Herpesviruses

The herpesviruses Ranid herpesvirus 1 (RaHV1), Ranid herpesvirus 2 (RaHV2), Ranid herpesviurs 3 (RaHV3) and Bufonid herpesvirus 1 (BfHV1) are known in frogs and toads. All four of these viruses belong to the genus Batrachovirus of the family Alloherpesviridae.
RaHV1 and RaHV2 are not visible to the eye, do not cause any skin changes and have so far only been detected in North America. RaHV3 and BfHV1 have been detected in Europe, both of which cause skin diseases.

Bufonid herpesvirus 1 (BfHV1)

BfHV1 was only described in 2018 (Origgi et al. 2018 ^[1]) and has been observed in common toads (Bufo bufo) in Switzerland every year since 2014. BfHV1 is found in the skin and sometimes in the brain. BfHV1 leads to a rampant skin disease in common toads with severe skin lesions and patchy brown discoloration of the skin (compare Figure 1). The skin lesions may become so large that they must be considered clinically relevant. BfHV1 was detected on dead common toads. However, it is unclear whether BfHV1 was the sole cause of death and what the mortality rate was.

The BfHV1 evidence from Switzerland comes from amphibian migration points managed during the spawning season and other observations from various regions of Switzerland (Anwil BL, Neuchâtel, Zurich area). In certain populations observed, approximately 10% of common toads were affected.

BfHV1 also in Germany

Another strain of BfHV1 or a herpesvirus closely related to BfHV1 was discovered in Germany (Eisenberg et al. 2020 ^[2]). In 2018, a few common toads with skin diseases were noticed at an amphibian migration site in Hesse. In March 2020, after over a third of all captured toads showed characteristic skin lesions, microbiological examinations were ordered. A larger fragment (1,552 bp) of the suspected DNA polymerase gene shows only four different nucleic acids compared to BfHV1. These four mutations cause only one amino acid exchange after translation. Because of the phylogenetic similarity, the virus is referred to as BfHV1_G/H/20_1. It may just be a different strain of BfHV1 (BfHV1_FO1_2015). Figures 2-5 show a common toad (Bufo bufo) with healthy skin (left) and a common toad with house lesions (middle & right).

BfHV1 probably widespread

Even outside Switzerland and Germany, toads are not spared from herpes viruses. More than 10 years ago, a common toad (Bufo bufo) with skin disease and BfHV1 symptoms tested positive for herpes viruses using molecular diagnostics (F. Pasmans, unpublished data). In the Netherlands, common toads with BfHV1 symptoms were also documented (compare Figure 6). The conclusion should be said: “The herpesvirus is not a new disease. The herpesvirus is likely to be widespread.” (Schmidt 2018 ^[3])

Confusion with natural staining

The natural staining of some common toads should not be confused with the sympotes of BfHV1 (compare Figure 7).

Confusion with “black fungi”

Black spots on common toads (Bufo bufo) can also be caused by various types of “black fungi”. There may have been confusion in the past. The few common toads that were found with skin diseases at an amphibian migration site in the Geldern/Lower Rhine (NRW) area in 2012 (David 2013 ^[4]) could also have been infected with a strain of BfHV1 (PD Dr. med. vet. Francesco Origgi, personal communication August 24, 2021).

Ranid herpesvirus 3 (RaHV3)

At the end of March 2015, common frogs (Rana temporaria) were observed with skin lesions in a pond near Zurich, which were showing symptoms that had already been observed 20 years earlier in European frogs. The common frogs showed white or especially gray skin spots (compare Figure 8). Some specimens from the pond in Zurich were collected and extensively examined. A new herpesvirus, RaHV3, was described (Origgi et al. 2017 ^[5]). It was found that RaHV3 infection in common frogs is usually not lethal. Furthermore, infestation of internal organs or tumor formation was not observed.

Detection also in other countries

In the UK, symptoms of RaHV3 have been observed since the early 1990s at several sites in England and Wales. All observations were made exclusively on common frogs. Investigations showed that it is RaHV3 or a virus similar to RaHV3, which is usually not lethal (Franklinas et al. 2018 ^[6]).

In Italy, symptoms of RaHV3 were described in agile frogs (Rana dalmatina) as early as 1994. For a long time the herpesvirus was only confirmed by histological examinations and it was not clear if it was RaHV3. Only a few years ago, a piece of the orginal tissue was analyzed and part of the genome was successfully sequenced, thus confirming RaHV3 or a closely related virus (Origgi et al. 2017 ^[5]). In a more recent publication only RaHV3 and no RaHV3 closely related viruses are mentioned in connection with agile frogs from Italy (Origgi et al. 2021 ^[7]). Upon request, it was confirmed that the infection of the spring frogs in Italy is RaHV3 (PD Dr. med. vet. Francesco Origgi, personal communication August 24, 2021).

In Germany in northeastern Berlin, moor frogs (Rana arvalis), common frogs (Rana temporaria), and spadefoot toads (Pelobates fuscus) were sighted with RaHV3 symptoms during spring from 2000 to 2003. However, the herpesviruses were only confirmed histologically. The skin of all species healed within 2-3 weeks. Five spadefoot toads were kept in the laboratory, and all five died a few weeks later. All five specimens were diagnosed with adenocarcinoma of the kidneys (Mutschmann et al. 2008 ^[8]). Unfortunately, there are no samples of either DNA or tissue, so not much more is known (PD Dr. med. vet. Francesco Origgi, personal communication August 24, 2021).

In Austria near Vienna more precisely in Bad Vöslau, common frogs with RaHV3 symptoms were documented in a private pond at the end of March 2021 (compare Figure 9-11). The photos were first shared on Instagram and later forwarded to involved researchers. Unfortunately, it was not possible to take skin swabs using sterile (DNAse-free) cotton swabs, as the common frogs had quickly migrated away after spawning.

In Sweden, an attentive resident also thought to have already seen common frogs with such symptoms (communicated March 2021). However, most of the common frogs in the wild were probably already healed when he went looking for them.

In Norway, there have also been sightings of infected common frogs. For the first time, tadpoles got examined for RaHV3. They tadpoles got collected and humanely euthanized. Five ponds in Lillehammer, Skytta, and Gjelleråsen got examined and RaHV3 DNA was detected in 7 tadpoles. No obvious tadpole die-offs or reduced fitness in tadpoles had been recently reported at the sampling sites; however, those sites are not presently monitored. (Origgi et al. 2023 ^[8B]).

Ranid herpesvirus 1 (RaHV1)

RaHV1, also known as Lucke’s herpesvirus, has been known for the longest time among the amphibian herpesviruses. RaHV1 is known to cause kidney tumors in leopard frogs (Lithobates pipiens) from North America. The tumor in leopard frogs was incorrectly described as an adrenal tumor by Smallwood as early as 1905 (Smallwood 1905 ^[9]). This mistake was denounced by Murray three years later (Murray 1908 ^[10]). Baldwin Lucké examined tumors in 158 leopard frogs, a sufficiently large number, and confirmed them as kidney tumors (Lucké 1934 ^[11]). Lucké later also confirmed using a light microscope the intranuclear inclusion bodies in the epithelium of the kidney tumors, which are similar to inclusion bodies from other herpesviruses (Lucké 1938 ^[12]). However, the virus itself, much too small for the light microscope, was only confirmed using an electron microscope in 1956 (Fawcett 1956 ^[13]). The complete genome sequencing of RaHV1 was carried out in 2006 (Davison et al. 2006 ^[14]). The phylogenetic relationships within the family Alloherpesviridae were then determined by comparing the DNA polymerase gene sequence and other well-conserved gene sequences (Waltzek et al. 2009 ^[15]). The proposal for the creation of a new genus “Batrachovirus” with RaHV1 and RaHV2 within the family Alloherpesviridae was submitted by Waltzek to the International Committee for Taxonomy of Viruses (ICTV) and accepted by it.

Detection of RaHV1 easiest after hibernation

RaHV1 repliziert resp. vermehrt sich im Tumor während den kälteren Temperaturen im Winter und wird besonders bei der Laichsession im Frühjahr abgegeben. Während den wärmeren Temperaturen im Sommer stellt sich die Virus Replikation ein. Umgekehrt verhält sich das Wachstum des Tumor, der bei wärmeren Temperatur stärker wächst und im Winter kaum (McKinnell 1967 ^[16], Mizell 1985 ^[17]; Williams et al. 1996 ^[18]). Der Nachweis von RaHV1 kann vor der Überwinterung negativ sein, während er danach positiv ist. In einem späteren Verlauf, kann der Virus neben dem Primärtumor in den Nieren, auch in den metastatischen Zellen der Blase, des Fettkörpers und der Leber nachgewiesen werden. In einem späteren Krankheitsverlauf ist es möglich, dass in beiden Nieren einzelne oder mehrere Tumore wachsen (Lucké 1934 ^[11]). Die RaHV1 Tumore können von anderen Tumoren mittels Licht- oder Elektronenmikroskop unterschieden werden, so gelingt die Ermittlung der Krankheitsursache auch ohne den direkten Nachweis von RaHV1 (Densmore et al. 2007 ^[19]). Betroffene Frösche zeigen möglicherweise keine Anzeichen, bis die Krankheit weit fortgeschritten ist. Eine Heilung ist nicht möglich, betroffene Frösche sollten von anderen Amphibien isoliert und erlöst werden.
RaHV1 replicates or multiplies in the tumor during the colder temperatures in winter and is released especially during the spawning session in spring. During the warmer temperatures in summer, virus replication stops. The reverse is true for tumor growth, which increases more during warmer temperatures and hardly grows at all during winter (McKinnell 1967 ^[16], Mizell 1985 ^[17]; Williams et al. 1996 ^[18]). Detection of RaHV1 may be negative before hibernation, while positive thereafter. In a later course, the virus can be detected in the metastatic cells of the bladder, fat body and liver, in addition to the primary tumor in the kidneys. In a later course of the disease, it is possible that single or multiple tumors grow in both kidneys (Lucké 1934 ^[11]). RaHV1 tumors can be distinguished from other tumors by light or electron microscopy, thus determination of the cause of disease is successful even without direct detection of RaHV1 (Densmore et al. 2007 ^[19]). Affected frogs may show no signs until the disease is well advanced. A cure is not possible, and affected frogs should be isolated from other amphibians and released from their pain.

Other species affected

Studies show that the American green frog (Lithobates clamitans) and the Pickerel frog (Lithobates palustris) are also susceptible to RaHV1 (Encyclopedia of Virology 4^th Edition ^[20]).

Ranid herpesvirus 2 (RaHV2)

RaHV2 ist auch als Frog Virus 4 (FV-4) bekannt und wurde aus dem Urin von Adenokarzinom-tragenden Leopardfröschen (Rana pipiens) isoliert (Rafferty 1965 ^[21]). RaHV2 unterscheidet sich deutlich von RHV1, da er bei Fröschen keine Tumore auslöst. Bis auf das Auftreten massiver Ödeme bei Fröschen, die dem Virus als Kaulquappen ausgesetzt waren, sind keine Ausbrüche oder Krankheiten im Zusammenhang mit einer RaHV2-Infektion dokumentiert worden (Rafferty 1967 ^[22], McKinnell 1973 ^[23]). RaHV2 wird bisher nur mit Leopardfröschen in Verbindung gebracht (Encyclopedia of Virology 4^th Edition ^[20]). Ohne RaHV1 wäre das Urin der Leopardfrösche nicht untersucht worden und RaHV2 heute vermutlich noch unbekannt.
RaHV2 is also known as Frog Virus 4 (FV-4) and has been isolated from the urine of adenocarcinoma-bearing leopard frogs (Rana pipiens) (Rafferty 1965 ^[21]). RaHV2 is distinctly different from RHV1 in that it does not cause tumors in frogs. Except for the occurrence of massive edema in frogs exposed to the virus as tadpoles, no outbreaks or disease associated with RaHV2 infection have been documented (Rafferty 1967 ^[22], McKinnell 1973 ^[23]). RaHV2 has only been associated with leopard frogs (Encyclopedia of Virology 4^th Edition ^[20]). Without RaHV1, the urine of leopard frogs would not have been studied and RaHV2 would probably be unknown today.

Interview with PD Dr. med. vet. F. Origgi (24. August 2021)

Is the ice frog (Lithobates sylvaticus) not affected by RaHV1?
“To my knowledge, no specific information is available on the susceptibility of the ice frog (L. sylvaticus) to RaHV1.”

Can the common frog (Rana temporaria) or other species in Europe be affected by RaHV1? Has this already been tested?
“This is a good question. I think the definitive answer would come from a transmission study.”

Have you already made a proposal to the ICTV (International Committee on the Taxonomy of Viruses) to list BfHV1 as a batrachovirus? If so, why hasn’t it been listed as a batrachovirus yet? If not, why haven’t you submitted the proposal yet?
“This is currently being worked out. I plan to submit the proposal to the ICTV shortly.”

What do you think about the risk of batrachoviruses recombining within the host?
“It is known that herpesviruses are capable of undergoing homologous recombination, however, to my knowledge, we have no evidence (yet) that this occurs with batrachoviruses.”

List of sources

[1] F. C. Origgi, B. R. Schmidt, P. Lohmann, P. Otten, R. K. Meier, S. R. R. Pisano, G, Moore-Jones, M. Tecilla, U. Sattler, T. Wahli, V. Gaschen, M. H. Stoffel, 2018: Bufonid herpesvirus 1 (BfHV1) associated dermatitis and mortality in free ranging common toads (Bufo bufo) in Switzerland. Sci Rep 8, 14737 (2018). link

[2] T. Eisenberg, HP. Hamann, C. Reuscher, A. Kwet, K. Klier-Heil, B. Lamp, 2021: Emergence of a bufonid herpesvirus in a population of the common toad Bufo bufo in Germany. Dis Aquat Organ. 2020 Jun 3;145:15-20. link

[3] B. R. Schmidt, 2018: Auch Frösche und Kröten können am Herpesvirus erkranken. PowerPoint Presentation der Koordinationsstelle für Amphibien- und Reptilienschutz in der Schweiz link

[4] M. David, 2013: Schwärzepilz bei Erdkröten am Niederrhein entdeckt. Feldherpetologisches Magazin. 20, 249 (2013).

[5] F. C. Origgi, B. R. Schmidt, P. Lohmann, P. Otten, E. Akdesir, V. Gaschen, L. Aguilar-Bultet, T. Wahli, U. Sattler, M. H. Stoffel, 2017: Ranid Herpesvirus 3 and Proliferative Dermatitis in Free-Ranging Wild Common Frogs (Rana Temporaria). Vet Pathol. 2017 Jul;54(4):686-694. link

[6] L. H. V. Franklinos, J. R. Fernandez, H. B. Hydeskov, K. P. Hopkins, D. J. Everest, A. A. Cunningham, B. Lawson, 2018: Herpesvirus skin disease in free-living common frogs Rana temporaria in Great Britain. Dis Aquat Organ. 2018 Aug 14;129(3):239-244. link

[7] F. C. Origgi, P. Otten, P. Lohmann, U. Sattler, T. Wahli, A. Lavazza, V. Gaschen, M. H. Stoffel, 2021: Herpesvirus-Associated Proliferative Skin Disease in Frogs and Toads: Proposed Pathogenesis. Vet Pathol. 2021 Jul;58(4):713-729. link

[8] F. Mutschmann, D. Schneeweiss, 2008: Herpes-Virus-Infektionen bei Pelobates fuscus und anderen Anuren im Berlin-Brandenburger Raum. Rana. 2008;5:113–118

[8B] F. C. Origgi, A. Taugbøl, 2023: Ranid Herpesvirus 3 Infection in Common Frog Rana temporaria Tadpoles. Emerging infectious diseases, 29(6), 1228–1231. link

[9] M. W. Smallwood, 1905: Adrenal tumors in the kidney of the frog. Anat. Anz. 26:652-658

[10] J. A. Murray, 1908: The zoological distribution of cancer. Imperial Cancer Research Fund, 3rd Scientific Report: 41-60

[11] B. Lucké, 1934: A neoplastic disease of the kidney of the frog, Rana pipiens. Am. J. Cancer 20:352-379 link

[12] B. Lucké, 1938: Carcinoma in the leopard frog: its probable causation by a virus. J. Exp. Med. 68:457-468 link

[13] D. W. Fawcett, 1956: Electron microscope observations on intracellular virus-like particles associated with the cells of the Lucké renal adenocarcinoma. J. Biophys. Biochem. Cytol. 2:725-741

[14] A. J. Davison, C. Cunningham, W. Sauerbier, R. G. McKinnell, 2006: Genome sequences of two frog herpesviruses. Journal of General Virology 87(12):1465-2099 link

[15] T. B. Waltzek, G. O. Kelley, M. E. Alfaro, T. Kurobe, A. J. Davison, R. P. Hedrick, 2009: Phylogenetic relationships in the family Alloherpesviridae. Diseases of Aquatic Organisms, 84:179-194 link

[16] R. G. McKinnell, 1967: Evidence for seasonal variation in incidence of renal adenocarcinoma in Rana pipiens. J. Minn. Acad. Sci. 34:173-175 link

[17] M. Mizell, 1985: Lucké frog carcinoma herpesvirus: transmission and expression during early development. In Advlances in Viral Oncology. Vol 5: Viruses as the Causative Agents of Naturally Occuring Tumors (Ed, G. Klein). Raven Press. New York. pp 129-146

[18] J. W. Williams, K. S. Tweedell, D. Sterling, N. Marshall, C. G. Christ, D. L. Carlson, R. G. McKinnell, 1996: Oncogenic herpesvirus DNA absence in kidney cell lines established from the northern leopard frog Rana pipiens. Dis Aquat Organ. 1996 Oct 14;27(1):1-4 link

[19] C. L. Densmore, D. E. Green, 2007: Diseases of Amphibians. ILAR Journal, Volume 48, Issue 3, 2007, Pages 235–254 link

[20] D. Bamford, M. Zuckermann, 2021: Encyclopedia of Virology 4^th Edition, Chapter: Fish and Amphibian Alloherpesviruses (Herpesviridae); Page 307 link

[21] K. A. Rafferty Jr., 1965: The cultivation of inclusion-associated viruses from Lucke ́tumor frogs. Ann N Y Acad Sci. 1965;126(1):3–21 link

[22] K. A. Rafferty Jr., 1967: The biology of spontaneous renal carcinoma of the frog. In: King JS, ed. Renal Neoplasia. Boston, MA: Little, Brown; 1967:311–315.

[23] R. G. McKinnell, 1973: The Lucke ́ frog kidney tumor and its herpesvirus. American Zoologist. 1973;13(1):97–114 link