Classification

Figure 2

Figure 2. A) Cladogram depicting relationships among viruses in the order Herpesvirales, based on the conserved regions of the terminase gene. The Bayesian maximum-likelihood tree was rooted by using bacteriophages T4 and RB69....

CyHV-3 is a member of the order Herpesvirales and newly designated family Alloherpesviridae (5,6) (Figure 2, panel A). Alloherpesviridae viruses infect fish and amphibians. The common ancestor of this family is thought to have diverged from the common ancestor of the family Herpesviridae (herpesviruses that infect reptiles, birds, and mammals) (6). According to phylogenetic analysis of specific genes, the family Alloherpesviridae seems to be subdivided into 2 clades (6) (Figure 2, panel B). The first clade comprises anguillid and cyprinid herpesviruses, which possess the largest genomes in the order Herpesvirales (245–295 kb). The second clade comprises ictalurid, salmonid, acipenserid, and ranid herpesviruses, which have smaller DNA genomes (134–235 kb).

Structure

Figure 3

Figure 3. Electron micrograph image of cyprinid herpesvirus 3 virion. Scale bar = 100 nm. Adapted with permission from Mettenleiter et al. (7).

The CyHV-3 structure is typical of viruses of the order Herpesvirales. An icosahedral capsid contains the genome, which consists of a single, linear, double-stranded DNA molecule. The capsid is covered by a proteinaceous matrix called the tegument, which is surrounded by a lipid envelope derived from host cell trans-golgi membrane (7) (Figure 3). The envelope contains viral glycoproteins (3). The diameter of the entire CyHV-3 particle is 170–200 nm (3,8).

Molecular Structure

In Vitro Replication

CyHV-3 is widely cultivated in cell lines derived from koi fin, C. carpio carp brain, and C. carpio carp gill (3,4,8,15–17) (Table 1). Other cell lines have been tested, but few have been found to be permissive for CyHV-3 infection (Table 1).

The CyHV-3 replication cycle was recently studied by use of electron microscopy (7). Its morphologic stages suggested that it replicates in a manner similar to that of members of the family Herpesviridae. Capsids leave the nucleus by budding at the inner nuclear membrane, resulting in formation of primary enveloped virions in the perinuclear space. The primary envelope then fuses with the outer leaflet of the nuclear membrane, thereby releasing nucleocapsids into the cytoplasm. Final envelopment occurs by budding into trans-golgi vesicles. Because CyHV-3 glycoproteins have little or no similarity with those of members of the family Herpesviridae, identification of the CyHV-3 glycoproteins involved in entry and egress will require further study.

Figure 4

Figure 4. Effects of temperature on cyprinid herpesvirus 3 replication in Cyprinus carpio carp brain cells. After infection, cells were kept at 22°C (A) or shifted to 30°C (B–D); some cells were returned...

Because fish are poikilotherms and because CyHV-3 only affects fish when the water temperature is 18°C–28°C, the effect of temperature on CyHV-3 replication growth in vitro has been investigated. Replication in cell culture is restricted by temperature; optimal viral growth is at 15°C–25°C. Virus propagation and virus gene transcription are turned off when cells are moved to a nonpermissive temperature of 30°C (18). Despite the absence of detectable virus replication, infected cells maintained for 30 days at 30°C preserve infectious virus, as demonstrated by viral replication when the cells are returned to permissive temperatures (18) (Figure 4). These results suggest that CyHV-3 can persist asymptomatically for long periods in the fish body when the temperature prevents virus replication; bursts of new infection occur after exposure to permissive temperatures.

History

In 1998, the first mass deaths of common and koi carp were reported in Israel and the United States (3). However, analyses of samples from archives determined that the virus had been in wild common carp since 1996 in the United Kingdom (19). Soon after the first report, outbreaks of CyHV-3 were identified in countries in Europe, Asia, and Africa. Currently, CyHV-3 has been identified everywhere in the world except South America, Australia, and northern Africa (20). Worldwide, CyHV-3 has caused severe financial and economic losses in the koi and common carp culture industries.

Host Range

Common and koi carp are the only species known to be affected by CyHV-3 infection (21). Numerous fish species, cyprinid and noncyprinid, were tested for their ability to carry CyHV-3 asymptomatically and to spread it to unexposed carp (21–23) (Table 2). CyHV-3 DNA was recovered from only 2 other fish species: goldfish and crucian carp. Cohabitation experiments suggest that goldfish, grass carp, and tench can carry CyHV-3 asymptomatically and spread it to unexposed common carp. Hybrids (koi–goldfish and koi–crucian carp) die of CyHV-3 infection (24).

Susceptibity

CyHV-3 affects carp of all ages, but younger fish (1–3 months, 2.5–6 g) seem to be more susceptible to infection than mature fish (1 year, ≈230 g) (16,21). Recently, the susceptibility of young carp to CyHV-3 infection was analyzed by experimental infection (25). Most infected juveniles (>13 days posthatching) died of the disease, but the larvae (3 days posthatching) were not susceptible.

Pathogenesis

Figure 5

Figure 5. Skin of carp as a portal of entry for cyprinid herpesvirus 3. A schematic representation of the system used to restrict viral inoculation to the fish skin is shown on the...

Several researchers have postulated that the gills might be the portal of entry for CyHV-3 (17, 26–28); however, this hypothesis was recently refuted (29). Bioluminescent imaging and an original system for performing percutaneous infection restricted to the posterior part of the fish showed that the skin covering the fin and body mediated entry of CyHV-3 into carp (29) (Figure 5). This study, together with an earlier study of the portal of entry of a rhabdovirus (infectious hematopoietic necrosis virus) in salmonids (30), suggests that the skin of teleost fish represents an efficient portal of entry for certain viruses. The skin of teleost fish is a stratified squamous epithelium that, unlike its mammalian counterpart, is living and capable of mitotic division at all levels, even the outermost squamous layer. The scales are dermal structures. More extensive studies are needed to demonstrate that the skin is the only portal of entry of CyHV-3 into carp.

After initial replication in the epidermis (29), the virus is postulated to spread rapidly in infected fish, as indicated by detection of CyHV-3 DNA in fish tissues (27). As early as 24 hours postinfection, CyHV-3 DNA was recovered from almost all internal tissues (including liver, kidney, gut, spleen, and brain) (27), where viral replication occurs at later stages of infection and causes lesions. One hypothesis regarding the rapid and systemic dissemination indicated by PCR is that CyHV-3 secondarily infects blood cells. Virus replication in organs such as the gills, skin, and gut at the later stages of infection represents sources of viral excretion into the environment. After natural infection under permissive temperatures (18°C–28°C), the highest mortality rates occur 8–12 days postinfection (dpi) (21). Gilad et al. suggest that death is due to loss of the osmoregulatory functions of the gills, kidneys, and gut (27).

All members of the family Herpesviridae exhibit 2 distinct life-cycle phases: lytic replication and latency. Latency is characterized by maintenance of the viral genome as a nonintegrated episome and expression of a limited number of viral genes and microRNAs. At the time of reactivation, latency is replaced by lytic replication. Latency has not been demonstrated conclusively in members of the family Alloherpesviridae. However, some evidence supports existence of a latent phase. CyHV-3 DNA has been detected by real-time PCR at 65 dpi in clinically healthy fish (27). Furthermore, the virus persisted in a wild population of common carp for at least 2 years after the initial outbreak (31). Finally, St-Hilaire et al. demonstrated the possibility of a temperature-dependent reactivation of CyHV-3 lytic infection several months after initial exposure to the virus (32). This finding suggests that the temperature of the water could control the outcome of the infection (replicative/nonreplicative). Whether the observations described above reflect latent infection, as described for the family Herpesviridae, or some type of chronic infection, remains to be determined. Similarly, the carp organs that support this latent or chronic infection still need to be identified.

Transmission

Horizontal transmission of CyHV-3 in feces (26) and secretion of viral particles into water (21) have been demonstrated. The skin of carp acts as the portal of entry of CyHV-3 and the site of early replication (29). The early replication of the virus at the portal of entry could contribute not only to the spread of the virus within infected fish but also to the spread of the virus throughout the fish population. As early as 2–3 dpi, infected fish rubbed against other fish or against objects. This behavior could contribute to a skin-to-skin mode of transmission. Later during infection, this mode of transmission could also occur when uninfected fish pick at the macroscopic herpetic skin lesions on infected fish. To date, no evidence of vertical transmission of CyHV-3 has been found.

Clinical Signs

Figure 6

Figure 6. Clinical signs in cyprinid herpesvirus 3–infected fish. A) Severe gill necrosis; B) hyperemia at the base of the caudal fin; C) herpetic skin lesions on the body and fin erosion.

The first signs appear at 2–3 dpi. The fish exhibit appetite loss and lethargy and lie at the bottom of the tank with the dorsal fin folded. Depending on the stage of the infection, the skin exhibits different clinical signs, such as hyperemia, particularly at the base of the fins and on the abdomen; mucus hypersecretion; and herpetic lesions (Figure 6). The gills frequently become necrotic and hypersecrete mucus, which suffocates the fish. Bilateral enophthalmia is observed in the later stages of infection. Some fish show neurologic signs in the final stage of the disease, when they become disoriented and lose equilibrium (3,19,21).

Histopathologic Findings

In CyHV-3 infected fish, prominent pathologic changes occur in the gill, skin, kidney, liver, spleen, gastrointestinal system, and brain (3,17,21,28). Histopathologic changes appear in the gills as early as 2 dpi and involve the epithelial cells of the gill filaments. These cells exhibit hyperplasia, hypertrophy, and/or nuclear degeneration (3,17,21,28). Severe inflammation leads to the fusion of respiratory epithelial cells with cells of the neighboring lamellae, resulting in lamellar fusion (17,28). In the kidney, a weak peritubular inflammatory infiltrate is evident as early as 2 dpi and, along with blood vessel congestion and degeneration of the tubular epithelium in many nephrons, increases with time (17). In the spleen and liver, splenocytes and hepatocytes, respectively, are the most obviously infected cells (28). In brain of fish that showed neurologic signs, congestion of capillaries and small veins are apparent in the valvula cerebelli and medulla oblongata, associated with edematous dissociation of nerve fibers (28).

Diagnosis

Diagnosis of CyHV-3 infection is described elsewhere (20). Suspicion of CyHV-3 infection is based on clinical signs and histopathologic findings. Since initial isolation of CyHV-3 in 1999, complementary diagnostic methods have been developed. Virus isolation from infected fish tissues in cell culture (C. carpio carp brain and koi fin cells) was the first method to be developed (3). This time-consuming approach is still the most effective method for detecting infectious particles during an outbreak of CyHV-3 infection. A complete set of techniques for detecting viral genes—including PCR (20), nested PCR (33), TaqMan PCR (27), and loop-mediated isothermal amplification (34)—has been developed. Real-time TaqMan PCR has been used to detect CyHV-3 in freshwater environments after concentration of viral particles (2). Finally, ELISAs have been developed to detect specific anti-CyHV-3 antibodies in the blood of carp (35) and to detect CyHV-3 antigens in samples (17,26).

Selection of CyHV-3–Resistant Carp

Carp resistance to CyHV-3 might be affected by host genetic factors. Shapira et al. demonstrated differential resistance to CyHV-3 (survival rates 8%–60%) by cross-breeding sensitive domesticate strains and a resistant wild strain of carp (38). Further supporting the role of host genetic factors in CyHV-3 resistance, major histocompatibility class II genes were recently shown to affect carp resistance (39).

Vaccination of Carp

Soon after the characterization of CyHV-3, a protocol to induce a protective adaptive immune response in carp was developed. This approach is based on the fact that CyHV-3 induces fatal infections only when the water temperature is 18°C–28°C.

According to this protocol, healthy, uninfected fish are exposed to CyHV-3 infected fish for 3–5 days at permissive temperature (22°C–23°C) and then transferred for 30 days to ponds at a nonpermissive temperature (≈30°C). After this procedure, 60% of fish become resistant to further challenge with CyHV-3 (4). Despite its ingenuity, this method has several disadvantages: 1) increasing the water temperature to 30°C makes the fish more susceptible to secondary infection by other pathogens and requires a large amount of energy in places where the water is naturally cool; 2) the protection is observed in only 60% of fish; 3) carp that are “vaccinated” by using this protocol have been exposed to wild-type virulent CyHV-3 and could therefore represent a potential source of CyHV-3 outbreaks if they later come into contact with an unexposed carp.

Attenuated live vaccine appears to be the most appropriate for mass vaccination of carp. Attenuated vaccine candidates have been produced by successive passages in cell culture (4). The vaccine strain candidate was further attenuated by UV irradiation to increase the mutation rate of the viral genome (4,37). A vaccine strain obtained by this process has been produced by KoVax Ltd. (Jerusalem, Israel) and has been shown to confer protection against a virulent challenge. However, this vaccine is available in only Israel and has 2 main disadvantages: 1) the molecular basis for the reduced virulence is unknown, and consequently, reversions to a pathogenic phenotype cannot be excluded; and 2) under certain conditions, the produced attenuated strain could retain residual virulence that could be lethal for a portion of the vaccinated fish (37).

An inactivated vaccine candidate was described by Yasumoto et al. (40). It consists of formalin-inactivated CyHV-3 trapped within a liposomal compartment. This vaccine can be used for oral immunization in fish food. Protection efficacy for carp is 70% (40).