Genetic Relatedness of Infectious Hypodermal and Hematopoietic Necrosis Virus Isolates, United States, 2019

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a nonenveloped, linear, single-stranded DNA virus belonging to the family Parvoviridae and is a World Organisation for Animal Health (OIE)–notifiable crustacean pathogen. During screening of Penaeus vannamei shrimp from 3 commercial shrimp facilities in the United States for a panel of OIE-listed (n = 7) and nonlisted (n = 2) crustacean diseases, shrimp from these facilities tested positive for IHHNV. Nucleotide sequences of PCR amplicons showed 99%–100% similarity to IHHNV isolates from Latin America and Asia. The whole genome of the isolates also showed high similarity to type 2 infectious forms of IHHNV. Phylogenetic analysis using capsid gene and whole-genome sequences demonstrated that the isolates clustered with an IHHNV isolate from Ecuador. The detection of an OIE-listed crustacean pathogen in the United States highlights the need for biosecurity protocols in hatcheries and grow-out ponds to mitigate losses.

IHHNV was fi rst reported in juveniles and subadults of blue shrimp (Penaeus stylirostris) in Hawaii in the early 1980s and caused mass deaths (5). The virus also caused large-scale deaths in blue shrimp in Mexico (6). The virus outbreak in P. stylirostris shrimp in Mexico in the early 1980s led to a transition in the captive breeding program from a more susceptible and physically larger shrimp species, P. stylirostris (a preferred cultured species in the mid-1980s), to the smaller but more tolerant P. vannamei shrimp (7). In the black tiger shrimp (P. monodon), another economically vital species, IHHNV is reported to cause asymptomatic infection and deformities (4,8). In a recent study, a farm-level IHHNV infection resulted in reduced growth performance, a higher food-conversion ratio, and a lower survival rate, which led to a production loss of approximately US $67,000 per hectare of farm gate value when ponds were stocked with IHHNV-infected postlarvae having high viral load compared with postlarvae with low viral load (9). This fi nding suggests that IHHNV remains an economically relevant viral pathogen in shrimp aquaculture.
Most of the genetic lines of P. vannamei shrimp farmed in Latin America and Asia today are claimed to be tolerant or resistant to the virus, and IHHNV is assumed to have no ill effect on the production parameters in these shrimp lines, although scientifi c evidence to support this assumption is lacking. Considering these genetic lines can tolerate high levels of IHHNV without displaying clinical manifestations of the disease, transboundary movement of shrimp broodstock and larvae from these lines might lead to widespread distribution of IHHNV unless rigorous biosecurity is practiced in hatcheries. Routine disease surveillance that adheres to the US Department of Agriculture and World Organisation for Animal Health (OIE) guidelines and diagnostics performed by the Aquaculture Pathology Laboratory (APL) and the National Veterinary Services Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a nonenveloped, linear, single-stranded DNA virus belonging to the family Parvoviridae and is a World Organisation for Animal Health (OIE)-notifi able crustacean pathogen. During screening of Penaeus vannamei shrimp from 3 commercial shrimp facilities in the United States for a panel of OIE-listed (n = 7) and nonlisted (n = 2) crustacean diseases, shrimp from these facilities tested positive for IHHNV. Nucleotide sequences of PCR amplicons showed 99%-100% similarity to IHHNV isolates from Latin America and Asia. The whole genome of the isolates also showed high similarity to type 2 infectious forms of IHHNV. Phylogenetic analysis using capsid gene and whole-genome sequences demonstrated that the isolates clustered with an IHHNV isolate from Ecuador. The detection of an OIE-listed crustacean pathogen in the United States highlights the need for biosecurity protocols in hatcheries and grow-out ponds to mitigate losses.
Laboratories (NVSL) have helped to mitigate the spread of major pathogens in the United States. This success is reflected by the 26-year absence of the virus from all US-based farms until now.
During June 2019, P. vannamei shrimp samples from broodstock and postlarvae originating in commercial shrimp facilities in Texas and Florida were submitted to the APL at the University of Arizona for routine screening for viral, bacterial, and fungal pathogens. IHHNV was the only pathogen identified in shrimp from both facilities. Trace testing resulted in additional IHHNV-positive animals. We further characterized the IHHNV strains detected in the United States by genome sequencing and determined their genetic similarities to other IHHNV strains. Considering the history of economic losses caused by IHHNV over the past 4 decades, the recent detection of IHHNV in commercial facilities in the United States highlights the need to enhance biosecurity to prevent spread and future disease outbreaks caused by IHH-NV or other diseases in shrimp aquaculture. The need to further enhance diagnostic testing and biosecurity measures was underscored by the subsequent detection of IHNNV in the United Kingdom and Canada, which was directly linked to commercial shrimp facilities in the United States (10,11). The objectives of this study were to elucidate the genomic characteristics of the virus to ensure that the IHHNV isolate is an infectious form of the virus and not a genome-integrated form, as well as to determine its genetic relatedness to IHHNV isolates described in the literature.
We performed testing for IHHNV first by using the OIE-recommended 389 F/R primer pair, followed by the 309 F/R primer pair to detect the presence of the infectious form of IHHNV (13). We visualized PCR products on a 1.5% agarose gel to confirm presence of viral DNA. We used DNA from samples testing positive for IHHNV by PCR screening to amplify the nonstructural (NS1) and capsid protein (CP) genes. We amplified the NS1 gene by using 2 sets of primers to generate 2 amplicons with a 66-bp overlap (Table 1) and the CP gene by using the method of Robles-Sikisaka et al. (21), which yields a 1,088-bp amplicon.

Sanger Sequencing and Data Analysis
We column purified PCR amplicons by using the GeneJET PCR Purification Kit (ThermoFisher Scientific, https://www.thermofisher.com) according to manufacturer instructions. Purified PCR products were submitted to the University of Arizona Genetics Core for Sanger sequencing ( Table 2). We aligned sequence data in Geneious Prime to generate a consensus sequence (22) and compared those sequences with published genomes using BLASTn (23).

Whole-Genome Sequencing and Bioinformatics Analyses
We extracted total nucleic acids from tissue homogenates for initial testing by using the MagMAX-Ambion kit 1836 on an automated MagMAX Express magnetic particle processor (ThermoFisher Scientific). We extracted additional DNA from tissue homogenates treated with Benzonase to reduce host DNA by using the DNeasy Blood and Tissue Kit (QIAGEN, https:// www.quiagen.com). We prepared libraries by using the Ion Xpress Plus Fragment Library Kit (Thermo-Fisher Scientific) according to manufacturer instructions and templates by using the Ion 520 on the Ion Chef System, then sequenced by using an Ion 520 Chip on the Ion S5 System (ThermoFisher Scientific). We performed reference-based mapping (GenBank accession no. NC_002190) by using SeqMan NGen version 14 (DNASTAR, https://www.dnastar.com) with default parameters for automatic read trimming and assembly and verified alignments resulting in the final consensus sequence. The genomes (minus ends because random priming was used) of IHHNV from Texas and Florida were deposited in GenBank (accession nos. MN968717.1 and MN968716.1).

Phylogenetic Analyses
Initially, we inferred the phylogenetic analysis by using the neighbor-joining method (24) for the CP gene of IHHNV with 1,000 bootstraps. The tree was drawn to scale; branch lengths were in the same units as those of the evolutionary distances used to infer the phylogenetic tree. We computed the evolutionary distances (base substitutions per site) by using the maximum composite likelihood method (25). This analysis involved 32 nt sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). A total of 995 positions were represented in the final dataset. We conducted evolutionary analyses in MEGA X (25).
We downloaded all available complete genomes from GenBank on November 1, 2019, for phylogenetic analysis. We inferred evolutionary history by using the maximum-likelihood method based on the Tamura-Nei model (24). We obtained initial tree(s) for the heuristic search automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated by using the maximum-likelihood approach and then by selecting the topology with superior log likelihood value. The tree is drawn to scale; branch lengths are measured in the number of substitutions per site. The analysis involved 28 nt sequences. Codon positions included were 1st+2nd+3rd+Noncoding. We eliminated all   ACT TGA TCC TTC GGC GTG TT  NS1  2540 IH_CP_S  AGG CCT CTT CCA AGA ATA CG  NS1  2682 IHHNV_NS1_AS  ACT TGA TCC TTC GGC GTG TT  NS1  IHHNV-1942_F  GTC ACT AAT TAC AAA CCT GCA G  NS1  IHHNV-2020R  GCA TAT TGT CGT AGT CTG GT  CP  IHHNV-CP_S  ATG TGC GCC GAT TCA ACA AG  CP  IH_CP_Seq_S  CAT AAT CAA CTA TCA ACT AA  CP  IH_CP_Seq_AS  TGC CAA TGT TAC GTC GGT TTC

Detection of IHHNV in Shrimp Hatchery-and Field-Collected Samples from Texas and Florida
We detected IHHNV in samples from Texas (case nos. 19-428 and 19-644) and Florida (case no. 19-490) by using the OIE-recommended conventional PCR-based diagnostic method (12) (Figure 1). These results confirmed the first detection of the virus in 2 commercial aquaculture facilities in the United States since 1993 (27).

Amplification and Sequencing of CP and NS1 Genes of IHHNV
The complete CP and NS1 genes of the IHHNV isolates from Texas and Florida were successfully amplified in overlapping fragments. The 2 amplicons together represent ≈88% of total length of IHHNV genome. We sequenced the NS1 and CP amplicons of both Texas and Florida IHHNV isolates. BLAST analysis demonstrated that both genes showed highest similarity to homologous genes of the IHHNV Ecuador strain (GenBank accession no. AY362548.1) ( Table 3).

Whole-Genome Sequencing of IHHNV Texas and Florida Isolates
For isolate 19

Phylogenetic Analysis
Phylogenetic analysis using only the CP gene sequences showed that IHHNV from both Texas and Florida formed a highly supported cluster with the Ecuador strain of IHHNV. This cluster represents the type 2 infectious form of IHHNV ( Figure 2). The IHH-NV in this cluster infect penaeid species including P. vannamei, P. stylirostris, and P. monodon shrimp. In the neighbor-joining phylogenetic tree, the type 3 clade is represented by IHHNV isolates from India, Tanzania, Madagascar, and Australia. These strains infect P. monodon shrimp, are noninfectious, and remain integrated into the P. monodon genome. The third genotype, type 1, is represented by isolates from the Philippines and Thailand that infect both P. monodon and P. vannamei shrimp. Whole-genome phylogeny showed equivalent results (tree not shown).

Predicted Tertiary Structure of CP of Texas and Florida IHHNV Isolates
The IHHNV Texas and Florida viruses detected have ≈88% amino acid sequence identity with the crystal structure of Penaeus stylirostris densovirus (PstDNV) capsid (PDB code 3N7X) (Figure 3, panel A). The crystal structure of PstDNV was determined to 2.5-Å resolution by x-ray crystallography and based on 299 out of 329 aa of capsid protein missed 30 N′-terminal aa (26) (Figure 3, panel B). Because of the high sequence similarity, the modeled structure showed key β-barrel motifs that are hallmarks of many icosahedral viruses.  encodes the viral capsid protein (3,4,7). Together, these 3 gene segments comprise ≈88% of the complete genome. In this study, after IHHNV was detected in the 3 samples, the CP (990 bp) and NS1 (2440 bp) genes were amplified and sequenced. Subsequently, the near full-length genome of 2 isolates originating from 2 shrimp facilities in Texas and Florida were sequenced by using next-generation sequencing, generating contigs of 3,750 bases in length for the Texas isolates and 3,749 bases in length for the Florida isolates. All 3 cases (19-428, 19-490, and 19-644) had high sequence identity (99%) among them, as well as to an IHHNV isolate originating in Ecuador (GenBank accession no. AY362548.1). In 2003, two genotypes of IHHNV were described. At that time, type 1 strains were found in the Americas and East Asia (primarily the Philippines), and type 2 strains were detected in Southeast Asia (29). Since then, an additional genotype, type 3, has been described, and the geographic range of type 1 and type 2 viruses has spread worldwide. Phylogenetic analysis using the CP gene and full-length genomes indicate the recently detected IHHNV viruses from the United States form a strongly supported cluster with type 2 strains from the Americas. These strains represent the infectious forms of IHHNV and are clearly distinct from type 1 (infectious) and type 3 (noninfectious). (Figure 2). Since the virus was first described in the early 1980s, <50 complete genomes have been published in GenBank, and many of the genomes have incomplete metadata, including date of isolation or detection, which confounds interpretation of phylogenetic analysis. The lack of these critical pieces of information limits the ability to infer transmission of the virus on the basis of phylogeny alone. Epidemiologic information for the Texas isolates and the phylogenetic analysis suggest a link between the detections of IHHNV in Texas and Florida, supporting their close genetic relationship.

Discussion
The close similarity at the whole-genome level and the phylogenetic data indicate that all sequences derive from a strain from Ecuador, suggesting a  (30,31). The transboundary movement of shrimp has gone hand-inhand with the expansion of shrimp farming and will not cease anytime soon. Investment in newer, more sensitive diagnostic methodologies based on CRISPR, digital droplet PCR, and next-generation sequencing approaches could help limit the spread of pathogens (32,33). In addition, rigorous screening of animals for IHHNV and other pathogens is necessary to make moving shrimp safer and thereby make the industry more sustainable and resilient in the long run. This rigorous screening would entail collaborative efforts between shrimp producers, diagnostic and research institutions, and corresponding government entities.
To determine whether the predicted amino acid sequences of the IHHNV capsid protein from Texas and Florida isolates can conform an icosahedral symmetry, homology modeling was performed with a reference tertiary structure of another IHHNV isolate, PstHPV-1, for which a crystal structure is available in the Protein Data Bank (PDB code 3N7X). Although a sequence identity of 88% between the reference strains and US strains might seem low, this identity is because of the absence of 20 aa from the N′-terminal of the reference strain. The analysis of the same sections (excluding the first 20 aa from the N′-terminal of US strains) of the CP of the 3 strains demonstrates a sequence identity of 97.5%. The tertiary structure showed that IHHNV Texas and Florida isolates can conform an icosahedral symmetry, as expected for a parvovirus infectious virion (26). Thus, the predicted tertiary structure analysis supports the phylogenetic data that IHHNV Texas and Florida isolates represent an infectious form of the virus and not a type 3 strain.
Although it might seem redundant at first sight to perform protein modeling, we considered it fundamental for the study to confirm the capsid protein coding regions were not endogenous viral elements (EVEs). These EVEs are well characterized in P. monodon shrimp, but they have not been reported as frequently in P. vannamei shrimp. Furthermore, the EVEs from P. vannamei shrimp might contain regions of the IHHNV genome that are different to the regions reported in P. monodon shrimp. Because of the lack of frozen shrimp samples, we could not delineate the pathogenicity of the virus in experimental bioassay, and hence could not perform histopathology to assess cellular manifestation of the viral infection; the in silico analysis of IHHNV capsid protein data gave further confidence that these strains represented an infectious form of IHHNV and not an EVE. A combination of protein modeling, whole-genome sequencing, PCR, and phylogenetic analysis are complementary in nature and sufficient to deem the IHHNV strain detected in the United States in 2019 an infectious type 2 strain of the virus.
IHHNV is well known to be endemic in shrimpproducing countries in the Americas, Asia, and Australia (7). IHHNV is also well established in wild shrimp populations in the Gulf of Mexico (21). In the United States, however, the virus had not been reported in farmed shrimp since 1993 (27). Over the past 4 decades, as shrimp farming evolved from subsistence levels of farming to an intensive culture system worldwide, infectious diseases have emerged and spread regularly. The spread of diseases has been further exacerbated by the movement of broodstock and post-larvae across countries and continents. The movement of infected shrimp has played a critical role in the spread of pathogens like white spot syndrome virus, Taura syndrome virus, and IHHNV (34). The origin of the IHHNV isolates described in this study remains unknown, but the data suggest these isolates could have originated in Latin America. This finding highlights the need to keep following strict biosecurity protocols, including disease surveillance in shrimp hatcheries and grow-out ponds, to prevent further introduction of IHHNV or other OIE-listed and nonlisted pathogens in shrimp facilities in the United States and elsewhere.