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Cowpox virus isolate virulent in humans shows attenuated phenotype in mice
Research in Veterinary Science 92 (2012) 333–337
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Cowpox virus isolate virulent in humans shows attenuated phenotype in mice Hartwig P. Huemer a,⇑, Caroline Lassnig b, Norbert Nowotny c a
Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Fritz-Pregl-Str. 3, R.301, A-6020 Innsbruck, Austria Institute of Animal Breeding and Genetics, Biomodels Austria, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Veterinärplatz 1, A-1210 Vienna, Austria c Zoonoses and Emerging Infections Group, Clinical Virology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Veterinärplatz 1, A-1210 Vienna, Austria b
a r t i c l e
i n f o
Article history: Received 2 February 2011 Accepted 11 March 2011
Keywords: Cowpox Vaccinia Virulence Human Mouse BALB/c
a b s t r a c t We have cultured Cowpox virus (CPXV) from skin lesion material of a human patient from Austria. Phylogenetic comparison of the HA-gene revealed a rather homogeneous cluster with other local isolates from recent years, the A36R-gene was mostly related to elephant derived strains from Germany. Despite causing disease in human, the isolate AT/Carinthia/788/07 surprisingly even at high titers showed a highly reduced virulence in BALB/c mice upon intranasal inoculation as compared to vaccinia virus. This contrasts earlier reports on other CPXV isolates. Using shotgun DNA sequencing several insertions and deletions were found in genes presumably involved in host range, immune regulation as well as established virulence factors. These preliminary data could be an indication that CPXV strains with proven pathogenicity for humans may have reduced virulence in mice and vice versa. Additionally strains with a reduced virulence may have an advantage in persisting in less dense rodent populations. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The Cowpox virus (CPXV), a member of the family of the Poxviridae (subfamily Chordopoxvirinae, genus: Orthopoxvirus), is a big brick shaped enveloped DNA viruses with a genome of about 220 kb. It is found in wild rodents of the old world (for review see Chantrey et al., 1999; Essbauer et al., 2009). There is only limited information about the natural course of CPXV in rodents, and the reported findings seem to vary between mouse species. Although earlier data suggested that most cases occur in younger ages and infections might be rather subclinical, CPXV has been found to lead to reduced fecundity in bank voles (Clethrionomys glareolus) and wood mice (Apodemus sylvaticus) in the UK (Feore et al., 1997). However, in field voles (Microtus agrestis) it seems to be quite lethal, reducing the overall survival rate significantly (Burthe et al., 2008). Humans can accidently acquire CPXV infection usually through direct skin contact with diseased cats (‘‘catpox’’) rather than cows, but recently increasingly infections associated with infected pet rats have been reported throughout Europe (Hönlinger et al., 2005; Becker et al., 2008; Ninove et al., 2009). Although the disease is mild and restricted to few skin lesions in most human cases, it is frequently associated with transient viremia (Nitsche et al., 2007) and more severe courses can occur in immunosuppressed patients (Hönlinger et al., 2005; Huemer et al., 2007), even leading to death under rare circumstances (Czerny et al., 1997). ⇑ Corresponding author. Tel.: +43 512 9003 70799; fax: +43 512 9003 73799. E-mail address: [email protected] (H.P. Huemer). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.03.011
Together with reports of high CPXV mortality in non-human primate species (Mätz-Rensing et al., 2006) or elephants (Baxby and Ghaboosi, 1977) this virulence would speak for a less well adaptation of CPXV in non-rodent species and especially to humans/primates than e.g. of the presumably horse derived Vaccinia virus (VACV). VACV has a long record of (relatively) safe use in humans as a vaccine against smallpox, but on the contrary is known since a long time to be rather virulent in mice (SvetMoldavskaya and Chimishkyan, 1969; Berhanu et al., 2008), thus supporting an early concept of adaptation of viral strains or isolates to their respective host environment (Dunlap and Barker, 1973). As an adaptation of CPXV to the mouse population would make sense in terms of the survival of the virus we therefore were interested how a virus with proven pathogenicity in humans would behave in mice. 2. Materials and methods 2.1. Virus strain, cell culture The human-derived CPXV strain AT/Carinthia/788/07 was isolated from crust material of a skin lesion of a 17 year old girl from South-Eastern Austria (Richter et al., 2008). Although it led to a favorable outcome this infection was considered systemic as it caused fever and malaise in a young healthy person (Strenger et al., 2009). The virus was grown in RK13 rabbit kidney cells (ATCC) cultured in DMEM supplemented with 5% FCS. VACV laboratory strain WR (western reserve; provided previously by Dr. B. Moss, Bethesda, MD, USA), was used for comparison for its
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proven virulence in mice. Viral preparations for both viruses were obtained on RK13 cells grown in roller bottles with enhanced surface. Cells were lysed by freeze-thawing, disrupted by hypotonic medium, briefly sonicated, and the nuclei were removed by low speed centrifugation (2000g). The supernates were concentrated at 20,000g and the virus was purified over sucrose gradients using ultracentrifugation (30,000g). Resulting viral stocks were dissolved in phosphate buffered saline (PBS) and the virus titers determined in 24 well plates by plaque assays using RK13 cells. 2.2. DNA analysis, sequencing The new CPXV Isolate was analysed by PCR using an A36R gene (Huemer et al., 2008) and hemagglutinin (HA) gene specific PCR (Ropp et al., 1995). Automated DNA sequencing of the amplified fragments was carried out on both strands, and the obtained A36R and HA sequences were deposited in GenBank (Accession # FM178393.1 and EU582018.1). Sequence comparison was performed by the BLAST algorithm (http://www.ncbi.nlm.nih.gov/ blast) using selected reference sequences from the GenBank database. Phylogenetic analysis was performed using Kimura twoparameter model of nucleotide substitution for calculation of the genetic distance of the DNA sequences aligned with MEGA 4 Molecular Evolutionary Genetics Analysis software. Phylogenetic trees were constructed using the Neighbour-Joining method with bootstrap analysis using 1000 pseudoreplicate datasets, and the trees finally plotted with Phylip 3.65. For a preliminary identification of genes possibly involved in altered virulence patterns we applied a shotgun sequencing approach using the GS FLX Titanium deep sequencing technology from Roche, Inc. Whole virus DNA was prepared from sucrose purified CPXV by digestion with proteinase K and subsequent phenol chloroform extraction. The virus genome was then incorporated into a Titanium 3kB paired end library using the materials and protocol of the manufacturer (Roche, Inc.). After PCR amplification with FLX Titanium emPCR it was finally sequenced on the FLX454 sequencer at the Medical University of Graz/Austria, ZMF – sequencing core facility and the obtained sequences compared to the published genome of CPXV strain Brighton as the reference sequence (GeneBank Accession # GI:308443369). 2.3. Animal experiments An early passage (P4) of the isolated CPXV was also tested in comparison with VACV in a mouse model studying virulence using the conditions described most recently (Huemer et al., 2010). Female inbred pathogen-free BALB/c mice (obtained from Charles River, Germany) with an age of 6 weeks and a weight of 16–17 g were used. For both viruses two different inoculation routes were chosen, intravenous (groups A, B) and intranasal (groups C, D). Five different infectious doses were applied ranging from 103 up to 107 plaque forming units (p.f.u.), thus resulting in a total of 20 subgroups with five animals in each group. On day 0 the animals were objected to short term anesthesia with ketamin/xylazin (75 and 7.5 mg/kg i.p.) and infected with 10 ll of the above purified viral stocks diluted in PBS via a nasal tube or by injection into the dorsal vein of the tail. The animals were controlled daily for disease symptoms and the body weights were determined. Severity of disease was evaluated according to a published scoring system (Berhanu et al., 2008), using disease index (DI) of 0 (normal), (1) (slightly ruffled skin), (2) (clearly ruffled skin and/or single sided conjunctivitis), (3) (hunched position and/or severe conjunctivitis of both eyes in addition to clear ruffling), (4) (score of three combined with difficulty in moving/socializing/breathing), and (5) (death). Animals with a DI of 4 and/or >30% weight loss were euthanized according to the prescriptions of the local ethics
committee. The animal experiments were approved under the permit # BMWF-68.205/0246-II/10b/2008 of the Austrian Federal Ministry of Research, Section for Animal Experiments. 3. Results 3.1. Genetic relationship of the isolated CPXV strain Both sequences were deposited in GenBank under the Accession Numbers FM178393.1 and EU582018.1 respectively. Sequence comparison as shown in Fig. 1, revealed that the A36R gene sequence of the isolate is most closely related to sequences EP1 and EP6 derived from infected elephants from Germany (Baxby and Ghaboosi, 1977), whereas CPXV isolates from the UK or Norway seem to form a distinct cluster. The HA gene was found closely related to Austrian CPXV isolates cpv-531, -667, and -610 from 1992, 1994 and 1997, which all have been isolated from diseased cats (Nowotny et al., unpublished), and a local human isolate from Styria from 2009, whereas the HA gene of the human isolate cpv-1218 from the Austrian federal state of Salzburg from the year 2000 (Hawranek et al., 2003) seems more distant. Interestingly other German or Italian isolates are rather unrelated supporting the facts of high variation among CPXV strains, and the finding of highly divergent regional clusters (Essbauer et al., 2009; Kaysser et al., 2010). 3.2. Reduced virulence of the CPXV strain in mice As demonstrated in Fig. 2, VACV led to severe disease in BALB/c mice upon intranasal (i.n.) infection with all studied viral loads. Rapid weight loss and disease symptoms were observed especially with inoculation doses above 104 p.f.u., requiring euthanasia of the animals about day 6. There was also significant disease and weight loss of about 20% in mice infected with 103 p.f.u. of VACV (Fig. 2C), which contrasts the outcome with the CPXV strain, which at the same dose showed no symptoms at all but a steady gain of weight. Even at high doses up to 107 p.f.u. of CPXV there was only a temporary stagnation of the weight gain until day 6 and a continuous increase in weight thereafter (Fig. 2D). Mice infected via the intravenous (i.v.) route remained rather unaffected with both viruses showing only marginal weight reduction around day 6 even at high VACV inoculation doses (Fig. 2A), which could be an indication that most virions may be rapidly inactivated in the serum (via complement?) before establishing more severe systemic infection. 3.3. Sequence differences as compared to virulent strain Brighton Although we only achieved about 25% coverage of the whole CPXV genome in the ‘‘shotgun’’ DNA sequencing approach chosen, which does not enable more extensive bioinformatic comparisons like investigation of whole reading frames, numerous differences were identified as compared to the sequence of the virulent CPXV strain Brighton. Regions showing insertions and deletions of several nucleotides are depicted in Table 1. The identified differences include also genes involved in host-range selection, virulence and immune modulation, e.g., ankyrin-like proteins, cytokine response modifying (crm) proteins like tumor necrosis factor (TNF) receptor homologue crmB, Semaphorin-like and lectin type receptors. 4. Discussion One of our aims was to study the virulence of a CPXV isolate with proven pathogenicity for humans in a mouse model. The reason for this resides in the varying severity of CPXV disease observed in different species as monkeys or elephants as has been
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Fig. 1. Phylogenetic analysis Comparisons of A36R gene (A) and HA gene (B) nucleotide sequences of the Austrian CPXV strain AT/Carinthia/788/07 with selected reference sequences is shown. The numbers attached to the strain names indicate the GenBank respective Accession Numbers. Scale bares represents the genetic distance (nucleotide substitutions per site).
Fig. 2. Infection of BALB/c mice Development of body weight of groups of BALB/c mice after infection with vaccinia virus (VACV) strain WR and cowpox virus (CPXV) strain AT/Carinthia/788/07 is shown. Mice were inoculated on day zero (d0) with the viral loads indicated on the right side. 103 up to 107 plaque forming units were applied either via the intravenous (i.v.) route (A and B) or by intranasal (i.n.) application (C and D). The days after infection are indicated on the x-axis, measured body weight in grams is shown on the y-axis. In the experimental setup ‘‘C’’ the animals subjected to VACV doses over 104 required euthanasia at day 6 due to the severity of disease symptoms and excessive weight loss.
outlined in the introduction. Additionally VACV, which due to its relatively low virulence in humans has been used for vaccination
purposes, is known since long time to have a rather high virulence in laboratory mice (Svet-Moldavskaya and Chimishkyan, 1969;
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Table 1 Identified insertions/deletions of the Austrian CPXV strain AT/Carinthia/788/07 This table shows some of the more extended mismatches found in the tested strain by shotgun sequencing as compared to the whole genome sequence of CPXV reference strain Brighton (Accession # GI:30844336). The nucleotides found at the indicated genomic positions in the reference sequence (Ref Nuc) and those of the investigated sequence (Var Nuc) are depicted. Numbering of the genomic positions (Start/End) and identified open reading frames (ORF) refers to CPXV reference strain Brighton. The name, as available, or the presumable functional property of the affected gene products (protein) are listed in the right hand column.
Berhanu et al., 2008). This would suggest that certain mechanisms of host adaptation may play a role in those viruses which do not easily cross the species barrier. The result observed in the present study of a rather reduced virulence of the used human CPXV isolate in BLAB/c mice was interesting because a variety of CPXV isolates has been found highly virulent especially upon intranasal infection. In the study of Smee et al. (2000) 100% mortality was observed upon intranasal infection with 5 105 p.f.u. of strain Brighton in female BALB/c
mice of similar age and weight. Kochneva et al. (2005) used CPXV strain GRI-90 in their study, and their BALB/c mice also died within 4–10 days when using 105 p.f.u. or more. Using 104 p.f.u. a weight loss of about 20% was observed their study with a maximum around day 9, which would correspond nicely to our findings with VACV but suggests a rather attenuated phenotype of our local CPXV strain AT/Carinthia/788/07. The reasons for this attenuation may reside in multiple genes. Removal of Kelch-like genes (D11L,C18L,G3L,A57R) from CPXV
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has been described to alter cell tropism, reduce pock formation on chorio-allantoic membrane and reduce mortality or weight loss in mice (Kochneva et al., 2005). The Kelch repeat motives predict a conserved tertiary structure used in many cellular and viral protein–protein interactions including HSV host cell factor 1 and 2, influenza NS1 binding protein and human serum proteins promoting monocyte spreading and T-cell clustering (for review see Adams et al., 2000). Interestingly some of the genes identified in our preliminary sequencing approach may be involved in immune response mechanisms and/or have been shown to be involved in virulence by several mechanisms. Cytokine response modifiers (crm) are suspected to act as virulence factors and e.g. CrmA has been found to increase severity of respiratory and intradermal CPXV infection (MacNeill et al., 2009). Deletion mutants of the A14.5L gene have been shown to reduce virulence of VACV in mice (Betakova et al., 2000), and B19R has been found to be a virulence gene in monkeypox virus (Chen et al., 2005). Orthopoxviruses use also strategies to evade the unspecific host immune response. This includes production of virally encoded chemokine receptors, viral effects on apoptosis, and interference with complement-mediated lysis (Smith and Kotwal, 2002; Seet et al., 2003), all those mechanisms could also contribute to an attenuated phenotype in mice. Summarizing we can state that we have isolated a CPXV strain causing symptomatic human infection, which was not only restricted to the skin but also associated with systemic infection/ fever in a young and otherwise healthy female (Strenger et al., 2009). This virus proved rather apathogenic in mice which presumably might be due to genetic changes involving numerous loci. Considering the decisive role of the immune system on the viral–host co-adaptation (for review see Zúñiga, 2002) it seems plausible that adaptations to mouse proteins not necessarily have to be effective also on the proteins of the human immune system. Thus a virus which is well adapted to rodents not necessarily has to be also well adapted and benign in another host. Repeated findings of severe CPXV or VACV disease in other species including nonhuman primates would support such a concept. Finally a possibly reduced virulence of CPXV field strains, although so far not an established fact, definitely would make sense. From the extensive field data with leporipoxviruses (Zúñiga, 2002) one can assume that, especially at low host population densities, less virulent strains may be preferentially selected also in CPXV infected animals. Considering a recent paper that virulent CPXV strains have a clear negative impact on the overall survival of field voles (Burthe et al., 2008), such attenuation would certainly be an advantage for viral persistence in the environment. 5. Conflict of interest statement There is no conflict of interest of any of the authors. Acknowledgments The work has been supported by the Austrian Federal Ministry of Health as a contribution to the smallpox preparedness program (Pockenalarmplan). References Adams, J., Kelso, R., Cooley, L., 2000. The kelch repeat superfamily of proteins: propellers of cell function. Trends in Cell Biology 10, 17–24. Baxby, D., Ghaboosi, B., 1977. Laboratory characteristics of poxviruses isolated from captive elephants in Germany. Journal of General Virology 37, 407–414. Becker, C., Kurth, A., Hessler, F., Kramp, H., Gokel, M., Hoffmann, R., Kuczka, A., Nitsche, A., 2008. Cowpox virus infection in pet rat owners: not always immediately recognized. Deutsches Ärzteblatt International 106, 329–334.
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Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Cowpox virus isolate virulent in humans shows attenuated phenotype in mice Hartwig P. Huemer a,⇑, Caroline Lassnig b, Norbert Nowotny c a
Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Fritz-Pregl-Str. 3, R.301, A-6020 Innsbruck, Austria Institute of Animal Breeding and Genetics, Biomodels Austria, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Veterinärplatz 1, A-1210 Vienna, Austria c Zoonoses and Emerging Infections Group, Clinical Virology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Veterinärplatz 1, A-1210 Vienna, Austria b
a r t i c l e
i n f o
Article history: Received 2 February 2011 Accepted 11 March 2011
Keywords: Cowpox Vaccinia Virulence Human Mouse BALB/c
a b s t r a c t We have cultured Cowpox virus (CPXV) from skin lesion material of a human patient from Austria. Phylogenetic comparison of the HA-gene revealed a rather homogeneous cluster with other local isolates from recent years, the A36R-gene was mostly related to elephant derived strains from Germany. Despite causing disease in human, the isolate AT/Carinthia/788/07 surprisingly even at high titers showed a highly reduced virulence in BALB/c mice upon intranasal inoculation as compared to vaccinia virus. This contrasts earlier reports on other CPXV isolates. Using shotgun DNA sequencing several insertions and deletions were found in genes presumably involved in host range, immune regulation as well as established virulence factors. These preliminary data could be an indication that CPXV strains with proven pathogenicity for humans may have reduced virulence in mice and vice versa. Additionally strains with a reduced virulence may have an advantage in persisting in less dense rodent populations. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The Cowpox virus (CPXV), a member of the family of the Poxviridae (subfamily Chordopoxvirinae, genus: Orthopoxvirus), is a big brick shaped enveloped DNA viruses with a genome of about 220 kb. It is found in wild rodents of the old world (for review see Chantrey et al., 1999; Essbauer et al., 2009). There is only limited information about the natural course of CPXV in rodents, and the reported findings seem to vary between mouse species. Although earlier data suggested that most cases occur in younger ages and infections might be rather subclinical, CPXV has been found to lead to reduced fecundity in bank voles (Clethrionomys glareolus) and wood mice (Apodemus sylvaticus) in the UK (Feore et al., 1997). However, in field voles (Microtus agrestis) it seems to be quite lethal, reducing the overall survival rate significantly (Burthe et al., 2008). Humans can accidently acquire CPXV infection usually through direct skin contact with diseased cats (‘‘catpox’’) rather than cows, but recently increasingly infections associated with infected pet rats have been reported throughout Europe (Hönlinger et al., 2005; Becker et al., 2008; Ninove et al., 2009). Although the disease is mild and restricted to few skin lesions in most human cases, it is frequently associated with transient viremia (Nitsche et al., 2007) and more severe courses can occur in immunosuppressed patients (Hönlinger et al., 2005; Huemer et al., 2007), even leading to death under rare circumstances (Czerny et al., 1997). ⇑ Corresponding author. Tel.: +43 512 9003 70799; fax: +43 512 9003 73799. E-mail address: [email protected] (H.P. Huemer). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.03.011
Together with reports of high CPXV mortality in non-human primate species (Mätz-Rensing et al., 2006) or elephants (Baxby and Ghaboosi, 1977) this virulence would speak for a less well adaptation of CPXV in non-rodent species and especially to humans/primates than e.g. of the presumably horse derived Vaccinia virus (VACV). VACV has a long record of (relatively) safe use in humans as a vaccine against smallpox, but on the contrary is known since a long time to be rather virulent in mice (SvetMoldavskaya and Chimishkyan, 1969; Berhanu et al., 2008), thus supporting an early concept of adaptation of viral strains or isolates to their respective host environment (Dunlap and Barker, 1973). As an adaptation of CPXV to the mouse population would make sense in terms of the survival of the virus we therefore were interested how a virus with proven pathogenicity in humans would behave in mice. 2. Materials and methods 2.1. Virus strain, cell culture The human-derived CPXV strain AT/Carinthia/788/07 was isolated from crust material of a skin lesion of a 17 year old girl from South-Eastern Austria (Richter et al., 2008). Although it led to a favorable outcome this infection was considered systemic as it caused fever and malaise in a young healthy person (Strenger et al., 2009). The virus was grown in RK13 rabbit kidney cells (ATCC) cultured in DMEM supplemented with 5% FCS. VACV laboratory strain WR (western reserve; provided previously by Dr. B. Moss, Bethesda, MD, USA), was used for comparison for its
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proven virulence in mice. Viral preparations for both viruses were obtained on RK13 cells grown in roller bottles with enhanced surface. Cells were lysed by freeze-thawing, disrupted by hypotonic medium, briefly sonicated, and the nuclei were removed by low speed centrifugation (2000g). The supernates were concentrated at 20,000g and the virus was purified over sucrose gradients using ultracentrifugation (30,000g). Resulting viral stocks were dissolved in phosphate buffered saline (PBS) and the virus titers determined in 24 well plates by plaque assays using RK13 cells. 2.2. DNA analysis, sequencing The new CPXV Isolate was analysed by PCR using an A36R gene (Huemer et al., 2008) and hemagglutinin (HA) gene specific PCR (Ropp et al., 1995). Automated DNA sequencing of the amplified fragments was carried out on both strands, and the obtained A36R and HA sequences were deposited in GenBank (Accession # FM178393.1 and EU582018.1). Sequence comparison was performed by the BLAST algorithm (http://www.ncbi.nlm.nih.gov/ blast) using selected reference sequences from the GenBank database. Phylogenetic analysis was performed using Kimura twoparameter model of nucleotide substitution for calculation of the genetic distance of the DNA sequences aligned with MEGA 4 Molecular Evolutionary Genetics Analysis software. Phylogenetic trees were constructed using the Neighbour-Joining method with bootstrap analysis using 1000 pseudoreplicate datasets, and the trees finally plotted with Phylip 3.65. For a preliminary identification of genes possibly involved in altered virulence patterns we applied a shotgun sequencing approach using the GS FLX Titanium deep sequencing technology from Roche, Inc. Whole virus DNA was prepared from sucrose purified CPXV by digestion with proteinase K and subsequent phenol chloroform extraction. The virus genome was then incorporated into a Titanium 3kB paired end library using the materials and protocol of the manufacturer (Roche, Inc.). After PCR amplification with FLX Titanium emPCR it was finally sequenced on the FLX454 sequencer at the Medical University of Graz/Austria, ZMF – sequencing core facility and the obtained sequences compared to the published genome of CPXV strain Brighton as the reference sequence (GeneBank Accession # GI:308443369). 2.3. Animal experiments An early passage (P4) of the isolated CPXV was also tested in comparison with VACV in a mouse model studying virulence using the conditions described most recently (Huemer et al., 2010). Female inbred pathogen-free BALB/c mice (obtained from Charles River, Germany) with an age of 6 weeks and a weight of 16–17 g were used. For both viruses two different inoculation routes were chosen, intravenous (groups A, B) and intranasal (groups C, D). Five different infectious doses were applied ranging from 103 up to 107 plaque forming units (p.f.u.), thus resulting in a total of 20 subgroups with five animals in each group. On day 0 the animals were objected to short term anesthesia with ketamin/xylazin (75 and 7.5 mg/kg i.p.) and infected with 10 ll of the above purified viral stocks diluted in PBS via a nasal tube or by injection into the dorsal vein of the tail. The animals were controlled daily for disease symptoms and the body weights were determined. Severity of disease was evaluated according to a published scoring system (Berhanu et al., 2008), using disease index (DI) of 0 (normal), (1) (slightly ruffled skin), (2) (clearly ruffled skin and/or single sided conjunctivitis), (3) (hunched position and/or severe conjunctivitis of both eyes in addition to clear ruffling), (4) (score of three combined with difficulty in moving/socializing/breathing), and (5) (death). Animals with a DI of 4 and/or >30% weight loss were euthanized according to the prescriptions of the local ethics
committee. The animal experiments were approved under the permit # BMWF-68.205/0246-II/10b/2008 of the Austrian Federal Ministry of Research, Section for Animal Experiments. 3. Results 3.1. Genetic relationship of the isolated CPXV strain Both sequences were deposited in GenBank under the Accession Numbers FM178393.1 and EU582018.1 respectively. Sequence comparison as shown in Fig. 1, revealed that the A36R gene sequence of the isolate is most closely related to sequences EP1 and EP6 derived from infected elephants from Germany (Baxby and Ghaboosi, 1977), whereas CPXV isolates from the UK or Norway seem to form a distinct cluster. The HA gene was found closely related to Austrian CPXV isolates cpv-531, -667, and -610 from 1992, 1994 and 1997, which all have been isolated from diseased cats (Nowotny et al., unpublished), and a local human isolate from Styria from 2009, whereas the HA gene of the human isolate cpv-1218 from the Austrian federal state of Salzburg from the year 2000 (Hawranek et al., 2003) seems more distant. Interestingly other German or Italian isolates are rather unrelated supporting the facts of high variation among CPXV strains, and the finding of highly divergent regional clusters (Essbauer et al., 2009; Kaysser et al., 2010). 3.2. Reduced virulence of the CPXV strain in mice As demonstrated in Fig. 2, VACV led to severe disease in BALB/c mice upon intranasal (i.n.) infection with all studied viral loads. Rapid weight loss and disease symptoms were observed especially with inoculation doses above 104 p.f.u., requiring euthanasia of the animals about day 6. There was also significant disease and weight loss of about 20% in mice infected with 103 p.f.u. of VACV (Fig. 2C), which contrasts the outcome with the CPXV strain, which at the same dose showed no symptoms at all but a steady gain of weight. Even at high doses up to 107 p.f.u. of CPXV there was only a temporary stagnation of the weight gain until day 6 and a continuous increase in weight thereafter (Fig. 2D). Mice infected via the intravenous (i.v.) route remained rather unaffected with both viruses showing only marginal weight reduction around day 6 even at high VACV inoculation doses (Fig. 2A), which could be an indication that most virions may be rapidly inactivated in the serum (via complement?) before establishing more severe systemic infection. 3.3. Sequence differences as compared to virulent strain Brighton Although we only achieved about 25% coverage of the whole CPXV genome in the ‘‘shotgun’’ DNA sequencing approach chosen, which does not enable more extensive bioinformatic comparisons like investigation of whole reading frames, numerous differences were identified as compared to the sequence of the virulent CPXV strain Brighton. Regions showing insertions and deletions of several nucleotides are depicted in Table 1. The identified differences include also genes involved in host-range selection, virulence and immune modulation, e.g., ankyrin-like proteins, cytokine response modifying (crm) proteins like tumor necrosis factor (TNF) receptor homologue crmB, Semaphorin-like and lectin type receptors. 4. Discussion One of our aims was to study the virulence of a CPXV isolate with proven pathogenicity for humans in a mouse model. The reason for this resides in the varying severity of CPXV disease observed in different species as monkeys or elephants as has been
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Fig. 1. Phylogenetic analysis Comparisons of A36R gene (A) and HA gene (B) nucleotide sequences of the Austrian CPXV strain AT/Carinthia/788/07 with selected reference sequences is shown. The numbers attached to the strain names indicate the GenBank respective Accession Numbers. Scale bares represents the genetic distance (nucleotide substitutions per site).
Fig. 2. Infection of BALB/c mice Development of body weight of groups of BALB/c mice after infection with vaccinia virus (VACV) strain WR and cowpox virus (CPXV) strain AT/Carinthia/788/07 is shown. Mice were inoculated on day zero (d0) with the viral loads indicated on the right side. 103 up to 107 plaque forming units were applied either via the intravenous (i.v.) route (A and B) or by intranasal (i.n.) application (C and D). The days after infection are indicated on the x-axis, measured body weight in grams is shown on the y-axis. In the experimental setup ‘‘C’’ the animals subjected to VACV doses over 104 required euthanasia at day 6 due to the severity of disease symptoms and excessive weight loss.
outlined in the introduction. Additionally VACV, which due to its relatively low virulence in humans has been used for vaccination
purposes, is known since long time to have a rather high virulence in laboratory mice (Svet-Moldavskaya and Chimishkyan, 1969;
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Table 1 Identified insertions/deletions of the Austrian CPXV strain AT/Carinthia/788/07 This table shows some of the more extended mismatches found in the tested strain by shotgun sequencing as compared to the whole genome sequence of CPXV reference strain Brighton (Accession # GI:30844336). The nucleotides found at the indicated genomic positions in the reference sequence (Ref Nuc) and those of the investigated sequence (Var Nuc) are depicted. Numbering of the genomic positions (Start/End) and identified open reading frames (ORF) refers to CPXV reference strain Brighton. The name, as available, or the presumable functional property of the affected gene products (protein) are listed in the right hand column.
Berhanu et al., 2008). This would suggest that certain mechanisms of host adaptation may play a role in those viruses which do not easily cross the species barrier. The result observed in the present study of a rather reduced virulence of the used human CPXV isolate in BLAB/c mice was interesting because a variety of CPXV isolates has been found highly virulent especially upon intranasal infection. In the study of Smee et al. (2000) 100% mortality was observed upon intranasal infection with 5 105 p.f.u. of strain Brighton in female BALB/c
mice of similar age and weight. Kochneva et al. (2005) used CPXV strain GRI-90 in their study, and their BALB/c mice also died within 4–10 days when using 105 p.f.u. or more. Using 104 p.f.u. a weight loss of about 20% was observed their study with a maximum around day 9, which would correspond nicely to our findings with VACV but suggests a rather attenuated phenotype of our local CPXV strain AT/Carinthia/788/07. The reasons for this attenuation may reside in multiple genes. Removal of Kelch-like genes (D11L,C18L,G3L,A57R) from CPXV
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has been described to alter cell tropism, reduce pock formation on chorio-allantoic membrane and reduce mortality or weight loss in mice (Kochneva et al., 2005). The Kelch repeat motives predict a conserved tertiary structure used in many cellular and viral protein–protein interactions including HSV host cell factor 1 and 2, influenza NS1 binding protein and human serum proteins promoting monocyte spreading and T-cell clustering (for review see Adams et al., 2000). Interestingly some of the genes identified in our preliminary sequencing approach may be involved in immune response mechanisms and/or have been shown to be involved in virulence by several mechanisms. Cytokine response modifiers (crm) are suspected to act as virulence factors and e.g. CrmA has been found to increase severity of respiratory and intradermal CPXV infection (MacNeill et al., 2009). Deletion mutants of the A14.5L gene have been shown to reduce virulence of VACV in mice (Betakova et al., 2000), and B19R has been found to be a virulence gene in monkeypox virus (Chen et al., 2005). Orthopoxviruses use also strategies to evade the unspecific host immune response. This includes production of virally encoded chemokine receptors, viral effects on apoptosis, and interference with complement-mediated lysis (Smith and Kotwal, 2002; Seet et al., 2003), all those mechanisms could also contribute to an attenuated phenotype in mice. Summarizing we can state that we have isolated a CPXV strain causing symptomatic human infection, which was not only restricted to the skin but also associated with systemic infection/ fever in a young and otherwise healthy female (Strenger et al., 2009). This virus proved rather apathogenic in mice which presumably might be due to genetic changes involving numerous loci. Considering the decisive role of the immune system on the viral–host co-adaptation (for review see Zúñiga, 2002) it seems plausible that adaptations to mouse proteins not necessarily have to be effective also on the proteins of the human immune system. Thus a virus which is well adapted to rodents not necessarily has to be also well adapted and benign in another host. Repeated findings of severe CPXV or VACV disease in other species including nonhuman primates would support such a concept. Finally a possibly reduced virulence of CPXV field strains, although so far not an established fact, definitely would make sense. From the extensive field data with leporipoxviruses (Zúñiga, 2002) one can assume that, especially at low host population densities, less virulent strains may be preferentially selected also in CPXV infected animals. Considering a recent paper that virulent CPXV strains have a clear negative impact on the overall survival of field voles (Burthe et al., 2008), such attenuation would certainly be an advantage for viral persistence in the environment. 5. Conflict of interest statement There is no conflict of interest of any of the authors. Acknowledgments The work has been supported by the Austrian Federal Ministry of Health as a contribution to the smallpox preparedness program (Pockenalarmplan). References Adams, J., Kelso, R., Cooley, L., 2000. The kelch repeat superfamily of proteins: propellers of cell function. Trends in Cell Biology 10, 17–24. Baxby, D., Ghaboosi, B., 1977. Laboratory characteristics of poxviruses isolated from captive elephants in Germany. Journal of General Virology 37, 407–414. Becker, C., Kurth, A., Hessler, F., Kramp, H., Gokel, M., Hoffmann, R., Kuczka, A., Nitsche, A., 2008. Cowpox virus infection in pet rat owners: not always immediately recognized. Deutsches Ärzteblatt International 106, 329–334.
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