Potential biosentinels of human waste in marine coastal waters: bioaccumulation of human noroviruses and enteroviruses from sewage-polluted waters by indigenous mollusks.

A major problem of existing methods to monitor for viral pathogens in large bodies of water (i.e., coastal waters) is in the initial viral recovery and concentration of these viruses. In this report, the indigenous filter-feeding bivalve mollusk, Isognomon sp., ubiquitous in the Indo-Pacific area, has been used successfully in this critical initial sequence (virus recovery) to bioaccumulate human enteropathogenic viruses from seawater seeded experimentally with either raw sewage or human norovirus-positive stool samples. Characteristic amplicons representing the human noroviruses (213 bp) and enteroviruses (196 bp) were detected by reverse transcription polymerase chain reaction (RT-PCR) employing specific primers. The data indicate the high feasibility of employing these mollusks to serve as practical biosentinels of waters contaminated with sewage in coastal and island communities.

White spot syndrome virus (WSSV) in cultured Penaeus monodon in the Philippines.

The prevalence and geographic distribution of white spot syndrome virus (WSSV) infection among cultured penaeid shrimp in the Philippines was determined from January to May, 1999, using PCR (polymerase chain reaction) protocol and Western blot assays. A total of 71 samples consisting of 18 post-larvae (PL) and 53 juvenile/adult shrimp samples (56 to 150 days-of-culture, DOC) were screened for WSSV. Of the 71 samples tested, 51 (72%) were found positive for WSSV by PCR: 61% (31/51) after 1-step PCR and 39% (20/51) after 2-step, non-nested PCR. Of the PL and juvenile/adult shrimp samples tested, 50 and 79% were positive for WSSV, respectively. By Western blot, only 6 of the 51 (12%) PCR-positive samples tested positive for WSSV. Of the 20 samples negative for WSSV by PCR, all tested negative for WSSV by Western blot assay. This is the first report of the occurrence of WSSV in the Philippines.

Dot-blot nitrocellulose enzyme immunoassays for the detection of white-spot virus and yellow-head virus of penaeid shrimp.

Dot-blot nitrocellulose enzyme immunoassays (DB-NC-EIA) were developed for the detection of white-spot virus (WSV) and yellow-head virus (YHV) in infected shrimp. The assays utilized HRP-conjugated virus-specific antibodies to detect virus antigen present in gill homogenates of infected shrimp spotted onto nitrocellulose membrane. The assays are by far the simplest and most rapid detection methods available for WSV and YHV.

Establishment, cryopreservation, and growth of 11 cell lines prepared from a juvenile Hawaiian monk seal, Monachus schauinslandi.

Eleven cell lines were prepared from skin, snout, liver, kidney, lung, heart, brain, spleen, thyroid, urinary bladder, and periorbital soft tissue of a juvenile Hawaiian monk seal (Monachus schauinslandi). The cell grew at 37 degrees C in RPMI 1640 medium supplemented with 20% fetal bovine serum. These cell lines have been subcultured 11-27 times since their initiation in May 1997. Growth of the monk seal cells was serum-dependent and plating efficiencies ranged from 4-24%. These monk seal cells grew well in M199, L-15 and MEM commonly used for cultivation of animal and mammalian cells and retained 87% cell viability following storage for 2.5 years in liquid nitrogen. Karyotyping indicated that these monk seal-derived cell lines remained diploid with a chromosome count of 34 at their early passage (passage 9-13). These cell lines were tested for herpesvirus by polymerase chain reaction using degenerate oligonucleotide primers designed from the highly conserved region of herpesviral DNA polymerase gene and no specific detection occurred. These newly established cell lines are currently being used for the investigation of an eye disease occurring in captive monk seal pups in Oahu and will be available for future isolation and study of monk seal viruses.

Molecular characterization of the glycoproteins from two warm water rhabdoviruses: snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS)/spring viremia of carp virus (SVCV).

We have determined the complete coding sequences for the glycoprotein (G) genes from two rhabdoviruses that infect warm water aquatic animals, the snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS). Surprisingly, the G nucleotide sequence from RPS, a virus which has been isolated from diseased shrimp in Hawaii on numerous occasions, was over 99% identical to the G nucleotide sequence from spring viremia of carp virus (SVCV), a fish virus from Europe and Asia. This is the first report of SVCV isolation outside of Europe and Asia, and it is also the first report of SVCV infecting a non-vertebrate species. The G gene from SHRV was most closely related to the G genes from the three Novirhabdoviruses, viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and hirame rhabdovirus (HIRRV), with 47, 37, and 36% amino acid identity, respectively. In addition, a phylogenetic analysis using the amino acid sequence from rhabdovirus G genes indicated that SHRV should be classified within the Novirhabdovirus genus. Finally, the SHRV-G gene was successfully expressed in mammalian cells under the control of the cytomegalovirus (CMV) promoter, establishing that it can potentially be used in the production of pseudotyped retroviruses designed to infect fish.

A polymerase chain reaction protocol for the detection of various geographical isolates of white spot virus.

Polymerase chain reaction (PCR) primers were designed based on the sequence of a cloned fragment of the white spot virus (WSV) genome and were used to detect at least four geographic isolates of WSV from both experimentally- and naturally-infected shrimp. In addition to high specificity, the one-step and two-step PCR protocols were determined to have sensitivities of 10-100 pg and 100 femtograms respectively. The two-step PCR protocol is recommended as a very sensitive and specific alternative protocol to Western blot assay for the detection of WSV.

Characterization of a non-occluded baculovirus-like agent pathogenic to penaeid shrimp.

A non-occluded baculovirus-like agent recently isolated by this laboratory from moribund Penaeus japonicus shrimps obtained from China and named Chinese baculovirus (CBV) was purified and some of its properties characterized. Under the electron microscope, negatively stained virus particles were rod-shaped, enveloped, and measured 322 to 378 nm in length and 130 to 159 nm in diameter. The nucleoprotein core exhibited a unique striated structure and measured 316 to 350 nm in length and 65 to 66 nm in diameter. The striations appear to be the result of the stacking of ring-like structures. These rings consisted of 2 rows of 12 to 14 globular subunits. Each globular subunit measured approximately 10 nm in diameter. SDS-PAGE gels of purified virus preparations showed, among several, 4 prominent protein bands with approximate molecular weights of 19, 23.5, 27.5 and 75 kDa. The structural viral proteins were identified by western blot analysis using polyclonal hyperimmune serum made against purified CBV. The 19, 27.5, and 75 kDa structural proteins were determined to be non-glycosylated components associated with the viral envelope. The 23.5 kDa protein, also non-glycosylated, was identified with the capsid structure. Viral genomic DNA digested with Hind III restriction endonuclease revealed at least 29 different fragments with a conservatively estimated total size of at least 183 kb.

A comparative study of three different isolates of white spot virus.

Three separate isolates of white spot virus (WSV) purified from 3 different penaeid shrimp species from different countries were compared morphologically, biochemically, and genomically using the following techniques; negative stain electron microscopy, sodium dodecyl-sulfate polyacrylamide gel electrophoresis/western blot, and restriction fragment length polymorphism (RFLP), respectively. Under the electron microscope, the 3 isolates were indistinguishable. Their nucleoprotein cores exhibited the unique striated structure characteristic of the baculovirus-like agents associated with white spot syndrome. The dimensions of the nucleoprotein cores were also identical for all 3 isolates. SDS-PAGE gels of purified virus preparations showed all 3 to be identical in the position of at least 3 of the most prominent protein bands of WSV, with approximate molecular weights of 19, 23.5, and 27.5 kDa. Western blot analyses also revealed these 3 same protein bands in identical positions for all 3 isolates. RFLP analyses of the viral genomes using Hind III and EcoR 1 enzymes revealed that although the 3 isolates were identical when cut with EcoR I, the isolate from Penaeus japonicus from China was distinguishable from the other 2 genomes (P. monodon from Indonesia and P. setiferus from the U.S.) when cut with Hind III.

Development of an in vitro quantal assay in primary cell cultures for a non-occluded baculo-like virus of penaeid shrimp.

An in vitro quantal assay (TCID50) for a non-occluded baculo-like virus isolate from naturally infected Penaeus japonicus obtained from China and experimentally infected P. stylirostris was developed using primary shrimp lymphoid cell cultures in Primaria 24-well tissue culture plates. The virus caused cytopathogenic effect (CPE) in the cell cultures as early as 2 day post-infection (p.i.). Initially, the cells rounded up and finally detached from the culture vessel as the infection progressed. At the present time, there is no established quantitative in vitro cell culture protocol for the assay of this baculo-like virus which has been reported by our laboratory to be highly pathogenic for P. stylirostris and P. vannamei, the two species of penaeid shrimp commercially cultured in Hawaii and the Western hemisphere. This quantal assay thus provides a simple and convenient method for the detection and assay of infectious virus in cultured penaeid shrimp.

Detection of yellowhead virus and Chinese baculovirus in penaeid shrimp by the Western blot technique.

The continuing threat posed by viral diseases in cultured shrimp calls for the development of detection technologies for monitoring the animals, especially broodstock. Two of the most highly pathogenic viruses of penaeid shrimp are the yellow-head virus (YHV) and Chinese baculovirus (CBV, also called white spot baculovirus). A Western blot (WB) protocol capable of detecting YHV and CBV in the hemolymph of infected shrimp was developed. The use of the hemolymph as material for virus detection allowed for sample collection without sacrificing the animals. This protocol was highly specific, rapid, and sensitive enough to detect the presence of the viruses before the appearance of overt symptoms. It was also useful for demonstrating the growth of both viruses in primary shrimp lymphoid cell cultures.

Transformation of primary cultures of shrimp (Penaeus stylirostris) lymphoid (Oka) organ with Simian virus-40 (T) antigen.

Primary cultures of lymphoid (Oka) organ from Penaeus stylirostris were transformed with naked or Lipofectin-mediated pSV-3 neo, a shuttle vector containing the tumor (T) antigen gene from Simian virus-40. The transformed cells, OKTr-1 and OKTr-23, exhibited the following characteristics: rounded morphology forming grapelike aggregates, loosely adhesive, increased growth rate in Medium-199, resistance to G-418 (a neomycin analog marker in the shuttle vector), cloning efficiencies of 68.7% and 36.7% in soft agarose, respectively, and stability in liquid nitrogen storage. Immunofluorescence staining (IFA) of the transformed cells using a monoclonal antibody against SV-40 tumor antigen showed positive results. In contrast, primary cell cultures exhibited fibroblast-like morphology and formed a tight, adhesive monolayer on the surface of the culture vessel. They were sensitive to G-418, and showed negative results with IFA. To date, OKTr-1 and OKTr-23 have undergone 44 and 18 passages, respectively. Primary cultures of the lymphoid organ have not been successfully passaged beyond the primary stage.

Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp.

A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.

A streptavidin-biotin-enhanced nitrocellulose enzyme immunoassay for the detection of rhabdovirus of penaeid shrimps from infected animals.

A streptavidin-biotin-enhanced nitrocellulose enzyme immunoassay was developed for the detection of the rhabdovirus of penaeid shrimps (RPS) in the tissues of infected animals. Initial tests indicate that the assay was capable of detecting as few as ten plaque-forming units of virus.

Production of high efficiency of plating hepatitis A virus in primary African green monkey kidney cells.

High-titered hepatitis A virus, strain HM-175, was produced in primary African green monkey kidney cells (5.5 x 10(10) tissue culture ID50/850 cm2 roller bottle). The virus preparation had an efficiency of plating of 15 particles per infectious unit. Single-cycle growth kinetics of the adapted virus indicated that after an eclipse period of 2 days, maximal yields were attained 6 days after infection.

Some biological properties of a rhabdovirus isolated from penaeid shrimps.

Some of the relevant biological properties of a rhabdovirus isolated from penaeid shrimps (RPS) were examined. The virus replicated in an established fish cell line, epithelioma papulosum cyprini (EPC) which allowed for the development of a quantitative plaque assay protocol. Virus replication was not inhibited by the DNA antagonist, 5-bromo-2'-deoxyuridine (20 micrograms/ml). Virus infectivity was sensitive to 20% ethyl ether, low pH, and to 37 degrees C. The virus showed marked lability to repeated freezing and thawing and storage at -10 degrees C, but was stable at -70 degrees C for several weeks. The virus particle to infectious unit ratio in EPC was found to be 30.

A new virus isolate from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps.

A new virus was isolated from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps. The virus was isolated from two species of penaeid shrimps obtained from three different sources employing a previously developed cell-culture assay. Electron-microscopical studies of both purified virus and infected cells showed bullet-shaped particles identifying it as a rhabdovirus.