1 National Center for Infectious Disease, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G43, Atlanta, GA 30333, USA
2 World Health Organization, Geneva, Switzerland
Correspondence
Inger K. Damon
iad7{at}cdc.gov
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ABSTRACT |
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Published online ahead of print on 25 July 2005 as DOI 10.1099/vir.0.81215-0.
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are DQ011153DQ011157.
Supplementary material is available in JGV Online.
These authors contributed equally to this work.
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INTRODUCTION |
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In 2003, an outbreak of febrile rash illness in the USA among humans and captive pet prairie dogs was attributed to MPXV-infected West African rodents imported from Ghana (CDC, 2003). Laboratory testing confirmed 37 human cases as of August 2003, all associated with ill prairie dogs (CDC, 2003
; Damon, 2003
); no human-to-human transmission was documented. Early observations suggested that the disease described during the USA outbreak was milder than had been previously described in DRC patients (Damon, 2003
; Peters, 2003
). Observations, which were based on single gene sequence phylogenies, indicated the monkeypox isolate identified in the USA belonged to a clade of MPXV distinct from that of the previously characterized DRC isolates (Reed et al., 2004
). Concurrent with the 2003 USA monkeypox outbreak, the Congo Basin nation Republic of Congo (RCG) reported 10 monkeypox cases and sustained human-to-human transmission (Learned et al., 2005
).
To discern potential pathogenic differences, we compared clinical and epidemiological features of confirmed human monkeypox disease, using retrospective datasets from outbreaks in the USA and DRC as well as laboratory detection of MPXV in blood samples from the concurrent USA and RCG outbreaks. We observed differences that, after controlling for population age and vaccination disparities, may be attributed to genetic differences in the geographically distinct viruses. Therefore, we sequenced and annotated five MPXV isolates to identify genomic and proteomic differences, to enable more robust depiction of MPXV clades and to allow the development of hypotheses regarding differences in pathogenesis.
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METHODS |
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Because age and prior vaccination for smallpox may affect the severity of human monkeypox illness, we stratified our analyses by these factors. We limited our analysis of DRC patients to the same age range as seen in the USA outbreak (648 years). This resulted in the exclusion of 200 cases (198<6 years of age, two >48 years of age) from the 327 laboratory-confirmed DRC cases. Stratification by vaccination status, however, was confounded by age, and stratification by both vaccination status and age resulted in empty cells since no children in the USA were vaccinated against smallpox, and there were no adults in DRC that had not been vaccinated.
Both datasets included information on rash burden (lesion counts), hospitalizations, case outcomes and types of complications and/or sequelae. Additionally, the USA dataset included a variable explaining the reason for hospitalization (e.g. for isolation, or severity of disease), while the WHO dataset scored illness severity as one, two or three on the basis of the number of lesions, degree of incapacitation and need for nursing care. A score of three was given to individuals with 100 lesions and severe incapacitation requiring medical care (Jezek & Fenner, 1988
), although individual scores for lesions and incapacitation occasionally differed (17 of 117 cases with known lesion counts were discordant). Therefore, for our comparisons, a severely ill individual from DRC was defined as anyone with
100 lesions who was hospitalized and had an illness severity score of three. A severely ill USA patient was similarly defined as having
100 lesions and was hospitalized and was noted to be hospitalized specifically for severity of disease.
Statistical methods.
Two-sided Fisher's exact tests were used to compare infection characteristics between DRC and USA case-patients, and a non-parametric test (Wilcoxon rank sum) was used to compare ages between the two populations. For ease of evaluation, comparisons of infection characteristics between DRC and USA cases are presented as relative risk ratios (RR) with 95 % confidence intervals (CI).
Strains.
The five sequenced MPXV strains were derived from isolates collected in Liberia, DRC, USA and RCG. MPXV-LIB-1970-184 (DQ011156; length, 200 263 bp; G+C content, 33·09 mol%) was collected from a 3-year-old girl in Liberia in 1970. A crust sample was processed and passaged once in chorioallantoic membrane (CAM) and twice in African green monkey kidney cells (BSC-40) prior to DNA isolation for sequencing. Descriptive and initial laboratory information has been published (Foster et al., 1972; Lourie et al., 1972
).
MPXV-ZAI-1979-005 (DQ011155; length, 196 967 bp; G+C content, 33·09 mol%) was isolated from a severely ill, 1-year-old boy living in Zaire in 1978 (Breman et al., 1980). The virus was isolated from a lesion scab, which was positive for poxvirus by electron microscopy and for monkeypox virus on CAM. DNA was derived from virus passaged five times on BSC-40 cells.
MPXV-USA-2003-044 (DQ011153; length, 198 780 bp; G+C content, 33·08 mol%) and MPXV-USA-2003-039 (DQ011157; length, 198 780 bp; G+C content, 33·08 mol%) were collected during the 2003 monkeypox outbreak in the USA from a prairie dog and human, respectively. The prairie dog isolate was from the lymph node of the animal associated with the initial (index) case recognized in Wisconsin. The human isolate was obtained from a skin vesicle of the exotic pet distributor who sold this prairie dog to the family that heralded the outbreak (Reed et al., 2004). Both were passaged twice on BSC-40 cells prior to DNA isolation for sequencing. The MPXV-USA-2003-044 and MPXV-USA-2003-039 genomes have a single nucleotide difference at base 31 in the inverted terminal repeat region prior to the leftmost open reading frame (ORF).
MPXV-RCG-2003-358 (DQ011154; length, 197 191 bp; G+C content, 33·09 mol%) was isolated from a 10-year-old girl infected in Impfondo, RCG, and admitted to the hospital on 9 June, 2003 (Learned et al., 2005). A bloody rash-derived sample was processed and passaged twice in BSC-40 cells, followed by DNA isolation.
Sequencing.
We sequenced the entire genome of five strains from USA and African MPXV isolates collected over 33 years and compared them to the published genome for MPXV-ZAI-1996-016 (Shchelkunov et al., 2002) (NC_003310). We compared genome nucleotide sequences to distinguish the clades, which we designated West African/USA and Congo Basin. Monkeypox virus DNA was prepared as previously described (Reed et al., 2004
). The genomic DNAs were used as templates for the production of 20 overlapping PCR amplicons designed to span the complete viral genome. Each amplicon (516 kb) was generated using the Expand High Fidelity PCR system (Roche Applied Science); for each DNA sequencing template, eight independent PCR reactions were pooled and treated with ExoSap-IT (USB). The templates were sequenced by primer-walking both strands, using ABI Big-Dye 3.1 dye chemistry and ABI 3730XL automated DNA sequencers (both from PE Biosystems). Sequencing primers were synthesized by Integrated DNA Technologies. Approximately 2500 reads were obtained for each genome, resulting in a ninefold mean redundancy at each base position. Chromatogram data were assembled using Seqmerge (Wisconsin Package version 10.3; Accelrys), Phred/Phrap base-calling and assembly software (Ewing et al., 1998
; Ewing & Green, 1998
) and Consed (Gordon et al., 2001
) for sequence editing.
Analysis of genomic sequences.
Sequence annotation was done through a locally modified version of Poxvirus Orthologous Clusters software (Ehlers et al., 2002). Genes were predicted using GeneMarkS (Besemer et al., 2001
) and Glimmer 2.02 (Delcher et al., 1999
) and then tested for the presence of regulatory elements and assigned an initial annotation by comparison to other poxvirus gene databases, using BLASTP (Altschul et al., 1997
). The remaining ORFs were then verified by manual inspection. Genome alignments were generated with Mavid (Bray & Pachter, 2004
) with default parameters and then edited by hand to remove errors. The predicted protein sequence alignments in Fig. 5
and Supplementary Fig. S1 (available in JGV Online) were constructed using CLUSTALW (Higgins & Sharp, 1988
). The diversity calculation,
, was calculated using a modification of the Nei & Miller method (1990)
in the DNAsp software package (Rozas et al., 2003
). MrBayes (Ronquist & Huelsenbeck, 2003
) was used to perform a maximum-likelihood Bayesian analysis of whole genomic nucleotide sequences from five MPXV isolates as well as from one isolate each of cowpox (CPXV-GRI; X94355) and vaccinia (VACV-COP; NC_001559). The program settings were as follows: the maximum-likelihood model utilized six substitution types, base frequencies were set to the empirically observed values, and rate variation across sites was modelled using a gamma distribution with a proportion of sites being invariant. The Markov Chain Monte Carlo search was run with four chains for 5 000 000 generations. Trees were sampled every 1000 generations and the first 200 trees were discarded.
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Analysis of clinical diagnostic samples.
A quantitative real-time PCR assay was used to assess the amount of viral DNA within clinical whole blood samples submitted from confirmed USA monkeypox cases, and from probable and confirmed RCG monkeypox cases to the Centers for Disease Control and Prevention (Atlanta, Georgia) during the summer of 2003. Each sample was confirmed as monkeypox, using a real-time PCR monkeypox-specific assay, and the DNA content quantified by triplicate runs, using an orthopox generic assay (Y. Li, V. Olson, T. Laue, M. Laker & I. Damon, unpublished data). Samples with MPXV DNA detected in none or only one of the triplicate runs were considered negative, and those with DNA in two of three runs were considered equivocal. A sample was considered positive only if all three runs crossed the threshold. A dilution series of purified vaccinia virus was used to create a reference standard curve for the orthopoxvirus generic real-time PCR assay. Using this standard curve, the viral load in femtograms for each sample was calculated on the basis of the mean cycle at which fluorescence from the sample crossed the threshold. To determine the length of viraemia, viral load was plotted against the number of days after onset of rash or fever.
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RESULTS |
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Although significantly more unvaccinated case-patients in the DRC than in the USA had pronounced rash (RR 5·6, 95 % CI 1·916·4; Table 2), they were not significantly more likely to be hospitalized. Overall, significantly more unvaccinated DRC than USA case-patients were severely ill; however, all unvaccinated, severely ill patients, regardless of their geographical origin, were <18 years old (Table 2
). No smallpox-vaccinated patients in the USA had pronounced rash or were severely ill, despite an apparent longer time period between onset of illness and last vaccination (>20 years for the 2 of 10 USA patients who could recall their last vaccination vs 9·4±4·3 years, range 118, in the DRC). In sum, human disease was more transmissible and more severe among DRC than USA case-patients, and this finding was independent of patient age and vaccination status.
Prolonged detection of viral nucleic acid
One hypothesis to explain the relative differences in disease severity and transmissibility would be a difference in the duration and magnitude of viraemia. We performed quantitative PCR for MPXV in those cases where whole blood samples were available from monkeypox patients in the USA and RCG (Fig. 1). Of 14 blood samples collected within 21 days of rash onset from 12 USA patients with confirmed infection, three were positive for MPXV DNA and two others were equivocal. All were negative past day 21, a time point likely exceeding the occurrence of viraemia in an otherwise healthy human host (Downie et al., 1950
). In contrast, two of three RCG samples obtained within 21 days of rash onset from three probable or confirmed patients were positive for MPXV DNA; one of five samples collected after day 21 (day 33) was positive. These results suggested a longer presence of virus in the blood of RCG patients. No USA or RCG samples contained viable virus at the time of testing.
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The remaining five proteins that consistently demonstrated geographically associated differences have been experimentally demonstrated to be involved with either immune evasion or host range determination in other poxviruses. These include the interleukin-1 (IL1
) receptor orthologue (affects the febrile response and virulence) (Smith & Chan, 1991
; Spriggs et al., 1992
; Alcami & Smith, 1992
, 1996
), the SPI-1 orthologue (apoptosis regulation, host range) (Smith et al., 1989b
; Kotwal & Moss, 1989
; Senkevich et al., 1993
; Thompson et al., 1993
; Ali et al., 1994
; Kettle et al., 1995
; Brooks et al., 1995
; Macen et al., 1996
; Shisler et al., 1999
; Moon et al., 1999
; Legrand et al., 2004
), the vaccinia C7L orthologue (a host range factor) (Perkus et al., 1990
; Oguiura et al., 1993
), the myxoma M-T4 orthologue (apoptosis regulation) (Barry et al., 1997
; Shchelkunov et al., 1998
; Hnatiuk et al., 1999
; Price et al., 2002
) and the orthologue of the complement control protein (CCP) (inhibits the classical and alternate complement pathways) (Isaacs et al., 1992
; Miller et al., 1997
; Smith et al., 2000
; Rosengard et al., 2002
; Isaacs et al., 2003
).
Fragmentation of the West African/USA MPXV IL1 receptor orthologue was observed. Either a single nucleotide deletion results in truncation after the signal peptide (Fig. 5a
) or, if the second methionine is used to initiate translation, only the first two immunoglobulin domains (Smith & Chan, 1991
) are translated. Within the Congo Basin MPXV clade, the MPXV-ZAI-1979-005 orthologue also lacked the third immunoglobulin domain, while MPXV-ZAI-1996-016 and MPXV-RCG-2003-358 orthologues had full-length sequences (Fig. 5a
).
Insertion/deletion events in the upstream regions of the West African/USA MPXV orthologues of vaccinia SPI-1 (C12L) and VAC-COP-C7L were predicted to affect these expressed proteins, in addition to amino acid changes. A 4 nt deletion in the variable repeat region upstream of the West African/USA MPXV vaccinia SPI-1 (C12L) orthologue may produce, dependent on promoter usage, an in-frame fusion resulting in a variable length string of amino-terminal IIY repeats (Supplementary Figs S1e, S1f, S2 and Online Supplementary Results and Discussion). Upstream of the West African/USA MPXV VAC-COP-C7L orthologue, a single nucleotide deletion was predicted to result in an amino-terminal fusion of 17 aa to the protein, depending upon which predicted promoter is used (Fig. 5b, Supplementary Fig. S3 and Online Supplementary Results and Discussion). A BIMAS-predicted 9 mer epitope of this gene product, conserved between vaccinia and variola, is protective as a single peptide vaccine against a lethal intranasal vaccinia mouse challenge model in HLA-A2.1 transgenic mice (Snyder et al., 2004
). In all monkeypox isolates sequenced, this epitope was perturbed (Fig. 5b
), such that the BIMAS-predicted dissociation half-life decreased three to fourfold (to 107 or 89 min). The West African/USA strain predicted protein sequences contained a unique 9 mer epitope with a predicted dissociation half-life even greater than that of the vaccinia/variola epitope (437 min vs 365, respectively).
Two immunomodulatory proteins of the Congo Basin clade viruses were fragmented or absent in the West African/USA MPXV clade. The West African/USA MPXV orthologue of the myxoma virus apoptotic regulator (M-T4) was predicted to be severely truncated (Fig. 3, box E), similar to the non-functional vaccinia B9R gene product (Price et al., 2002
). Although there was minimal overall amino acid conservation (
24 %), Congo Basin orthologues retained the conserved cysteine residues and, thus, may exhibit anti-apoptotic function and permit infection of lymphocytes (Fig. 5c
) (Barry et al., 1997
). Lastly, the MPXV orthologue of the vaccinia CCP and smallpox virus inhibitor of complement enzymes was completely absent in West African/USA isolates (Fig. 3
, box B). The orthopoxvirus orthologues interfere with the classical and alternative pathways of complement activation and prevent complement-mediated virus neutralization. The Congo Basin strains are predicted to express a CCP, albeit with a truncated fourth short consensus repeat (Fig. 5d
). This MPXV protein is reported to inhibit the classical complement pathway (Smith et al., 2000
).
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DISCUSSION |
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Whole genome analysis confirmed the existence of two monkeypox clades, which previously was suggested by single-gene phylogenies (Reed et al., 2004) and whole-genome RFLP analysis (Mackett & Archard, 1979
; Esposito & Knight, 1985
), and provided clues to understanding the differences in human monkeypox disease pathology. More nucleotide divergence was seen at the right and left termini of the genome, a finding consistent with previous observations of general orthopoxvirus genome structure (Esposito & Knight, 1985
; Shchelkunov et al., 2002
). Although the effect of various selective pressures has not been extensively evaluated, our observations from the USA outbreak suggested MPXV was stably replicated because only a single nucleotide substitution existed between human and prairie dog MPXV isolates. Although the in vitro and in vivo significances of proteins predicted to differ between the two clades remain to be validated, it is noteworthy that these changes have been reproducibly maintained for over 30 years among MPXV isolates obtained from the two regions.
A number of proteins were identified as candidates that might affect the observed different human disease manifestations. Their orthologues in other orthopoxviruses have been shown to promote viral persistence, and/or to evade immune recognition and clearance. The clade-specific orthologues may modulate viral pathogenesis or host response, perhaps playing a role in the observed differential clearance of virus from the blood of individuals infected with these strains.
ORF comparisons clearly predicted loss of function for the West African/USA MPXV CCP. Both vaccinia and variola orthologues interfere with the classical and alternate complement pathways (Rosengard et al., 2002). If Congo Basin MPXV CCP is effective in blocking complement-enhanced viral neutralization, as observed in vaccinia (Isaacs et al., 1992
), one would expect prolongation of viraemia; this may partly explain the ability to detect MPXV nucleic acid by PCR in blood specimens collected at longer intervals following onset of rash in individuals infected with the Congo Basin MPXV clade. The haemorrhagic manifestation of skin lesions observed in the USA outbreak (Reed et al., 2004
) may correlate with a lack of complement inhibition (Miller et al., 1997
).
A novel prediction, based on previous studies with vaccinia and on sequence comparative analysis with the BIMAS program, was the potential for a peptide of the West African/USA clade to contain a unique 9 aa sequence. This new epitope, not present in the Congo Basin clade and derived from the C7L-orthologue predicted amino-terminal fusion, is expected to facilitate efficient immune recognition and clearance of West African/USA MPXV-infected host cells.
VAC-WR IL1 receptor inhibits IL1-induced murine T- and B-lymphocyte proliferation in vitro (Spriggs et al., 1992
), which is anticipated to diminish host immune recognition and viral clearance. The West African/USA isolates lack a predicted IL1
receptor orthologue. In vivo models of systemic disease suggest functional IL1
receptor decreases the pathogenic febrile (cytokine-induced) response (Alcami & Smith, 1996
); febrile response has not been systematically evaluated in either human disease population and may be of interest for further study.
The myxoma virus protein M-T4 is important for host range and lymphocyte infection (Barry et al., 1997). Absence of M-T4 attenuates disease in both intranasal and intradermal rabbit models and enhances the inflammatory response (Barry et al., 1997
). Therefore, absence of this protein in West African/USA strains may also contribute to our observed decrease in viraemia and ultimately effect a milder disease presentation.
Combined, these observations suggest that the effect of changes among a relatively small number of ORFs could account for the differences in viral clearance and pathogenesis of human infections with West African/USA and Congo Basin MPXVs. If transmission correlates with duration and magnitude of viral presence and shedding, the observations reported here may explain why no human-to-human transmissions have been observed in the West African/USA case series.
It is possible that the clade-specific orthologues may also influence disease in reservoir or susceptible species. Host species-specific effects of the monkeypox virally encoded IL1 receptor are suggested by studies demonstrating the cowpox virus orthologue has different affinities for mouse and human IL1 (Alcami & Smith, 1996
). Similar adaptations of the Congo Basin MPXV orthologue could affect disease presentation in different host species, and the absence of a West African/USA orthologue could differentially affect presentation of the virus in different hosts, influencing viral host range.
On the basis of comparisons of the entire genome of five different MPXV isolates, we have verified and elaborated on the preliminary observation of the existence of two well-developed, geographically distinct clades of MPXV, suggesting significant divergence in their evolutionary histories. Evolutionary divergence may also correlate with subtle differences in the natural histories of reservoir host species or subspecies as has been observed with other viruses (Plyusnin & Morzunov, 2001; Gonzalez, 1996
), and this possibility will be the subject of future studies.
In an era of heightened awareness to the potential of nefarious bioterrorist events, the emergence of MPXV in the USA serves as a timely reminder that orthopoxviruses continue to naturally exploit novel ecological and geographical niches. A better appreciation for the steps involved in the evolution of zoonotic orthopoxviruses, including MPXV, may well be relevant to understanding the events that led to the evolution of Variola virus, a pathogen with severe human pathogenicity, efficient transmission and highly specialized (human) host range.
Further efforts to understand the contribution of these distinct MPXV clades to human disease will continue to influence and contribute to informed decision-making relevant to interruption of monkeypox transmission to humans, possible future outbreak responses, diagnostic test deployment and even possible outbreak-related decisions regarding vaccination and therapeutics.
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ACKNOWLEDGEMENTS |
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Received 27 May 2005;
accepted 18 July 2005.
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