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Journal of Virology, May 2009, p. 4631-4641, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.02085-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cardioviruses Are Genetically Diverse and Cause Common Enteric Infections in South Asian Children

Olga Blinkova,^1,2 Amit Kapoor,^1,2 Joseph Victoria,^1,2 Morris Jones,³ Nathan Wolfe,^4,5 Asif Naeem,⁶ Shahzad Shaukat,⁶ Salmaan Sharif,⁶ Muhammad Masroor Alam,⁶ Mehar Angez,⁶ Sohail Zaidi,⁶ and Eric L. Delwart^1,2*

Blood Systems Research Institute, San Francisco, California,¹ Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California,² Clinical Investigation Facility, David Grant USAF Medical Center, Travis, California 94535,³ Global Viral Forecasting Initiative, San Francisco, California 94105,⁴ Program in Human Biology, Stanford University, Stanford, California,⁵ National Institute of Health, Department of Virology, Islamabad, Pakistan⁶

Received 3 October 2008/ Accepted 27 January 2009

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Cardioviruses cause enteric infections in mice and rats which when disseminated have been associated with myocarditis, type 1 diabetes, encephalitis, and multiple sclerosis-like symptoms. Cardioviruses have also been detected at lower frequencies in other mammals. The Cardiovirus genus within the Picornaviridae family is currently made up of two viral species, Theilovirus and Encephalomyocarditis virus. Until recently, only a single strain of cardioviruses (Vilyuisk virus within the Theilovirus species) associated with a geographically restricted and prevalent encephalitis-like condition had been reported to occur in humans. A second theilovirus-related cardiovirus (Saffold virus [SAFV]) was reported in 2007 and subsequently found in respiratory secretions from children with respiratory problems and in stools of both healthy and diarrheic children. Using viral metagenomics, we identified RNA fragments related to SAFV in the stools of Pakistani and Afghani children with nonpolio acute flaccid paralysis (AFP). We sequenced three near-full-length genomes, showing the presence of divergent strains of SAFV and preliminary evidence of a distant recombination event between the ancestors of the Theiler-like viruses of rats and those of human SAFV. Further VP1 sequencing showed the presence of five new SAFV genotypes, doubling the reported genetic diversity of human and animal theiloviruses combined. Both AFP patients and healthy children in Pakistan were found to be excreting SAFV at high frequencies of 9 and 12%, respectively. Further studies are needed to examine the roles of these highly common and diverse SAFV genotypes in nonpolio AFP and other human diseases.

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Some of the recent advances in characterizing new human viruses have come from shotgun sequencing of randomly PCR amplified viral nucleic acids (i.e., viral metagenomics) (2, 3, 11-13, 20-22, 48, 49) and the application of viral oligonucleotide microarrays (6, 25, 47). While most of the newly identified human viruses were initially found in patients with clinical symptoms, not all have been causally linked or even associated with particular diseases or symptoms. Viral metagenomics is therefore resulting in a growing list of "orphan" human viruses whose pathogenicity, if any, remains unknown. One component of testing linkage between a new viral species and human disease is an appreciation of the full range of genetic diversity of the viral species since it is well established that distinct viral genotypes or even minor genetic variations can result in large changes in viral pathogenicity. Specific variants belonging to the two currently recognized species of the Cardiovirus genus, Theilovirus and Encephalomyocarditis virus (EMCV), have been shown to induce other demyelinating diseases (an animal model of multiple sclerosis), encephalitis, myocarditis, or type 1 diabetes in rodents (4, 10, 33, 50, 55). Cardioviruses typically replicate asymptomatically in the gastrointestinal tract following fecal-oral transmission, and some strains can replicate in the central nervous system following intracerebral injection, resulting in neurological symptoms. Prior to 2007, the sole known human virus belonging to the Cardiovirus genus was the Vilyuisk virus, isolated from the cerebrospinal fluid of an adult patient with a neurodegenerative disease from an area with a high prevalence of encephalomyelitis (5, 31, 32). Because the Vilyuisk virus was isolated following extensive in vivo mouse and cell culture passages and is phylogenetically closely related to the rodent theiloviruses, the possibility remains that it is a contaminating animal cardiovirus (40).

In 2007, Jones et al. reported the sequence of a divergent cardiovirus in the stool sample of a febrile infant, which when cultured in 1981 had caused unidentified cytopathic effects, and named it Saffold virus, or SAFV (GenBank accession no. NC_009448) (21). In 2008, a virus closely related to SAFV (GenBank accession no. AM922293) was independently characterized, using a similar metagenomics approach, from another cell line supernatant showing cytopathic effects following inoculation with the respiratory secretions of a Canadian child with respiratory symptoms (1). SAFV variants were also detected by reverse transcription-PCR (RT-PCR) in two other similarly affected children (1). Six more infections with SAFV were then detected by PCR in stool samples from children with gastroenteritis in Germany and Brazil (8). More recently, a human cardiovirus closely related to the Canadian SAFV variant was identified in the respiratory secretions of a child with influenza-like symptoms, using a ViroChip microarray, and then by PCR in six stool samples from U.S. children both with and without gastroenteritis. This virus was named human TMEV-like cardiovirus (HTCV) (GenBank accession no. NC_010810) (6).

In this study, we detected multiple highly divergent variants of the new Saffold human cardiovirus species at high frequencies in stool samples from South Asian children with nonpolio acute flaccid paralysis (AFP), as well as healthy children. We characterized three nearly full-length genomes and the VP1 regions of another seven SAFV strains. Phylogenetic analyses and genetic distance measurements confirmed that SAFVs qualify as divergent members of the Theilovirus species, most closely related to rat theiloviruses (Theiler-like virus [TLV] of rats and rat theilovirus 1). Genetic distance-based classification of VP1 sequences revealed the presence of seven SAFV genotypes, bringing the known total to eight SAFV genotypes, including five new ones reported here. Fully characterizing the range of genetic diversity of these new human cardioviruses and their incidence in different demographic and patient groups will assist future studies of the associations between particular viral genotypes and human diseases.

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Biological samples. Stool samples from South Asian children <15 years old who either had nonpolio AFP (n = 57; mean age, 54.6 months) or were healthy household contacts of AFP patients (n = 9; mean age, 27 months) or unrelated healthy children (n = 41; mean age, 39.8 months) were collected in Pakistan and Afghanistan. All studies were reviewed and approved by the University of California, San Francisco, Committee on Human Research. Stool samples were collected between November 2006 and April 2008.

Viral metagenomics. The method used for viral metagenomics has been described previously (23) and was used with the following modifications. Stool was suspended in Hanks buffered salt solution, subjected to a vigorous vortex, and then centrifuged at 12,000 rpm for 2 min in a microfuge. Supernatants were then filtered through an Ultrafree-MC HV 0.45-µm sterile filter (catalog no. UFC30HV0S; Millipore), and the flowthrough fraction was ultracentrifuged at 30,000 rpm for 3 h at 8°C in a Beckman Coulter Optima LE-80 ultracentrifuge to pellet viral particles. Pellets were resuspended in 100 µl of Hanks buffered salt solution, which was then treated with 14 U of Turbo DNase (Ambion) and 2 µl of 10 mg/ml RNase A at 37°C for 2 h to digest non-particle-protected nucleic acids. A total of 140 µl of capsid-protected viral nucleic acids (both RNA and DNA) were extracted with a QIAamp viral RNA minikit (Qiagen) and eluted into 50 µl of water with 20 U of recombinant RNase inhibitor (Roche).

Random amplification, subcloning, and sequencing. Ten microliters of purified viral nucleic acids was mixed with 50 pmol (1 µl) of random primer RA01 (GTTTCCCAGTCACGATANNNNNNNNNN), denatured at 85°C for 2 min, and chilled on ice. A reaction mixture of 9 µl containing 4 µl of 5x SuperScript III buffer (Invitrogen), 2 µl of 100 mM dithiothreitol, 1.25 µl of 10 mM deoxynucleoside triphosphate (dNTP) solution, 0.75 µl of diethyl pyrocarbonate (DEPC)-treated water, and 1 µl of SuperScript III reverse transcriptase (200 U) was added. The reaction mixture was incubated at 25°C for 10 min and then at 42°C for 60 min and 70°C for 5 min and chilled on ice for 2 min. For the second-strand cDNA synthesis, 0.5 µl of 100 µM RA01 primer was added. The reaction mixture was incubated at 95°C for 2 min and chilled on ice for 2 min. A volume of 2.5 U of 3'-to-5' exo Klenow DNA polymerase (New England Biolabs) was added to extend RA01, and the mixture was incubated at 37°C for 60 min and then subjected to enzyme inactivation at 75°C for 10 min. Five microliters of the reaction product was then used as a template for PCR with a mixture of 5 µl of 10x AmpliTaq Gold DNA polymerase buffer (Applied Biosystems), 8 µl of 25 mM MgCl₂, 1.25 µl of 10 mM dNTP, 5 µl of 10 µM specific primer RA02 (GTTTCCCAGTCACGATA), and 2.5 U of AmpliTaq Gold DNA polymerase (low DNA; Applied Biosystems). The conditions for PCR were denaturation at 95°C for 5 min, 5 cycles (95°C for 1 min, 55°C for 1 min, and 72°C for 1 min 30 s), 35 cycles (95°C for 30 s, 55°C for 30 s, and 72°C for 1 min 30 s, with the extension time increasing by 2 s per cycle), and final extension at 72°C for 10 min. Random PCR products were separated on 1.5% agarose gel, and DNA smears ranging in size from 500 to 1,500 bp were extracted using the QIAquik gel extraction kit (Qiagen). The purified PCR product was ligated to the vector pGEM-T Easy (Promega Inc.) and introduced into chemically competent Escherichia coli cells. Bacteria were plated onto Luria-Bertani agar plates containing ampicillin, X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), and IPTG (isopropyl-β-D-thiogalactopyranoside). Ninety-six white colony inserts were sequenced using the T-7 forward primer. Sequences were assembled in Sequencher 4.1 (Gene Codes), and sequence similarity searching was performed using tBLASTx (http://www.ncbi.nlm.nih.gov/blast/).

Direct PCR screening for human cardioviruses. Stool supernatants were tested for Saffold-like viruses using RT-nested PCR directed toward the conserved 2C (helicase) region. cDNA was first generated from viral nucleic acid extracted directly from stool supernatants by using the Qiagen viral RNA minikit. Fifty picomoles of random octamer oligonucleotide was added to 10 µl of each nucleic acid, and the sample was denatured at 65°C for 5 min and then chilled on ice. A reaction mixture of 9 µl containing 4 µl of 5x SuperScript buffer (Invitrogen), 1 µl of 100 mM dithiothreitol, 1.25 µl of 10 mM dNTP, and 200 U of SuperScript III reverse transcriptase (Invitrogen) was added, and the resulting mixture was incubated at 25°C for 10 min, 42°C for 60 min, and 70°C for 5 min and then chilled. PCR primers HelF1 (5'-GYCAAATCATCGCHCAAGCAGT-3') and HelR1 (5'-TGATTCTYCTATCAACAGCTGG-3') were used for the first round of nested PCR; HelF2 (5'-CAATCRGTTTAYTCTCTYCCACC-3') and HelR2 (5'-TATCAACAGCTGGRTAGTGWGC-3') were used for the second round of nested PCR. For the first round of nested PCR, 3 µl of each specimen cDNA was mixed with 5 µl of 10x TermoPol reaction buffer (New England Biolabs), 5 µl of 10 µM dNTP, 2.5 µl of each primer (HelF1 and HelR1) at 10 µM, 1 µl of Taq DNA polymerase (New England Biolabs), and 31 µl of DEPC-treated water. The reaction was performed using denaturation at 95°C for 3 min, 5 cycles (95°C for 1 min, 55°C for 1 min, and 72°C for 1 min 30 s), 35 cycles (95°C for 30 s, 52°C for 30 s, and 72°C for 45 s), and 72°C for 10 min. For the second round of nested PCR, identical cycling conditions were used for reaction mixtures containing 1.5 µl of the PCR product from the first round mixed with 5 µl of 10x TermoPol reaction buffer (New England Biolabs), 5 µl of 10 µM dNTP, 2.5 µl of each primer (HelF2 and HelR2) at 10 µM, 1 µl of Taq DNA polymerase (New England Biolabs), and 32.5 µl of DEPC-treated water. Products were visualized following electrophoresis on 1.5% agarose gel. PCR products showing positive bands corresponding to the 265-bp amplicon were purified using a PCR purification kit (Qiagen) and directly sequenced.

PCR analysis of the VP1 region. Two sets of degenerate primers were designed to amplify the VP1 region using RT-nested PCR: VP1F1 (5'ACWCTTGGTTTCDGGHGG3') and VP1R1 (5'TCGCCCATRCASACRAGRA3') for the first PCR round and VP1F2 (5'-GACTTYACYCTBAGAATGCC-3') and VP1R2 (5'-ACTGTTCTAYCRTGAACTTTGTA-3') for the second PCR round. The first round of PCR was performed with a 50-µl reaction mixture containing 3 µl of cDNA, 5 µl of 10x PCR buffer (Takara Bio Inc.), 5 µl of 25 mM MgCl₂, 5 µl of a mixture of dNTPs (2.5 mM each), 2.5 µl of 10 mM VP1F1, 2.5 µl of 10 mM VP1R1, and 3 U of Ex Taq polymerase (Takara Bio Inc.). PCR conditions were 95°C for 3 min, 4 cycles (95°C for 1 min, 55°C for 1 min, and 68°C for 1 min 30 s), 35 cycles (95°C for 30 s, 53°C for 30 s, and 68°C for 1 min 30 s), and 68°C for 10 min. The same PCR conditions were used for the second round of PCR. A 1.5-µl sample of the first-round PCR product was used to initiate the second PCR round.

Phylogenetic analyses. Sequences from the indicated cardioviruses with the following accession numbers were retrieved from GenBank and aligned by the CLUSTALW program with default settings (16): SAFV prototype, NC_009488; SAFV Canadian isolate Can112051-06, AM922293; SAFV strain BR/118/2006 (SAFV-BR/118/2006), EU681177; SAFV-D/VI2273/2004, EU681178; SAFV-D/VI2223/2004, EU681179; SAFV-D/VI2229/2004, EU681176; human Theiler murine encephalomyelitis virus (TMEV)-like cardiovirus (HTCV)-UC1, EU376394; HTCV-UC2, EU604745; HTCV-UC3, EU604750; HTCV-UC4, EU604747; HTCV-UC5, EU60746; HTCV-UC6, EU604748; HTCV-UC7, EU604749; TLV-NGS910, AB090161; rat theilovirus 1, EU815052; EMCV, X87335; EMCV-EMC-B (nondiabetogenic), M22457; EMCV-EMC-D (diabetogenic), M22458; porcine EMCV, DQ517424; TMEV-GDVII, X56019; TMEV-DA, M20301; TMEV-BeAn 8386, M16020; Mengo virus isolate M, L22089; and Vilyuisk human encephalomyelitis virus (VHEV, or Vilyuisk virus), M94868, M80888, and EU723237. A neighbor-joining tree based on nucleotide distances was constructed using the MEGA4 software (28) by a bootstrapping approach (with 1,000 replicates).

Recombination analyses. Similarity plots showing the relationships among the aligned nucleotide sequences were generated using SimPlot, version 3.5.1 (http://sray.med.som.jhmi.edu/SCRoftware) (34). The level of similarity in each window of 600 nucleotides was calculated by the Kimura two-parameter method, which was developed to estimate evolutionary distances in terms of the number of nucleotide substitutions (24). We increased the size of the sliding window compared to the default settings in SimPlot (200 to 600 nucleotides) because large windows give a clearer signal and can detect significance in long sections of weak similarity, while smaller windows tend to produce more noise (41). The overall transition/transversion ratio was calculated using MEGA 4 software (46). To assess potential recombinational relationships, aligned sequences were subsequently analyzed by using the bootscanning method implemented in SimPlot.

Nucleotide sequence accession numbers. Sequences were deposited in GenBank. The following accession numbers were assigned to near-full-length genome sequences of the indicated viruses: SAFV-Pak5003, FJ463615; SAFV-Pak5152, FJ463616; and SAFV-Pak6572, FJ463617. The following accession numbers were assigned to VP1 region sequences of the indicated viruses: SAFV-Pak12_VP1, FJ463600; SAFV-Pak971_VP1, FJ463601; SAFV-Afg1449_VP1, FJ463602; SAFV-Pak962_VP1, FJ463603; SAFV-Pak1141_VP1, FJ463604; SAFV-Pak2578_VP1, FJ463605; and SAFV-Pak5842_VP1, FJ463606. The following accession numbers were assigned to 2C region sequences of the indicated viruses: SAFV-Pak971_2C, FJ463607; SAFV-Pak962_2C, FJ463608; SAFV-Pak9_2C, FJ463609; SAFV-Pak12_2C, FJ463610; SAFV-Pak5842_2C, FJ463611; SAFV-Pak2578_2C, FJ463612; SAFV-Afg1449_2C, FJ463613; and SAFV-Pak1141_2C, FJ463614.

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Identification and sequencing of three new human SAFV genomes. Sequence-independent RT-PCR amplification was first performed on nucleic acid extracted from partially purified viral particles in stool samples from children with nonpolio AFP (see Materials and Methods). Random PCR amplification products were then subcloned and sequenced. Three of 57 stool samples from children with AFP showed the presence of multiple nucleic acid fragments whose in silico translation products showed 67 to 80% identity to SAFV-encoded proteins (corresponding to GenBank accession no. NC_009448) (21). In order to sequence the genomes of these three viruses, the gaps between the SAFV-like fragments were filled by RT-PCR using primers based on the initial homologous fragments and directly sequenced. To acquire the 5' and 3' extremities, PCR primers based on an alignment of the two human cardiovirus genome sequences available at the time (GenBank accession no. NC_009448 and AM922293) were used (1, 21). Three nearly full-length genomes were sequenced. We tentatively named these viruses SAFV-Pak5003, SAFV-Pak5152, and SAFV-Pak6572. The organizations of the three genomes were typical of the Cardiovirus genus, including the leader (L) protein gene and expected proteolytic cleavage sites resulting in the typical L, VP4, VP3, VP2, VP1, 2A, 2B, 2C, 3A, 3B, 3C, and 3D cleavage product order.

Phylogenetic analyses of human cardioviruses. Phylogenetic analyses of various viral regions (the full polyprotein, P1 capsid proteins, and P2 and P3 replicative function proteins) confirmed that all three viruses clustered with the original SAFV and the other two human SAFVs with fully sequenced genomes (strains HTCV-UC1 and SAFV-112051-06) (Fig. 1). The three Pakistani strains (Pak5003, Pak5152, and Pak6572) also clustered together. To determine whether SAFV was sufficiently divergent from existing theiloviruses to qualify as a distinct Cardiovirus species, we tested the criteria of the International Committee on Taxonomy of Viruses (<70% amino acid identity in P1, <70% amino acid identity in the 2C-3CD region, and a different natural host range). P1 alignments showed all six available SAFV sequences to be >70% identical to that of mouse theilovirus TMEV-DA (73 to 77%) and slightly closer to that of rat TLV (74 to 79%). Among SAFVs, the level of P1 identity ranged from 83 to 91%. Protein distance measurements based on 2C-3CD alignment also showed all six available SAFV sequences to be >70% identical to that of mouse theilovirus TMEV-DA (79 to 80%) and slightly closer to that of TLV (87 to 88%). Among SAFVs, the level of 2C-3CD identity ranged from 97 to 98%. Levels of P1 and 2C-3CD identity to the corresponding regions in EMCV were <70% (64 to 68% and 62 to 63%, respectively). Therefore, according to the genetic distance-based International Committee on Taxonomy of Viruses definition, SAFVs do not qualify as members of a new species in the Cardiovirus genus but rather represent new genotypes of theiloviruses. Human SAFV does have a distinct host range relative to those of other theiloviruses (mouse, rat, and possibly human for Vilyuisk virus) and EMCVs (mouse).

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FIG. 1. Phylogenetic analysis of cardioviruses based on full-length genomes (A) and on amino acid sequences of P1 (B), P2 (C), and P3 (D) regions of polyproteins. Bootstrap values from 1,000 replicates are shown for the nodes.

Recombination among theiloviruses. Based on analysis of the P1 region, TLV and rat theilovirus 1 clustered with the mouse TMEVs (Fig. 1B), while based on analyses of the P2 and P3 regions (Fig. 1C and D), the rat viruses clustered with the human SAFVs. The phylogenetic relationship of SAFV to the rat and mouse theiloviruses therefore varied depending on the genomic region analyzed. SimPlot recombination analysis was performed to compare the sequence of the rat TLV against the available full genome sequences of the other human and animal cardioviruses (Fig. 2A). While TLV of rats and rat theilovirus 1 were most similar to murine TMEVs in the P1 region, they were more closely related to SAFVs in the P2 and P3 regions (Fig. 2A), as also seen by phylogenetic analysis (Fig. 1). SimPlot analysis therefore provided preliminary evidence of a possible ancient recombination event in the 2A protein at the beginning of the P2 region in the progenitors of rat theiloviruses and those of the SAFVs (Fig. 2A). Bootscan analysis supported this conclusion when the sequence of TLV of rats was used as a query against those of other cardioviruses (Fig. 2B). To further test the possibility of recombination between the ancestors of the rat TLVs and SAFVs, the grouping scan analysis test for recombination (29, 43) was also used. This method failed to find evidence for recombination between TLV of rats and SAFV above the threshold grouping score of 0.5 (data not shown). The conclusion that ancient recombination occurred between the ancestors of TLV of rats and SAFV must therefore be regarded as preliminary.

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FIG. 2. Recombination analyses. (A) Sliding-window SimPlot graph generated by using the sequence of the TLV of rats as a query against all other cardiovirus and EMCV sequences. (B) Bootscan analysis of the sequence of the TLV of rats in comparison to those of SAFV, TMEV-GDVII, and EMCV. (C) Sliding-window SimPlot graph generated by using the SAFV-Pak5152 sequence as a query against all other available SAFV sequences. (D) Bootscan analysis of the sequence of SAFV-Pak5152 in comparison to other available SAFV sequences. The virus closest to TLV of rats is rat theilovirus 1. The nucleotide position is given on the x axis, and the percent nucleotide sequence similarity is given on the y axis. The positions relative to viral genes are shown by genome diagrams above the plots and scans.

The SAFV genomes were also examined for recombination among themselves. Recombination between the genotype 5 SAFV-Pak5152 and the genotype 6 SAFV-Pak6572 strains, occurring between the structural and nonstructural genes, was seen (Fig. 2B). The Pak5152 and Pak5003 strains were very similar in their P1 regions, while in the P3 region, Pak5152 was most closely related to Pak6572, which carried a very divergent P1 (Fig. 2C). Bootscan analysis supported the conclusion that recombination between SAFV-Pak5152 and SAFV-Pak6572 occurred (Fig. 2D). Evidence of recombination among SAFV genotypes was therefore also observed.

Frequency of SAFV infections in the nonpolio AFP patients, their contacts, and healthy children from South Asia. Feces from 107 South Asian children were analyzed for the presence of SAFV by using an RT-nested PCR assay of a conserved part of the 2C (helicase)-encoding region. Eleven (10%) of 107 samples (including the three samples in which SAFV sequences were originally detected by shotgun sequencing) were positive (Table 1). The helicase PCR products were confirmed to be from SAFV by direct amplicon sequencing. Five SAFV-positive children had nonpolio AFP (9% of nonpolio AFP patients were infected), one SAFV-positive child was healthy but had been in recent close contact with a nonpolio AFP patient (11% of contacts were infected), and the last five SAFV-positive children were asymptomatic (12% of healthy controls were infected). The ages of the SAFV-positive children varied from 7 months to 13 years (average, 4.1 years) (Table 2). The symptoms and demographics of SAFV-positive and SAFV-negative AFP children were compared and showed no significant difference in age, the province of residence, the month of sample collection, the presence or absence of fever, the character of the paralysis, the number of prior polio vaccinations, or the duration of the paralysis (more or less than 2 weeks).

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TABLE 1. Prevalence of SAFV-positive specimens from AFP patients, healthy AFP patient contacts, and healthy control children

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TABLE 2. Clinical and demographic characteristics of SAFV-positive childrena

VP1 sequencing and classification of new genotypes of human cardioviruses. The VP1 protein, a primary determinant of viral tropism and antibody neutralization sensitivity and the most diverse protein of picornaviruses, was targeted for more extensive sequencing. Since genotype classification criteria for SAFVs have not been defined, we used those defined by Oberste et al. for human enteroviruses, in which VP1 variants showing >88% amino acid similarity are highly likely to belong to the same antibody neutralization serotype and those with a lower level of VP1 similarity are not cross-neutralized (38). Complete VP1 sequences from 10 of the 11 SAFVs detected using the helicase PCR primers (i.e., all except SAFV-Pak9 yielded amplification products with the VP1 primers) were acquired (see Materials and Methods). Pairwise amino acid identities for all available SAFV VP1 sequences were then calculated. VP1 protein distance measurements revealed a total of eight VP1 genotypes (Fig. 3). VP1 sequences were also used for phylogenetic analysis and showed the same clustering by genotypes (Fig. 4). No Pakistani VP1 sequences clustered with that of the original U.S. SAFV genotype 1 variant isolated in 1981. One virus, SAFV-Pak971, clustered with the largest group of eight closely related variants previously identified in Canada, the United States, Germany, and Brazil (genotype 2). Another Pakistani variant, SAFV-Pak2578, clustered with four previously reported variants from the United States and Germany (genotype 3). Three new VP1 variants clustered together into a new genotype group (genotype 4), while two others formed another genotype group (genotype 5). The other three VP1 variants were each distinct enough to qualify as prototypes of new genotypes (genotypes 6 to 8) (Fig. 3 and 4). SAFV VP1 sequencing also showed that the diversity of the human SAFV cardioviruses was greater than that seen in the rest of the theiloviruses, with an average and upper range of SAFV VP1 divergence of 25 and 38%, compared to 22 and 30% for the animal theiloviruses (including mouse TMEVs, rat theilovirus 1, TLV, and Vilyuisk viruses). The much more genetically homogeneous EMCV VP1 regions varied by 3 and 4%.

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FIG. 3. Pairwise amino acid identity levels (in percentages) of available human cardiovirus VP1 sequences.

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FIG. 4. Phylogenetic analysis of the VP1 proteins of human and animal theiloviruses and EMCVs.

Characteristics of L protein domains and EF and CD loop structures of capsid proteins. The L peptide and EF and CD loops of picornaviral capsids play important roles in the persistence and pathogenicity of cardioviruses (26, 53). Protein L inhibits interferon production in infected cells. All sequenced genomes of SAFVs, including those described here, encode the conserved putative zinc finger and acidic domains of other L proteins, but the SAFV proteins have shorter Ser/Thr phosphorylation domains than those of the theiloviruses, missing five of six conserved Ser/Thr residues (Fig. 5A). EMCVs are missing all six Ser/Thr residues in that domain and may instead be phosphorylated at the acidic domain Y and T residues (9, 56), which are largely missing in animal theiloviruses but conserved in all SAFVs (Fig. 5A). Like theiloviruses, the SAFVs retain a 7-amino-acid (aa) stretch with a conserved MEWTD/NLP sequence (19) deleted in the EMCV Ser/Thr-rich domain (Fig. 5A). The SAFV L protein therefore shows properties of both theiloviruses (a conserved MEWTD/NLP sequence) and EMCVs (deleted Ser/Thr residues and potentially phosphorylated Y and T residues in the acidic domain).

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FIG. 5. Alignments of SAFV proteins with homologues from other theiloviruses and EMCVs. (A) Alignments of the L proteins showing different domains with conserved amino acid motifs (highlighted). The Y and T residues in the acidic domain thought to be phosphorylated in EMCVs are labeled with a star. (B) Alignments of potential L* proteins encoded by SAFV with those of other theiloviruses. SAFV proteins are depicted as being initiated by an ACG (Thr) codon and terminated by premature stop codons. The carboxy-terminus 72 aa of the other theilovirus L* proteins are not shown. (C) Alignments of the EF loops (in VP2 proteins) of theiloviruses and EMCVs. (D) Alignments of the CD loops (in VP1 proteins) of theiloviruses and EMCVs. Columns highlighted in black show absolute amino acid conservation, while those highlighted in gray show amino acids with highly similar properties. Terminal residue positions of the aligned L protein and EF and CD loop structures according to those in a reference strain (TMEV-DA) are used.

The L* protein is a unique picornavirus protein that appears to prevent apoptosis and facilitate macrophage infections and is translated from an alternative reading frame (14, 27, 45, 51). A potential ACG-initiated L* protein (35 aa long) that was much shorter than the similarly positioned L* found in theiloviruses (156 aa) was found in all human cardioviruses (Fig. 5B). The original SAFV strain had a considerably longer L* protein (57 aa) than the other SAFVs (34 aa) (Fig. 5B). Whether the L* proteins of SAFVs are expressed and retain any activity remains to be determined, although it should be noted that the L* in TMEV-GDVII is indeed translated from an ACG threonine start codon also seen in the SAFVs (27, 52).

EF and CD loop structures, located in the VP2 and VP1 proteins, respectively, are associated with tropism and virulence (17-19, 36, 54). Loop sequences in both regions were highly diverse among SAFVs, with levels of amino acid identity ranging upward from 55% in EF loops and 45% in CD loops (Fig. 5C and D). Pairwise comparisons of amino acids of SAFV versus animal cardioviruses revealed levels of identity ranging from 30 to 38% in EF loops and from 25 to 50% in CD loops. The SAFV loops in both the EF and CD regions were also distinct in length from those of both theiloviruses and EMCVs (except for EF loop 1 of theiloviruses and CD loop 2 of EMCVs). Within the VP1 region-defined SAFV genotypes, the pairwise identity of CD and EF loops ranged from 98 to 100%.

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We report here a high degree of viral genetic diversity and the frequent detection of SAFV in the feces of South Asian children. The range of genetic diversity for SAFV is now greater than that reported for either the rest of the theiloviruses combined or the EMCVs. Multiple new genotypes, encoding highly divergent VP1 proteins, were identified, bringing the number of SAFV genotypes from three to eight. A recent phylogenetic analysis of theiloviruses (30) reclassified them according to five genotypes, the TMEV, Vilyuisk virus (or VHEV), rat theilovirus 1, SAFV1 (21), and SAFV2 (1) genotypes. Using the same VP1 genetic distance-based criteria, we show the presence of a further 6 theilovirus genotypes, for a current total of 11 theilovirus genotypes (1 in rat, and 1 to 2 in mouse, and 8 to 9 in human depending on the natural tropism of VHEV). Preliminary evidence of recombination between the rat theiloviruses and SAFV was detected using SimPlot and bootscan analyses, indicating that the 3' half of the ancestor of the worldwide-distributed SAFVs (now reported in the United States, Canada, Germany, Brazil, Pakistan, and Afghanistan) may have once replicated in rats. The recombination event appeared to have taken place near a region separating structural from nonstructural genes and generally favored for recombination in other genera of picornaviruses (15, 42, 44). The possibility of an ancient recombination event between the ancestors of rat TLVs and SAFV must be considered preliminary since a more stringent test for recombination described by Kurbanov et al. and Simmonds and Midgley (29, 43) failed to detect this recombination. It is expected that an ancient recombination event will be more difficult to document by all methods than a more recent one, possibly accounting for the failure of the more stringent grouping scan test to detect it. Recombination between SAFV genotypes, again with a breakpoint between structural and nonstructural regions, was also observed. Recombination between the structural and nonstructural regions of different serotypes of the same species of enterovirus is an extensively documented phenomenon (7, 35, 42).

To date, SAFVs have been found in the feces of 1 febrile child (21), the respiratory secretions of 4 children with flu-like symptoms (1, 6), and the stools of 10 children with gastroenteritis (2 of which provided samples months after symptom resolution) (6, 8) and 2 healthy children (6). SAFV was not detected in further screenings of 428 samples of respiratory secretions from people with flu-like symptoms or 400 cerebrospinal fluid samples from U.S. residents with encephalitis, meningitis, or multiple sclerosis (6). In the two largest surveys (6, 8), SAFV was detected in feces from children with gastroenteritis in German day cares and in Salvador de Bahia, Brazil, at frequencies of 7.8% (4 of 51 samples) and 1.1% (2 of 188 samples), respectively, and in 1.2% (6 of 498 samples) of feces from U.S. children, with only two of the six children with positive samples having current gastroenteritis. We report here on the prevalence of SAFV detection in the stools of children with AFP (6 of 57 samples, or 9%) and without any overt neurological symptoms (5 of 41 samples, or 12%). Based on these early epidemiological surveys, it is therefore premature to associate SAFV with either AFP or gastroenteritis. The highest frequencies of SAFV shedding have been found in Pakistani children (9 to 12%) and German children in child care centers (7.8%) (8), likely reflecting high levels of exposure or susceptibility at a young age. To date, of the 28 cases of reported SAFV shedding, all but 2 (in 8- and 13-year-old children) have been detected in children under 6 years of age (1, 6, 8, 21).

Picornavirus pathogenicity is highly variable, depending on the condition of the host and viral genetics. Different serotypes or genotypes within the same enterovirus species have been associated with very different symptoms (39). Genetic variants belonging to the same serotype can also result in serious disease outbreaks, while closely related variants appear much less virulent (37). The outcome of infection in infants is also likely to be strongly influenced by maternal antibodies, and in older children by previous exposure to related viral strains. The overall health and immunocompetency of children, as well as the presence of coinfections, may also influence outcome (39). Because only a small fraction of these very common picornavirus infections cause any symptoms, it will be difficult to link infection to any particular disease without the inclusion of large, demographically matched, healthy control groups. It is also conceivable that SAFV may trigger autoimmune responses so that the viruses will no longer be detected by the time overt disease develops, requiring the use of serology to link disease with past infections.

The high degree of genetic diversity of SAFV will also complicate studies of association with disease. Larger numbers of cases and controls will be needed to analyse sufficient numbers of cases involving any particular genotypes. We were unable here to associate SAFV infection in general with nonpolio AFP. Larger genotype-specific studies of viral prevalence and of seroprevalence will be required to determine the likely diverse and complex pathogenic potential of the highly diverse SAFVs. Besides respiratory symptoms and gastroenteritis, other symptoms associated with cardiovirus infection in mice, such as encephalitis, myocarditis, type 1 diabetes, and demyelinating diseases, make up some of the candidate diseases for future studies of associations with the numerous genotypes of SAFV.

 
We thank Michael Busch and Blood Systems for continued support and Peter Simmonds for the grouping scan analysis.

This work was supported by National Heart, Lung, and Blood Institute grant R01HL083254 (to E.L.D.).

 
* Corresponding author. Mailing address: BSRI, 270 Masonic Ave., San Francisco, CA 94118. Phone: (415) 923-5763. Fax: (415) 567-5899. E-mail: delwarte{at}medicine.ucsf.edu

Published ahead of print on 4 February 2009.

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1
1. Abed, Y., and G. Boivin. 2008. New Saffold cardioviruses in 3 children, Canada. Emerg. Infect. Dis. 14:834-836.[Medline]
2
2. Allander, T., K. Andreasson, S. Gupta, A. Bjerkner, G. Bogdanovic, M. A. Persson, T. Dalianis, T. Ramqvist, and B. Andersson. 2007. Identification of a third human polyomavirus. J. Virol. 81:4130-4136.[Abstract/Free Full Text]
3
3. Allander, T., M. T. Tammi, M. Eriksson, A. Bjerkner, A. Tiveljung-Lindell, and B. Andersson. 2005. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc. Natl. Acad. Sci. USA 102:12891-12896.[Abstract/Free Full Text]
4
4. Brahic, M., J. F. Bureau, and T. Michiels. 2005. The genetics of the persistent infection and demyelinating disease caused by Theiler's virus. Annu. Rev. Microbiol. 59:279-298.[CrossRef][Medline]
5
5. Casals, J. 1963. Immunological characterization of Vilyuisk human encephalomyelitis virus. Nature 200:339-341.[CrossRef][Medline]
6
6. Chiu, C. Y., A. L. Greninger, K. Kanada, T. Kwok, K. F. Fischer, C. Runckel, J. K. Louie, C. A. Glaser, S. Yagi, D. P. Schnurr, T. D. Haggerty, J. Parsonnet, D. Ganem, and J. L. DeRisi. 2008. Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections. Proc. Natl. Acad. Sci. USA 105:14124-14129.[Abstract/Free Full Text]
7
7. Domingo, E., V. Martin, C. Perales, and C. Escarmis. 2008. Coxsackieviruses and quasispecies theory: evolution of enteroviruses. Curr. Top. Microbiol. Immunol. 323:3-32.[CrossRef][Medline]
8
8. Drexler, J. F., L. K. de Souza Luna, A. Stocker, P. S. Almeida, T. C. M. Ribeiro, N. Petersen, P. Herzog, C. Pedroso, H. I. Huppertz, H. da Costra Ribeiro, Jr., S. Baumgarte, and C. Drosten. 2008. Circulation of 3 lineages of a novel Saffold cardiovirus in humans. Emerg. Infect. Dis. 14:1398-1405.[CrossRef][Medline]
9
9. Dvorak, C. M., D. J. Hall, M. Hill, M. Riddle, A. Pranter, J. Dillman, M. Deibel, and A. C. Palmenberg. 2001. Leader protein of encephalomyocarditis virus binds zinc, is phosphorylated during viral infection, and affects the efficiency of genome translation. Virology 290:261-271.[CrossRef][Medline]
10
10. Fauquet, C. M., M. A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball (ed.). 2005. Virus taxonomy. Eighth report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego, CA.
11
11. Feng, H., M. Shuda, Y. Chang, and P. S. Moore. 2008. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 319:1096-1100.[Abstract/Free Full Text]
12
12. Fouchier, R. A., N. G. Hartwig, T. M. Bestebroer, B. Niemeyer, J. C. de Jong, J. H. Simon, and A. D. Osterhaus. 2004. A previously undescribed coronavirus associated with respiratory disease in humans. Proc. Natl. Acad. Sci. USA 101:6212-6216.[Abstract/Free Full Text]
13
13. Gaynor, A. M., M. D. Nissen, D. M. Whiley, I. M. Mackay, S. B. Lambert, G. Wu, D. C. Brennan, G. A. Storch, T. P. Sloots, and D. Wang. 2007. Identification of a novel polyomavirus from patients with acute respiratory tract infections. PLoS Pathog. 3:e64.[CrossRef][Medline]
14
14. Ghadge, G. D., L. Ma, S. Sato, J. Kim, and R. P. Roos. 1998. A protein critical for a Theiler's virus-induced immune system-mediated demyelinating disease has a cell type-specific antiapoptotic effect and a key role in virus persistence. J. Virol. 72:8605-8612.[Abstract/Free Full Text]
15
15. Heath, L., E. van der Walt, A. Varsani, and D. P. Martin. 2006. Recombination patterns in aphthoviruses mirror those found in other picornaviruses. J. Virol. 80:11827-11832.[Abstract/Free Full Text]
16
16. Higgins, D. G., A. J. Bleasby, and R. Fuchs. 1992. CLUSTAL V: improved software for multiple sequence alignment. Comput. Appl. Biosci. 8:189-191.[Abstract/Free Full Text]
17
17. Jarousse, N., R. A. Grant, J. M. Hogle, L. Zhang, A. Senkowski, R. P. Roos, T. Michiels, M. Brahic, and A. McAllister. 1994. A single amino acid change determines persistence of a chimeric Theiler's virus. J. Virol. 68:3364-3368.[Abstract/Free Full Text]
18
18. Jnaoui, K., and T. Michiels. 1998. Adaptation of Theiler's virus to L929 cells: mutations in the putative receptor binding site on the capsid map to neutralization sites and modulate viral persistence. Virology 244:397-404.[CrossRef][Medline]
19
19. Jnaoui, K., M. Minet, and T. Michiels. 2002. Mutations that affect the tropism of DA and GDVII strains of Theiler's virus in vitro influence sialic acid binding and pathogenicity. J. Virol. 76:8138-8147.[Abstract/Free Full Text]
20
20. Jones, M. S., A. Kapoor, V. V. Lukashov, P. Simmonds, F. Hecht, and E. Delwart. 2005. New DNA viruses identified in patients with acute viral infection syndrome. J. Virol. 79:8230-8236.[Abstract/Free Full Text]
21
21. Jones, M. S., V. V. Lukashov, R. D. Ganac, and D. P. Schnurr. 2007. Discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin. J. Clin. Microbiol. 45:2144-2150.[Abstract/Free Full Text]
22
22. Jones, M. S., II, B. Harrach, R. D. Ganac, M. M. Gozum, W. P. dela Cruz, B. Riedel, C. Pan, E. L. Delwart, and D. P. Schnurr. 2007. New adenovirus species found in a patient presenting with gastroenteritis. J. Virol. 81:5978-5984.[Abstract/Free Full Text]
23
23. Kapoor, A., J. Victoria, P. Simmonds, C. Wang, R. W. Shafer, R. Nims, O. Nielsen, and E. Delwart. 2008. A highly divergent picornavirus in a marine mammal. J. Virol. 82:311-320.[Abstract/Free Full Text]
24
24. Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120.[CrossRef][Medline]
25
25. Kistler, A., P. C. Avila, S. Rouskin, D. Wang, T. Ward, S. Yagi, D. Schnurr, D. Ganem, J. L. Derisi, and H. A. Boushey. 2007. Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity. J. Infect. Dis. 196:817-825.[CrossRef][Medline]
26
26. Kong, W. P., G. D. Ghadge, and R. P. Roos. 1994. Involvement of cardiovirus leader in host cell-restricted virus expression. Proc. Natl. Acad. Sci. USA 91:1796-1800.[Abstract/Free Full Text]
27
27. Kong, W. P., and R. P. Roos. 1991. Alternative translation initiation site in the DA strain of Theiler's murine encephalomyelitis virus. J. Virol. 65:3395-3399.[Abstract/Free Full Text]
28
28. Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245.[Abstract/Free Full Text]
29
29. Kurbanov, F., Y. Tanaka, A. Kramvis, P. Simmonds, and M. Mizokami. 2008. When should "I" consider a new hepatitis B virus genotype? J. Virol. 82:8241-8242.[Free Full Text]
30
30. Liang, Z., A. S. Kumar, M. S. Jones, N. J. Knowles, and H. L. Lipton. 2008. Phylogenetic analysis of the species Theilovirus: emerging murine and human pathogens. J. Virol. 82:11545-11554.[Abstract/Free Full Text]
31
31. Lipton, H. L. 2008. Human Vilyuisk encephalitis. Rev. Med. Virol. 18:347-352.[CrossRef][Medline]
32
32. Lipton, H. L., A. Friedmann, P. Sethi, and J. R. Crowther. 1983. Characterization of Vilyuisk virus as a picornavirus. J. Med. Virol. 12:195-203.[CrossRef][Medline]
33
33. Lipton, H. L., Z. Liang, S. Hertzler, and K. N. Son. 2007. A specific viral cause of multiple sclerosis: one virus, one disease. Ann. Neurol. 61:514-523.[CrossRef][Medline]
34
34. Lole, K. S., R. C. Bollinger, R. S. Paranjape, D. Gadkari, S. S. Kulkarni, N. G. Novak, R. Ingersoll, H. W. Sheppard, and S. C. Ray. 1999. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J. Virol. 73:152-160.[Abstract/Free Full Text]
35
35. Lukashev, A. N. 2005. Role of recombination in evolution of enteroviruses. Rev. Med. Virol. 15:157-167.[CrossRef][Medline]
36
36. McCright, I. J., I. Tsunoda, F. G. Whitby, and R. S. Fujinami. 1999. Theiler's viruses with mutations in loop I of VP1 lead to altered tropism and pathogenesis. J. Virol. 73:2814-2824.[Abstract/Free Full Text]
37
37. Modlin, J. F. 2007. Enterovirus deja vu. N. Engl. J. Med. 356:1204-1205.[Free Full Text]
38
38. Oberste, M. S., K. Maher, D. R. Kilpatrick, and M. A. Pallansch. 1999. Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. J. Virol. 73:1941-1948.[Abstract/Free Full Text]
39
39. Pallansch, M. A., and R. P. Roos. 2001. Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses, p. 723-776. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
40
40. Pritchard, A. E., T. Strom, and H. L. Lipton. 1992. Nucleotide sequence identifies Vilyuisk virus as a divergent Theiler's virus. Virology 191:469-472.[CrossRef][Medline]
41
41. Siepel, A. C., A. L. Halpern, C. Macken, and B. T. Korber. 1995. A computer program designed to screen rapidly for HIV type 1 intersubtype recombinant sequences. AIDS Res. Hum. Retrovir. 11:1413-1416.[Medline]
42
42. Simmonds, P. 2006. Recombination and selection in the evolution of picornaviruses and other mammalian positive-stranded RNA viruses. J. Virol. 80:11124-11140.[Abstract/Free Full Text]
43
43. Simmonds, P., and S. Midgley. 2005. Recombination in the genesis and evolution of hepatitis B virus genotypes. J. Virol. 79:15467-15476.[Abstract/Free Full Text]
44
44. Simmonds, P., and J. Welch. 2006. Frequency and dynamics of recombination within different species of human enteroviruses. J. Virol. 80:483-493.[Abstract/Free Full Text]
45
45. Takata, H., M. Obuchi, J. Yamamoto, T. Odagiri, R. P. Roos, H. Iizuka, and Y. Ohara. 1998. L* protein of the DA strain of Theiler's murine encephalomyelitis virus is important for virus growth in a murine macrophage-like cell line. J. Virol. 72:4950-4955.[Abstract/Free Full Text]
46
46. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596-1599.[Abstract/Free Full Text]
47
47. Urisman, A., R. J. Molinaro, N. Fischer, S. J. Plummer, G. Casey, E. A. Klein, K. Malathi, C. Magi-Galluzzi, R. R. Tubbs, D. Ganem, R. H. Silverman, and J. L. Derisi. 2006. Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog. 2:e25.[CrossRef][Medline]
48
48. van den Hoogen, B. G., J. C. de Jong, J. Groen, T. Kuiken, R. de Groot, R. A. Fouchier, and A. D. Osterhaus. 2001. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat. Med. 7:719-724.[CrossRef][Medline]
49
49. Van Der Hoek, L., K. Pyrc, M. F. Jebbink, W. Vermeulen-Oost, R. J. Berkhout, K. C. Wolthers, P. M. Wertheim-Van Dillen, J. Kaandorp, J. Spaargaren, and B. Berkhout. 2004. Identification of a new human coronavirus. Nat. Med. 10:368-373.[CrossRef][Medline]
50
50. van der Werf, N., F. G. Kroese, J. Rozing, and J. L. Hillebrands. 2007. Viral infections as potential triggers of type 1 diabetes. Diabetes Metab. Res. Rev. 23:169-183.[CrossRef][Medline]
51
51. van Eyll, O., and T. Michiels. 2000. Influence of the Theiler's virus L* protein on macrophage infection, viral persistence, and neurovirulence. J. Virol. 74:9071-9077.[Abstract/Free Full Text]
52
52. van Eyll, O., and T. Michiels. 2002. Non-AUG-initiated internal translation of the L* protein of Theiler's virus and importance of this protein for viral persistence. J. Virol. 76:10665-10673.[Abstract/Free Full Text]
53
53. van Pesch, V., O. van Eyll, and T. Michiels. 2001. The leader protein of Theiler's virus inhibits immediate-early alpha/beta interferon production. J. Virol. 75:7811-7817.[Abstract/Free Full Text]
54
54. Wada, Y., I. J. McCright, F. G. Whitby, I. Tsunoda, and R. S. Fujinami. 1998. Replacement of loop II of VP1 of the DA strain with loop II of the GDVII strain of Theiler's murine encephalomyelitis virus alters neurovirulence, viral persistence, and demyelination. J. Virol. 72:7557-7562.[Abstract/Free Full Text]
55
55. Yoon, J. W., and H. S. Jun. 2006. Viruses cause type 1 diabetes in animals. Ann. N. Y. Acad. Sci. 1079:138-146.[CrossRef][Medline]
56
56. Zoll, J., W. J. Melchers, J. M. Galama, and F. J. van Kuppeveld. 2002. The mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of NF-B. J. Virol. 76:9664-9672.[Abstract/Free Full Text]

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Journal of Virology, May 2009, p. 4631-4641, Vol. 83, No. 9
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