COMMON MIDWIFE TOAD VIRUS: OUTBREAKS IN EUROPE, PATHOLOGY AND

GENOMIC ANALYSESSERIDA

GENOMIC ANALYSESAna Balseiro1, Carla Mavián2, Alberto López-Bueno2, Antonio Alcamí2, Alí Alejo3, Debra Miller4, Rosa Casais1

Centro de Biotecnología Animal, SERIDA, 33394 Gijón, Spain. Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain. Centro de investigación en sanidad animal, Instituto Nacional de Investigación y Tecnología Agraria y

Ana Balseiro , Carla Mavián , Alberto López-Bueno , Antonio Alcamí , Alí Alejo , Debra Miller , Rosa Casais1Centro de Biotecnología Animal, SERIDA, 33394 Gijón, Spain. 2Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain. 3Centro de investigación en sanidad animal, Instituto Nacional de Investigación y Tecnología Agraria y

Alimentaria, Valdeolmos, Spain.4Center for Wildlife Health, University of Tennessee, Knoxville, Tennessee, USA.

In Europe, the first known outbreak of common midwife toad virus (CMTV) occurred in northern Spain in 2007

(Balseiro et al., 2009). Since then, three more outbreaks have occurred in Spain and in The Netherlands (Balseiro et

OUTBREAKS IN EUROPECommon midwife toad (Alytes obstetricans)

Picos de Europa (Spain)

(Balseiro et al., 2009). Since then, three more outbreaks have occurred in Spain and in The Netherlands (Balseiro et

al., 2010; Kik et al., 2011, 2012), affecting both wild and captive amphibians, and indicating that the host range and

geographic distribution of CMTV are much wider than previously suspected. CMTV is known to infect wild tadpolesgeographic distribution of CMTV are much wider than previously suspected. CMTV is known to infect wild tadpoles

of the common midwife toad (Alytes obstetricians) and juvenile alpine newts (Triturus alpestris) in Spain; and adult

wild water frogs (Pelophylax spp.) and common newts (Lissotriton vulgaris) in The Netherlands. Recently it has been

reported in captive frogs in the former country.reported in captive frogs in the former country.

GENOMIC ANALYSES

Outbreaks of CMTV in Europe:

Spain and The Netherlands

Phylogenetic analysis of CMTV using the sequence of the major capsid protein, placed this virus as a

close relative of FV3 and other amphibian-like ranaviruses (ALRVs) (Mavian et al., 2012), clustering with

isolates from different hosts (fish and amphibian) and geographic origin (Fig. 4A and 4B). Analysis of the

gene content of CMTV (Fig. 4C), showed that it contained all the ALRV specific genes, confirming its

PATHOLOGYCommon gross lesions include systemic hemorrhagic disease (Fig. 1).

Spain and The Netherlands gene content of CMTV (Fig. 4C), showed that it contained all the ALRV specific genes, confirming its

adscription to this group. Further, phylogenetic analysis of the concatemeric sequence of the iridoviral

core protein sequences of all completely sequenced ranaviruses, using the LCDV China sequence as a

distantly related outgroup, positioned CMTV as a closer relative of the FV3-like virus group within theCommon gross lesions include systemic hemorrhagic disease (Fig. 1).

Electron microscopy of virus particles demonstrated enveloped iridovirus-

like virus particles with hexagonal nucleocapsid morphology and

distantly related outgroup, positioned CMTV as a closer relative of the FV3-like virus group within the

ranaviruses with high confidence (Fig. 4D, upper panel). Dot plot analysis comparing CMTV to FV3 or

EHNV viruses (Fig. 4D, lower panel), however, showed that its genome was not fully collinear with

either of them. Particularly, the position of the observed genomic rearrangements suggest that CMTVlike virus particles with hexagonal nucleocapsid morphology and

approximately 160-180 nm in diameter (Fig. 2). Histological findings

revealed the presence of intracytoplasmic inclusion bodies and the

necrosis of epithelial and endothelial cells, which result in destruction of

either of them. Particularly, the position of the observed genomic rearrangements suggest that CMTV

might constitute the type isolate of an evolutionary intermediate between both ALRV groups. Inversion

of one segment from an EHNV-like precursor might have given rise to the CMTV-like structure, while a

further large genomic inversion might have generated the precursor of current FV3-like viruses. The usenecrosis of epithelial and endothelial cells, which result in destruction of

several organs, including skin, liver, pancreas, spleen, kidney and intestine

(Fig. 3).

further large genomic inversion might have generated the precursor of current FV3-like viruses. The use

of primers specific for the flanking sequences produced a larger PCR amplification product on CMTV

DNA than on FV3 DNA, and that it can therefore be used for differentiation of these viruses (Fig. 4E).

Figure 2. Electron micrograph

of the CMTV in the

glomerulus from an affected

kidney from a CMT tadpole.

DA

C:

A

C:

ESV

(Silurus glanis)

C EB

(Silurus glanis)

1R 4R 5R 6R 8R 10R 13R 14R 19R 20R

10kb0 20kb 30kb

27R

Figure 1. A) Adult live frog with multiple haemorrhages in the C

v2L 3L 7L 9L 11L 12L 15L 16L 17L 18L 21L 22L 23L 24L 25L 26L

27R 29R 30R 32R 33R 34R 35R 39R 40R 41R 42R 46R 48R 53R 54R 55R

*

Figure 1. A) Adult live frog with multiple haemorrhages in the

skin, including around the eardrums (arrows) (Kik et al., 2011). B)

Dead metamorphosing tadpole with haemorrhages within the

skin (black arrow) and subcutaneous oedema of the legs (white

arrow) (Kik et al., 2011) C) Tadpole of the CMT showing systemic

haemorrhages.

CFigure 3. A) Viral inclusions (arrows) and necrotic cells in renal

glomeruli. Haematoxylin-Eosin, 200x. Inset: Necrotic tubule

epithelial cells with viral inclusions (arrow) in renal tubule.

Haematoxylin-Eosin, 400x. B) Areas of focal necrosis with v28L 31L 35L 36L 37L 38L 43L 44L 45L 47L 49L 50L 51L 52L 54L 56L

40kb 50kb 60kb

57R 59R 60R61R 62R 64R 71R 72R 73R63R

*haemorrhages.Haematoxylin-Eosin, 400x. B) Areas of focal necrosis with

hepatocytes containing viral inclusions (arrows). Haematoxylin-

Eosin, 200x.

DISCUSSION

As we have shown here, CMTV is an emerging pathogen infecting

57R 59R 60R61R 62R 64R 71R 72R 73R63R

58L 65L 67L 69L 70L 74L 76L 77L 78L 79L 80L 81L 82L 83L 84L68L66L 75L

70kb 80kb 90kb

56L

As we have shown here, CMTV is an emerging pathogen infecting

numerous species within continental Europe. Additionally, we have shown

it to be a novel ALRVs that could represent an evolutionary intermediate 85L 87L 88L 91L

92R

93L 94L 95L 99L 100L

86R 89R 90R 97R 98R 101R 102R

96L

100kb 106878 bp

84Lit to be a novel ALRVs that could represent an evolutionary intermediate

between the two previously recognized groups within ALRVs, namely FV3-

like and EHNV-like viruses. This information will be of importance both to

further understand the evolutionary relationship among ranaviruses as

Figure 4: A) The CMTV is a novel amphibian-like ranavirus isolated in Europe. B) Name, location and host of isolation of currently fully sequenced ranavirus genomes. C) Linear

systematic organization of the CMTV genome. Known conserved genes are labelled in red (iridovirus core genes), green (ranavirus-specific genes) and blue (ALRV-specific

genes). D) The CMTV is an evolutionary intermediate in ALRV. E) PCR amplification of CA microsatellites in FV3 (lane2) and CMTV (lane 3). The rest of the lanes correspond to

known size markers which were used for determination of the actual size of the CA-microsatellite in CMTV.

REFERENCES

further understand the evolutionary relationship among ranaviruses as

well as to understand their mechanims of spread and host shifts. Also, it

should prove helpful to define the species and strain structure of

ranaviruses, which will be crucial to determine the mechanisms of• Balseiro A, Dalton KP, del Cerro A, Marquez I, Cunningham AA, Parra F, Prieto JM, Casais R (2009). Pathology, isolation and molecular

characterisation of a ranavirus from the common midwife road Alytes obstetricans on the Iberian Peninsula. Dis Aquat Organ, 84: 95-104.ranaviruses, which will be crucial to determine the mechanisms of

emergence of CMTV-caused disease and whether it may be linked to

environmental factors, virus spread, or both. Better knowledge of the

ranavirus species and strain structure will be crucial to set up detection

characterisation of a ranavirus from the common midwife road Alytes obstetricans on the Iberian Peninsula. Dis Aquat Organ, 84: 95-104.

• Balseiro A, Dalton KP, del Cerro A, Márquez I, Parra F, Prieto JM, Casais R (2010). Outbrealk of common midwife toad virus (Mesotriton

alpestris cyreni) and common midwife toads (Alytes obstetricans) in northern Spain: a comparative pathological study of an emerging. Vet

J, 186: 256-8.

• Kik M, Martel A, Sluijs AS, Pasmans F, Wohlsein P, Gröne A, Rijks JM (2011). Ranavirus-lassociated mass mortality in wild amphibians, theranavirus species and strain structure will be crucial to set up detection

methods and surveillance strategies in order to minimize their impact on

amphibian wildlife and cultivated species. It is possible that the

emergence of ranavirus disease on Europe is human or bird-mediated,

• Kik M, Martel A, Sluijs AS, Pasmans F, Wohlsein P, Gröne A, Rijks JM (2011). Ranavirus-lassociated mass mortality in wild amphibians, the

Netherlands, 2010: a first report. Vet J, 90: 284-6.

• Kik M, Stege M, Boonyarittichaikij R, van Asten A (2012). Concurrent ranavirus and Batrachochytrium dendrobatidis infection in captive

frogs (Phyllobates and Dendrobates species), The Netherlands, 2012: a first report. Vet J,194: 247-9.

• Mavian C, López-Bueno A, Balseiro A, Casais R, Alcamí A, Alejo A (2012). The genome sequence of the emerging common midwife toad

virus identifies an evolutionary intermediate within ranaviruses. J Virol, 86: 3617-25.emergence of ranavirus disease on Europe is human or bird-mediated,

and that the virus was naturally introduced from another region. It is

important to remain vigilant in order to detect new outbreaks and to

monitor the possible spread of this pathogen and its possible impact onACKNOWLEDGEMENTS

The authors thank the Veterinary Services of the National Park of the “Picos de Europa” for bringing the samples to the laboratory and P. Solano for helping with the processing

virus identifies an evolutionary intermediate within ranaviruses. J Virol, 86: 3617-25.

monitor the possible spread of this pathogen and its possible impact on

wildlife biodiversity.

The authors thank the Veterinary Services of the National Park of the “Picos de Europa” for bringing the samples to the laboratory and P. Solano for helping with the processing

of samples. This work was supported by grant AGL 2009-08711 from the Spanish Ministerio de Ciencia e Innovación. Alberto López-Bueno and Carla Mavián are recipients of

the Ramón y Cajal and Formación del Personal Investigador fellowships, respectively, from the same institution. Ana Balseiro is recipient of a Contrato de Investigación from

INIA.