Frog virus 3 is the best characterized species within the genus Ranavirus, family Iridoviridae. FV3's large (∼105 kbp) dsDNA genome encodes 98 putative open reading frames (ORFs) that are expressed in a coordinated fashion leading to the sequential appearance of immediate early (IE), delayed early (DE) and late (L) viral transcripts. As a step toward elucidating molecular events in FV3 replication, we sought to identify the temporal class of viral messages. To accomplish this objective an oligonucleotide microarray containing 70-mer probes corresponding to each of the 98 FV3 ORFs was designed and used to examine viral gene expression. Viral transcription was initially monitored during the course of a productive replication cycle at 2, 4 and 9 hours after infection. To confirm results of the time course assay, viral gene expression was also monitored in the presence of cycloheximide (CHX), which limits expression to only IE genes, and following infection with a temperature sensitive (ts) mutant which at non-permissive temperatures is defective in viral DNA synthesis and blocked in late gene expression. Subsequently, microarray analyses were validated by RT-PCR and qRT-PCR. Using these approaches we identified 33 IE genes, 22 DE genes and 36 L viral genes. The temporal class of the 7 remaining genes could not be determined. Comparison of putative protein function with temporal class indicated that, in general, genes encoding putative regulatory factors, or proteins that played a part in nucleic acid metabolism and immune evasion, were classified as IE and DE genes, whereas those involved in DNA packaging and virion assembly were considered L genes. Information on temporal class will provide the basis for determining whether members of the same temporal class contain common upstream regulatory regions and perhaps allow us to identify virion-associated and virus-induced proteins that control viral gene expression.

A virus, designated Rana catesbeiana virus Z (RCV-Z), was isolated from the visceral tissue of moribund tadpoles of the North American bullfrog Rana catesbeiana. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of viral proteins and sequence analysis of the amino terminal end of the major capsid protein showed that RCV-Z was similar to frog virus 3 (FV3) and other ranaviruses isolated from anurans and fish. However, analysis of restriction fragment profiles following digestion of viral genomic DNA with Xba I and Bam HI indicated that RCV-Z was markedly different from FV3. Moreover, in contrast to FV3, RCV-Z contained a full-length copy of the viral homolog of eukaryotic initiation factor 2 alpha (eIF-2α). Experimental infection of bullfrog tadpoles with FV3 and RCV-Z demonstrated that RCV-Z was much more pathogenic than FV3, and that prior infection with FV3 protected them from subsequent RCV-Z induced mortality. Collectively, these results suggest that RCV-Z may represent a novel species of ranavirus capable of infecting frogs and that possession of a viral eIF-2α homolog (vIF-2α) correlates with enhanced virulence. KEY WORDS: Iridovirus · Ranavirus · Frog virus 3 · Rana catesbeiana virus Z · eIF-2α homolog · Virulence Resale or republication not permitted without written consent of the publisherDis Aquat Org 73: [1][2][3][4][5][6][7][8][9][10][11] 2006 disease in mature frogs was linked to immune suppression. In addition, these investigators showed that FV3 has a strong tropism for the proximal tubular epithelium of the kidney, but rarely disseminates beyond that tissue unless host defenses are experimentally impaired or developmentally immature as in larvae (Robert et al. 2005). Collectively, these results indicate that FV3 possesses low virulence or pathogenicity in vivo when inoculated into immunocompetent adult frogs, but readily infects and kills frog embryos, larvae, and immunocompromised adults.In contrast to the above studies, other members of the genus Ranavirus have been linked to die-offs of cultured frogs in China and Thailand, garden frogs in the UK, and wild frogs in Europe, South America, and North America (Cunningham et al. 1996, Hyatt et al. 2000, Zhang et al. 2001, Chinchar 2002, Pearman & Garner 2005. Moreover, ranaviruses have been linked to die-offs of adult salamanders and fish, indicating that ranaviruses possess considerable pathogenic potential in cold-blood animals (Langdon et al. 1986, Jancovich et al. 2005. It is unclear whether recent die-offs among various frog species are due to one or more novel ranavirus species, or to pathogenic strains of FV3. Furthermore, it is not known if enhanced pathogenicity is inherent in the virus itself or reflects the effects of environmental insults that depress immunity and predispose to viral disease, e.g. crowding among farmed animals or exposure to toxic chemicals or other pollutants. Herein we report the isolation and characterization of a ranavirus that was responsible for a die-off among cultured Ame...

Frog virus 3 (FV3) is a large DNA virus that encodes approximately 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIalpha). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIalpha triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.