Volume 20, Number 12—December 2014
Gouleako and Herbert Viruses in Pigs, Republic of Korea, 2013
Several viruses in the family Bunyaviridae are pathogenic to animals and cause vector-borne zoonoses. In 2013, investigation of cause of death of 9 pigs on 1 farm in the Republic of Korea found infection with Gouleako and Herbert viruses. Subsequent investigation revealed high prevalence of these viruses among pigs throughout the country.
Several viruses in the family Bunyaviridae, such as severe fever thrombocytopenia syndrome virus, sandfly fever Naples virus, and La Crosse virus, cause vector-borne zoonotic problems (1–7). Recently, outbreaks of severe disease caused by Rift Valley fever virus and Schmallenberg virus produced abortion storms, resulting in a high mortality rate among newborn lambs and calves (4,8). Gouleako virus (GOLV) and Herbert virus (HEBV) have been isolated from mosquitoes (Culex spp.) trapped in Côte d'Ivoire (9,10); however, their infectivity or virulence have not been proven. Investigation of the cause of death of pigs in the Republic of Korea identified GOLV and HEBV infection.
In March 27, 2013, a piglet, ≈8 weeks of age, on a 150-sow farm in Gyeonggi, Republic of Korea, died after onset of high fever (40°C), wasting, respiratory disease, and diarrhea. The carcass was sent to the Department of Veterinary Medicine Virology Laboratory, Seoul National University, Seoul, Republic of Korea, for diagnostics. Necropsy and microscopic examinations revealed greenish lung tissue with lymphoid depletion, consistent with severe bronchopneumonia. Despite the presence of multiple clinical signs, the results of routine tests for major pathogens in pigs (e.g., porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, Escherichia coli, Streptococcus spp., and Salmonella spp.) were negative.
To further explore cause of the death, we used the particle-associated nucleic acid –random PCR method (Technical Appendix). Sequencing and BLAST analysis (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) of the agent-specific amplicon simultaneously detected 2 viruses in lung tissue RNA samples. One partial sequence had 100% identity with 63 nt of the GOLV strain F23/CI/2004 glycoprotein gene (GenBank accession no. FJ765411). Another sequence had 97% similarity with 66 nt of the HEBV strain F23-K4 RNA-dependent RNA polymerase (RdRp) gene (GenBank accession no. EF423168).
Results were validated with reverse transcription PCR (RT-PCR) (Technical Appendix). We obtained partial sequences of 235 nt of GOLV and 324 nt of HEBV. These sequences had 97.1% and 96.9% similarity with GOLV and HEBV, respectively, previously isolated from mosquitoes (9,10). The sequences were registered as GenBank accession nos. KF361520 and KF361522 and designated as GOLV/P1 and HEBV/P1, respectively.
During March–May 2013, we received a total of 9 dead pigs from the same farm; they had displayed various clinical signs. We further screened these pigs for the presence of GOLV and HEBV by using the same primer sets (Technical Appendix) selective for their glycoprotein and RdRp genes, respectively. The results showed that the pigs were infected with GOLV and HEBV at a prevalence of 83.3% and 100%, respectively, mostly in lung samples (Table 1). The sequences obtained from this assay were registered as GenBank accession nos. KF361521 and KF361523 and designated GOLV/P8 and HEBV/P9, respectively.
Because of the high rate of GOLV positivity, we conducted a histopathologic RNA in situ hybridization study (Technical Appendix). Hybridization signal was positive in lung and lymph node tissues and negative in intestine and control tissues. Hybridization was strong in the cytoplasm of mononuclear cells (deep blue color) (Technical Appendix Figure 1).
Using the same RT-PCR method (Technical Appendix), we investigated the prevalence of GOLV and HEBV in other swine populations in the Republic of Korea; we used the existing primer sets: (GOLV-NCF and GOLV-NCR) and (HEBV-F and HEBV-R). During March–September 2013, a total of 461 serum samples were randomly collected from 40 commercial swine farms in 9 provinces. Of these, 204 (44.3%) samples were positive for GOLV and 26 (5.6%) samples were positive for HEBV (Table 2). The rates of positivity for the investigated provinces are shown in Figure 1. When examined according to season, positive samples were more frequently found in the summer than in spring. For example, during July–August, rates were ≈65% (for GOLV) and 10% (for HEBV), but in March, rates for each virus were <10%. Rates for GOLV and HEBV positivity were higher among sows (1–4 years of age) than among pigs in other age groups (Table 2).
The study was extended to include sows on other farms in the Republic of Korea because of the major role of sows on a commercial swine farm. Pigs were divided into 3 groups and the following samples were collected: blood from healthy sows >1 year of age (n = 76), blood from abortion-problem sows (n = 13), and tissue from aborted fetuses (n = 42). Rates of virus positivity for GOLV and HEBV were higher for the 42 fetuses (33 [78.6%] and 11 [26.2%]) than for pigs in the healthy group (42 [55.3%] and 12 [15.8%]), respectively. Of the 13 abortion-problem sows, GOLV and HEBV, respectively, were found in 10 (76.9%) and 3 (23%) samples. The rates of GOLV and HEBV positivity among the healthy and abortion groups (abortion-problem sows and fetuses) were statistically compared by using the Pearson χ2 test in SPSS version12.0 (SPSS Inc., Chicago, IL, USA). The only significant correlation found was for HEBV infection in the abortion group; p<0.05.
Within the abortion group, pooled tissues from the 42 fetuses were screened for other pathogens (Technical Appendix). The highest rate of positivity for the fetuses was for GOLV; 17 (40.5%) of the 42 samples were positive for GOLV only. Concurrent GOLV and HEBV infection was found in 9 (21.4%) samples and GOLV and swine influenza virus in 4 (9.5%) samples; no specific pathogens were detected in 7 (16.7%) samples (Technical Appendix Table).
The phylogenetic relationships of GOLV and HEBV isolated from swine in the Republic of Korea (Technical Appendix) with the other members of family Bunyaviridae were analyzed; analyses were based on genes that encode the nucleocapsid protein (for GOLV) and RdRp (for HEBV). Of the GOLVs, the result showed that samples from the swine farms examined (KJ830623, KJ830624) clustered with GOLV strains from Africa (9) and showed high similarities (98.34%–98.98%) with a strain of GOLV from mosquitoes in western Africa (HQ541736) (Figure 2, panel A). Of the HEBV viruses, samples from swine farms examined (KJ830625, KJ830626) formed a branch with existing strains (KF590583, JQ659256) from mosquitoes in Côte d'Ivoire (10) (Figure 2, panel B). Similarities with each other were 94.46%–97.23%.
Two field GOLV strains (CP-1/2013 and CP-2/2013) used in this study were isolated from pig kidney (PK15) cells. Detailed information about the methods used to prove the results are shown in Technical Appendix Figure 2.
We demonstrated that GOLV and HEBV are prevalent on swine farms in the Republic of Korea. Prevalence of these viruses was first suspected after particle-associated nucleic acid–random PCR of tissue from dead pigs, and it was proven by RT-PCR screening of a large collection of samples (serum, fetal tissue) from healthy and sick pigs throughout the country. The in situ hybridization method detected GOLV RNA in pig tissues and provided evidence in support of the presence of GOLV in the infected tissues of pigs. The findings of this study indicate that GOLV and HEBV may be associated with disease in pigs; investigation of the pathogenicity of the viruses in pigs, as well as their relation to other emerging viruses of swine, is needed.
Mr Chung is a virologist at the Department of Veterinary Medicine Virology Lab, College of Veterinary Medicine, Seoul National University, Republic of Korea. His research interests include human infectious disease, zoonoses, vector-borne diseases, novel viruses, swine virology, transgenic pigs, endogenous retroviruses, viral enteritis of pigs, and viral diseases of animals.
We thank Su Hee Yun for expert technical assistance.
This study was supported by a grant (PJ009015) from BioGreen 21 Program, Republic of Korea.
- Wu Y, Gao GF. Severe fever with thrombocytopenia syndrome virus expands its borders. Emerging Microbes & Infections. 2013;2:e36.
- Chang MS, Woo JH. Severe fever with thrombocytopenia syndrome: tick-mediated viral disease. J Korean Med Sci. 2013;28:795–6.
- Lambert AJ, Blair CD, D'Anton M, Ewing W, Harborth M, Seiferth R, La Crosse virus in Aedes albopictus mosquitoes, Texas, USA, 2009. Emerg Infect Dis. 2010;16:856–8.
- Coetzer JA. The pathology of Rift Valley fever. II. Lesions occurring in field cases in adult cattle, calves and aborted foetuses. Onderstepoort J Vet Res. 1982;49:11–7 .
- Zhioua E, Moureau G, Chelbi I, Ninove L, Bichaud L, Derbali M, Punique virus, a novel phlebovirus, related to sandfly fever Naples virus, isolated from sandflies collected in Tunisia. J Gen Virol. 2010;91:1275–83.
- Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, Schirrmeier H, Novel orthobunyavirus in cattle, Europe, 2011. Emerg Infect Dis. 2012;18:469–72.
- Kim KH, Yi J, Kim G, Choi SJ, Jun KI, Kim NH, Severe fever with thrombocytopenia syndrome, South Korea, 2012. Emerg Infect Dis. 2013;19:1892–4.
- Bilk S, Schulze C, Fischer M, Beer M, Hlinak A, Hoffmann B. Organ distribution of Schmallenberg virus RNA in malformed newborns. Vet Microbiol. 2012;159:236–8.
- Marklewitz M, Handrick S, Grasse W, Kurth A, Lukashev A, Drosten C, Gouleako virus isolated from West African mosquitoes constitutes a proposed novel genus in the family Bunyaviridae. J Virol. 2011;85:9227–34.
- Marklewitz M, Zirkel F, Rwego IB, Heidemann H, Trippner P, Kurth A, Discovery of a unique novel clade of mosquito-associated bunyaviruses. J Virol. 2013;87:12850–65 .