Porcine Reproductive and Respiratory Syndrome Virus, Thailand, 2010-2011.
Subject: Porcine reproductive and respiratory syndrome (Genetic aspects)
Infection (Genetic aspects)
Swine
Pork industry
Authors: Nilubol, Dachrit
Tripipat, Thitima
Hoonsuwan, Tawatchai
Kortheerakul, Khampee
Pub Date: 12/01/2012
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: Dec, 2012 Source Volume: 18 Source Issue: 12
Product: Product Code: 0213000 Hogs NAICS Code: 11221 Hog and Pig Farming SIC Code: 0213 Hogs; 2011 Meat packing plants; 2013 Sausages and other prepared meats
Geographic: Geographic Scope: Thailand Geographic Code: 9THAI Thailand
Accession Number: 313345658
Full Text: 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 (7-3). 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[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.

[FIGURE 2 OMITTED]

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.

[FIGURE 2 OMITTED]

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.

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.

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.

Author affiliation: Chulalongkorn University, Bangkok, Thailand

DOI: 10.3201/eid1811.111105

References

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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: dachrit@gmail.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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