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ANALYSES OF VARIABLE NUMBERS OF TANDEM REPEATS IN ORF 94 OF WHITE
SPOT SYNDROME VIRUS (WSSV) ISOLATES FROM OUTBREAKS IN MEXICAN SHRIMP
FARMS
Lucio Galaviz-Silva*, José Reyes González-Galavíz, José C. Ibarra-Gámez and Zinnia J. Molina-Garza
Facultad de Ciencias Biológicas
Universidad Autónoma de Nuevo León
Nuevo León, México
lgs12167@yahoo.com
White spot syndrome virus (WSSV) causes the most serious losses to commercial shrimp farming worldwide. Since its discovery
in Fujian, China (1991) the WSSV spread quickly from South-East-Asia to United States in 1995; Central, South America
and Mexico since 1999; and in 2002 the virus was also reported in France and Iran. Sequence comparisons of genomic regions
have shown very little variation in widely separated geographical isolates of WSSV. Recent publications have revealed a variable
numbers of tandem repeats (VNTRs) in the region encoding ORF94. In all WSSV isolates from Thailand or Vietnam,
ORF94 has tandem repeat units (RUs) of 54 bp, with a single nucleotide polymorphisms at position 48 (G or T). This genetic
variation may be useful as a genetic marker for strain identification and epidemiological studies and could be correlated with
the spread of WSSV.
The origin of the collected WSSV-infected shrimps (Litopenaeus vannamei) was shrimp ponds experiencing outbreaks of
WSSV, located in Sinaloa (Tarahumara Duran) and Sonora (Gutierrez Acuicola, Acuaproductiva and Remanentes). Infected tissues
were homogenized in TN buffer and DNA was isolated with proteinase K and sarkosyl, followed by phenol-chlorophorm
extraction. The primers used were derived from ORF94 of WSSV (Wongteerasupaya et al. 2003. DAO 54:253-257). PCR products
were purified and cloned in the pGEM-T easy vector system (Promega). Plasmids were prepared with DNA purification
system (Promega) and sequenced using an automated ABI 3130 Sequenced system (Applied Biosystems). Sequence alignment
and phylogenetic analysis were conducted using Clustal W and GeneDoc software. Our results from 4 outbreaks ponds shrimp
indicated the evolutionary variability of the WSSV. Based on this test, we assumed that each pond outbreak was caused by 4
different isolates of WSSV. An example is showing in Table 1 for fragments representing 2, 4, 6 and 7 repeats. Six and seven
RUs of 54 bp have been reported in Thai isolates, but the presence of 2 and 4 RU´s could suggest two WSSV isolates natives
from Mexico, in accordance a phylogenetic analysis (Fig. 1).
Table 1. Number of ORF94 repeats found in DNA extracts of samples from 4 WSSV
outbreaks ponds.
Samples Amplicon
size (bp)
No. of
repeats
T. DURAN 235 2
ACUAP 398 4
REM 506 6
GEZ
ACUICOLA
560 7
Fig 1. Bootstrap analysis of unrooted
phylogenetic trees of an isolate of WSSV from
Sonora (508) was located in a separated cluster.