Identification of a Novel Solinvivirus with Nuclear Localization Associated with Mass Mortalities in Cultured Whiteleg Shrimp (Penaeus vannamei).

The emergence and spread of disease-causing viruses in shrimp aquaculture is not uncommon. Since 2016, unusual mortalities have been affecting the Brazilian shrimp industry and we have associated these unusual mortalities with a novel variant of infectious myonecrosis virus (IMNV). The transcriptome analysis of these diseased shrimp showed an additional divergent viral sequence that we have assigned to the family Solinviviridae. The novel virus has been tentatively termed Penaeus vannamei solinvivirus (PvSV) (GenBank accession: OP265432). The full-length genome of the PvSV is 10.44 kb (excluding the poly A tail) and codes for a polyprotein of 3326 aa. Five conserved domains coding for a helicase, RdRp, calicivirus coat protein, G-patch and tegument protein were identified. The genome organization of the PvSV is similar to other (Nylan deria fulva virus 1) solinvivirus. A unique feature of this virus that differs from other members of the Solinviviridae is the presence of putative nuclear localization signals. The tissue tropism of this virus is wide, infecting cells of the hepatopancreas, gastrointestinal tract, lymphoid organ and muscle tissue. Another unique feature is that it is the only RNA virus of penaeid shrimp that shows a nuclear localization by in situ hybridization. The PvSV has a wide distribution in Brazil and has been found in the states of Maranhão State (Perizes de Baixo), Piaui State (Mexeriqueira), Ceará State (Camocim, Jaguaruana, Aracati and Alto Santo) and Pará State where it has been detected in coinfections with IMNV. The diagnostic methods developed here (real-time RT-PCR and in situ hybridization) are effective for the detection of the pathogen and should be employed to limit its spread. Furthermore, the identification of the PvSV shows the increasing host range of the relatively new family Solinviviridae.

Current status of infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) in the Peruvian and Ecuadorian shrimp industry.

Infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a crustacean disease that caused large-scale mortality in Penaeus stylirostris, deformity and growth retardation in Penaeus vannamei and Penaeus monodon. We surveyed the presence of IHHNV in three major shrimp-producing regions in Ecuador, namely Guayas, El Oro, and Esmeralda. The data show that IHHNV is endemic (3.3-100% prevalence) to shrimp farms in these regions. The whole genome sequences of representative circulating IHHNV genotypes in Ecuador and Peru showed that these genotypes formed a separate cluster within the Type II genotypes and were divergent from other geographical isolates of IHHNV originating in Asia, Africa, Australia, and Brazil. In experimental bioassays using specific pathogen-free (SPF) P. vannamei, P. monodon, and P. stylirostris and representative IHHNV isolates from Ecuador and Peru, the virus did not cause any mortality or induce clinical signs in any of the three penaeid species. Although IHHNV-specific Cowdry type A inclusion bodies were histologically detected in experimentally challenged P. vannamei and P. monodon and confirmed by in situ hybridization, no such inclusions were observed in P. stylirostris. Moreover, P. vannamei had the highest viral load, followed by P. monodon and P. stylirostris. Based on IHHNV surveillance data, we conclude that the currently farmed P. vannamei lines in Ecuador are tolerant to circulating IHHNV genotypes. The genome sequence and experimental bioassay data showed that, although the currently circulating genotypes are infectious, they do not induce clinical lesions in the three commercially important penaeid species. These findings suggest a potentially evolving virus-host relationship where circulating genotypes of IHHNV co-exist in equilibrium with P. vannamei raised in Peru and Ecuador.

The emerging pathogen Enterocytozoon hepatopenaei drives a degenerative cyclic pattern in the hepatopancreas microbiome of the shrimp (Penaeus vannamei).

The microsporidian Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that causes substantial economic losses in shrimp (Penaeus spp.) aquaculture worldwide. To prevent diseases in shrimp, the manipulation of the gut microbiota has been suggested. However, prior knowledge of the host-microbiome is necessary. We assessed the modulation of the microbiome (bacteria/fungi) and its predicted functions over the course of disease progression in shrimp experimentally challenged with EHP for 30 days using high throughput 16S rRNA and ITS amplicon sequencing. Infection grade was assessed for the first time by quantitative digital histopathology. According to the infection intensity, three disease-stages (early/developmental/late) were registered. During the early-stage, EHP was not consistently detected, and a high diversity of potentially beneficial microorganisms related to nutrient assimilation were found. In the development-stage, most of the shrimp start to register a high infection intensity related to a decrease in beneficial microorganisms and an increase in opportunistic/pathogenic fungi. During late-stage, animals displayed different infection intensities, showed a displacement of beneficial microorganisms by opportunistic/pathogenic bacteria and fungi related to pathogen infection processes and depletion of energetic reserves. The degenerative cyclic pattern of EHP infection and its effects on beneficial microorganisms and beneficial functions of the shrimp hepatopancreas microbiome are discussed.

Evidences supporting Enterocytozoon hepatopenaei association with white feces syndrome in farmed Penaeus vannamei in Venezuela and Indonesia.

White feces syndrome (WFS) is an emerging and poorly described disease characterized by the presence of floating white fecal strings in shrimp (Penaeus monodon and P. vannamei) grow-out ponds. WFS has been associated with several pathogens, including Enterocytozoon hepatopenaei. This association is based on the fact that in areas where E. hepatopenaei has been reported, there was also a high WFS prevalence. E. hepatopenaei is an emerging pathogen that has affected cultured shrimp in Indonesia, Vietnam, China, Thailand, and India. In 2016, we reported the presence of E. hepatopenaei in farmed P. vannamei in Venezuela. In this study, we describe the first case of WFS in Venezuela associated with E. hepatopenaei. The white fecal strings and shrimp displaying white feces along the gastrointestinal tract observed in this study were similar to the gross signs found in WFS-impacted P. vannamei in SE Asian countries. Furthermore, we describe a strong association between WFS and E. hepatopenaei in the samples obtained from Venezuela and Indonesia. Quantification of E. hepatopenaei in WFS-affected ponds, ponds with a history of WFS, and ponds with no WFS showed that E. hepatopenaei loads were significantly higher in WFS-affected ponds. Furthermore, these findings constitute the first report of WFS being associated with E. hepatopenaei in farmed shrimp in Latin America. Additionally, we propose that the gross signs of WFS such as floating whitish fecal strings can be used as an indicator of the presence of E. hepatopenaei in countries where E. hepatopenaei is endemic.

Novel infectious myonecrosis virus (IMNV) genotypes associated with disease outbreaks on Penaeus vannamei shrimp farms in Indonesia.

Infectious myonecrosis virus (IMNV) is one of the most pathogenic viruses that affect Penaeus vannamei shrimp. In 2018, IMNV was reported in grow-out ponds of P. vannamei in Situbondo, Indonesia. Diseased animals displayed clinical signs of infectious myonecrosis (IMN) characterized by white discoloration of skeletal muscle. Histopathology of affected shrimp revealed lesions that are pathognomonic of IMNV infection. The major capsid protein (MCP) gene was amplified and sequenced from representative samples showing IMN pathology. Multiple alignment of predicted amino acid sequences of the MCP gene with known IMNV genotypes in the GenBank database revealed three unique genotypes, SB-A, SB-B and SB-C,in Situbondo samples. The number of amino acid changes in SB-A, SB-B and SB-C compared to known IMNV genotypes ranged from 7-710, including the isolate SB-B, which contains deletion of 622 aa. A phylogenetic analysis using homologous sequences from Brazil and Indonesia showed that these three isolates represent new IMNV genotypes.

Genetic signature of rapid IHHNV (infectious hypodermal and hematopoietic necrosis virus) expansion in wild Penaeus shrimp populations.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a widely distributed single-stranded DNA parvovirus that has been responsible for major losses in wild and farmed penaeid shrimp populations on the northwestern Pacific coast of Mexico since the early 1990's. IHHNV has been considered a slow-evolving, stable virus because shrimp populations in this region have recovered to pre-epizootic levels, and limited nucleotide variation has been found in a small number of IHHNV isolates studied from this region. To gain insight into IHHNV evolutionary and population dynamics, we analyzed IHHNV capsid protein gene sequences from 89 Penaeus shrimp, along with 14 previously published sequences. Using Bayesian coalescent approaches, we calculated a mean rate of nucleotide substitution for IHHNV that was unexpectedly high (1.39x10(-4) substitutions/site/year) and comparable to that reported for RNA viruses. We found more genetic diversity than previously reported for IHHNV isolates and highly significant subdivision among the viral populations in Mexican waters. Past changes in effective number of infections that we infer from Bayesian skyline plots closely correspond to IHHNV epizootiological historical records. Given the high evolutionary rate and the observed regional isolation of IHHNV in shrimp populations in the Gulf of California, we suggest regular monitoring of wild and farmed shrimp and restriction of shrimp movement as preventative measures for future viral outbreaks.

Quantitative detection of hepatitis a virus and enteroviruses near the United States-Mexico border and correlation with levels of fecal indicator bacteria.

For decades, untreated sewage flowing northward from Tijuana, Mexico, via the Tijuana River has adversely affected the water quality of the recreational beaches of San Diego, California. We used quantitative reverse transcription-PCR to measure the levels of hepatitis A virus (HAV) and enteroviruses in coastal waters near the United States-Mexico border and compared these levels to those of the conventional fecal indicators, Escherichia coli and enterococci. Over a 2-year period from 2003 to 2005, a total of 20 samples were assayed at two sites during both wet and dry weather: the surfzone at the mouth of the Tijuana River and the surfzone near the pier at Imperial Beach (IB), California (about 2 km north of the mouth of the Tijuana River). HAV and enterovirus were detected in 79 and 93% of the wet-weather samples, respectively. HAV concentrations in these samples ranged from 105 to 30,771 viral particles/liter, and enterovirus levels ranged from 7 to 4,417 viral particles/liter. The concentrations of HAV and enterovirus were below the limit of detection for all dry weather samples collected at IB. Regression analyses showed a significant correlation between the densities of both fecal bacterial indicators and the levels of HAV (R2>0.61, P<0.0001) and enterovirus (R2>0.70, P<0.0001), a finding that supports the use of conventional bacterial indicators to predict the levels of these viruses in recreational marine waters.