During June 17–August 6, 2008, an outbreak of an unknown disease was observed among adults in Yonezawa, Yamagata, Japan. A total of 22 patients who lived in the city of Yonezawa who had myalgia, muscular weakness, sore throat, and orchiodynia (among men) sought treatment at Yonezawa City Hospital. Because all had symptoms of severe myalgia, these patients were given a diagnosis of with acute myalgia syndrome of unknown cause. The patients consisted of 15 men and 7 women ages 25–66 years (mean 37 years). Because several patients had contact with other persons who had similar symptoms, the outbreak was considered to be associated with an infectious agent. Virologic and serologic analyses were carried out to find the associated agent. This study was approved by the Ethics Committee of the Yonezawa City Hospital.

Throat swab and stool specimens were collected from 14 patients (Table 1). Throat swab specimens were placed immediately in tubes containing 3 mL of transport medium, and stool specimens were put in a stool container and transported to the Department of Microbiology, Yamagata Prefectural Institute of Public Health, Yamagata, to undergo virus isolation and reverse transcription PCR (RT-PCR) for enteroviruses, HPeV1, and HPeV2.

Virus isolation was carried out using a described microplate method (15). In brief,, HEF, HEp-2, Vero E6, MDCK, RD-18S, and GMK cell lines were prepared in the wells of a 96-well microplate (Greiner Bio-One, Frickenhausen, Germany). After a medium change, throat swabs and 10% stool suspension specimens were centrifuged at 1,500 × g for 20 min, and 75 μL of the supernatant was added to 2 wells of each of the cell lines. The inoculated plates were centrifuged for 20 min at 450 × g, incubated at 33°C in a 5% CO₂ incubator, and assessed for cytopathic effect.

RNA was extracted from 200 μL of each throat swab specimen or 10% stool suspension using a High Pure Viral RNA Kit (Roche Diagnostics, Manheim, Germany) according to the manufacturer’s instructions and then transcribed into complementary DNA (cDNA) as described (16). PCR was performed as described (17,18), except that we used a mixture of 224 and 222 primers for the first PCR and a mixture of AN89 and AN88 primers for the nested PCR to detect enteroviruses and K28, K29, and K30 primers to detect HPeV1 and HPeV2. The remainder of each specimen and the cDNA specimens were stored at −80°C.

Ultra-high throughput direct sequencing analysis was carried out as described (19). Total DNA or RNA was prepared from the specimens of case-patient 11 by using a viral nucleic acid purification kit (Roche Diagnostics). Double-stranded cDNA (ds-cDNA) was prepared from 1 µg of total RNA using the random priming method with the SuperScript Choice System for cDNA synthesis (Invitrogen, Carlsbad, CA, USA). A mixture of DNA and ds-cDNA was purified by using a QIAquick PCR Purification kit (QIAGEN, Hilden, Germany).

An ≈300-bp length DNA library was prepared from a mixture of 2 µg of total DNA and ds-cDNA by using a genomic DNA sample prep kit (Illumina, San Diego, CA, USA), and DNA clusters were generated on a slide using a Single Read Cluster Generation kit version 4 on an Illumina cluster station (Illumina) according to the manufacturer’s instructions. To obtain ≈1.0 × 10⁷ clusters for 1 lane, the general procedure as described in the manufacturer’s standard recipe was performed. All sequencing runs for 83-mers were performed with GA II using the Illumina Sequencing Kit version 5. Fluorescent images were analyzed using the Illumina SCS2.8/RTA1.8 to obtain FASTQ formatted sequence data. The obtained DNA sequence reads were investigated by using a MEGABLAST search (20) with an e^–5 e-value cutoff against the nonredundant nt database nt, followed by taxonomic classification using MEGAN version 3.9.0 (21) with the following parameters: minimum support 1; minimum score 35.0.

RT-PCR was performed by using ≈100 ng of total RNA, the appropriate primer pair, and the PrimeScript II High Fidelity One-Step RT-PCR Kit (TaKaRa, Shiga, Japan). The following quantitative RT-PCR program was used: reverse transcription reaction 45°C for 10 min; initial denaturation 94°C for 2 min; and 3 steps of amplification (× 35 cycles) at 98°C for 10 s, 55°C for 15 s, and 68°C for 1 min. PCR products were resolved and purified by agarose gel electrophoresis and then sequenced by Sanger sequencing by using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

After HPeV3 was detected in the specimens from case-patient 11 by ultra-high throughput direct sequencing analysis, to investigate whether HPeV3 was associated with the myalgia epidemic, we repeated the virus isolation focusing on HPeV3, using GMK, Vero, and LLC-MK2 cell lines using the 28 stocked throat swab and stool samples shown in Table 1. We used 2 LLC-MK2 cell lines, one provided by the National Institute of Infectious Diseases Japan and the other by the Niigata Prefectural Institute of Public Health and Environmental Sciences. We also attempted to specifically amplify the HPeV3 genome via RT-PCR using frozen cDNA and our original primers (Parecho3-VP1F1Y and Parecho3-VP1R1Y for the first PCR and Parecho3-VP1F2Y and Parecho3-VP1R2Y for the nested PCR [Table 2]), using the same conditions as in the preliminary screening procedure. When the RT-PCR result was positive, PCR products were purified and sequenced on a Sanger sequencer using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the primers listed in Table 2. We also attempted to amplify the HPeV3 genome using RT-PCR from all serum specimens that were initially collected for serologic analysis.

To further evaluate the role of HPeV3 in this outbreak of myalgia, we measured the neutralizing antibody response against HPeV3 in 1–5 serum samples from 20 of the patients (Table 1). To observe seroconversion and changes in antibody titers, we measured neutralization antibodies using a microneutralization test on a 96-well microplate. Sample serum samples were inactivated at 56°C for 30 min and then diluted from 1:8 to 1:4,096 by serial 2-fold dilution. One HPeV3 isolate in this outbreak in Yamagata (1356-Yamagata-2008) was used as a challenge virus antigen. We mixed and incubated (37°C, 60 min) 60 μL of each diluted serum sample with 60 μL of virus fluid containing ≈10² 50% tissue culture infectious dose. We prepared confluent monolayers of the LLC-MK2 cell line provided by the Niigata Prefectural Institute of Public Health and Environmental Sciences, washed the cells with phosphate-buffered saline without calcium or magnesium, added 50 μL of maintenance media, and injected 50 μL of each incubated virus–serum mixture into each of 2 cultures. The plates were then incubated in a 5% CO₂ incubator at 33°C for cytopathic effect observation. The reciprocal value of the highest dilution of serum neutralizing the virus compared to the control was taken to be the titer. Seropositivity was defined as titer >8. Virus infection was considered confirmed if seroconversion from negative to positive could be documented. Alternatively, if all serum samples were positive, a 4-fold increase in antibody titer was considered serologic evidence of infection with HPeV3.

The 22 case-patients in this outbreak had similar symptoms (Table 1); all had severe myalgia involving mainly the proximal muscles of the upper and lower extremities. The next most common symptom was muscular weakness in the arms and legs (21, 95.5%), followed by fever (19, 86.4%), pharyngitis (15, 68.2%), orchiodynia (4, 18.2%), and seizures (1, 4.5%). Symptoms worsened rapidly within 1–2 days after onset. Eight patients were hospitalized on a visit to Yonezawa City Hospital and remained hospitalized for 5 (4 patients), 6 (1 patient), 8 (2 patients), or 10 days (1 patient); mean hospitalization was 6.5 days. Case-patient 14 had seizures and subsequent disturbance of consciousness. For all patients, muscular weakness improved as muscle pain decreased.

Creatinine phosphokinase (CPK), C-reactive protein, and myoglobin levels were higher than reference ranges in 12, 19, and 16 patients, respectively (Table 1). Elevated CPK levels returned to normal within several days. An examination of cerebrospinal fluid showed no sign of inflammation.

Magnetic resonance imaging (MRI) of the muscles of case-patients 4, 13, and 15 showed increased signal intensity on T2-weighted images. We suspected that case-patient 14 had encephalitis on the basis of clinical symptom, but results of MRI of the brain and cerebrospinal fluid examination did not support this diagnosis. Clinical symptoms and laboratory findings for all patients were consistent with inflammatory myositis.

No viruses were detected by screening steps at the Yamagata Prefectural Institute of Public Health using virus isolation and RT-PCR targeting enteroviruses, HPeV1, and HPeV2. Thus, we investigated possible uncharacterized pathogens by using direct DNA sequencing. To determine potential pathogens for the patients, we performed direct sequencing of a mixture of purified DNA and ds-cDNA from the total RNA extracted from either the throat swab or stool specimens of case-patient 11. No other possible viral sequences were detected by the high-throughput sequencing for case-patient 11.

Next-generation DNA sequencer GA II produced ≈1.5 × 10⁷ 83-mer short reads from the mixed DNA library. To exclude the human-derived read sequences, all obtained reads were initially aligned to a reference sequence of human genomic DNA, followed by quality trimming to remove low-quality reads and exclude reads with similarities to ambiguous human sequences. All remaining possible pathogen reads were further analyzed using a MegaBLAST search against nonredundant databases.

One type of HPeV was found in the analyzed specimens. Three HPeV reads were identified from the throat swab specimen and 1,505 HPeV reads from the stool specimen of case-patient 11. To further characterize the type of HPeVs detected, de novo assembly was performed by using Euler-SR version 1.0 (22); the resulting partial contigs showed higher similarity to HPeV3 than to other HPeVs.

To determine the whole HPeV sequence for the isolates we obtained, RT-PCR was performed (Figure 1), and the products were sequenced. The whole coding nucleotide sequence of the polyprotein in the HPeV detected in the stool sample from case-patient 11 was aligned against all available HPeV complete genomes, including HPeV1–8. A phylogenetic tree was generated for the VP1 region (Figure 2), and the HPeV in the stool specimen of case-patient 11 was identified as HPeV3. All detected HPeV3 VP1 sequences among the 2008 myalgia cases were identical (Table 1), except that we found a single nucleotide substitution in sequences from the serum samples from case-patients 4 and 22.

We passaged isolates 6 times using GMK and LLC-MK2 cell lines, but all strains except 1 (isolated after 5 passages) were recovered within 3–4 passages. We could not isolate HPeV3 using the LLC-MK2 cell line provided by the National Institute of Infectious Diseases Japan, but we were successful when using LLC-MK2 cell line from the Niigata Prefectural Institute of Public Health and Environmental Sciences. In total, we isolated HPeV3 strains from either the throat swab or stool specimen of 7/14 patients analyzed (Table 1).

RT-PCR was successful in detecting the HPeV3 genome in the throat swabs or stool specimens from 9/14 patients (Table 1). We also detected the HPeV3 genome in 3 serum specimens collected within 3 days after the onset of illness (Table 1). Sequence data were registered under GenBank accession nos. AB668029–AB668045.

Of 20 analyzed patients, seroconversion was observed in 6 patients. A 4-fold increase in neutralization antibodies against HPeV3 was confirmed in 5 patients (Table 1).