Three WSSV isolates from Mexican shrimp farms were characterized by bioassay and through whole genome sequencing. WSSV isolates tested in bioassays generally induce the following clinical signs in infected white shrimp; lethargy, decreased food intake, discoloration of the hepatopancreas, cuticular loosening and red uropods [12]. Apart from these general clinical signs Shrimp infected with the CIAD isolate in the challenge bioassay showed 19% survival at 10 days post infection similar to the percentage obtained by Sekar, with 100% mortality until day 20 [34]. In contrast the, isolates from Angostura and Sonora induced 100% mortality between 3- and 10-days post-infection [35, 2].
The Angostura isolate produced the highest viral load of the three isolates (Fig. 1B). The differences between isolates however were in the range of published data reporting the viral load of WSSV in three Penaeus species (P. vannamei, P. stylirostris and P. monodon) obtained by real-time PCR in the range of 2 x 104 – 2 x 109 WSSV copies µg− 1 of total DNA, in infected shrimp [25]. A review to evaluate the risk associated for the shrimp trade, data on WSSV concentrations obtained from tissues of different species, including P. vannamei, indicated high viral load discharges at the beginning of mortality, with concentrations of 1 x 109 – 1 x 1010 [36].
The CIAD isolate presented a mortality risk percentage between 95% and 19% with a probability of 50% at 131 h and an overall survival rate of 19%. Isolate Angostura presented a constant 65% mortality risk percentage between 50 and 150 h, for a 50% survival at 150 h (Fig. 1A). For isolate Sonora, three drops in survival were observed, with risks of death between 64% and 43% from 50 to 150 h, and a probability of survival of 50% at 99 h. This is a possible sign of a faster progressing replication and therefore increased virulence of the isolate Sonora compared to isolate Angostura. Both isolates Angostura and Sonora induced 100% mortality observed at 168 HPI. Therefore, we tentatively classified the isolates as low virulence (CIAD), moderate virulence (Angostura) and high virulence (Sonora) from the mean post-infection lethal time according to [37].
The genomes of the isolates from Angostura and Sonora, presented a length within the estimated range for Mexican isolates from localities between Sinaloa and Nayarit (257,675 − 290,879 bp) [7]. To assess genomic differences of the isolates we were able to identify specific sites with differences to isolates from foreign and domestic outbreaks (Fig. 3A, B). Deletions in the WSSV genome are considered as enhancements of the adaptive response by WSSV as it is reasoned that deleted regions, do not encode proteins vital for WSSV pathogenicity or virulence [38]. The differences are possibly caused by recombination events mediated by transposons causing insertions or deletions [39]. An inversion is apparent among the 2008 Sonora and 2013 Angostura isolate as spotted in the MAUVE alignment of motives (Fig S2).
Since the isolate with the shortest sequence was the most virulent (Sonora), we analyzed the deletions. In our comparative analysis between the genome of the isolates of the present study, with sequences of international and national isolates, a group of deleted genes stands out: wsv237(VP41A) [40], wsv238(VP52A-VP51A) [41]. wsv242(VP41B) [42] and wsv493(ICP35-VP35) [43].
The VP41A envelope protein is known to interact with VP26, VP56 and VP28 envelope proteins [40, 44] and with the viral envelope protein VP51A (VP52A for AF332093) [45]. It has been suggested that VP26 is anchored to the envelope with its N-terminal hydrophobic region, as a C-terminal region binds to the nucleocapsid [46], while VP51A is a type II transmembrane protein, with a transmembrane domain highly hydrophobic at its N-terminus and an exposed C-terminus on the virion surface [47]. On the other hand, VP41A has only one transmembrane region at its C-terminal end, and this envelope protein is part of a group of 11 proteins that interacts with the chitin-binding protein of P. monodon (PmCBP) [16] which are known to participate in shrimp immune response [48]. It also interacts with preitrophin-like protein in L. vannamei (LvPT). LvPT is involved in the formation of the peritrophic membrane [49] and contains a chitin-binding domain facilitating binding of WSSV to the intestinal epithelium. It has been shown to promote infection, as silencing of LvPT decreased the viral load in pleopods, stomach and intestines in previously WSSV infected shrimp [50].
Therefore, the absence of the envelope proteins VP41A and VP51A could modify the usual conformation of the protein complex composed of VP26, VP28 and VP56, as well as the interactions of VP26 with VP51A reducing viral recognition by chitin-binding proteins such as PmCBP and LvPT. The observed deletions in Sonora and Angostura isolates could contribute to changes in the WSSV viral envelope and enhance virulence causing high mortality.
VP38 acts as a repressor of Penaeus japonicus caspase (Pjcaspase) transcription in M. japonicus, while VP41B activates its expression, regulating the apoptosis mechanism used by shrimp to control viral load [42]. Elevated Pjcaspasa gene expression of M. japonicus was observed in shrimp survival challenges while siRNA silencing of Pjcaspasa increased post-infection viral load [51]. So, deletion of VP41B would not allow timely induction of apoptosis of infected cells allowing the virus to replicate without limitation and thus having a major impact on the viral load control mechanism employed by WSSV.
The Sonora isolate has noticeable modifications on wsv129, wsv178, wsv204 and wsv249 as gain or loss of specific sequences equivalent to Repeated Motifs (RM) that strongly suggest a role in the infection process. A high variation from 3 to 20 in the number of repetitive sequences in WSV178 has been related to virulence [52, 53]. In contrast a lower mean number of repeated sequences was observed in outbreaks, where WSSV genotypes with 5, 6 and 7 of a 54 bp RM were those that predominated in semi-intensive shrimp crops [54]. In our study, we identified 4 and 3 repetitions of a 54 bp RM in WSV178 in the Angostura and Sonora genomes respectively. Previous studies identified wsv178 between rr1 and rr2, coding for enzymes that catalyze the formation of deoxyribonucleotide precursors, for the DNA replication process [55–57]. A study conducted in outbreaks which occurred in Sonora during 2005 and between 2010–2013, indicated a higher frequency of genotypes with less than 8 RMs in WSV178. In addition, during mortality events in Tastiota and Atanasia regions in 2008, WSSV genomes were observed which showed between 1 and 4 RM in WSV178. However, the mortality of these events was low at about 1.5% [58]. In outbreaks on farms in Culiacán Sinaloa in 2000, WSSV genomes with 4, 8, 9 and 12 repetitions in WSV178 were identified. WSSV genomes with 4 repetitions detected on a particular farm in Cruz Blanca, suggested the emergence of WSSV genomes with 4 repetitions in Mexico [59]. A study analyzing possible variations in WSV178, suggested that VNTRs in the different genotypes are stable during multiple infection cycles in crustaceans [60]. Therefore, our observation of RM distribution in the Angostura and Sonora isolates in combination with the reported observations on shrimp farms in Mexico could suggest that if the frequency of WSSV genomes with varying and in particular lower number of repetitions in WSV178 reaches statistically significant frequencies in a given population, WSSV outbreaks may develop [58].
A similarity difference < 30% was observed in wsv204 between the Sonora and Angostura isolates in comparison to CN01, with Angostura being distinctly different from Sonora and CN01. The gene wsv204 was identified in isolates CN01 and CN03, but not in CN02 who were respectively shown to be high, low and moderate virulent in bioassays by Li, et al (2016) [14].
We compared the most variable regions at the protein level from the sequence of isolate CN01 (KT995472.1) [61], identifying that wsv129, wsv178 and wsv497 could be encoding structure- and packaging-related proteins, and appear to be involved in enhancing WSSV virulence by promoting viral replication.
A difference in similarity < 30% in wsv204 was also observed between Sonora and Angostura isolates compared to CN01, with Angostura being clearly different from Sonora and CN01. wsv204 gene was identified in CN01 and CN03 isolates, but not in CN02, which respectively proved to be of high, low and moderate virulence in bioassays performed by Li, et al (2016)[14]. The function of wsv204, which contains RMs, is currently unknown, but its function could be related to transcription factors, as the RMs were similar to those found in the SOX gene member (wsv129), the EST domain transcription factor (wsv178), the tryptophan cluster factors (wsv129, wsv204 and wsv 249), the homeodomain motif (wsv 249) or the paired box factor motif (wsv497).
In eukaryotic organisms, sequences similar to these are found in transcriptional regulatory sites or factors that bind to DNA and control the activity of other genes. They are involved in the developmental process, as modulators or architectural components [62], in genome accessibility, DNA shape, and interact with many transcriptional partners, in activation of transcriptional programs [63], in neighbor cell communication, in cellular secretion of abrogation [64], and in tryptophan regulation. Tryptophan has a vital role in serotonin (5-HT) synthesis that regulates homeostasis, stress and cannibalism among other vital functions in shrimp [65]. Therefore, it is not entirely clear how wsv204 might be involved in regulatory processes that have an influence on WSSV replication success in host cells.