Paula M. Kinnunen

Orthopox virus infections in Eurasian wild rodents.

The genus Orthopoxvirus includes variola (smallpox) virus and zoonotic cowpox virus (CPXV). All orthopoxviruses (OPV) are serologically cross-reactive and cross-protective, and after the cessation of smallpox vaccination, CPXV and other OPV infections represent an emerging threat to human health. In this respect CPXV, with its reservoir in asymptomatically infected wild rodents, is of special importance. In Europe, clinical cowpox has been diagnosed in both humans and animals. The main objective of this study was to elucidate the prevalence of OPV infections in wild rodents in different parts of Eurasia and to compare the performance of three real-time polymerase chain reaction (PCR) methods in detecting OPV DNA in wildlife samples. We investigated 962 wild rodents from Northern Europe (Finland), Central Europe (Germany), and Northern Asia (Siberia, Russia) for the presence of OPV antibodies. According to a CPXV antigen-based immunofluorescence assay, animals from 13 of the 17 locations (76%) showed antibodies. Mean seroprevalence was 33% in Finland (variation between locations 0%-69%), 32% in Germany (0%-43%), and 3.2% (0%-15%) in Siberia. We further screened tissue samples from 513 of the rodents for OPV DNA using up to three real-time PCRs. Three rodents from two German and one Finnish location were OPV DNA positive. The amplicons were 96% to 100% identical to available CPXV sequences. Further, we demonstrated OPV infections as far east as the Baikal region and occurring in hamster and two other rodent species, ones previously unnoticed as possible reservoir hosts. Based on serological and PCR findings, Eurasian wild rodents are frequently but nonpersistently infected with OPVs. Results from three real-time PCR methods were highly concordant. This study extends the geographic range and wildlife species diversity in which OPV (or CPXV) viruses are naturally circulating.

Molecular epidemiology of Aleutian mink disease virus in Finland.

Aleutian mink disease virus (AMDV) is a parvovirus that causes an immune complex-mediated disease in minks. To gain a more detailed view of the molecular epidemiology of mink AMDV in Finland, we phylogenetically analysed 14 new Finnish strains from 5 farms and all 40 strains with corresponding sequences available in GenBank. A part of the major non-structural (NS1) protein gene was amplified and analysed phylogenetically. A rooted nucleotide tree was constructed using the maximum parsimony method. The strains described in this study showed 86-100% nucleotide identity and were nearly identical on each farm. The ratio of synonymous to non-synonymous substitutions was approximately 2.7, indicating a mild purifying selection. Phylogenetic analysis confirmed that AMDV strains form three groups (I-III), all of which contained Finnish strains. The tree inferred that the three lineages of AMDV have been introduced to Finland independently. The analysis suggested that AMDV strains do not cluster into genotypes based on geographical origin, year of isolation or pathogenicity. Based on these data, the molecular clock is not applicable to AMDV, and within this gene area no recombination was detected.

Experimental Infection of Voles with Francisella tularensis Indicates Their Amplification Role in Tularemia Outbreaks

Tularemia outbreaks in humans have been linked to fluctuations in rodent population density, but the mode of bacterial maintenance in nature is unclear. Here we report on an experiment to investigate the pathogenesis of Francisella tularensis infection in wild rodents, and thereby assess their potential to spread the bacterium. We infected 20 field voles (Microtus agrestis) and 12 bank voles (Myodes glareolus) with a strain of F. tularensis ssp. holarctica isolated from a human patient. Upon euthanasia or death, voles were necropsied and specimens collected for histological assessment and identification of bacteria by immunohistology and PCR. Bacterial excretion and a rapid lethal clinical course with pathological changes consistent with bacteremia and tissue necrosis were observed in infected animals. The results support a role for voles as an amplification host of F. tularensis, as excreta and, in particular, carcasses with high bacterial burden could serve as a source for environmental contamination.

Detection of Francisella tularensis in voles in Finland.

Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis-infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals.

Intracerebral Borna Disease Virus Infection of Bank Voles Leading to Peripheral Spread and Reverse Transcription of Viral RNA

Bornaviruses, which chronically infect many species, can cause severe neurological diseases in some animal species; their association with human neuropsychiatric disorders is, however, debatable. The epidemiology of Borna disease virus (BDV), as for other members of the family Bornaviridae, is largely unknown, although evidence exists for a reservoir in small mammals, for example bank voles (Myodes glareolus). In addition to the current exogenous infections and despite the fact that bornaviruses have an RNA genome, bornavirus sequences integrated into the genomes of several vertebrates millions of years ago. Our hypothesis is that the bank vole, a common wild rodent species in traditional BDV-endemic areas, can serve as a viral host; we therefore explored whether this species can be infected with BDV, and if so, how the virus spreads and whether viral RNA is transcribed into DNA in vivo. We infected neonate bank voles intracerebrally with BDV and euthanized them 2 to 8 weeks post-infection. Specific Ig antibodies were detectable in 41%. Histological evaluation revealed no significant pathological alterations, but BDV RNA and antigen were detectable in all infected brains. Immunohistology demonstrated centrifugal spread throughout the nervous tissue, because viral antigen was widespread in peripheral nerves and ganglia, including the mediastinum, esophagus, and urinary bladder. This was associated with viral shedding in feces, of which 54% were BDV RNA-positive, and urine at 17%. BDV nucleocapsid gene DNA occurred in 66% of the infected voles, and, surprisingly, occasionally also phosphoprotein DNA. Thus, intracerebral BDV infection of bank vole led to systemic infection of the nervous tissue and viral excretion, as well as frequent reverse transcription of the BDV genome, enabling genomic integration. This first experimental bornavirus infection in wild mammals confirms the recent findings regarding bornavirus DNA, and suggests that bank voles are capable of bornavirus transmission.

Serological Survey of Rodent-Borne Viruses in Finnish Field Voles

In northern Europe, rodent populations display cyclic density fluctuations that can be correlated with the human incidence of zoonotic diseases they spread. During density peaks, field voles (Microtus agrestis) become one of the most abundant rodent species in northern Europe, yet little is known of the viruses they host. We screened 709 field voles, trapped from 14 sites over 3 years, for antibodies against four rodent-borne, potentially zoonotic viruses or virus groups-hantaviruses, lymphocytic choriomeningitis virus (LCMV), Ljungan virus (LV), and orthopoxviruses (OPV). Antibodies against all four viruses were detected. However, seroprevalence of hantaviruses, LV, and LCMV was low. OPV antibodies (most likely cowpox) were more common but restricted geographically to southeastern Finland. Within these sites, antibody prevalence showed delayed density dependence in spring and direct density dependence in fall. Higher seroprevalence was found in spring than fall. These results substantially increase knowledge of the presence and distribution of viruses of field voles in Finland, as well as CPXV infection dynamics.

Phylogeography of Francisella tularensis subspecies holarctica in Finland, 1993-2011.

Abstract Background: Finland repeatedly reports some of the highest incidences of tularaemia worldwide. To determine genetic diversity of the aetiologic agent of tularaemia, Francisella tularensis, a total of 76 samples from humans (n = 15) and animals (n = 61) were analysed. Methods: We used CanSNPs and canINDEL hydrolysis or TaqMan MGB probes for the analyses, either directly from the clinical tissue samples (n = 21) or from bacterial isolates (n = 55). Results: The genotypes of the strains were assigned to three previously described basal subspecies holarctica clades. The majority of strains (n = 67) were assigned to B.12, a clade reported to dominate in Scandinavia and Eastern Europe. A single strain was assigned to clade B.4, previously reported from North America, Europe and China. The remaining strains (n = 8) were members of clade B.6. Importantly, new diversity was discovered in clade B.6. We describe two newly designed TaqMan MGB probe assays for this new B.6 subclade B.70, and its previously identified sister clade B.11, a clade dominantly found in Western Europe. Conclusions: The high genetic diversity of F. tularensis subspecies holarctica present in Finland is consistent with previous findings in Sweden. The results suggest a northern and southern division of the B.6 subclade B.10, where B.11 predominates in Western and Central Europe and B.70 is found in Fennoscandia. Further research is required to define whether the vast diversity of genotypes found is related to different habitats or reservoir species, their different postglacial immigration routes to Fennoscandia, or dynamics of the reservoir species.

Phylogeography of Francisella tularensis subspecies holarctica in Finland, 1993-2011

Abstract Background: Finland repeatedly reports some of the highest incidences of tularaemia worldwide. To determine genetic diversity of the aetiologic agent of tularaemia, Francisella tularensis, a total of 76 samples from humans (n = 15) and animals (n = 61) were analysed. Methods: We used CanSNPs and canINDEL hydrolysis or TaqMan MGB probes for the analyses, either directly from the clinical tissue samples (n = 21) or from bacterial isolates (n = 55). Results: The genotypes of the strains were assigned to three previously described basal subspecies holarctica clades. The majority of strains (n = 67) were assigned to B.12, a clade reported to dominate in Scandinavia and Eastern Europe. A single strain was assigned to clade B.4, previously reported from North America, Europe and China. The remaining strains (n = 8) were members of clade B.6. Importantly, new diversity was discovered in clade B.6. We describe two newly designed TaqMan MGB probe assays for this new B.6 subclade B.70, and its previously identified sister clade B.11, a clade dominantly found in Western Europe. Conclusions: The high genetic diversity of F. tularensis subspecies holarctica present in Finland is consistent with previous findings in Sweden. The results suggest a northern and southern division of the B.6 subclade B.10, where B.11 predominates in Western and Central Europe and B.70 is found in Fennoscandia. Further research is required to define whether the vast diversity of genotypes found is related to different habitats or reservoir species, their different postglacial immigration routes to Fennoscandia, or dynamics of the reservoir species.

Severe Ocular Cowpox in a Human, Finland

To the Editor: We describe cowpox with corneal involvement in a 31-year-old atopic woman who lived in southern Finland and was unvaccinated for smallpox. In August 2009, she noticed irritation and edema in her right eye and sought care from a local physician; she started topical antimicrobial drug therapy and oral cephalexin 2 days later. Over the following week, fever developed (37.6°C –39.0°C), edema developed on half her face, the eye became increasingly painful, and visual acuity decreased. The conjunctiva was severely chemotic and hyperemic, but the cornea was clear and the other eye unaffected. Microbiologic samples taken from the eye 11 days after onset showed neither bacteria nor respiratory viruses. Orbital tomography results were normal. The patient was hospitalized, and broad-spectrum intravenous antimicrobial treatment (meropenem, vancomycin, valacyclovir, and fluconazole) was started, combined with topical corticosteroids and antimicrobial drugs. Within 2 weeks, the conjunctiva showed necrosis, and epithelial erosions appeared in the lower cornea, but visual acuity normalized (Technical Appendix Figure, panels A, B). A strong cytopathic effect was observed in Vero cells infected with conjunctival swab (Technical Appendix Table 1), but the virus was unidentifiable by routine methods. In electron microscopy, cell culture and tear fluid samples contained particles with typical orthopoxvirus (OPV) morphology. PCRs for hemagglutinin (1) and 14-kDa genes (2) verified OPV infection. Additional PCRs and sequencing confirmed zoonotic cowpox virus (CPXV) with strain designation FIN/K2009. Nucleotide sequences of the hemagglutinin, thymidine kinase, and A-type inclusion body protein genes were identical to those of CPXV strains T2000 and E1989 previously identified in Finland (3). In phylogenetic analysis (Figure), CPXV/FIN/K2009 clustered with strains from Austria and shared ancestry with vaccinia virus. OPV IgG and IgM were detected by immunofluorescence assay (3) in serum samples up to 5 months after symptom onset (Technical Appendix Table 1). Figure Phylogenetic tree of orthopoxviruses constructed on the basis of the hemagglutinin gene; boldface indicates the CPXV strain infecting the patient described in this article. The phylogeny shows that the sequence derived from this patient represents a locally ... The patient was started on intravenous polyclonal gammaglobulin and topical trifluorothymidine with in vitro anti-OPV effects; nevertheless, corneal erosions enlarged, corneal stromal edema ensued, and intraocular pressure increased (online Technical Appendix Figure, panel C), suggesting trabeculitis. Topical autologous serum drops had no effect. Periorbital edema slowly resolved, but corneal erosions persisted. Amniotic membrane transplantation (AMT) (4) was performed 2.5 months after onset. The inferior cornea melted, and the cornea lost transparency (online Technical Appendix Figure, panel D). AMT was repeated twice at 1-month intervals because of corneal thinning. At 3.5 months after symptom onset, tecovirimat (400 mg 2×/d) was given orally for 14 days. Despite treatment, ocular OPV PCR test results remained positive until 9 months after onset (Technical Appendix Table 1), and corneal melting progressed (Technical Appendix Figure, panel E). Corneal collagen cross-linking and a fourth AMT were performed at 5 months after onset with partial success (Technical Appendix Figure, panel F). At 1 year after symptom onset, corneal limbal stem cell deficiency with deep corneal neovascularization was evident. Autologous limbal stem cell transplantation from the patient’s other eye and another AMT were performed, resulting in stable corneal surface 2 months later (Technical Appendix Figure, panel G). Neovascularization regressed, the cornea cleared, and vision improved (Technical Appendix Figure, panels H, I). Cowpox is transmitted to humans sporadically from rodents or cats (5). We snap-trapped 23 wild rodents from the yard of the patient’s home and from an adjacent meadow and trapped 136 rodents from 3 other regions 30–100 km from the patient’s home (Technical Appendix Table 2). We also collected 8 environmental samples from the patient’s storehouse. In accordance with the Finnish Act on Use of Animals for Experimental Purposes (62/2006) and the Finnish Animal Experiment Board’s later decision (May 16, 2007), the animal capture technique used is not an animal experiment and requires no ethics license. Diluted blood for IFA was collected from all rodents (6), and DNA was extracted from rodent liver and lungs and from environmental samples. One vole and 1 mouse from the meadow were seropositive for OPV; however, no CPXV DNA was amplifiable in the samples from the liver, lungs, or environment (online Technical Appendix Table 2). CPXV infection may manifest in severe ocular forms along with self-limiting cutaneous pocks (5). Our patient had keratitis with no other identifiable cause but CPXV. Culture and PCR from early conjunctival samples and serology confirmed the etiologic diagnosis. Our case and that of another report (7) highlight the challenges of treating cowpox keratitis. Topical and systemic antiviral drugs and AMT appear ineffective during the acute phase. Corneal melting and scarring continued as long as CPXV was observed and until combined limbal stem cell and AMT treatment had favorable outcomes. Anamnesis of therapy-resistant keratitis should include information on rodent contacts. We dated the infection to mid-August (incubation 7–21 days). Catching OPV-IgG–positive rodents close to the patient’s home 2 months after onset showed that OPVs were circulating in the local rodent population and indicated the putative role of CPXV-infected voles as the source of infection. The latest cowpox outbreak in Central Europe involved several humans and pets (8). This patient was born in 1977, after Finland ceased smallpox vaccinations. Declining cross-reactive smallpox-vaccination immunity enables emergence of unusual cowpox infections in humans (9). Technical Appendix.Diagnostic findings of patient, laboratory findings of sampling from rodents and environment, and images of progressive disease in eye of patient with ocular cowpox, Finland. Click here to view.(150K, pdf)

Characterization of a New Epidemic Necrotic Pyoderma in Fur Animals and Its Association with Arcanobacterium phocae Infection

A new type of pyoderma was detected in Finnish fur animals in 2007. The disease continues to spread within and between farms, with severe and potentially fatal symptoms. It compromises animal welfare and causes considerable economic losses to farmers. A case-control study was performed in 2010–2011 to describe the entity and to identify the causative agent. Altogether 99 fur animals were necropsied followed by pathological and microbiological examination. The data indicated that the disease clinically manifests in mink (Neovison vison) by necrotic dermatitis of the feet and facial skin. In finnraccoons (Nyctereutes procyonoides), it causes painful abscesses in the paws. Foxes (Vulpes lagopus) are affected by severe conjunctivitis and the infection rapidly spreads to the eyelids and facial skin. A common finding at necropsy was necrotic pyoderma. Microbiological analysis revealed the presence of a number of potential causative agents, including a novel Streptococcus sp. The common finding from all diseased animals of all species was Arcanobacterium phocae. This bacterium has previously been isolated from marine mammals with skin lesions but this is the first report of A. phocae isolated in fur animals with pyoderma. The results obtained from this study implicate A. phocae as a potential causative pathogen of fur animal epidemic necrotic pyoderma (FENP) and support observations that the epidemic may have originated in a species -shift of the causative agent from marine mammals. The variable disease pattern and the presence of other infectious agents (in particular the novel Streptococcus sp.) suggest a multifactorial etiology for FENP, and further studies are needed to determine the environmental, immunological and infectious factors contributing to the disease.