Generation of an infectious clone of HuN4-F112, an attenuated live vaccine strain of porcine reproductive and respiratory syndrome virus

Background Nowadays, PRRS has become one of the most economically important infectious diseases of pig worldwide. To better characterize and understand the molecular basis of PRRSV virulence determinants, it would be important to develop the infectious cDNA clones. In this regard, HuN4-F112, a live-attenuated North-American-type PRRSV vaccine strain, could serve as an excellent model. Results In the study, genomic sequence of HuN4-F112, an attenuated vaccine virus derived from the highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) HuN4 strain, was determined and its full-length cDNA was cloned. Capped RNA was transcribed in vitro from the cDNA clone and transfected into BHK-21 cells. The supernatant from transfected monolayers were serially passaged in Marc-145 cells. The rescued virus exhibited a similar growth pattern to its parental virus in Marc-145 cells with peak titers at 48 h post-infection. Conclusion In conclusion, we rescued virus from an infectious cDNA clone of attenuated vaccine. It is possible in the future that a new attenuated PRRSV vaccine with broader specificity and good immunogenicity can be designed in vitro via an infectious cDNA clone platform coupled with validated information on virulence determinants.


Background
Porcine reproductive and respiratory syndrome (PRRS) was first reported in late 1980s in North America and shortly thereafter in Europe. The disease is characterized by reproductive failure in late gestation in sows and respiratory symptoms in pigs of all ages [1][2][3]. Nowadays, PRRS has become one of the most economically important infectious diseases of pig worldwide [4,5]. The PRRS virus (PRRSV) is a small enveloped positive-strand RNA virus belonging to the family Arteriviridae in the order Nidovirales together with equine arteritis virus (EAV), simian hemorrhagic fever virus, and lactate dehydrogenase-elevating virus (LDV) of mice [6,7]. PRRSV is further classified into two distinct genotypes, the North American type and the European type [8,9].
Since May 2006, an atypical PRRS (also known as porcine high fever syndrome) has been pandemic in China. Several studies have confirmed that the causative agent of the outbreaks was highly pathogenic PRRSV (HP-PRRSV) and several HP-PRRSV strains were isolated [10][11][12]. The genome sequences of several strains representing PRRSV have been determined and infectious cDNA clones were developed [13,14]. However, few of these came from a strain with attenuated virulence. To better characterize and understand the molecular basis of PRRSV virulence determinants, it would be important to develop infectious clone of a virulence-attenuated strain. In this regard, HuN4-F112, a live-attenuated North-American-type PRRSV vaccine strain, could serve as an excellent model.
The present article describes the genomic sequence of a virulence-attenuated PRRSV strain HuN4-F112 and the successful rescuing of the virus from its infectious full-length cDNA clone.

Virus strain and cell line
HuN4-F112 is a virulence-attenuated North-Americantype PRRSV strain derived via consecutive passage in Marc-145 cells. This virus strain was maintained in our laboratory as a live-attenuated vaccine [15]. BHK-21 cells were used to rescue virus by transfection with in vitrotranscribed RNA. Marc-145 cells were used for virus rescue and subsequent experiments. Cells were maintained as previously described [10].

RNA extraction and molecular cloning of viral genomic cDNA fragments
Total RNAs were isolated from the supernatants of MARC-145 cells culture at 48 h post infection (PI) by using a QIAamp viral RNA kit (QIAGEN). The cDNA synthesis was performed with SupersciptIII reverse transcriptase (Invitrogen) and reverse primers of each fragment ( Table 1). The PCR primers in Table 1 were designed mainly according to the sequence of PRRSV HuN4 strain with the GeneBank accession number of EF635006 [10]. Six fragments (designated as A, B, C, D, E and F. Figure 1). spanning the full-length genome, were subsequently generated by PCR amplification with Platinum ® Taq DNA polymerase. Each PCR product was cloned into pCR-Blunt II-TOPO vector (Invitrogen). Nucleotide sequencing of the clones was performed by Shanghai Auke Inc. (Shanhai, China) using an ABI 377 automatic sequencer. The sequence data were analyzed using the Lasergene Software Package (DNAstar Inc., USA).

Construction of full-length cDNA clone of HuN4-F112
The strategy for construction of full-length cDNA clone of HuN4-F112 is illustrated in Figure 1. A total of six fragments, covering the complete HuN4-F112 genome, were subsequently PCR amplified with Platinum ® pfx DNA polymerase according to the manufacturer's protocol. The A at position 14680 was mutated to G by PCR to create a restriction enzyme site MluI, a translationally silent substitution, both for fragment E and F ligation and as a genetic marker to differentiate the cloned virus and the parental virus. The bacteriophage SP6 RNA polymerase promoter (underlined sequence) and two non-viral guanosine residues were engineered into primer F16 (Table 1.).
The vector, pBluescript II SK(+) (Stratagene) was modified, the T7 RNA promoter in the vector was changed into the SP6 RNA promoter by PCR mutagenesis. The fragment between the XhoI and NotI sites were replaced by a stuffer fragment, which was prepared as a synthetic gene containing the restriction enzyme sites as shown in Figure 1. The PCR-amplified fragments were gel purified, digested with enzymes as indicated in Figure 1, and cloned into the modified pBluescript II SK(+) vector. After each ligation step, the constructed plasmid was transformed into Escherichia coli DH5α cells and grown overnight at 37°C in the presence of ampicillin. Over-lapped cDNA fragments were joined by using shared restriction sites ( Figure 1). Finally, a full-length HuN4-F112 cDNA clone, pHuN4-F112, was obtained ( Figure 1). The completely assembled full-length cDNA clone was sequenced.

In vitro transcription and transfection
The full-length cDNA clone was linearized by cleavage with restriction enzyme SwaI, which cuts downstream of the poly (A) tail. Linearized plasmid DNA was used for in vitro transcription of capped RNA with the mMessage High Yield Capped RNA Transcription kit (Ambion) according to the manufacturer's instructions and including treatment of the RNA with DNase to remove input plasmid. The transcribed RNA was purified with a MEGA clear kit (Ambion) following the manufacturer's instruction and quantified by spectrophotomery. The synthetic RNA was transfected into BHK-21 cells using DMRIE-C reagent (Invitrogen) according the protocol recommended by the manufacturer. To rescue the virus, cell culture supernatant obtained 24 h post-transfection was serially passaged on MARC-145 cells. Rescue of infectious virus was confirmed by indirect immunofluorescence assay (IFA) using anti-N monoclonal antibody N3H2 [16].

Sequence determination of the PRRSV HuN4-F112 genome
The PRRSV HuN4-F112 strain genome was determined to be 15352 nucleotides in length. The genome contain nine deduced open reading frames (ORF) which is flanked by a 5'untranslated region (5'UTR) of 189 nt and a 3'untranslated region (3'UTR) of 150 nt excluding the poly(A) tail, respectively.

Construction of full-length cDNA clone of HuN4-F112 and determination of its infectivity
The plasmid pHuN4-F112 that contained the full-length cDNA of the entire viral genome of the HuN4-F112 was linearized by digestion with SwaI, and used for in vitro transcription by SP6 RNA polymerase to synthesize capped

Discrimination between cloned virus and parental virus
To differentiate the rescued viruses and parental viruses, a genetic marker, MluI site at nt 14680, was engineered into the genome length cDNA. As expected, the RT-PCR fragment derived from the cloned virus was cleaved by MluI, generating two fragments of 360 bp and 92 bp (Figure 4). In contrast, the PCR amplification products derived from the parental isolate was not cleaved by MluI (Figure 4).

Characterization of the cloned virus
Growth kinetics studies were performed to compare the growth of rescued virus and parental virus in MARC-145 cells. Results ( Figure 5) show that growth kinetics of rescued virus and parental virus was not significantly different from each other and that titers peaked at 48 h pi for both viruses. The result indicates that the rescued virus possesses growth characteristics similar to the parental virus.

Discussion and conclusions
In this study, we described the complete nucleotide sequence of the viral genome and the construction of infectious cDNA clone of HuN4-F112, a live-attenuated  North-American-type PRRSV vaccine strain. Transfection of capped in vitro transcripts, derived from this clone, into BHK-21 cells and Marc-145 cells led to efficient recovery of infectious virus from cultured cells. Indeed, at 3 days post-transfection, CPE was observed in transfected cells. The rescued virus displayed similar growth properties to its parent virus in MARC-145 cells. Detection of the MluI genetic marker indicates that the rescued virus remained genetically stable during passage in cells.
Since May 2006, atypical PRRS (so-called porcine high fever syndrome (PHFS) in china) was pandemic in china. Several studies have confirmed that the causative agent of this outbreak was highly pathogenic PRRSV (HP-PRRSV), several HP-PRRSV strains were isolated and their reverse genetic systems were developed by different labs [13,[17][18][19][20]. We also isolated an atypical PRRS strain named HuN4 and attenuated this strain in vitro by passaging on Marc-145 cells, the attenuated strain was designated HuN4-F112 (112 th passage). Study shows that HuN4-F112 was sufficiently attenuated and antigenic enough to confer protection against a lethal wild-type challenge [10,15].
Sequence alignment between HuN4-F112 and its parental virulent isolate HuN4 showed that both the 5'UTR and 3'UTR are identical and mutations are presented in both nonstructural and structural protein regions. We surmise that the basis of PRRSV attenuation may be multifactor. A recent study also confirmed this by constructing of a series of chimeric viruses where specific genomic regions of a highly virulent PRRSV infectious clone (FL12) were replaced with their counterparts of an attenuated vaccine strain PrimePac. Results indicate that NSP3-8 and GP5 are the location of major virulence determinants, while other virulence determinants may also be contained in NSP1-3, NSP10-12 and GP2 [21]. The availability of the complete genome sequence and infectious clone of the virulence-attenuated strain HuN4-F112 would make it possible to use a comparative virology approach to dissect the virulence determinants of PRRSV. This approach will likely yield new information on PRRSV virulence regulation and virus-host interaction in the future.
In conclusion, we rescued virus from an infectious cDNA clone of attenuated vaccine. It is possible in the future that a new attenuated PRRSV vaccine with broader specificity and good immunogenicity can be designed in vitro via an infectious cDNA clone platform coupled with validated information on virulence determinants.