Pro) in ORF2, and nt
12697 (Ser→Arg) in ORF3.
To differentiate the recombinant virus from the parental virus, and to exclude the possibility of a
contamination, pJX143M was constructed containing a translationally silent substitution, A14680G,
which created a novel MluI restriction site. As described previously (Yuan & Wei, 2008), 3 µg in
vitro-transcribed RNA was mixed with 2 µl DMRIE-C (Invitrogen) and transfected into a
subconfluent monolayer of MA-104 cells. At 72 h post-transfection (h p.t.), cytopathic effect (CPE)
was observed in cells transfected with both pJX143 (Fig. 1a, panel i) and pJX143M (data not
shown), while mock-transfected cells remained normal (Fig. 1a, panel ii). To characterize the
rescued viruses, immunofluorescence assays (IFA) were conducted using a monoclonal antibody
recognizing the viral nucleocapsid (N) protein (a kind gift from Dr Kegong Tian). Briefly, passage
3 material of parental JX143 and the rescued viruses (vJX143 and vJX143M) was used for
inoculation of MA-104 cells at an m.o.i. of 0.1. At 36 h post-infection (h p.i.), the infected cells
were fixed and used for IFA as previously described (Sun et al., 2007). As shown in Fig. 1(b), at 36
h p.i., about 20% of the cells stained positive, indicating that the rescued viruses displayed infection
kinetics similar to those of the parental virus.
The growth kinetics of the rescued viruses were evaluated by inoculating MA-104 cells with P3
viruses and at m.o.i. of 0.1, collecting the supernatant of the infected cells and virus progeny
titration by determining the 50% tissue culture infectious dose (TCID50 ml−1) (Pizzi, 1950). As
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shown in Fig. 1(c), the growth curves of the rescued viruses were similar to those of the parental
virus. At 56 h p.i., all three viruses reached their peak titre, which was around 1.5×106.5 TCID50
ml−1, indicating that the growth properties of the recombinant viruses on MA-104 cells were
To investigate if the rescued viruses remained stable during further passaging, both vJX143 and
vJX143M were serially passaged on MA-104 cells using low infection doses (0.1 m.o.i.). The
culture supernatant of the tenth passage was used for viral RNA isolation, followed by single-tube
RT-PCR amplification (Tiangen) with primer pair SF13851 and Qst (Supplementary Table S1). The
nucleotide sequence of the genomic region from nt 13851 to the end of the poly(A) tail revealed
that the engineered MluI site and its flanking sequences had remained unchanged. We next
evaluated the viral RNA profiles in infected cells using Northern blot as described previously (Sun
et al., 2007). The rescued viruses displayed the same genomic and subgenomic mRNA profiles as
parental JX143 (Fig. 1d), demonstrating that the recombinant viruses had retained the molecular
biological properties of the parental JX143 virus. Taken together, our data showed that we
developed the first infectious cDNA clones for the PRRSV isolate associated with the PHFS
outbreak in China.
The potential role of other unknown pathogen(s) in PHFS pathogenesis has been debated, since
other viral and even bacterial pathogens were isolated from PHFS-like cases (Ning et al., 2006).
Therefore, the question remained whether PHFS is caused by a single highly virulent pathogen or
may result from a co-infection by multiple aetiological agents. To further define the aetiological
agent of PHFS, and the pathogenicity of our recombinant PRRSV, we conducted animal challenge
experiments. A group of 35 day-old pigs were purchased from a PRRSV-free farm and were further
screened using serological tests and RT-PCR to exclude the presence of PRRSV, PCV2, CSFV,
pseudorabies virus (PRV) and the pathogenic bacteria Streptococcus, Staphylococcus and
Mycoplasma, as described by Tian et al.(2007). The twelve pigs were randomly divided into 4
groups and housed in isolation in a BL-2 animal facility (Zhejiang Yebio Biotech). The animals in
groups A, B and C were inoculated with 2 ml 3×104.5 TCID50 of parental JX143, recombinant
vJX143 or recombinant vJX143M, respectively, with group D forming the mock-inoculated control.
The injected pigs were observed daily for clinical symptoms and rectal temperatures were recorded
until the end of the experiment. Blood samples were collected from all the animals at 0, 3, 5, 7, 14
and 16 days p.i.
All virus-inoculated groups developed high fever, a typical PHFS sign, with rectal temperatures up
to 41 °C, starting from 4 days p.i. and sustained for at least 8 days (Fig. 2a). The temperature peak
and infection kinetics of the recombinant virus-inoculated groups were similar to those of the
parental virus-infected group (P<0.05). All virus-inoculated pigs developed PHFS clinical
symptoms beginning at 5 days p.i., displaying lethargy, lack of appetite, coughing and often
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paralysis, as reported by others (An et al., 2007; Li et al., 2007; Tian et al., 2007). Remarkably, all
virus-inoculated pigs had died by 13 to 16 days p.i. Specifically, pigs in group A died at 13, 14 and
16 days p.i., while the pigs of group B and C died at 14–15 days p.i. Post-mortem necropsy revealed
pathological changes including interstitial pneumonia with lung hyperplasia, lung oedema, blood
spots or ecchymosis, splenitis, sporadic blood spots in the kidneys, perivasculitis, cerebral oedema,
meningorrhagia and suppurative encephalitis, haemorrhagic spots in the lymph nodes and
lymphadenectasis (data not shown), as previously described by Tian et al. (2007). Although the
numbers of animals used were limited by the availability of qualified pathogen-free pigs, this in
vivo study demonstrated that the recombinant viruses retained the in vivo pathogenicity of the
The humoral immune response in the infected animals was measured by ELISA (IDEXX HerdChek
PRRS 2X52). As shown in Fig. 2(b), the serum antibody level began to rise at 5 days p.i. and all
virus-inoculated pigs had seroconverted by 14 days p.i. The overall serological responses of the
virus-inoculated pigs were indistinguishable from each other, demonstrating that the recombinant
viruses retained the immunological properties of the parental JX143 isolate.
Virus isolation on MA-104 cells, RT-PCR and nucleotide sequencing were conducted to evaluate
virus replication and tissue distribution in the infected animals. Viruses were detected in multiple
tissues including lymph nodes, lungs, spleens and kidneys, which were tested by inoculating MA-
104 cells with homogenized tissues. There were no significant differences in virus distribution
among different tissues and between the recombinant and parental viruses (data not shown). These
results established that all inoculated pigs developed a viraemia starting at 3 days p.i. and lasting
until the day of the animal’s death, as summarized in Table 1, and that the recombinant viruses
displayed virtually the same in vivo infection kinetics as the parental virus.
Taken together, both recombinant viruses (vJX143 and vJX143M) reproduced the clinical,
pathological, immunological and virological parameters observed in clinical cases of PHFS, thus
fulfilling Koch’s postulates and unambiguously identifying HP PRRSV as the causative agent of the
devastating PHFS outbreak in China. However, it should be pointed out that PHFS has remained a
poorly defined clinical term and that there is no generally accepted description of the clinical
symptoms associated with PHFS, although ‘high fever’ was often observed in field cases. The
experiments with the molecularly cloned virus described in this paper demonstrated that PRRSV is
the major culprit of the PHFS epidemic, which in the field often presents as a variety of secondary
infections due to HP PRRSV-induced immunosuppression. PRRSV shows a high degree of genetic,
antigenic and virulence variations (Ellis et al., 1999; Meng, 2000; Mengeling et al., 1998;
Sutherland et al., 2007; Yuan et al., 1999), and the cross-protection efficacy of the current vaccines
based on classical PRRSV strains needs to be improved (Cano et al., 2007; Charerntantanakul et al.,
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2006). The development of the HP PRRSV reverse genetics system described in this study may
contribute to the improvement of such a live attenuated vaccine.
This project was co-sponsored by the Natural Science Foundation of China (#30530580), the
National Basic Research Program (2005CB523202), the National non-profit institute funding
program MOST (#2005DIB4J051), the Ministry of Human Resources of China (#2006-Z3), and the
Shanghai Pujiang Talent program (06PJ14118).
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Fig. 1. Virological characterization of recombinant HP PRRSV. Subconfluent MA-104 cell
monolayers were inoculated with the recombinant viruses vJX143 and vJX143M, respectively,
while cells infected with the parental virus and mock-infected cells served as controls. (a) CPE was
observed at 72 h p.i. with vJX143 (i), while mock-infected cells remained normal (ii). (b) IFA was
conducted to stain MA-104 cell monolayers at 36 h p.i. Cells were fixed and stained with an N
protein-specific mAb, followed by a FITC-labelled secondary Ab, and were viewed by
immunofluorescence microscopy (×200 magnification). (i) vJX143, (ii) vJX143M, (iii) parental
JX143; (iv) negative control. (c) Multi-step growth curve of the P3 viruses on MA-104 cells.
Aliquots of the supernatants of infected cells were collected at the indicated time points and virus
titration (TCID50 ml−1) was conducted to determine the growth curves. ○, vJX143; □, vJX143M; ▲,
parental JX143. (d) Northern blot analysis of RNA from infected cells. Total intra cellular RNA
was isolated from cells infected with vJX143, vJX143M and parental JX143 at 36 h p.i. RNA was
separated in a 1% agarose gel, followed by blotting onto NC membranes and hybridization with a
Bright-Star-labelled oligonucleotide probe complementary to the ORF7 sequence (Supplementary
Table S1). The viral genome (RNA 1) and subgenomic mRNAs (RNAs 2–7) are indicated.
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Fig. 2. Overview of rectal temperatures and immune responses of piglets inoculated with
recombinant and parental HP PRRSV. Four groups of three pigs were mock-inoculated ( ) or
inoculated with 2 ml 3×104.5 TCID50 ml−1 of vJX143 (○), vJX143M (□) and parental JX143 (▲),
respectively. (a) Rectal temperatures were recorded daily from 0 to 17 days p.i. Average
temperatures from the three animals in each group are shown. Note that from 13 days p.i. infected
animals began to die (see text). (b) Humoral immune responses in infected animals as measured by
ELISA (S/P ratio) using serum samples of the inoculated pigs collected at the indicated times.
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Table 1. Viraemia in animals infected with recombinant or parental HP PRRSV-inoculated pigs, as
analysed by RT-PCR and virus isolation
RT-PCR results using serum samples of the inoculated pigs. Virus isolation results based on the
induction by serum samples of CPE on MA-104 cells.
Group Virus Number of RT-PCR positive pigs / total Number of virus isolation positive pigs /
number of pigs* (days p.i.) total number of pigs* (days p.i.)
0 3 5 7 14 0 3 5 7 14
A parental 0/3 3/3 3/3 3/3 1/1 0/3 3/3 3/3 3/3 1/1
B vJX143 0/3 3/3 3/3 3/3 2/2 0/3 2/3 3/3 3/3 2/2
C vJX143M 0/3 3/3 3/3 3/3 1/1 0/3 3/3 3/3 3/3 1/1
D None† 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
*The numbers of dead pigs at the indicated time points were deducted, and the remaining pigs which died later were all
†Negative control group inoculated with PBS.
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Supplementary Fig. S1. The viral genomic organization and cDNA cloning strategy. Five overlapping regions of the
viral genome were amplified to generate the cDNA fragments A, B, C, D, and E. Fragment E was amplified to contain a
NotI recognition site following the poly(A) tail. The T7 RNA polymerase promoter was inserted immediately upstream
of the 5′-terminal sequences in fragment A. The PCR products were cloned, sequenced and verified, followed by
ligation via single restriction enzyme site in the overlapping regions. The complete genomic cDNA was finally
assembled into the pBlueScript II SK (+) vector to generate the full-length JX143 cDNA clone, designated pJX143.
An MluI site was created in ORF5 region as described in the text, and the mutant plasmid was designated pJX143M.
Page 13 of 17
Supplementary Table S1 Oligonucleotides used for RT-PCR and as probe
Name* Sequence† Location‡
STL ACATGCATGCTAATACGACTCACTATAGGTATGACGTATAGGTGTTGGC 1–49
SF1392 GCCGCGCTTTGCCCGTTCGT 1392–1411
SR2573 CTGCCCAGGCCATCATGTCCGAAGTC 2573–2548
SF4344 GCCCCGTCGGTCTCAGTCTTGCCATTTTT 4344–4373
SR6589 ACCGAGGCTGTAAAAGGCAAGTGACC 6589–6573
SF7682 CTTTCCGTTGAGCAGGCCCTTGGTATGA 7682–7609
SR9033 GGCGGCAAACCAGCGGACAA 9033–9014
SF11422 TTTATAAGGCCACTGCCACC 11422–11442
SR13334 TTGCCGCCGTCGACTTGATGCTGGTAAT 13334–13306
SF13851 TTACCCTGTCTTTTTGCCATTC 13851–13872
SR15369§ TCATGCTGAGGGTGATGCTGTGACGCGAATCAGGCGCACAGTATGATGCG 15319–15369
Qst GAGTGACGAGGACTCGAGCGCATGCTTTTTTTTTTTTTT 15506–15545
*Primer names are organized in groups. Prefixes: SF, forward PCR primer; SR, reverse PCR primer.
†Specific primer pairs were designed according to the conserved regions of the type II strains SP (GenBank accession no. AF184212).
‡The nucleotide positions within the viral sequence are based on AF184212.
§The ORF7-specific RNA probe used in the Northern blot identification.
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Supplementary Table S2 Comparison of the nucleotides and amino acid sequences of strain JX143
with those of strains VR2332 and LV
Nt (%), percentage of identity of nucleotide sequences; Aa (%), percentage of identity of amino
Similarity JX143 vs LV JX143 vs VR2332
Nt (%)* Aa (%)† Nt (%)* Aa (%)†
Overall 61.6 89.5
ORF1a 59.1 46.1 85.9 86.7
ORF1b 61.4 68.1 91.5 96.8
ORF2 66.8 62.2 93.3 93
ORF3 61.3 55.1 88.6 85.4
ORF4 66.9 69.7 89.8 89.9
ORF5 63.5 57.7 89.1 88.1
ORF6 69.0 79.2 95.2 97.7
ORF7 65.9 58.5 94.1 95.1
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Supplementary Table S3. Nucleotide differences between the parental virus and the full-length
Nucleotide position Nucleotides in Nucleotides in the Change
within the cDNA parental virus cDNA clone
232 A G Silent
328 C† T Silent
334 C T Silent
336 A T Y→F
381 A T Silent
421 C T F→L
458 G A Silent
625 C A H→N
2124 C T Silent
2227 G C A→P
2312 A G D→G
2549 C A T→N
2613 G T Silent
3136 G T V→F
3176 T C I→T
3191 T C L→P
3278 C T V→A
3509 A T D→V
4113 A G Silent
5565 C T Silent
5877 T C Silent
6267 C† T Silent
6990 C T Silent
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Nucleotide position Nucleotides in Nucleotides in the Change
within the cDNA parental virus cDNA clone
7188 G A Silent
7688 C T Silent
8938 A G K→R
9812 C T Silent
11223 G A Q→K
12309 A G N→D
12697 C A S→R
13037 C T Silent
13423 A G Silent
13573 T C Silent
13766 C T Silent
*The viral genomic sequence was generated by sequencing the RT-PCR fragments, and multiple
clones were subjected to if ambiguity occurs; the final nucleotide sequence were generated by
comparing five parallel sequence files at each genomic location.
†Among the five different clones RT-PCR fragment A (Fig. S1), nucleotide sequence showed that
4/5 were the cytosine(C) and only 1/5 was thymine(T).
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