Co-Infection of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in the Wild Crustaceans of Andaman and Nicobar Archipelago, India

The present study was intended to screen the wild crustaceans for co-infection with Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Andaman and Nicobar Archipelago, India. We screened a total of 607 shrimp and 110 crab samples using a specific polymerase chain reaction, and out of them, 82 shrimps (13.5%) and 5 (4.5%) crabs were found positive for co-infection of IHHNV and WSSV. A higher rate of co-infection was observed in Penaeus monodon and Scylla serrata than other shrimp and crab species. The nucleotide sequences of IHHNV and WSSV obtained from crab in this present study exhibited very high sequence identity with their counterparts retrieved from various countries. Histopathological analysis of the infected shrimp gill sections further confirmed the eosinophilic intra-nuclear cowdry type A inclusion bodies and basophilic intra-nuclear inclusion bodies characteristics of IHHNV and WSSV infections, respectively. The present study serves as the first report on co-infection of WSSV and IHHNV in Andaman and Nicobar Archipelago, India and accentuates the critical need for continuous monitoring of wild crustaceans and appropriate biosecurity measures for brackishwater aquaculture.


Introduction
Viruses pose a major threat to human and animal health in comparison to other pathogens across the globe. The rapid growth of the aquaculture sector was stagnated by the incidence of diseases mainly caused by infectious viruses leading to crop losses and economic impacts. Globally, more than 20 viruses have so far been recorded for shrimp and some of these are found to be responsible for epidemics severely impacting its health [1,2]. White Spot Syndrome Virus (WSSV) and Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) are the widespread and most prevalent pathogens causing mass mortality and growth retardation leading to huge economic losses in crustaceans [3][4][5]. WSSV was known to incur losses to the tune of $10 billion in penaeid culture [6,7] whereas IHHNV is known to cause runting syndrome and reduce the market value of penaeid shrimps up to 10% to 50% [8,9]. Due to the worldwide impact, both were collected with mean carapace length (cm) and mean weight (g) ranging from 4.8 ± 0.2 to 8.4 ± 1.0 and 71.4 ± 6.7 to 350.9 ± 71.2, respectively. Tissues such as pleopod and gill in case of shrimp samples and cheliped muscle and gill in case of crab samples were dissected out and preserved in 90% ethanol for further use. Similarly, the tissue samples were also preserved in Davidson's fixative for histopathology.

DNA Isolation and PCR Amplification of Target Viruses
DNA was isolated from the tissue samples using DNAzol reagent (Invitrogen, Waltham, MA, USA) by following the manufacturer's instructions. The extracted DNA was used for the PCR detection of IHHNV and WSSV as per the protocols of Tang et al. [42] and Kimura et al. [43], respectively. Internal positive and negative controls were included in all the reactions. The amplified products were resolved in agarose gel electrophoresis and visualised using a gel documentation system (Bio-Rad, Hercules, CA, USA).

Sequencing of PCR Products and Bioinformatic Analysis
IHHNV and WSSV positive PCR products obtained from crab samples were purified and sequenced using ABI 3730xl DNA analyzer (Eurofins Genomics India Pvt. Ltd., Bengaluru, India). The obtained sequences were analysed for finding homology with other sequences using BLAST program in NCBI database (Bethesda, MD, USA). The phylogenetic tree was constructed for the nonstructural protein 1 (NS1) gene of IHHNV (309 bp) and WSV285 gene of WSSV (570 bp) separately by using MEGA X software version 10.1.7 [44]. The multiple sequence alignment of nucleic acid sequences of the viruses was generated by using the MUSCLE program with default settings. Then 'Find Best DNA Models' tool in the MEGA X software version 10.1.7 was used to identify the best-fitting model for

DNA Isolation and PCR Amplification of Target Viruses
DNA was isolated from the tissue samples using DNAzol reagent (Invitrogen, Waltham, MA, USA) by following the manufacturer's instructions. The extracted DNA was used for the PCR detection of IHHNV and WSSV as per the protocols of Tang et al. [42] and Kimura et al. [43], respectively. Internal positive and negative controls were included in all the reactions. The amplified products were resolved in agarose gel electrophoresis and visualised using a gel documentation system (Bio-Rad, Hercules, CA, USA).

Sequencing of PCR Products and Bioinformatic Analysis
IHHNV and WSSV positive PCR products obtained from crab samples were purified and sequenced using ABI 3730xl DNA analyzer (Eurofins Genomics India Pvt. Ltd., Bengaluru, India). The obtained sequences were analysed for finding homology with other sequences using BLAST program in NCBI database (Bethesda, MD, USA). The phylogenetic tree was constructed for the nonstructural protein 1 (NS1) gene of IHHNV (309 bp) and WSV285 gene of WSSV (570 bp) separately by using MEGA X software version 10.1.7 [44]. The multiple sequence alignment of nucleic acid sequences of the viruses was generated by using the MUSCLE program with default settings. Then 'Find Best DNA Models' tool in the MEGA X software version 10.1.7 was used to identify the best-fitting model for constructing a phylogenetic tree. Based on the lowest Bayesian information criterion score, the Kimura 2-parameter model with discrete gamma distribution (K2 + G) and the Tamura 3-parameter (T92) models were identified as the best model for IHHNV and WSSV, respectively. Then the phylogenetic tree was constructed with the maximum likelihood method using the selected models. The neighbour-joining and BioNJ algorithms were used for obtaining the initial tree required for the heuristic search. Missing data and gaps were completely deleted, and the test of phylogeny was done using the bootstrap method with 1000 replications. The online tool interactive tree of life (iTOL, http://itol.embl.de/ accessed on 7 July 2021) was used to visualize the final tree [45].

Histopathological Analysis
Tissue samples were processed for histopathology as per the standard protocol [46]. Finally, the tissue sections (5 µ) were stained by using hematoxylin and eosin.

Analysis of Data
Statistical software SPSS 16.0 was used to calculate the descriptive statistics such as mean and standard error of length (cm) and weight (g) of the collected shrimp and crab samples. The odds ratio is used to measure the strength of association between the exposure and an outcome. The odds ratio values for co-infection of IHHNV and WSSV was computed by using MedCalc statistical software version 20.009 at 95% confidence interval with z statistics and significance level [47].

Co-Infection of IHHNV and WSSV in Wild Shrimps
A total of 607 shrimp samples comprising of four species were collected from seven fish landing centres located in three districts of Andaman and Nicobar Archipelago (Table 1  and Supplementary Table S1). The PCR analysis revealed that out of 607 shrimp sampled, 123 (20.3%) were found positive for IHHNV alone, 46 (7.6%) were positive for WSSV alone and co-infection of both IHHNV and WSSV was found in 82 (13.5%). Varying degrees of co-infection with IHHNV and WSSV were found across the landing centres and among the shrimp species. Among the districts, the number of shrimp samples with co-infection was higher in South Andaman (n = 52) followed by North and Middle Andaman (n = 20) and Nicobar (n = 10) whereas the positivity rate of co-infection in relation to the total number of shrimp samples collected in that particular district was found higher in Nicobar (40%) followed by South Andaman (18.8%) and North and Middle Andaman (6.5%). Among the shrimp species, co-infection was higher in Penaeus monodon (n = 72) followed by P. merguiensis (n = 6), P. indicus (n = 3) and P. penicillatus (n = 1). Within P. monodon, the positivity rate of co-infection in relation to the total number of P. monodon samples collected in that particular district was found higher in Nicobar (50%) followed by South Andaman (22.7%) and North and Middle Andaman (10.8%). Among the WSSV positive shrimp samples, only 12 samples were found to be positive by first step PCR comprising of P. monodon (n = 8), P. merguiensis (n = 3) and P. penicillatus (n = 1) and the remaining samples were nested PCR positive for WSSV.

Co-Infection of IHHNV and WSSV in Wild Crabs
Altogether, 110 crab samples comprising of four species were collected from a total of eight fish landing centres ( Table 2 and Supplementary Table S2). The PCR analysis revealed that out of 110 crabs sampled, 19 (17.3%) were found positive for IHHNV, 9 (8.2%) were positive for WSSV and co-infection of both IHHNV and WSSV was found in 5 (4.5%). Varying degrees of co-infection with IHHNV and WSSV were found across the landing centres and among the crab species. Among the districts, the number of crab samples with co-infection was higher in South Andaman (n = 2) and Nicobar (n = 2) followed by North and Middle Andaman (n= 1) whereas the positivity rate of co-infection in relation to the total number of crab samples collected in that particular district was found higher in Nicobar (11.8%) followed by South Andaman (3.2%) and North and Middle Andaman (3.2%). Among the crab species, co-infection was higher in S. serrata (n = 3) followed by S. tranquebarica (n = 1) and P. pelagicus (n = 1) whereas co-infection was not observed in Viruses 2021, 13, 1378 5 of 15 P. reticulatus. On the other hand, P. reticulatus was tested positive for IHHNV only. Within S. serrata, the positivity rate of co-infection in relation to the total number of S. serrata samples collected in that particular district was found higher in Nicobar (10%) followed by North and Middle Andaman (3.2%) and South Andaman (2.6%). All the WSSV positive crab samples were found to be detected only by nested PCR.

Analysis of Odds Ratio
The odds ratio was evaluated in order to ascertain the strength of association between IHHNV and WSSV incidences during the co-infection. In P. monodon and P. merguiensis, the incidences of IHHNV infection are significantly associated with the incidences of WSSV infection whereas no significant association was found in P. indicus and P. penicillatus (Table 3). Among all the crab species, no significant association was found between the incidences of IHHNV and WSSV (Table 3). Subsequently, a significant association between the incidences of IHHNV and WSSV was observed in shrimp sampling locations such as Junglighat and Lohabarrack landing centres from South Andaman, Kalighat from North and Middle Andaman and Campbell Bay from Nicobar (Table 4). No significant association was observed in other shrimp sampling locations. Consequently, no significant association between the incidences of IHHNV and WSSV was observed in any of the crab sampling locations (Table 4). Table 3. Odds ratio values for co-infection of IHHNV and WSSV in different wild shrimp and crab species. The confidence interval was calculated by using MedCalc statistical software. If the 95% confidence interval excludes 1.0, the association is statistically significant at p < 0.05. If the 95% confidence interval includes 1.0, the association is not statistically significant at p < 0.05.

Sequence Analysis of IHHNV and WSSV
The BLAST analysis of the nucleic acid sequence of IHHNV obtained from crab of Andaman and Nicobar Archipelago (GenBank accession number MZ098150) in the present study revealed 99.68% sequence identity to the sequences of IHHNV reported from Andaman AN-01 (KU992382), China (KU373072), Australia (KM272863), Peru (MW357700), Vietnam (JX840067), Texas (MN968717), Florida (MN968716), Taiwan (AY355308) and Ecuador (AY362548), 99.67% with Philippines (KY273368), 99.35% with Venezuela (KM485615) and Brazil (KJ862253), 99.03% with South Korea (JN377975), 96.44% with India (MH252959), 96.12% with Thailand (AY362547) and 95.78% with Indonesia (KU215793). Phylogenetic analysis showed that the IHHNV sequence obtained from the present study revealed the closest relationship with other IHHNV sequences retrieved from various countries (Figure 2 and Supplementary Table S3). Similarly, the IHHNV sequence derived from the present study has emerged in the same clade as the earlier reported IHHNV sequence (Genbank accession number KU992382) obtained from the wild shrimps of Andaman and Nicobar Islands [41].  Similarly, the BLAST analysis of WSSV sequence obtained from S. serrata of Andaman and Nicobar Archipelago (GenBank accession number MZ098151) in the present study showed 100% sequence identity with the WSSV sequence reported earlier from wild shrimps of Andaman AN-01 (KX980155), 99.82% with USA (MN840357), Mexico (KU216744), Brazil (MG264599), China (KY827813), South Korea (JX515788), Thailand (AF369029), Taiwan (AF440570) and India (MH883319), 99.65% with Ecuador (MH090824) and Australia (MF768985). Phylogenetic analysis revealed that WSSV obtained from the present study exhibited the closest relationship with WSSV sequences obtained from other countries (Figure 3 and Supplementary Table S4). Moreover, as mentioned earlier, the WSSV sequence obtained from this present study clade with the WSSV sequence (Genbank accession number KX980155) reported earlier from the wild shrimps of Andaman and Nicobar Islands [40].

Histopathology
Histopathology analysis of the infected animals revealed a severe infection with hypertrophied and pyknotic nuclei with eosinophilic, basophilic and cowdry type A intranuclear inclusion bodies. The infected shrimp gill section revealed the eosinophilic intranuclear cowdry type A inclusion bodies inside the hypertrophied nucleus pathognomonic to IHHNV and eosinophilic to basophilic intra-nuclear inclusion bodies suggestive of WSSV infection (Figure 4). Further, the infected shrimp gill section showed severe infection of WSSV with characteristics intra-nuclear inclusion bodies inside the hypertrophied nucleus ( Figure 4). The WSSV characteristic inclusion bodies were fully developed, more basophilic and granular in texture. No occlusion bodies were observed in the gill sections.

Histopathology
Histopathology analysis of the infected animals revealed a severe infection with hypertrophied and pyknotic nuclei with eosinophilic, basophilic and cowdry type A intranuclear inclusion bodies. The infected shrimp gill section revealed the eosinophilic intranuclear cowdry type A inclusion bodies inside the hypertrophied nucleus pathognomonic to IHHNV and eosinophilic to basophilic intra-nuclear inclusion bodies suggestive of WSSV infection ( Figure 4). Further, the infected shrimp gill section showed severe infection of WSSV with characteristics intra-nuclear inclusion bodies inside the hypertrophied nucleus ( Figure 4). The WSSV characteristic inclusion bodies were fully developed, more basophilic and granular in texture. No occlusion bodies were observed in the gill sections.

Discussion
Among the pathogens, WSSV is considered as the most devastating pathogen as it brings mass mortality and crop losses in the crustacean aquaculture sector. The WSSV was first reported in 1992, since then it has emerged as the dreadful pathogen known to infect a majority of the shrimp and crab species in various countries and remains a global challenge to the aquaculture industry [24,48]. Whereas IHHNV may not cause mass mortality but known to incur slow and stunted growth, resulting in severe economic losses [49]. As compared with the research efforts on crustacean viral diseases in the aquaculture sector, limited reports are available on the spread or contamination of viruses in wild crustaceans [26]. Further, the studies on the co-infection of viruses in wild crustaceans are still very limited. The summary on the single and co-infection of various viruses in the wild crustaceans reported from different countries is provided in Table 5. The present study has been instigated in order to understand the level of co-infection of IHHNV and WSSV in wild crustaceans with a focus on shrimps and crabs in the Andaman and Nicobar Archipelago.

Discussion
Among the pathogens, WSSV is considered as the most devastating pathogen as it brings mass mortality and crop losses in the crustacean aquaculture sector. The WSSV was first reported in 1992, since then it has emerged as the dreadful pathogen known to infect a majority of the shrimp and crab species in various countries and remains a global challenge to the aquaculture industry [24,48]. Whereas IHHNV may not cause mass mortality but known to incur slow and stunted growth, resulting in severe economic losses [49]. As compared with the research efforts on crustacean viral diseases in the aquaculture sector, limited reports are available on the spread or contamination of viruses in wild crustaceans [26]. Further, the studies on the co-infection of viruses in wild crustaceans are still very limited. The summary on the single and co-infection of various viruses in the wild crustaceans reported from different countries is provided in Table 5. The present study has been instigated in order to understand the level of co-infection of IHHNV and WSSV in wild crustaceans with a focus on shrimps and crabs in the Andaman and Nicobar Archipelago.  Infections of IHHNV and WSSV in the wild crustaceans of Andaman and Nicobar Archipelago have been reported by few researchers with various levels of prevalence [39][40][41]. A study conducted by ICAR-Central Institute of Brackishwater Aquaculture during 2004 in the South Andaman district revealed the presence of WSSV in wild crustaceans such as P. monodon, P. semisulcatus, F. merguiensis and Portunus pelagicus by nested PCR (unpublished). WSSV was also detected by either first step or nested PCR in wildcaught P. monodon, F. merguiensis and Scylla serrata samples collected from South and North Andaman districts during the period 2006 to 2007 [39]. In our earlier studies, WSSV was detected in wild P. monodon samples collected from South Andaman and Nicobar districts whereas IHHNV was detected in wild P. monodon samples collected from South Andaman, North and Middle Andaman, and Nicobar districts during the period 2015 to 2016 [40,41]. However, there is no information available on the co-infection of crustacean viruses, namely, IHHNV and WSSV from Andaman and Nicobar Archipelago. In the present study conducted during 2018 to 2020 by covering all the three districts of Andaman and Nicobar Archipelago, co-infection of IHHNV and WSSV was detected in all the tested shrimp species viz., P. monodon, P. merguiensis, P. indicus and P. penicillatus whereas in the case of crab, co-infection was observed only in S. serrata, S. tranquebarica and P. pelagicus. These findings clearly indicate that long-term surveillance with more samples, species and geographical coverage provides the tangible indication of the establishment of co-infection of IHHNV and WSSV in the wild crustaceans of Andaman and Nicobar Archipelago. Similarly, various levels of prevalence of these viruses were recorded elsewhere in wild crustaceans [26,[55][56][57][58] and shrimps can also serve as asymptomatic carriers which may remain infected for a long period of time [6,55]. Disease surveillance is a continuous process which requires regular monitoring in order to understand the prevalence of viruses in the wild population which may vary over the period of time based on various factors [50,54,55]. It is noteworthy to mention that in the present study, co-infection of IHHNV and WSSV was found in wild crustaceans of all the three districts of Andaman and Nicobar Archipelago which provides further insights on the spread of these viruses in the wild conditions.
In this present study, the prevalence of IHHNV was higher than that of WSSV in both wild shrimps and crabs. It is further substantiated that the low level of WSSV infections may be due to the season, temperature, co-infection, or pre-infection with IHHNV and host as evidenced from the earlier study [9]. The IHHNV infection mechanism and its virulence varies with different species and developmental stages of the host [59]. Further, the majority of the obtained WSSV infections in wild shrimp and crab species are restricted to nested PCR positive which may be due to the fact that it is hard to find acute infection and dead animals as weak and moribund animals are certainly susceptible prey for various predators in the wild environment [9]. Further, the shrimp and crab samples detected with WSSV by nested PCR did not show any clinical white spots. It has been reported that the host affected with low level of infection may not show any clinical signs and non-penaeid species such as crab mostly have sub-clinical infection in the natural environment [10]. The earlier reports on WSSV infection also substantiates the low level of infection or nested PCR positive as obtained in this present study [39,40]. Co-infection was found higher in P. monodon than any other shrimp species which is also validated with the earlier reports in which the infection of WSSV and IHHNV was higher in P. monodon [39][40][41] and clearly indicates that these Islands alike Southeast Asian countries are within the geographical range of these viruses [3,17,60]. Natural viral infections with a wide range of prevalence were also observed in wild crabs of the Asian region [13,24,51,61]. On the other hand, the low level of co-infection in wild crabs observed in the present study cannot be overlooked as the crabs may serve as reservoirs to spread these viruses [23,48]. It is reported that both horizontal and vertical transmissions are responsible for these viral disease transmissions in the natural environment [3,55,58,[62][63][64] and it is difficult to gauge the impact of these viral diseases in wild crustaceans [54]. The odds ratio analysis revealed that the presence or incidence of IHHNV infection was found to exacerbate the incidence of WSSV infection and vice versa in shrimp species such as P. monodon and P. merguiensis and also for the sampling locations such as Junglighat, Lohabarrack, Kalighat and Campbell Bay. However, preinfection with IHHNV was reported to reduce the WSSV incidences in wild crustaceans [9], which warrants further investigation on the biological association between IHHNV and WSSV infection in wild crustaceans.
The high identity between IHHNV and WSSV sequences derived from crab samples in the present study and IHHNV and WSSV sequences reported from wild shrimps of Andaman and Nicobar Islands further confirmed that these particular strains of IHHNV and WSSV are widely prevalent among wild crustaceans of Andaman and Nicobar Archipelago. The IHHNV and WSSV infections were also confirmed by histopathology which further supported the PCR results. Histopathology of the infected shrimp gill tissues revealed the eosinophilic intra-nuclear cowdry type A inclusion bodies confirming the IHHNV infection and eosinophilic to basophilic and more basophilic intra-nuclear inclusion bodies denoting the WSSV infection as reported in earlier studies to support the specific infections [55,65]. It is speculated that the possible establishment of IHHNV and WSSV infections in wild crustaceans of Andaman and Nicobar Archipelago, India might be due to natural oceanic currents or ballast/ bilge water from the ships navigating through the Islands [9,28]. As Andaman and Nicobar Archipelago is located in close proximity to the Southeast Asian countries and sharing the Andaman Sea, there may be possibilities that the infected host or vectors might be carried away by the water currents. This speculation is in line with the previous study, where the alongshore currents from Brazil transported the infectious agents up to 5500 km, and also the fluctuation in current impacts the prevalence of WSSV in Brazil [9,66,67]. Similarly, the ballast/ bilge water from the transporting vessels might have contributed to the transmission of infections through the infected host or vectors. However, further studies are also recommended in order to validate these possibilities to arrive at a definitive conclusion.

Conclusions
This is the first report on the co-infection of IHHNV and WSSV in wild crustaceans of Andaman and Nicobar Archipelago, India, which provides further information on the extent of the spread of these viruses and biogeography among the wild populations. It is noteworthy to mention that the research on viral infections in wild crustaceans should be given due priority as such viral infections could possibly endanger the potential of brackishwater aquaculture sector in these Islands. Therefore, continuous disease surveillance or monitoring is the need of the hour to gauge the dynamics of the prevalence of pathogens in wild crustaceans and their associated ecosystems. Further, biosecurity measures should be considered as the cardinal sign before venturing into the brackishwater aquaculture sector in Andaman and Nicobar Archipelago towards sustainable aquaculture and ecosystem health management.