Complete nucleic acid sequence of Penaeus monodon densovirus (PmDNV) from India

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Abstract

The complete nucleic acid sequence of the Penaeus monodon densovirus (PmDNV) from India was characterized. Analysis of the whole genome, consisting of 6310 bp revealed the presence of three open reading frames (ORFs), comprising 1281 bp, 1734 bp and 2460 bp, respectively. The complete genome and amino acid sequences of three proteins viz., NS1, NS2 and VP were compared with PmDNV from Thailand, PmergDNV from Australia and other partial sequences in GenBank, respectively. Highest nucleotide similarity was observed with the Thai strain (88%), while 33, 32 and 91 amino acid substitutions were observed in the NS2, NS1 and VP, respectively. Phylogenetic analysis of shrimp, insect and vertebrate parvovirus sequences revealed that the Indian PmDNV is more closely related to Thai isolates than all other parvoviruses reported so far.

Introduction

Penaeus monodon densovirus (PmDNV) or hepatopancreatic parvovirus (HPV) of penaeid shrimp is an emerging shrimp virus that causes considerable economic loss in shrimp culture all over the world (Flegel, 2006). It was first reported in 1984 in wild Penaeus merguiensis and Fenneropenaeus indicus from Singapore (Chong and Loh, 1984). Later, Lightner and Redman (1985) reported the presence of P. monodon densovirus (PmDNV) in postlarvae of the penaeid shrimp and Fenneropenaeus chinensis from Korea (HPVchin). HPV infected shrimp shows reduced growth rates without showing any visible signs of disease (Flegel et al., 1999). The known host range of hepatopancreatic parvovirus (HPV) encompasses at least 10 penaeid shrimps around the world and the freshwater prawn, Macrobrachium rosenbergii (Lightner and Redman, 1985, Anderson et al., 1990, Lightner, 1996, Gangnonngiw et al., 2009). It is believed to be transmitted both vertically and horizontally (Lightner and Redman, 1992). In India heavy mortality of P. monodon postlarvae in hatcheries was reported due to multiple virus infection that included HPV infection along with monodon baculovirus (MBV) and white spot syndrome virus (WSSV) infection (Manivannan et al., 2002, Umesha et al., 2006).

HPV of penaeid shrimps are included in the family Parvoviridae under the sub-family Densovirinae. It is non-enveloped icosahedral virions, averaging 22–24 nm in diameter and containing linear, minus single-stranded DNA (Bonami et al., 1995). It generally infects epithelial cells of anterior midgut and hepatopancreas of shrimp (Lightner et al., 1993). Diagnosis of HPV infection includes histology (Lightner, 1996), transmission electron microscopy (TEM), in situ hybridization (Pantoja and Lightner, 2001, Phromjai et al., 2002), gene probes (Lightner et al., 1994, Mari et al., 1995), polymerase chain reaction (PCR) (Sukhumsirichart et al., 1999, Pantoja and Lightner, 2000, Phromjai et al., 2001), nested PCR (Manjanaik et al., 2005), real-time PCR (La Fauce et al., 2007a), polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) (Sukhumsirichart et al., 2002) and loop-mediated isothermal amplification (LAMP) (Nimitphaka et al., 2008). However, the absence of gross signs specific for HPV makes the diagnosis difficult particularly in the presence of other pathogens that may mask the effect (Manivannan et al., 2002, Chayaburakul et al., 2004). Even under TEM, particles of HPV are very difficult to visualize because of their size and shape which is very similar to normal cellular components such as the ribosome (Pantoja and Lightner, 2001).

Till date only two completely sequenced genomes of HPV are available, and these have been reported from Thailand (P. monodon densovirus – PmDNV) (GenBank Accession No. DQ002873) and Australia (P. merguiensis densovirus – PmergDNV) (GenBank Accession No. DQ458781) (Sukhumsirichart et al., 2006, La Fauce et al., 2007b). Partial sequences are available from Korea (HPVchin) (GenBank Accession No. AY008257), India (HPVsemi) (Manjanaik et al., 2005), Tanzania (GenBank Accession No. EU588991), New Caledonia (GenBank Accession No. EU346369) and Madagascar (GenBank Accession No. EU247528) (Bonami et al., 1995, Phromjai et al., 2002, Manjanaik et al., 2005, Tang et al., 2008). Comparison of these sequences from GenBank suggests that HPV isolated from different shrimp species and/or different geographical region is genetically different (La Fauce et al., 2007b). Complete nucleotide analysis of HPV of P. monodon from Thailand (PmDNV) revealed it be of 6321 nucleotides, suggesting it to be the largest among the parvoviruses reported so far. The genome contained three major open reading frames (ORFs), all in the complementary strand (plus) of viral genome and two non-coding termini (Sukhumsirichart et al., 2006). The HPV genome from Australia was 6299 kb long (La Fauce et al., 2007b) and differed considerably from that of PmDNV genome from Thailand. The Thai genome differed from HPVchin approximately 30% (Phromjai et al., 2002). These findings suggest that HPV strains may vary based on their host shrimp species. Here we report the complete nucleotide sequence of P. monodon densovirus (PmDNV) from India and its relationship to other known arthropod parvoviruses. The present study clearly shows that the P. monodon densovirus genome from India and Thailand showed sequence variation of 22% though it is from the same host species.

Section snippets

Sample collection

Eighty-three shrimp samples (P. monodon), which showed a reduced growth rate, were collected from grow out farms on the east coast (Andhra Pradesh and Tamil Nadu) and the west coast (Karnataka, Kerala) of India during the interval between December 2006 and January 2008. Three shrimps from each of the slow growth affected ponds were pooled and considered as an individual sample. Ninety-four postlarval (PL-10 to PL-20) samples (pool of 30) collected from different hatcheries were also analyzed

Results

177 samples were collected and analyzed for the presence of PmDNV by nested PCR. The first step PCR generated an amplicon of 441 bp, where as the second step PCR generated an amplicon size of 265 bp. Out of the 94 P. monodon postlarval samples collected from different hatcheries in India, six (6.3%) were positive for PmDNV by first step PCR and eight (8.5%) were positive for PmDNV by nested PCR. However, none of the 83 shrimp samples collected from ponds showing slow growth along the east and

Discussion

This is the first report of complete nucleotide sequence (6310 bp) of P. monodon densovirus (PmDNV) from India. The Indian isolate of PmDNV is the third strain of HPV to be sequenced completely. The other two strains are PmDNV of P. monodon from Thailand (Sukhumsirichart et al., 2006) and PmergDNV of P. merguensis from Australia (La Fauce et al., 2007b). Analysis of the complete genome sequences of the Indian isolate of PmDNV suggests that they are closely related to Thai PmDNV isolate (6321 bp)

Acknowledgement

The authors are grateful to the “Indo-Australia Biotechnology Fund”, for financial support for this work.

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