Philip C. Loh

The preferential cytotoxicity of reovirus for certain transformed cell lines

SummaryThe susceptibility of a variety of cell lines of different mammalian origin to cytotoxic (CT) induction by either ultraviolet light-irradiated reovirus type 2 (UVR2) or viable reovirus type 2 plus the protein synthesis inhibitor, cycloheximide, was examined. The following groups of cells were found to be susceptible to CT-induction: certain tumor cells and spontaneously transformed cell lines of human origin and certain virally and spontaneously transformed cell lines of murine origin. The following groups of cells were found to be resistant: normal human diploid cell lines, primary and continuous cell cultures of subhuman primates, primary mouse cells, normal rat kidney cells and baby hamster kidney cells. Susceptibility to CT-induction could not be related to the adsorption of virus to cells, nor to the capacity of the cell to support virus replication.

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.

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.

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.

Seroepidemiology of Human Syncytial Virus: Antibody Prevalence in the Pacific

A seroepidemiological survey of the human syncytial (foamy) virus was done by means of an indirect immunofluorescence test on 1,717 sera from nine different Pacific island territories. The specificity of the reaction was verified by neutralization tests. The study indicated that the virus is ubiquitous in this part of the world, with no region being entirely free of antibody. The antibody prevalence ranged from a low of 1.2% in Ponape to a high of 15.6% in the Cook Islands. The average prevalence for the nine insular communities was 6.9%.

Viral pathogens of the penaeid shrimp.

Publisher Summary While significant advances that have been made in determining the role of viruses involved in various epizootics occurring in penned shrimp aquaculture, viral diseases will continue to plague the industry. A major obstacle to the study of these diseases is the lack of convenient and quantitative methodologies, such as in vitro cell culture systems to grow and study (characterize) the virus. A beginning has been made with the recent development of protocols for the consistent preparation of primary shrimp lymphoid cells, which were employed for the quanta1 assay of some of the shrimp viral pathogens. The primary cell lines have also been used to analyze the synthesis of viral proteins at the cellular level and to study viral pathogenesis. With the further successful development of additional primary cell lines from other shrimp tissues and the establishment of continuous diploid and transformed shrimp cell lines, this problem is being solved. The value of cell culture systems is becoming increasingly clear. They present several obvious advantages because (1) they are more cost effective, sensitive, and convenient than whole animals, particularly for rapid monitoring of infectivity, (2) they yield quantitatively reproducible results, and (3) viral growth kinetics, biochemical and genetic characteristics, and so on can be studied more easily. Their biggest potential use is in future molecular biology and genetic studies of shrimp viruses.

Reovirus Type 2: Induction of Viral Resistance and Interferon Production in Fathead Minnow Cells

Summary Reovirus type 2 was found to adsorb to cells derived from a poikilothermic animal, the fathead minnow. Although this interaction between cell and virus did not result in the production of infectious virus, viral antigen, or viral inclusion, a state of viral resistance was developed and an antiviral substance was produced. This viral inhibitor was shown to be interferon based on the possession of common characteristics.

Cytomegalovirus isolations from cell cultures of human adenocarcinomas of the colon.

Cytomegalovirus was isolated from cell cultures derived from 3 of 16 surgical specimens of adenocarcinomas of the colon. Virus identification was accomplished through electron microscopical, cytochemical, and immunofluorescent procedures.

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.

Use of the nitrocellulose-enzyme immunosorbent assay for rapid, sensitive and quantitative detection of human enteroviruses

A modified enzyme immunosorbent assay (EIA) employing nitrocellulose (NC) membrane as a high-capacity solid phase was successfully employed for the sensitive and rapid detection of human enteric viruses, poliovirus and Coxsackievirus B-5. The sensitivity of the NC-EIA ranged from 7 to 70 pg of viral antigen diluted in phosphate-buffered saline. When virus was added to crude supernatants of mollusc tissue homogenates prepared by the standard procedure for the recovery of viruses in molluscs, the sensitivity was reduced by approximately 10-fold. The sensitivity of the NC-EIA for the detection of viral antigens was 10- to 100-fold higher than conventional EIA using polystyrene microtiter plates as solid phase supports. This simple, rapid and sensitive assay using minimal amounts of antibodies should prove to be a useful and practical diagnostic tool to augment infectivity assays currently employed by various virus monitoring procedures. The method also may be applicable for the detection of difficult to grow and/or non-cultivatable enteric viruses which may be present in sewage-contaminated environments.