Reston virus (RESTV) is the sole member of the species Reston ebolavirus, which is included in the genus Ebolavirus, family Filoviridae, order Mononegavirales. It was discovered in 1989 in cynomolgus macaques that had been imported into the United States from the Philippines [1]. Subsequently, three outbreaks of disease in nonhuman primates in the United States and Italy were also traced back to imported cynomolgus macaques from the same facility in the Philippines, which had been reported to have RESTV-infected animals [2–4]. In 2008, RESTV was isolated for the first time from domestic pigs (Sus scrofa) during an outbreak of porcine reproductive respiratory syndrome (PRRS) [5].

In the present study, three farms, located 35 km from each other in Shanghai, with a total of 137 pigs, were studied from February to September 2011. A total of 137 spleen specimens were collected from pigs that had died after showing typical clinical signs of PRRS, such as high fever or blue ears, and infection by PRRSV was confirmed using RT-PCR as described previously [6]. These spleen specimens (20 to 40 mg) were homogenized separately in 500 μl PBS and centrifuged, and 200 μl of supernatant of the centrifuged homogenized spleen was used for RNA extraction. Total RNA was extracted using TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The primers RESTV-ER3-F and RESTV-ER3-R were designed [5], and RESTV was detected by RT-PCR. The amplified products were analyzed by electrophoresis in 1.2 % agarose gels containing ethidium bromide (0.5 μg/ml), and a 385-bp amplicon was purified using a QIAquick Gel Extraction Kit (QIAGEN, Germany) following the manufacturer’s instructions. The purified PCR products were ligated into the PMD18-T vector (TaKaRa, Japan) at 16 °C overnight using T4 DNA ligase (TaKaRa, Japan). Competent E. coli DH5a cells (TaKaRa, Japan) were transformed with the recombinant plasmid. Four clones were sequenced for each sample, and similarity searches using these sequences were carried out using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). After multiple alignment with CLUSTAL W (version 1.4), four 385-bp nucleotide sequences obtained in the present study were used for phylogenetic analysis. The reliability of the different phylogenetic groupings was evaluated by using the bootstrap test (1,000 bootstrap replications) available in MEGA4.0 [7].

RT-PCR results indicated that four specimens were positive for RESTV (2.92 %, 4/137), suggesting for the first time the presence of RESTV infection in domestic pigs in Shanghai. All four of the positive spleen specimens were from piglets that were under the age of 8 weeks and were co-infected with PRRSV. The nucleotide sequences were 95.1 % to 97.2 % similar to each other (Table 1). The four L gene sequences amplified in the present study, together with their primer sequences, have been submitted to GenBank under accession numbers JNB872215–JNB872218. A phylogenetic tree was constructed based on 385-bp L gene sequences from the RESTV variants from the current study and 15 reference viruses (Figure 1). The present four RESTV variants were closely related, with 96.1 %-98.9 % sequence similarity, to two RESTV variants (FJ621585 and AB050936) that were discovered previously in domestic pigs and cynomolgus macaques from the Philippines [5, 8], but they formed a separate branch in the phylogenetic tree.

Table 1 Differences in the nucleotide sequences of the four RESTV variants in the present study and those of published RESTV reference variants, Ebola virus, Bundibugyo virus, Taï Forest virus and Sudan virus reference variants. The evolutionary distances were computed by the ClustalW method using DNAMAN 6.0 software
Full size table
Fig. 1
figure 1

Phylogenetic analysis of partial 385-bp nucleotide sequences of the L gene of our RESTV variants together with those of published RESTV reference variants, Ebola virus, Bundibugyo virus, Taï Forest virus and Sudan virus reference variants, using the neighbor-joining method with Mega 4.0 software. Bootstrap values >50 % are indicated for the corresponding nodes based on 1000 bootstrapping replicates. GenBank accession numbers for the reference strains are shown at each branch point. The four newly identified RESTV strains from the present study (GenBank accession numbers: JNB872215- JNB872215) are indicated with ‘‘♦’

Full size image

It is unlikely that the RT-PCR sequences that match the earlier RESTV sequences were derived from another as yet undescribed filovirus. Ebolavirus L genes are characteristically well conserved within members of each ebolaviral species (typically >96 %) as compared to between members of ebolaviral species (typically <80 %). Furthermore, while some ebolavirus- and marburgvirus-like sequences have been found integrated into some mammalian genomes [9], this has thus far not been demonstrated for pigs, and no sequences with this level of similarity to those of RESTV have been found. It is therefore likely that the detection of these sequences was due to the presence of RESTV in the animals.

An asymptomatic experimental infection of pigs with RESTV has been shown recently by Marsh et al. [10], while experimental infection with Ebola virus (EBOV) in pigs by Kobinger et al. [11] produced severe respiratory disease and virus shedding. While the impact of RESTV by itself on public and animal health remains to be determined, it is possible that asymptomatic infections in pigs could contribute to disease severity in the context of other infections such as PRRSV. In this study, the detection of RESTV in pigs was associated with the presence of PRRSV, and the pigs that were infected with RESTV were under 8 weeks old, which is consistent with the results from the Philippines [5], where it was demonstrated that pigs infected with RESTV were immunocompromised or were coinfected with a relative of PRRSV, simian hemorrhagic fever virus (SHFV).

Of the four RESTV sequences, the JNB8782215 sequence was most closely related to the previously reported RESTV sequences from isolates from pigs and cynomolgus macaques originally from the Philippines, while the other three sequences were more distantly related to the previously reported Reston virus sequences, and also to each other. This indicates that the Reston virus genes sequenced in this study had mutated. Because all of the pigs in this study were bred and raised in Shanghai, and bats of the species Rousettus amplexicaudatus in the Philippines were recently shown to have antibodies against RESTV [12], it cannot be ruled out that bats are a possible vehicle for spread of RESTV, but further study is needed.

In conclusion, the outbreaks of RESTV infection in nonhuman primates and domestic pigs suggest the zoonotic potential of this virus [13, 14]. We report the molecular detection and phylogenetic analysis of RESTV in domestic pigs in China. Detection and further analysis of RESTV variants from different geographic areas will be helpful for understanding the worldwide distribution and heterogeneity of RESTV variants in domestic pigs and their potential in zoonotic infection. This study indicates the presence of RESTV in domestic pigs in Shanghai, China. It provides additional information on its genetic diversity, possible coinfections, and infection of immunocompromised pigs. It is expected that future epidemiology and pathogenesis studies of RESTV will shed light on its potential reservoirs, mode(s) of transmission, mechanisms of pathogenesis, prevalence in nature, and consequences for the agricultural industry and trade.