The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Detection of a new microsporidium Perezia sp. in shrimps Penaeus monodon and P. indicus by histopathology, in situ hybridization and PCR.

Samples of microsporidia-infected shrimps exhibiting clinical signs of cotton shrimp disease were collected from Madagascar, Mozambique, and the Kingdom of Saudi Arabia from 2005 to 2014. The tails of the infected shrimps appeared opaque and whitish; subsequent histological examination revealed the presence of cytoplasmic inclusions and mature spores in tissues of the muscle, hepatopancreas, gills, heart, and lymphoid organ. PCR analysis targeting the small subunit rDNA (SSU rDNA) from infected samples resulted in the amplification of a 1.2 kbp SSU rDNA sequence fragment 94% identical to the corresponding region in the genome of the microsporidian Perezia nelsoni, which infects populations of Penaeus setiferus in the USA. Its SSU rDNA sequence was 100% identical among isolates from Madagascar and Saudi Arabia, indicating that shrimps from the Red Sea and Indian Ocean were infected with the same microsporidium, the novel Perezia sp. A 443 bp fragment of the SSU rDNA sequence was cloned, labeled with digoxigenin and subjected to an in situ hybridization assay with tissue sections of Perezia sp.-infected Penaeus monodon from Madagascar and Mozambique, and P. indicus from Saudi Arabia. The probe hybridized to the mature spores in the hepatopancreas and muscle from which the spores had been obtained for DNA isolation. This assay was specific, showing no reaction to another microsporidium, Enterocytozoon hepatopenaei (EHP), infecting the hepatopancreas of shrimp P. stylirostris cultured in SE Asian countries. We also developed an SSU rDNA-based PCR assay, specific for the novel Perezia sp. This PCR did not react to EHP, nor to genomic DNA of shrimp and other invertebrates.

Extended genome sequences of penaeid shrimp infectious myonecrosis virus strains from Brazil and Indonesia.

New sequencing studies of the nonsegmented dsRNA genome of penaeid shrimp infectious myonecrosis virus (IMNV), a tentatively assigned member of the family Totiviridae, identified previously unread sequences at both genome termini in three previously analyzed IMNV strains, one from Brazil (the prototype strain of IMNV) and two from Indonesia. The new sequence determinations add >600 nt to the 5' end of the genomic plus strand of each strain, increasing the length of the 5' nontranslated region to at least 469-472 nt and the length of the upstream open reading frame (ORF1) translation product by at least 48 aa. These new findings are similar to recent ones for two other IMNV strains (GenBank KF836757.1 and KJ556923.1) and thereby corroborate important amendments to the full-length IMNV genome sequence.

Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp.

A microsporidian parasite, Enterocytozoon hepatopenaei (abbreviated as EHP), is an emerging pathogen for penaeid shrimp. EHP has been found in several shrimp farming countries in Asia including Vietnam, Thailand, Malaysia, Indonesia and China, and is reported to be associated with growth retardation in farmed shrimp. We examined the histological features from infected shrimp collected from Vietnam and Brunei, these include the presence of basophilic inclusions in the hepatopancreas tubule epithelial cells, in which EHP is found at various developmental stages, ranging from plasmodia to mature spores. By a PCR targeting the 18S rRNA gene, a 1.1kb 18S rRNA gene fragment of EHP was amplified, and this sequence showed a 100% identity to EHP found in Thailand and China. This fragment was cloned and labeled with digoxigenin-11-dUTP, and in situ hybridized to tissue sections of infected Penaeus vannamei (from Vietnam) and P. stylirostris (Brunei). The results of in situ hybridization were specific, the probe only reacted to the EHP within the cytoplasmic inclusions, not to a Pleistophora-like microsporidium that is associated with cotton shrimp disease. Subsequently, we developed a PCR assay from this 18S rRNA gene region, this PCR is shown to be specific to EHP, did not react to 2 other parasitic pathogens, an amoeba and the cotton shrimp disease microsporidium, nor to genomic DNA of various crustaceans including polychaetes, squids, crabs and krill. EHP was detected, through PCR, in hepatopancreatic tissue, feces and water sampled from infected shrimp tanks, and in some samples of Artemia biomass.

Genotyping of virulence plasmid from Vibrio parahaemolyticus isolates causing acute hepatopancreatic necrosis disease in shrimp.

Acute hepatopancreatic necrosis disease (AHPND) has caused severe mortalities in farmed penaeid shrimp throughout SE Asia and Mexico. The causative agent of AHPND is the marine bacterium Vibrio parahaemolyticus, which secretes PirA- and PirB-like binary toxin that caused deterioration in the hepatopancreas of infected shrimp. The genes responsible for the production of this toxin are located in a large plasmid residing within the bacterial cells. We analyzed the plasmid sequence from the whole genome sequences of AHPND-V. parahaemolyticus isolates and identified 2 regions that exhibit a clear geographical variation: a 4243-bp Tn3-like transposon and a 9-bp small sequence repeat (SSR). The Tn3-like transposon was only found in the isolates from Mexico and 2 unspecified Central American countries, but not in SE Asian isolates from China, Vietnam, and Thailand. We developed PCR methods to characterize AHPND-V. parahaemolyticus isolates as either Mexican-type or SE Asian-type based on the presence of the Tn3-like transposon. The SSR is found within the coding region of a hypothetical protein and has either 4, 5, or 6 repeat units. SSRs with 4 repeat units were found in isolates from Vietnam, China, and Thailand. SSRs with 5 repeat units were found in some Vietnamese isolates, and SSRs with 6 repeat units were only found in the Mexican isolates.

Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp.

The 69 kb plasmid pVPA3-1 was identified in Vibrio parahaemolyticus strain 13­028/A3 that can cause acute hepatopancreatic necrosis disease (AHPND). This disease is responsible for mass mortalities in farmed penaeid shrimp and is referred to as early mortality syndrome (EMS). The plasmid has a GC content of 45.9% with a copy number of 37 per bacterial cell as determined by comparative quantitative PCR analyses. It consists of 92 open reading frames that encode mobilization proteins, replication enzymes, transposases, virulence-associated proteins, and proteins similar to Photorhabdus insect-related (Pir) toxins. In V. parahaemolyticus, these Pir toxin-like proteins are encoded by 2 genes (pirA- and pirB-like) located within a 3.5 kb fragment flanked with inverted repeats of a transposase-coding sequence (1 kb). The GC content of these 2 genes is only 38.2%, substantially lower than that of the rest of the plasmid, which suggests that these genes were recently acquired. Based on a proteomic analysis, the pirA-like (336 bp) and pirB-like (1317 bp) genes encode for 13 and 50 kDa proteins, respectively. In laboratory cultures of V. parahaemolyticus 13-028/A3, both proteins were secreted into the culture medium. We developed a duplex PCR diagnostic method, with a detection limit of 10(5) CFU ml(-1) and targeting pirA- and pirB-like genes in this strain of V. parahaemolyticus. This PCR protocol can reliably detect AHPND-causing strains of V. parahaemolyticus and does not cross react with non-pathogenic strains or with other species of Vibrio isolated from shrimp ponds.

Detection of acute hepatopancreatic necrosis disease (AHPND) in Mexico.

Acute hepatopancreatic necrosis disease (AHPND), which has also been referred to as early mortality syndrome (EMS), initially emerged as a destructive disease of cultured shrimp species in Asia in 2009. The pathogen associated with the disease, Vibrio parahaemolyticus, subsequently spread to the Western Hemisphere and emerged in Mexico in early 2013. The spread to the Western Hemisphere is a major concern to shrimp producers in the region. To date, the only peer-reviewed published method for determining whether mortalities are due to AHPND is through histological examination. A novel PCR detection method was employed to assess samples from Mexico in order to confirm the presence of the pathogen in this country. This manuscript details the detection methods used to confirm the presence of AHPND in Mexico. Both immersion and per os challenge studies were used to expose the Penaeus vannamei to the bacteria in order to induce the disease. Histological analysis confirmed AHPND status following the challenge studies. Also provided are the details of the molecular test by PCR that was used for screening candidate V. parahaemolyticus isolates. A rapid PCR assay for detection of AHPND may help with early detection and help prevent the spread of AHPND to other countries.

"Candidatus Hepatobacter penaei," an intracellular pathogenic enteric bacterium in the hepatopancreas of the marine shrimp Penaeus vannamei (Crustacea: Decapoda).

The bacteria that cause necrotizing hepatopancreatitis in Penaeus vannamei adversely affect penaeid shrimp cultured in the western hemisphere. 16S rRNA and gyrase B gene analyses determined the taxonomic position of these bacteria. The name "Candidatus Hepatobacter penaei" is proposed for these pathogenic bacteria, which are members of the Rickettsiales order.

Characterization of a new strain of Taura syndrome virus (TSV) from Colombian shrimp farms and the implication in the selection of TSV resistant lines.

Prior to 2004, Colombian shrimp farming benefited from a selection program in which Penaeus vannamei stocks were developed with resistance to Taura syndrome disease (TS). However since 2004, TS reappeared as a significant disease. In 2010, an apparently new strain of TSV (designated as CO 10) was collected in Colombia. Its genome was sequenced and compared with six other fully sequenced isolates. This analysis revealed that the TSV CO 10 is closely related to the isolates from Hawaii and Venezuela. Phylogenetic analysis based on capsid protein 2 (CP2) region from 59 TSV isolates shows that the recent Colombian isolates (2006-2010) form a new cluster and differ from the previous Colombia isolates (1994-1998) by 4% in nucleotide sequence. The virulence of this CO 10 isolate was similar to a Belize TSV determined through experimental infection in P. vannamei showing 100% mortalities and similar survival curves. By RT-qPCR for TSV, the viral loads were also close in the infected shrimp from both CO 10 and Belize at the order of 1×10(10) copies per µl RNA. To develop TSV-resistant lines, the candidate shrimp should be challenged with virus strains that have been isolated most recently from the regions where they will be cultured. This study suggests that the TSV present in Colombian shrimp farms during the last 5 years is a new TSV strain with high virulence.

A histological variant of white spot syndrome virus (WSSV) from the Kingdom of Saudi Arabia.

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp. The major targets of WSSV infection are tissues of ectodermal and mesodermal embryonic origin, predominantly the cuticular epithelium and subcuticular connective tissues. Recently, we discovered a WSSV variant in Penaeus indicus that heavily infects the subcuticular connective tissue, with very slight indications in the cuticular epithelium. The variant was also unusual in that WSSV accumulations were found in the interstitial spaces of both the subcuticular connective tissue and the lymphoid organ. This WSSV variant was confirmed through immunohistochemistry with an anti-WSSV VP28 monoclonal antibody, and also by in situ hybridization with a VP28 DNA probe. By in situ hybridization, shrimp with variant and typical histology were shown a deletion in ORF94, which is characteristic of a new type of WSSV found in Saudi Arabia; apparently, the loss of this ORF is not associated with the variant's reduced capability of infecting the cuticular epithelium cells.

Novel, closely related, white spot syndrome virus (WSSV) genotypes from Madagascar, Mozambique and the Kingdom of Saudi Arabia.

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the aquaculture industry around the world. During 2010 to 2012, WSSV caused severe mortalities in cultured penaeid shrimp in Saudi Arabia, Mozambique and Madagascar. To investigate the origins of these WSSV, we performed genotyping analyses at 5 loci: the 3 open reading frames (ORFs) 125, 94 and 75, each containing a variable number of tandem repeats (VNTR), and deletions in the 2 variable regions, VR14/15 and VR23/24. We categorized the WSSV genotype as {N125, N94, N75, ΔX14/15, ΔX23/24} where N is the number of repeat units in a specific ORF and ΔX is the length (base pair) of deletion within the variable region. We detected 4 WSSV genotypes, which were characterized by a full-length deletion in ORF94/95, a relatively small ORF75 and one specific deletion length in each variable region. There are 2 closely related genotypes in these 3 countries: {6125, del94, 375, Δ595014/15, Δ1097123/24} and {7125, del94, 375, Δ595014/15, Δ1097123/24}, where del is the full-length ORF deletion. In Saudi Arabia, 2 other related types of WSSV were also found: {6125, 794, 375, Δ595014/15, Δ1097123/24} and {8125, 1394, 375, Δ595014/15, Δ1097123/24}. The identical patterns of 3 loci in these 4 types indicate that they have a common lineage, and this suggests that the WSSV epidemics in these 3 countries were from a common source, possibly the environment.

Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp.

A new emerging disease in shrimp, first reported in 2009, was initially named early mortality syndrome (EMS). In 2011, a more descriptive name for the acute phase of the disease was proposed as acute hepatopancreatic necrosis syndrome (AHPNS). Affecting both Pacific white shrimp Penaeus vannamei and black tiger shrimp P. monodon, the disease has caused significant losses in Southeast Asian shrimp farms. AHPNS was first classified as idiopathic because no specific causative agent had been identified. However, in early 2013, the Aquaculture Pathology Laboratory at the University of Arizona was able to isolate the causative agent of AHPNS in pure culture. Immersion challenge tests were employed for infectivity studies, which induced 100% mortality with typical AHPNS pathology to experimental shrimp exposed to the pathogenic agent. Subsequent histological analyses showed that AHPNS lesions were experimentally induced in the laboratory and were identical to those found in AHPNS-infected shrimp samples collected from the endemic areas. Bacterial isolation from the experimentally infected shrimp enabled recovery of the same bacterial colony type found in field samples. In 3 separate immersion tests, using the recovered isolate from the AHPNS-positive shrimp, the same AHPNS pathology was reproduced in experimental shrimp with consistent results. Hence, AHPNS has a bacterial etiology and Koch's Postulates have been satisfied in laboratory challenge studies with the isolate, which has been identified as a member of the Vibrio harveyi clade, most closely related to V. parahemolyticus.

The role of selective breeding and biosecurity in the prevention of disease in penaeid shrimp aquaculture.

About 3.5 million metric tons of farmed shrimp were produced globally in 2009 with an estimated value greater than USD$14.6 billion. Despite the economic importance of farmed shrimp, the global shrimp farming industry continues to be plagued by disease. There are a number of strategies a shrimp farmer can employ to mitigate crop loss from disease, including the use of Specific Pathogen Free (SPF), selectively bred shrimp and the adoption of on-farm biosecurity practices. Selective breeding for disease resistance began in the mid 1990s in response to outbreaks of Taura syndrome, caused by Taura syndrome virus (TSV), which devastated populations of farmed shrimp (Litopenaeus vannamei) throughout the Americas. Breeding programs designed to enhance TSV survival have generated valuable information about the quantitative genetics of disease resistance in shrimp and have produced shrimp families which exhibit high survival after TSV exposure. The commercial availability of these selected shrimp has benefitted the shrimp farming industry and TSV is no longer considered a major threat in many shrimp farming regions. Although selective breeding has been valuable in combating TSV, this approach has not been effective for other viral pathogens and selective breeding may not be the most effective strategy for the long-term viability of the industry. Cost-effective, on-farm biosecurity protocols can be more practical and less expensive than breeding programs designed to enhance disease resistance. Of particular importance is the use of SPF shrimp stocked in biosecure environments where physical barriers are in place to mitigate the introduction and spread of virulent pathogens.

Protection from yellow head virus (YHV) infection in Penaeus vannamei pre-infected with Taura syndrome virus (TSV).

Pacific white shrimp Penaeus vannamei that were pre-exposed to Taura syndrome virus (TSV) and then challenged with yellow head virus (YHV) acquired partial protection from yellow head disease (YHD). Experimental infections were carried out using specific-pathogen-free (SPF) shrimp which were first exposed per os to TSV; at 27, 37 and 47 d post infection they were then challenged by injection with 1 × 104 copies of YHV per shrimp (designated the TSV-YHV group). Shrimp not infected with TSV were injected with YHV as a positive control. Survival analyses comparing the TSV-YHV and YHV (positive control) groups were conducted, and significant survival rates were found for all the time groups (p < 0.001). A higher final survival was found in the TSV-YHV group (mean 55%) than in the positive control (0%) (p < 0.05). Duplex reverse transcription quantitative PCR was used to quantify both TSV and YHV. Lower YHV copy numbers were found in the TSV-YHV group than in the positive control in pleopods (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.001) and lymphoid organ (LO) samples (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.01). In situ hybridization assays were conducted, and differences in the distribution of the 2 viruses in the target tissues were found. The foci of LO were infected with TSV but were not infected with YHV. This study suggests that a viral interference effect exists between TSV and YHV, which could, in part, explain the absence of YHD in the Americas, where P. vannamei are often raised in farms where TSV is present.

New genotypes of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) from the Kingdom of Saudi Arabia.

White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) are highly pathogenic to penaeid shrimp and have caused significant economic losses in the shrimp culture industry around the world. During 2010 and 2011, both WSSV and TSV were found in Saudi Arabia, where they caused severe mortalities in cultured Indian white shrimp Penaeus indicus. Most outbreaks of shrimp viruses in production facilities can be traced to the importation of infected stocks or commodity shrimp. In an attempt to determine the origins of these viral outbreaks in Saudi Arabia, we performed variable number of tandem repeat (VNTR) analyses for WSSV isolates and a phylogenetic analysis for TSV isolates. From the WSSV genome, the VNTR in open reading frames (ORFs) 125 and 94 were investigated with PCR followed by DNA sequence analysis. The genotypes were categorized as {N125, N94} where N is the number of repeat units in a specific ORF, and the subscript indicates the ORF (i.e. ORFs 125 and 94 in this case). From 15 Saudi Arabia WSSV isolates, we detected 3 genotypes: {6125, 794}, {7125, del94}, and {8125, 1394}. The WSSV genotype of {7125, del94} appears to be a new variant with a 1522 bp deletion encompassing complete coding regions of ORF 94 and ORF 95 and the first 82 bp of ORF 93. For TSV genotyping, we used a phylogenetic analysis based on the amino acid sequence of TSV capsid protein 2 (CP2). We analyzed 8 Saudi Arabian isolates in addition to 36 isolates from other areas: SE Asia, Mexico, Venezuela and Belize. The Saudi Arabian TSV clustered into a new, distinct group. Based on these genotyping analyses, new WSSV and TSV genotypes were found in Saudi Arabia. The data suggest that they have come from wild shrimp Penaeus indicus from the Red Sea that are used for broodstock.

Histopathological characterization and in situ detection of Callinectes sapidus reovirus.

A reovirus (tentatively designated as Callinectes sapidus reovirus, CsRV) was found in the blue crabs C. sapidus collected in Chesapeake Bay in 2005. Histological examination of hepatopancreas and gill from infected crabs revealed eosinophilic to basophilic, cytoplasmic, inclusions in hemocytes and in cells of connective tissue. A cDNA library was constructed from total RNA extracted from hemolymph of infected crabs. One clone (designated as CsRV-28) with a 532-bp insert was 75% identical in nucleotide sequence (and 95% similar in translated amino acid sequence) to the quanylytransferase gene of the Scylla serrata reovirus (SsRV). The insert of CsRV-28 was labeled with digoxigenin-11-dUTP and hybridized to sections of hepatopancreas and gill of infected C. sapidus, this probe reacted to hemocytes and cells in the connective tissue. No reaction was seen in any of the tissues prepared from uninfected crabs. Thus, this in situ hybridization procedure can be used to diagnose CsRV.

Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon.

We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.

Ultrastructural and sequence characterization of Penaeus vannamei nodavirus (PvNV) from Belize.

The Penaeus vannamei nodavirus (PvNV), which causes muscle necrosis in Penaeus vannamei from Belize, was identified in 2005. Infected shrimp show clinical signs of white, opaque lesions in the tail muscle. Under transmission electron microscopy, the infected cells exhibit increases in various organelles, including mitochondria, Golgi stacks, and rough endoplasmic reticulum. Cytoplasmic inclusions containing para-crystalline arrays of virions were visualized. The viral particle is spherical in shape and 19 to 27 nm in diameter. A cDNA library was constructed from total RNA extracted from infected shrimp. Through nucleotide sequencing from the cDNA clones and northern blot hybridization, the PvNV genome was shown to consist of 2 segments: RNA1 (3111 bp) and RNA2 (1183 bp). RNA1 contains 2 overlapped open reading frames (ORF A and B), which may encode a RNA-dependent RNA polymerase (RdRp) and a B2 protein, respectively. RNA2 contains a single ORF that may encode the viral capsid protein. Sequence analyses showed the presence of 4 RdRp characteristic motifs and 2 conserved domains (RNA-binding B2 protein and viral coat protein) in the PvNV genome. Phylogenetic analysis based on the translated amino acid sequence of the RdRp reveals that PvNV is a member of the genus Alphanodavirus and closely related to Macrobrachium rosenbergii nodavirus (MrNV). In a study investigating potential PvNV vectors, we monitored the presence of PvNV by RT-PCR in seabird feces and various aquatic organisms collected around a shrimp farm in Belize. PvNV was detected in mosquitofish, seabird feces, barnacles, and zooplankton, suggesting that PvNV can be spread via these carriers.

Infectious myonecrosis virus has a totivirus-like, 120-subunit capsid, but with fiber complexes at the fivefold axes.

Infectious myonecrosis virus (IMNV) is an emerging pathogen of penaeid shrimp in global aquaculture. Tentatively assigned to family Totiviridae, it has a nonsegmented dsRNA genome of 7,560 bp and an isometric capsid of the 901-aa major capsid protein. We used electron cryomicroscopy and 3D image reconstruction to examine the IMNV virion at 8.0-A resolution. Results reveal a totivirus-like, 120-subunit T = 1 capsid, 450 A in diameter, but with fiber complexes protruding a further 80 A at the fivefold axes. These protrusions likely mediate roles in the extracellular transmission and pathogenesis of IMNV, capabilities not shared by most other totiviruses. The IMNV structure is also notable in that the genome is centrally organized in five or six concentric shells. Within each of these shells, the densities alternate between a dodecahedral frame that connects the threefold axes vs. concentration around the fivefold axes, implying certain regularities in the RNA packing scheme.

Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis.

A pathogenic Spiroplasma penaei strain was isolated from the hemolymph of moribund Pacific white shrimp, Penaeus vannamei. The shrimp sample originated from a shrimp farm near Cartagena, Colombia, that was suffering from high mortalities in ponds with very low salinity and high temperatures. This new emerging disease in a marine crustacean in the Americas is described as a systemic infection. The multilocus phylogenetic analysis suggests that S. penaei strain has a terrestrial origin. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S rDNA and 23S-5S rDNA intergenic spacer region, were reconstructed using the distance-based Neighboring-Joining (NJ) method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborate the observations by other investigators using the 16S rDNA gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to Spiroplasma insolitum and distantly related to Spiroplasma sp. SHRIMP from China.