Porcine Reproductive and Respiratory Syndrome Virus, Thailand, 2010-2011.
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.
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[degrees]C-42[degrees]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 [approximately equals] 4,000 pigs; nearly all pigs were sick within 1 week, and the mortality rate approached 60% within 2 weeks.
We performed PCR on serum samples from sick pigs to determine the presence of PRRSV; previously reported primers (7,70) were used to amplify partial Nsp2 and complete ORF5 genes. Products were cloned and sequenced at Bio Basic Inc. (Markham, Ontario, Canada). ClustalW (77) was used to align nucleotide and deduced amino acid sequences; 18 partial Nsp2 and 58 complete ORF5 genes were analyzed (Table 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).
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 (72). 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.
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 HPPRRSV (Figure 2). CH-1R is a classical PRRSV from PRC that does not possess the 2 discontinuous 30-aa deletions in Nsp2 (75). 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.
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.
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.
Author affiliation: Chulalongkorn University, Bangkok, Thailand
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Address for correspondence: Dachrit Nilubol, Chulalongkorn University, Veterinary Microbiology, Faculty of Veterinary Science, Henry Dunant Rd, Pathumwan, Bangkok 10230 Thailand; email: email@example.com
Table 1. Characteristics of pig farms with herds infected by PRRSV, Thailand, 2010-2011 * Herd ID, geographic Production location in country system UDT, northeast Farrow-to-wean UD, north Farrow-to-wean SCP, west Farrow-to-finish FDT, central Farrow-to-finish Herd ID, geographic Herd size, Used location in country no. sows attenuated North American PRRSV vaccine UDT, northeast 500 No UD, north 1,500 NK SCP, west 500 Yes FDT, central 1,200 Yes Herd ID, geographic No. (%) sow losses ([dagger]) location in country Died Culled UDT, northeast 48 (9.6) 102 (20.4) UD, north ND ND SCP, west 153 (30.6) 68 (13.6) FDT, central 29 (2.4) 80 (6.7) Herd ID, geographic No. (%) sows location in country that aborted UDT, northeast 264 (52.8) UD, north ND SCP, west 42 (8.4) FDT, central 104 (8.7) * PRRSV, porcine reproductive and respiratory syndrome virus; ID, identification; NK, not known; ND, no data available. ([dagger]) Data are for the 4 weeks following the start of the outbreak on each farm. ([double dagger]) Data are for the 4 weeks following the start of the outbreak on each farm. Table 2. PRRSV isolates obtained for sequence analysis from infected pig herds, Thailand, 2010-2011 * Isolate Isolate name no. 1 UD1210EU24/3 2 UD1210EU23/2 3 UD1210EU24/1 4 SCP1210EU7/79-A07 5 UD1210EU24/2 6 UD1210EU24/1 7 UD1210EU25/2 8 UD1210EU25/1 9 SCP0311EU1/3 10 SCP0311EU1/2 11 FDT0111EU2/3 12 FDT0111EU2/2 13 UD1210EU23/3 14 SCP0311EU1/1 15 FDT0111EU1/2 16 SCP0311EU3/1 17 FDT0111EU1/1 18 FDT0111EU2/1 19 SCP0311EU3/2 20 UDT0810US 5/28-160 21 UDT0810US 5/28-161 22 UDT0810US 5/28-162 23 UDT0810US 5/28-163 24 UDT0810US 5/28-164 25 UDT0810US 5/28-165 26 UDT0810US 5/28-166 27 UDT0810US 5/28-167 28 UD1210US/61-E03 29 UD1210US/61-F03 30 UD1210US/61-G03 31 UD1210US/62-H03 32 UD1210US/62-A04 33 UD1210US/62-B04 34 UD1210US-25-1 35 FDT10US-2-1 36 FDT10US-2-2 37 FDT10US-2-3 38 SCP1210-U.S.-7-79-1 39 SCP1210-U.S.-7-79-2 40 UDT0810 E02 41 UDT0810 C02 42 SCP1210 H02 43 SCP1210 B03 44 FST0311 C03 45 UD1210 (31)14-1 46 UD1210 (31)14-2 47 1-13(30)UD-1 48 UD1210 (31)13-2 49 FDT10 3/2 50 FDT 3/1 51 FDT 2/1 52 FDT 2/2 53 FDT 2/3 54 FST0311 54-4.1 55 FST0611 G03 56 FST0611 F03 57 FST0611 E03 58 US65DPI-2 Isolate Year and month no. collected 1 2010 Dec 2 2010 Dec 3 2010 Dec 4 2010 Dec 5 2010 Dec 6 2010 Dec 7 2010 Dec 8 2010 Dec 9 2011 Mar 10 2011 Mar 11 2011 Mar 12 2011 Mar 13 2010 Dec 14 2011 Mar 15 2011 Mar 16 2011 Mar 17 2011 Mar 18 2011 Mar 19 2011 Mar 20 2010 Dec 21 2010 Dec 22 2010 Dec 23 2010 Dec 24 2010 Dec 25 2010 Dec 26 2010 Dec 27 2010 Dec 28 2010 Dec 29 2010 Dec 30 2010 Dec 31 2010 Dec 32 2010 Dec 33 2010 Dec 34 2010 Dec 35 2010 Dec 36 2010 Dec 37 2010 Dec 38 2010 Dec 39 2010 Dec 40 2010 Dec 41 2010 Dec 42 2010 Dec 43 2010 Dec 44 2010 Dec 45 2010 Dec 46 2010 Dec 47 2010 Dec 48 2010 Dec 49 2010 Dec 50 2010 Dec 51 2010 Dec 52 2010 Dec 53 2010 Dec 54 2010 Dec 55 2010 Dec 56 2010 Dec 57 2010 Dec 58 2010 Dec Isolate Genotype no. 1 I 2 I 3 I 4 I 5 I 6 I 7 I 8 I 9 I 10 I 11 I 12 I 13 I 14 I 15 I 16 I 17 I 18 I 19 I 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Isolate Genes no. analyzed 1 ORF5 2 ORF5 3 ORF5 4 ORF5 5 ORF5 6 ORF5 7 ORF5 8 ORF5 9 ORF5 10 ORF5 11 ORF5 12 ORF5 13 ORF5 14 ORF5 15 ORF5 16 ORF5 17 ORF5 18 ORF5 19 ORF5 20 ORF5 21 ORF5 22 ORF5 23 ORF5 24 ORF5 25 ORF5 26 ORF5 27 ORF5 28 ORF5 29 ORF5 30 ORF5 31 ORF5 32 ORF5 33 ORF5 34 ORF5 35 ORF5 36 ORF5 37 ORF5 38 ORF5 39 ORF5 40 Partial Nsp2 41 Partial Nsp2 42 Partial Nsp2 43 Partial Nsp2 44 Partial Nsp2 45 Partial Nsp2 46 Partial Nsp2 47 Partial Nsp2 48 Partial Nsp2 49 Partial Nsp2 50 Partial Nsp2 51 Partial Nsp2 52 Partial Nsp2 53 Partial Nsp2 54 Partial Nsp2 55 Partial Nsp2 56 Partial Nsp2 57 Partial Nsp2 58 Partial Nsp2 Isolate GenBank no. accession no. 1 JX183110 2 JX183111 3 JX183112 4 JX183113 5 JX183114 6 JX183115 7 JX183116 8 JX183117 9 JX183118 10 JX183119 11 JX183120 12 JX183121 13 JX183122 14 JX183123 15 JX183124 16 JX183125 17 JX183126 18 JX183127 19 JX183128 20 JN255819 21 JN255820 22 JN255821 23 JN255822 24 JN255823 25 JN255824 26 JN255825 27 JN255826 28 JN255827 29 JN255828 30 JN255829 31 JN255830 32 JN255831 33 JN255832 34 JN255833 35 JN255834 36 JN255835 37 JN255836 38 JN255837 39 JN255838 40 JN255839 41 JN255840 42 JN255842 43 JN255841 44 JN255843 45 JN255844 46 JN255845 47 JN255846 48 JN255847 49 JN255848 50 JN255849 51 JN255852 52 JN255851 53 JN255850 54 JN255853 55 JN255854 56 JN255855 57 JN255856 58 JN255857 * PRRSV, porcine reproductive and respiratory syndrome virus; ORF, open reading frame; Nsp2, nonstructural protein 2.
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|Author:||Nilubol, Dachrit; Tripipat, Thitima; Hoonsuwan, Tawatchai; Kortheerakul, Khampee|
|Publication:||Emerging Infectious Diseases|
|Date:||Dec 1, 2012|
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