Fatal Outbreak in Tonkean Macaques Caused by Possibly Novel Orthopoxvirus, Italy, January 2015 1

Abstract

In January 2015, during a 3-week period, 12 captive Tonkean macacques at a sanctuary in Italy died. An orthopoxvirus infection was suspected because of negative-staining electron microscopy results. The diagnosis was confirmed by histology, virus isolation, and molecular analysis performed on different organs from all animals. An epidemiologic investigation was unable to define the infection source in the surrounding area. Trapped rodents were negative by virologic testing, but specific IgG was detected in 27.27% of small rodents and 14.28% of rats. An attenuated live vaccine was administered to the susceptible monkey population, and no adverse reactions were observed; a detectable humoral immune response was induced in most of the vaccinated animals. We performed molecular characterization of the orthopoxvirus isolate by next-generation sequencing. According to the phylogenetic analysis of the 9 conserved genes, the virus could be part of a novel clade, lying between cowpox and ectromelia viruses.

Keywords: Italy; Macaca tonkeana; Tonkean macaque; cluster analysis; cowpox virus; diagnosis; ectromelia virus; epidemiology; metagenomics; orthopoxvirus infection; phylogeny; vaccination; viruses; zoonoses.

Figure 1

Crater-shaped skin lesions at inguinal region of Tonkean macaque (Macaca tonkeana) housed at animal sanctuary, Italy, January 2015.

Figure 2

Crater-shaped skin lesions on face of Japanese macaque (Macaca fuscata), Italy, 2003.

Figure 3

Results from necropsy of Tonkean macaque (Macaca tonkeana) from animal sanctuary, Italy, January 2015, showing severe congestion in the lungs (A) and erythematous papules and pustular lesions on the buccal and tongue mucosae (B).

Figure 4

Results from necropsy of Tonkean macaque (Macaca tonkeana) from animal sanctuary, Italy, January 2015. A) Hematoxylin and eosin stain of cutaneous lesion. Focal epidermal necrosis, acanthosis ballooning degeneration, and acantholysis of keratinocytes was observed. Staining shows early vesiculation with eosinophilic intracytoplasmic inclusion bodies (arrows) in enlarged degenerated cells. B) Electron micrograph of skin lesion sample showing negatively stained brick-shaped viral particle of ≈160–220 nm, consistent with orthopoxvirus. Scale bar = 100 nm. C) SYBR Green (ThermoFisher Scientific, Waltham, MA, USA) real-time PCR melting curve of all tested samples. The y-axis shows the ratio of the change in fluorescence over the change in temperature. The average melting temperature (80.8°C ± 1°C) was consistent with that for the orthopoxvirus genome.

Figure 5

Identity between OPV Abatino, obtained from skin lesion of Tonkean macaque during outbreak at animal sanctuary, Italy, January 2015, and available OPV genomes on the basis of 9 concatenated conserved genes: A7L, A10L, A24R, D1R, D5R, H4L, E6R, E9L, and J6R. Red indicates more similarity, green less similarity. Sequences shown (GenBank accession nos.): 1) OPV Tena Dona AK2015 (KX914668–76); 2) OPV GCP2013 Akhmeta (KM046934–42); 4–6) ECTV-Moscow (AF012825.2), ECTV-Naval (KJ563295.1), ECTV-VR-1431 (JQ410350.1); 7) VARV-Bangladesh-1975 (L22579.1); 8–14) CPXV-HumLue09–1 (KC813494.1), CPXV-Germany1990–2 (HQ420896.1), CPXV-Francy2001-Nancy (HQ420894.1), CPXV-MarLei07–1 (KC813499.1), CPXV-Norway1994-MAN (HQ420899.1), CPXV-UK2000-K2984 (HQ420900.1), CPXV-BrightonRed (AF482758.2); 15–16) MPXV-Congo2003–358 (DQ011154.1), MPXV-Liberia-1970–184 (DQ011156.1); 17–18) VACV-IOC-B141 (KT184690.1), VACV-Lister (KX061501.1); 19–22) CPXV-Austria1999 (HQ407377.1), CPXV-HumLit08–1 (KC813493.1), CPXV-GRI90 (X94355.2), CPXV-Finland2000 (HQ420893.1); 23) TATV-Dahomey-1968 (DQ437594.1); 24–25) CMLV-0408151v (KP768318.1), CMLV-M96 (AF438165.1); 26‒31) CPXV-HumGra07–1 (KC813510.1), CPXV-RatKre08–2 (KC813505.1), CPXV-RatGer09–1 (KC813503.1), CPXV-RatAac09–1 (KC813501.1), CPXV-HumAac09–1 (KC813508.1), CPXV-HumKre08–1 (KC813512.1); 32) CPXV-Germany1998–2 (HQ420897.1); and 33–46) CPXV-Germany1980-EP4 (HQ420895.1), CPXV-HumPad07–1 (KC813496.1), CPXV-HumLan08–1 (KC813492.1), CPXV-RatHei09–1 (KC813504.1), CPXV-MonKre08–4 (KC813500.1), CPXV-JagKre08–1 (KC813497.1), CPXV-JagKre08–2 (KC813498.1), CPXV-Germany2002-MKY (HQ420898.1), CPXV-HumGri07–1 (KC813511.1), CPXV-HumMag07–1 (KC813495.1), CPXV-CatBer07–1 (KC813502.1), CPXV-HumBer07–1 (KC813509.1), CPXV-CatPox07–1 (KC813506.1), CPXV-BeaBer04–1 (KC813491.1). CMLV, camelpox virus; CPXV, cowpox virus; ECTV, ectromelia virus; MPXV, monkeypox virus; OPV, orthopoxvirus; TATV, taterapox virus; VACV, vaccinia virus; VARV, variola virus.

Figure 6

Phylogenetic analysis of OPV Abatino obtained from skin lesion of Tonkean macaque during outbreak at animal sanctuary, Italy, January 2015. Nine conserved genes (GenBank accession nos. KY100107–KY100115) obtained with next-generation sequencing were concatenated and aligned with the homologous concatenated sequences from representative OPV strains (GenBank accession no.): TATV-Dahomey-1968 (DQ437594.1), VARV-Bangladesh-1975 (L22579.1), CMLV-M96 (AF438165.1), CPXV-HumAac09–1 (KC813508.1), CPXV-Germany2002-MKY (HQ420898.1), CPXV-Germany1998–2 (HQ420897.1), CPXV-MarLei07–1 (KC813499.1), MPXV-Congo2003–358 (DQ011154.1), CPXV-Finland2000 (HQ420893.1), VACV-Lister (KX061501.1), ECTV-Moscow (AF012825.2), OPV GCP2013 Akhmeta (KM046934–42), and OPV Tena Dona AK2015 (KX914668–76). New World strain RACV-MD19 (GenBank accession no. FJ807746–54) was added to the analysis as an outgroup. We generated multiple alignments with MUSCLE version 3.8.31 (30) and built the phylogenetic tree by using the Bayesian Markov chain Monte Carlo model with MRBAYES version 3.2.5 (31) using the general time-reversible plus gamma model with 1 million generations, retaining a minimum of 10,000 posterior probabilities, and maximum-likelihood model RaxML version 8.1.24 (32) using the general time-reversible plus gamma with 1,000 pseudoreplicates. Numbers represent the reliability of the nodes with the minimum probability of 75% and minimum bootstrap value of 75. Scale bar indicates nucleotide substitutions per site. CMLV, camelpox virus; CPXV, cowpox virus; ECTV, ectromelia virus; MPXV, monkeypox virus; OPV, orthopoxvirus; RACV, raccoonpox virus; TATV, taterapox virus; VACV, vaccinia virus; VARV, variola virus.