Emerg Infect DisEmerging Infect. DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention23171619355786811-110510.3201/eid1812.111105DispatchDispatchPorcine Reproductive and Respiratory Syndrome Virus, Thailand, 2010–2011Porcine Reproductive and Respiratory Syndrome VirusNilubolDachritTripipatThitimaHoonsuwanTawatchaiKortheerakulKhampeeAuthor affiliation: Chulalongkorn University, Bangkok, ThailandAddress for correspondence: Dachrit Nilubol, Chulalongkorn University, Veterinary Microbiology, Faculty of Veterinary Science, Henry Dunant Rd, Pathumwan, Bangkok 10230 Thailand; email: dachrit@gmail.com122012181220392043

Characterization of porcine reproductive and respiratory syndrome virus (PRRSV) isolates from pigs in Thailand showed 30-aa discontinuous deletions in nonstructural protein 2, identical to sequences for highly pathogenic PRRSV. The novel virus is genetically related to PRRSV from China and may have spread to Thailand through illegal transport of infectious animals from bordering countries.

Keywords: porcine reproductive and respiratory syndrome virusnonstructural protein 2ThailandArteriviridaevirusespigsporcine reproductive and respiratory syndromePRRSPRRSV

Porcine reproductive and respiratory syndrome (PRRS) has a substantial economic effect on the swine industry worldwide. PRRS virus (PRRSV), a member of the family Arteriviridae, is the etiologic agent of the syndrome. PRRSVs are divided into 2 distinct genotypes: type 1 and type 2. The genotypes have a similar genomic organization, and 10 open reading frames (ORFs) have been identified (13). Nonstructural protein 2 (Nsp2) and ORF5 are the most variable regions (4,5), coding for replicase protein and neutralizing epitope, respectively.

In general, PRRSV causes a disease characterized by reproductive failure in sows and respiratory infection in growing pigs. However, in June 2006, a disease characterized by high fever and associated with a high mortality rate emerged in the People’s Republic of China (PRC), resulting in the death of >20 million pigs (6). The disease, referred to as porcine high fever disease (PHFD), was caused by a new PRRSV variant with a unique hallmark: 2 discontinuous 30-aa deletions in Nsp2. The variant, identified as a highly pathogenic (HP) PRRSV, has subsequently become endemic in PRC (7), and it has spread to other countries, including Vietnam (8) and Lao People’s Democratic Republic (Lao PDR) (9).

It is thought that HP-PRRSV spread to Thailand early in 2010. Pigs on a small farm in Nong Khai, a border province in northeastern Thailand located near Lao PDR, showed signs of illness identical to those for PHFD. Within 2 weeks of the initial outbreak, similar clinical features were observed in pigs on 19 small farms in a nearby village. Since then, pigs exhibiting similar clinical signs have been observed in >100 herds in >20 provinces throughout Thailand. The causative agent was isolated from sick pigs and determined to be PRRSV.

To further our knowledge about PRRSV in Thailand, we genetically characterized partial Nsp2 and complete ORF5 genes of PRRSV isolates. In addition, we determined sickness and mortality rates on affected farms.

The Study

During August 2010–June 2011, outbreaks of disease consistent with PHFD were investigated on 4 pig farms located in geographically separate regions of Thailand (Table 1). Herds were selected for study if farm owners agreed to participate. Pigs in all 4 herds had similar clinical signs. In 3 herds, the outbreak was initially observed in the breeding herd and lasted for ≈1 month; most deaths occurred in the third week. In those 3 herds, the initial signs of illness in sows were inappetence and high fever (40°C–42°C), followed by reddened skin and abortion. Illness rates among sows were 100%, 50%, and 60%, respectively for the 3 herds. The highest number of deaths among the sows occurred within 1 week of onset of the first symptoms. The percentage of culled sows on the 3 farms was 20.4%, 13.6%, and 6.7%, respectively; abortion rates were 52.8%, 8.4%, and 8.7%, respectively (Table 1). The outbreak in the fourth herd was confined to nursery facilities housing ≈4,000 pigs; nearly all pigs were sick within 1 week, and the mortality rate approached 60% within 2 weeks.

Characteristics of pig farms with herds infected by PRRSV, Thailand, 2010–2011*
Herd ID, geographic location in countryProduction systemHerd size, no. sowsUsed attenuated North American PRRSV vaccineNo. (%) sow losses†
No. (%) sows that aborted
DiedCulled
UDT, northeastFarrow-to-wean500No48 (9.6)102 (20.4)264 (52.8)
UD, northFarrow-to-wean1,500NK NDNDND
SCP, westFarrow-to-finish500Yes153 (30.6)68 (13.6)42 (8.4)
FDT, centralFarrow-to-finish1,200Yes29 (2.4)80 (6.7)104 (8.7)

*PRRSV, porcine reproductive and respiratory syndrome virus; ID, identification; NK, not known; ND, no data available.
†Data are for the 4 weeks following the start of the outbreak on each farm.

We performed PCR on serum samples from sick pigs to determine the presence of PRRSV; previously reported primers (7,10) were used to amplify partial Nsp2 and complete ORF5 genes. Products were cloned and sequenced at Bio Basic Inc. (Markham, Ontario, Canada). ClustalW (11) was used to align nucleotide and deduced amino acid sequences; 18 partial Nsp2 and 58 complete ORF5 genes were analyzed (Table 2).

PRRSV isolates obtained for sequence analysis from infected pig herds, Thailand, 2010–2011*
Isolate no.Isolate nameYear and month collectedGenotypeGenes analyzedGenBank accession no.
1UD1210EU24/32010 DecIORF5JX183110
2UD1210EU23/22010 DecIORF5JX183111
3UD1210EU24/12010 DecIORF5JX183112
4SCP1210EU7/79-A072010 DecIORF5JX183113
5UD1210EU24/22010 DecIORF5JX183114
6UD1210EU24/12010 DecIORF5JX183115
7UD1210EU25/22010 DecIORF5JX183116
8UD1210EU25/12010 DecIORF5JX183117
9SCP0311EU1/32011 MarIORF5JX183118
10SCP0311EU1/22011 MarIORF5JX183119
11FDT0111EU2/32011 MarIORF5JX183120
12FDT0111EU2/22011 MarIORF5JX183121
13UD1210EU23/32010 DecIORF5JX183122
14SCP0311EU1/12011 MarIORF5JX183123
15FDT0111EU1/22011 MarIORF5JX183124
16SCP0311EU3/12011 MarIORF5JX183125
17FDT0111EU1/12011 MarIORF5JX183126
18FDT0111EU2/12011 MarIORF5JX183127
19SCP0311EU3/22011 MarIORF5JX183128
20UDT0810US_5/28–1602010 DecIIORF5JN255819
21UDT0810US_5/28–1612010 DecIIORF5JN255820
22UDT0810US_5/28–1622010 DecIIORF5JN255821
23UDT0810US_5/28–1632010 DecIIORF5JN255822
24UDT0810US_5/28–1642010 DecIIORF5JN255823
25UDT0810US_5/28–1652010 DecIIORF5JN255824
26UDT0810US_5/28–1662010 DecIIORF5JN255825
27UDT0810US_5/28–1672010 DecIIORF5JN255826
28UD1210US/61-E032010 DecIIORF5JN255827
29UD1210US/61-F032010 DecIIORF5JN255828
30UD1210US/61-G032010 DecIIORF5JN255829
31UD1210US/62-H032010 DecIIORF5JN255830
32UD1210US/62-A042010 DecIIORF5JN255831
33UD1210US/62-B042010 DecIIORF5JN255832
34UD1210US-25–12010 DecIIORF5JN255833
35FDT10US-2–12010 DecIIORF5JN255834
36FDT10US-2–22010 DecIIORF5JN255835
37FDT10US-2–32010 DecIIORF5JN255836
38SCP1210-U.S.-7–79–12010 DecIIORF5JN255837
39SCP1210-U.S.-7–79–22010 DecIIORF5JN255838
40UDT0810_E022010 DecIIPartial Nsp2JN255839
41UDT0810_C022010 DecIIPartial Nsp2JN255840
42SCP1210_H022010 DecIIPartial Nsp2JN255842
43SCP1210_B032010 DecIIPartial Nsp2JN255841
44FST0311_C032010 DecIIPartial Nsp2JN255843
45UD1210 (31)14–12010 DecIIPartial Nsp2JN255844
46UD1210 (31)14–22010 DecIIPartial Nsp2JN255845
471–13(30)UD-12010 DecIIPartial Nsp2JN255846
48UD1210 (31)13–22010 DecIIPartial Nsp2JN255847
49FDT10_3/22010 DecIIPartial Nsp2JN255848
50FDT_3/12010 DecIIPartial Nsp2JN255849
51FDT_2/12010 DecIIPartial Nsp2JN255852
52FDT_2/22010 DecIIPartial Nsp2JN255851
53FDT_2/32010 DecIIPartial Nsp2JN255850
54FST0311_54–4.12010 DecIIPartial Nsp2JN255853
55FST0611_G032010 DecIIPartial Nsp2JN255854
56FST0611_F032010 DecIIPartial Nsp2JN255855
57FST0611_E032010 DecIIPartial Nsp2JN255856
58
US65DPI-2
2010 Dec
II
Partial Nsp2
JN255857
*PRRSV, porcine reproductive and respiratory syndrome virus; ORF, open reading frame; Nsp2, nonstructural protein 2.

To determine the relationship of PRRSV from herds in Thailand to HP-PRRSV, we compared the partial Nsp2 amino acid sequences corresponding to aa 404–640 of ORF1a from the isolates from Thailand with sequences for HP-PRRSV from PRC and Vietnam and for strain VR2332. PRRSV isolates from Thailand possess 2 discontinuous 30-aa deletions (aa 482 and 534–562) that are identical to those in HP-PRRSV (Figure 1).

Alignment of amino acid sequences of partial nonstructural protein 2 corresponding to aa 404–640 of ORF1a for porcine reproductive and respiratory syndrome virus (PRRSV) isolates. Sequences are for PRRSV from infected herds in Thailand; highly pathogenic PRRSV isolates from the People’s Republic of China and Vietnam; and strain VR2332, the North American PRRSV prototype. Dashes represent deletions of amino acid residues.

To analyze the ORF5 genes of isolates from Thailand, PRC, and Vietnam, we constructed a phylogenetic tree by using the distance-based neighbor-joining method as implemented in MEGA4 (12). Bootstrap analysis was performed with 1,000 replicates. The tree showed the co-existence of HP-PRRSV types 1 and 2 in pigs in Thailand (Figure 2). Type 1 isolates from all 4 examined herds clustered with previously reported clusters (13,14) distinct from type 1 modified live vaccine viruses (Porcillis PRRSV and Amervac PRRS). In contrast, some of the type 2 isolates from affected herds in Thailand had formed a novel cluster distinct from previously reported clusters (13,14). The novel type 2 isolates from Thailand clustered with isolates from PRC and Vietnam that were associated with PHFD. Genetic similarities between the novel type 2 isolates and HP-PRRSV were 97.8%–98.5% and 96.5%–99.0% homologous at the nucleotide and amino acid levels, respectively. However, the novel type 2 isolates from Thailand were more closely related to the 07QN isolate from Vietnam (98.5% nt and 99.0% aa similarities) than to the isolates from PRC.

Phylogenetic analysis of types 1 and 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolates constructed by the neighbor-joining method and based on the nucleotide sequences of complete ORF5 genes. The analysis included the following: previous and recent isolates (solid red circles) from herds in Thailand that had an outbreak of HP-PRRSV; European references, including Lelystad virus (solid triangle) and 2 type 1 modified live vaccines (Porcilis PRRS, MSD Animal Health, Boxmeer, the Netherlands; and AMERVAC PRRS, Hipra, Spain) from Europe (open triangles); North American references, including VR2332 (solid diamond) and North American modified live vaccines (Ingelvac PRRS MLV, Boehringer Ingelheim, USA) (open diamonds); modified live vaccines from the People’s Republic of China (CH1R) (open square); isolates from the People’s Republic of China (boldface); and isolate from Vietnam (purple font). Scale bar indicate nucleotide substitutions per site; numbers at nodes represent the percentage of 1,000 bootstrap replicates.

We further investigated routes by which the virus spread. Before the outbreaks in Thailand, dead pigs were illegally transported from Lao PDR to an illegal slaughterhouse located not far from the farm where the first outbreak occurred, and the owner of the farm often visited the slaughterhouse. These findings suggest the movement of infected pigs in neighboring countries might play a role in introducing HP-PRRSV to new regions.

Infected pigs that were transported across the country and illegal slaughterhouses were the most likely routes of the spread of PRRSV within Thailand. The owners of several of the herds we investigated reported that pigs showed clinical signs within 1–2 days after trucks hauling dead pigs arrived at their farms. It was reported that dead pigs from herds in outbreak areas had been loaded on the trucks the day before they arrived at these farms. In Thailand, unlike in the United States, dead pigs are not composted, buried, or incinerated; instead, they are sold to feed catfish. Truckers associated with this trade visit pig farms to buy and transport dead pigs. These trucks are not washed, so they are a potential source of contamination on farms.

Another source for the introduction of the novel PRRSV into Thailand could be an unapproved vaccine from PRC. The phylogenetic tree demonstrated that 3 recent isolates from Thailand (UD1210US/61-F03, UD1210US/61-G03, and UD1210US/61-E03) were more genetically related to CH-1R (an attenuated vaccine strain used in PRC) than HP-PRRSV (Figure 2). CH-1R is a classical PRRSV from PRC that does not possess the 2 discontinuous 30-aa deletions in Nsp2 (15). Furthermore, CH-1R is an attenuated PRRSV vaccine strain in PRC, and there is evidence that it has been illegally smuggled into Thailand. Thus, it is possible that this modified live virus from PRC may have been administered to the herd involved in the initial outbreak in Thailand and may have been the source of the novel PRRSV strain that caused the outbreak.

Conclusions

A novel PRRSV, which is genetically related to PRRSV isolates from PRC, has been introduced into Thailand. Sequences of Nsp2 revealed a unique 30-aa discontinuous deletion in the novel virus, a hallmark of HP-PRRSV. The virus may have been introduced into Thailand through the illegal transport of infected materials from bordering countries, more specifically, from Vietnam to Thailand through Lao PDR. This scenario is supported by our finding that PRRSV isolates from Thailand are more homologous with an isolate from Vietnam than with isolates from PRC. The cause of viral spread within Thailand may have been the movement of infected live and dead pigs across the country.

Suggested citation for this article: Nilubol D, Tripipat T, Hoonsuwan T, Kortheerakul K. Porcine reproductive and respiratory syndrome virus, Thailand, 2010–2011. Emerg Infect Dis [Internet]. 2012 Dec [date cited]. http://dx.doi.org/10.3201/eid1811.111105

Acknowledgments

We thank Brad and Eileen Thacker for reviewing the manuscript.

Funds for this research were provided by the Thailand Research Fund (project nos. MRG5080323 and IUG5080001) and by the government of Thailand (budget year 2010).

Dr Nilubol is an assistant professor in the Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University. His research interests are PRRSV and porcine epidemic diarrhea.

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