Introduction

It is realized that the prime purpose of this symposium is to exchange information and thoughts pertinent to those diseases associated with marine mammals; however, in programming this symposium it was felt by some that it would be of general interest to briefly consider virus diseases in a related area of aquatic life, that of the bony fishes. In this review I will discuss the work done by several different groups, as well as some studies done by myself both here at Florida Atlantic University and in the laboratory of Dr. Michael Sigel at the University of Miami. All of this work has been directed toward developing a better understanding of viruses as etiological agents of disease in both fresh water and marine environments. Since tumor production of possible viral origin will be discussed in another paper, they will not be considered in this presentation.

The rather rapid acceleration of our knowledge of the viruses of fish which has occurred in recent years is actually a result of the interplay of two factors. First, fish which were considered to be of importance for either economic reasons or as game fish, were being raised under hatchery conditions, which meant a high fish population density. Moreover, they were confined to an area where man could make careful and deliberate observations of naturally occurring biological phenomena. Second, the development of both fresh water and marine fish tissue culture techniques as established by Wolf et al. (1960), Clem et al. (1961), and others.

It is of added interest to note that tissue culture and virus interactions are being studied in marine mammal cells such as the spotted dolphin (Stenella plagiodon) and the sea lion (Eumetopias jubata). In these studies by Kniazeff and Groyon (1966), herpes simplex, vaccinia, vesicular stomatitis, and Coxsackie B5 viruses were found to replicate in the above mentioned cell cultures.

A listing of the known and suspected viral agents of fish would read as follows, infectious pancreatic necrosis (IPN), lymphocystis, viral hemorrhagic septicemia (VHS), Chinook salmon virus, sockeye salmon virus or viruses, contagious stomatitis virus, fish pox virus and the grunt fin orphan agent (GFA). Time prevents a review of all of these viruses. Moreover, with respect to several of these agents, there is a definite need for further study to establish firmly that the infectious factor is truly a viral agent.

Lymphocystis

One could not consider the viruses of fish without a discussion of lymphocystis virus. This agent, as mentioned by Wissenberg (1965) in his review of the disease, was considered to be viral in nature as far back as 1911. Although at that time this was somewhat controversial and many felt that the responsible agent was a protozoan. Those cells on the fish which are infected with the virus create an epidermal papilloma-like growth on the surface of the animal. However, this phenomenon is due to the growth of the single infected cell rather than cell proliferation. The infected cell may increase in size until it measures hundreds of microns in diameter. Wolf et al. (1966) succeeded in infecting for the first time, in vitro cell cultures of the largemouth bass (Micropterus salmoides) and bluegill (Lepomis macrochirus) with lymphocystis virus. The infected cells developed the typical hyaline capsule, and became Feulgen positive with cytoplasmic basophilic inclusions. In three to four weeks the cells became mature giant sized cells. Using the virus produced by these infected cells for direct nucleic acid determinations, the DNA nature of the virus was confirmed. Furthermore, it has been shown by Walker (1962) that the virus is about 200 mµ in diameter and possesses icosahedral symmetry. The virus infects both fresh water and marine species, and as a further extension of the above mentioned work, Sigel et al. (1966), have shown that lymphocystis virus from infected marine fish will grow and produce a similar phenomenon in marine tissue culture cells in vitro.

Viral Hemorrhagic Septicemia

VHS is a disease associated with salmonid fishes in Europe. It manifests itself as a problem in fish hatcheries in different sections of Europe and it is for this reason that the same etiological agent has been known by many names, depending on the country and the particular researcher involved. The principal pathological features of the disease in the fish are sluggishness, appetite loss, edema, especially in the abdominal area, exopthalmia, and a darkening of the skin. Hemorrhagic areas are apparent in many of the internal organs especially the kidney and muscle tissues. Fingerling fish are the prime victims of this disease (Rasmussen, 1965). Jensen (1965) mentioned growth of the virus in the RTG-2 cell line. In that same year Drs. Jensen and Christensen provided our laboratory at the University of Miami with some of the virus. In some initial probes with this virus, I found that it would grow and produce a cellular degeneration in the marine GF 11 cell line, provided that young cultures in an alkaline environment were used.

Infectious Pancreatic Necrosis

Just as VHS mentioned above presents a hatchery problem in trout in Europe, IPN is a contagious and frequently fatal disease of young brook trout (Salvelinus fontanelles) here in the United States. Wolf et al. (1963) has shown that this virus can be contracted by feeding infected material, injection, and by environmental contact. The infected fish swim in a corkscrew fashion. There are petechiae in the pyloric caeca along with a pale liver and spleen, and destruction of splenic tissue can be evidenced. Work done by Malsberger and Cerini (1963, 1965), indicate that this virus is 18 to 20 mµ in diameter and possesses an RNA core. It has also been shown that this virus will grow in marine fish cell lines as well as those derived from fresh water fish (Sigel, 1967).

Grunt Fin Orphan Agent

The last virus to be considered is more of an enigma and object of speculation than a known disease agent in fish. A few years ago, the GF I cell line initiated by Clem et al. (1961) was being used in the laboratory of Dr. Sigel as an indicator system for the detection of possible orphan viruses in marine fish. Intestinal extracts from several hundred fish samples produced no suggestion of viral isolation. However, an un-inoculated bottle of GF I cells gave rise to foci of necrotic cells similar to that produced by polio virus in mammalian cell cultures (Clem et al. 1965). These foci of a virus-like cytopathogenic effect (CPE) spread until they were confluent and destroyed the cell monolayer. The infectious factor was found to be resistant to antibiotics, incapable of growth in mammalian cells, non-hemagglutinating, highly labile in the absence of protective colloids, such as serum, and ether sensitive. Attempts to grow the agent in RTG-2 cells were also negative, as were attempts to obtain growth in media used for the isolation of mycoplasma. Furthermore, the agent showed no serological cross reactivity with any of the mammalian viruses present in the laboratory at that time, and growth was obtained only in cell cultures prepared from marine species. It has been possible to establish a single step growth curve for this agent, and a typical virus eclipse and latent stage in its replicative cycle is demonstrable. Although no disease has been associated with this infectious agent, it in a sense appears to be the first isolation of a marine fish orphan virus which may have been either latent in the cells or introduced as a contaminant (Clem et al., 1965). While working in the laboratory of Dr. Sigel, Dr. Clem most generously provided me with some of the virus stocks so that I might further these studies. So as to avoid any question which might arise concerning the possible latency phenomenon and its effect on subsequent observations with the use of the same cell line from which the virus spontaneously appeared, new cell lines from grunt (Heamulon sciurus) (Figure 1) and schoolmaster snapper (Litjuanus apodus) were initiated, using fish provided by the Miami Seaquarium. This second grunt fin cell line (GF II), appears to grow somewhat faster and has an optimal temperature at about 25 C , as opposed to the optimal temperature of about 20 C for the GF I cell line. The maximal temperature for the GF II cells is about 28 C; whereas, the schoolmaster snapper fin (SSF) cells have a maximal temperature of 30 C , with optimal growth at 25 C At 25 C virus CPE progresses much more rapidly than at 20 C. It is also of interest to note that the SSF cells are more fibroblastic in their morphological appearance than the GF II cells, but that they support virus CPE production equally well. As previously mentioned, the CPE with adequate dilution appears as a focus phenomenon, ultimately spreading throughout the cell sheet. In the GF II cells 24 hours after inoculation with an appropriate dilution of GFA, foci of pyknotic refractile cells, as opposed to the normal epithelial cells, are apparent (Figure 2). With continued incubation, the focus may be shown to spread (Figure 3), ultimately destroying the cell monolayer. Un-inoculated monolayer cultures still retain their normal epithelial-like appearance (Figure 4). By employing a gelatin overlay (Beasley, personal communication) and incubating at 20 C , virus plaquing and therefore quantitation, is very readily accomplished. In Figure 5, two cell monolayers infected with different virus dilutions are shown. The bottle showing the I mm plaques was stained at 4 days post infection, The second bottle, showing the larger plaques, was inoculated with a more dilute virus preparation and stained 6 days after infection. Clem et al. (1965) has shown that the number of plaque forming units is linear with dilution of the virus. As mentioned in the discussion of VHS and IPN viruses, the fingerling stage of the fish appears to be more susceptible to overt virus infection. Clem et al. (1965) has shown that the virus rapidly disappears from adult fish without causing infection. It was felt that perhaps young juvenile Haemulon sciurus might prove more responsive to this virus in terms of infection. When juvenile fish 15 to 25 mm long were inoculated with the virus no obvious disease pattern was observed for a period of up to one month post inoculation; however, it was possible to recover virus from the infected fish for up to 12 days post inoculation. This recoverable virus was no higher in titer than the initial input inoculum, which suggests no apparent virus replication.

Figure 1. A species of the grunt (Haemulon sciurus)used in preparing the GF II cell line which has now been passed in vitro well over one hundred times.

Figure 2. GF II cells 24 hours post infection. A focus of rounded infected cells is apparent against a background of normal looking cells.

Figure 3. After 48 hours the focus has spread until most of the cell sheet is infected. Many of the infected cells have rounded and fallen off the glass.

Figure 5A and 5B.. Plaquing of the grunt fin orphan agent in GF II cells. The bottle on the left (5A) was inoculated with a high virus concentration and stained 4 days post inoculation. The bottle on the right (5B) was inoculated with a lower virus concentration and stained 6 days post inoculation. Note the differences in plaque size and number dependent upon virus concentration and incubation time.

Biochemical and biophysical characterization necessitating large quantities of virus, have been impaired to an extent by the extreme lability of the virus, which is especially true in its more purified forms. However, these studies are being currently undertaken. Work done by Clem et al. (1965) indicates that the agent will pass through a 0.22µ porosity filter; at present more precise size determinations are being performed. As a presumptive test to determine the nature of the nucleic acid present in this agent, pyrimidine nucleotide analogs which act to inhibit or alter DNA replication by more than one mode of action, have been used in the cultivation of this virus. It appears that neither fluoro-, iodo- or bromodeoxyuridine inhibit virus replication in mycoplasma-free GF II cells when used in concentrations of up to 100 Y per ml . On the basis of this presumptive test it is suggested that the agent is an RNA virus which is independent of de novoDNA synthesis.

At present, additional work is being done at Florida Atlantic University to further characterize this agent. The origin of the virus is still an enigma. Sigel (1967) feels that there is evidence which suggests that this virus may have been reactivated after a prolonged latent period in tissue culture. This is apparently based upon the relative ease with which one can establish chronically infected carrier cell cultures. This may also involve an interferon-like substance. One can still but speculate on the origin of this agent. However, its sudden appearance in a "normal" culture, in addition to its apparent affinity for marine cells, leads to a feeling that this virus warrants further study and may provide man with added knowledge of virus-cell and virus-host interactions in a marine environment.

References

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