One more piece in the VACV ecological puzzle: could peridomestic rodents be the link between wildlife and bovine vaccinia outbreaks in Brazil?

Abstract

Background: Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks - BV), affecting dairy cattle and milkers. Little is known about VACV's natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated.

Methodology/principal findings: In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks.

Conclusion/significance: These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks?

Figure 1. Location and phytoecological characteristics of Mariana county BV outbreak area.

The outbreak region is located in a biome transition area, between the Mata Atlântica (Atlantic rainforest) and the Cerrado (Savanna), with accentuated anthropogenic disturbance.

Figure 2. Immunofluorescence assay of infected BSC-40 cells revealed that MARV-M, MARV-H and MARV-B are OPV.

BSC-40 cells were infected with MARV isolates at a MOI of 3. After 24 h, an anti-OPV antibody revealed the presence of the B5R protein which was punctually distributed, especially in cytoplasmic membranes and the Golgi complex, similarly to the pattern observed in VACV-WR-infected cells. The DAPI staining highlights the DNA contained in viral factories (white arrows).

Figure 3. MARV-M, MARV-B and MARV-H induced the formation of white pocks in CAM and presented a similar profile in plaque phenotype and one-step growth curve assays.

(A) CEF monolayers infected with MARV isolates were harvested after 72 h and inoculated onto the CAM of embryonated chicken eggs. Typical VACV white pocks were observed in inoculated CAM after 72 hours. (B) MARV isolates presented small plaques in BSC-40 cells (average diameter: 0.6 mm), distinct from the prototypical VACV-WR plaque phenotype (average diameter: 1.4 mm). (C) One-step growth curve assays in BSC-40 cells showed that MARV-B, MARV-H and MARV-M present a similar replication profile, which was statically distinct from that observed for VACV-WR (p<0.04).

Figure 4. MARV is grouped phylogenetically with several Brazilian VACV strains isolated during BV outbreaks, but is segregated from VACV vaccine strains.

(A) Amino acid inferred sequence of the MARV hemagglutinin (HA) gene and comparison to the homologous sequence from several OPV. The box with an asterisk indicates the deletion region conserved in the sequences of MARV, several Brazilian field VACV samples and the IOC vaccine strain. (B) Phylogenetic tree constructed based on the nucleotide sequence of the OPV ha gene. The ha tree shows MARV grouping with VACV field samples. Despite the presence of the 6 aa deletion, the IOC strain grouped with other vaccine strains. Nucleotide sequences were obtained from GenBank (VACV-ARAV:AY523994; VACV-LOR:DQ810281; VACV-RIA:DQ810280; VACV-CTGV:AF229247; VACV-PSTV:DQ070848; VACV-MURV:DQ247770; VACV-GP2V:DQ206437; VACV-Acambis clone3:AY313848; VACV-DUKE:DQ439815; VACV-MALBRAN:AY146624; VACV-SV2:EF063677; Rabbitpox:AF37511; VACV-LST:AY678276; VACV-SPAn232:DQ222922; VACV-BeAn58058:DQ206442; VACV-GP1V:DQ206436; VACV-WR: AY243312; VACV-Ankara:U944848; VACV-VBH:DQ206435; VACV-Lister-Butantan:EF175985; VACV-IOC:AF225248; VACV-Bfl3906:AF375077; VACV-Bfl81:AF375078; HSPV:DQ792504; MPXV-ZAIRE:DQ011155; MPXV-mpv3945:AF375098; MPXV-Sierra Leone:AY741551; CPXV-Germany: DQ437593; CPXV-cowHA12:AY902253; CPXV-GRI90:AF375087; VARV:DQ437589. Bootstrap confidence intervals are shown on branches (1,000 replicates).

Figure 5. A hypothetical model of the VACV transmission cycle.

Peridomestic rodents could promote the transmission of VACV between wild animals and cattle or humans, since they circulate both in farm buildings and their surroundings. This diagram was proposed based on published epidemiological and laboratory data on VACV and the behavioral characteristics of Brazilian rodents and wild animals. Solid lines indicate experimentally determined data, and dashed lines represent hypothetical propositions still under investigation.