Asao Itagaki

Molecular survey of Babesia microti, Ehrlichia species and Candidatus Neoehrlichia mikurensis in wild rodents from Shimane Prefecture, Japan

A significant number of patients are diagnosed with “fevers of unknown origin” (FUO) in Shimane Prefecture in Japan where tick‐borne diseases are endemic. We conducted molecular surveys for Babesia microti, Ehrlichia species, and Candidatus Neoehrlichia mikurensis in 62 FUO cases and 62 wild rodents from Shimane Prefecture, Japan. PCR using primers specific for the Babesia 18S small‐subunit rRNA (rDNA) gene and Anaplasmataceae groESL amplified products from 45% (28/62) and 25.8% (16/62) of captured mice, respectively. Of the 28 18S rDNA PCR positives, 23 and five samples were positive for Hobetsu‐ and Kobe‐type B. microti, respectively. In contrast, of the 16 groESL PCR positives, eight, one and seven samples were positive for Ehrlichia muris, Ehrlichia sp. HF565 and Candidatus N. mikurensis, respectively. Inoculation of selected blood samples into Golden Syrian hamsters indicated the presence of Hobetsu‐ and Kobe‐type B. microti in four and one sample, respectively. Isolation of the latter strain was considered important as previous studies suggested that the distribution of this type was so far confined to Awaji Island in Hyogo Prefecture, where the first case of transfusion‐associated human babesiosis originated. DNA samples from 62 FUO human cases tested negative for B. microti 18S rDNA gene, Anaplasmataceae groESL gene, Rickettsia japonica 17K genus‐common antigen gene and Orientia tsutsugamushi 56K antigen gene by PCRs. We also conducted seroepidemiological surveys on 62 human sera collected in Shimane Prefecture from the FUO patients who were suspected of carrying tick‐borne diseases. However, indirect immunofluorescent antibody tests using B. microti‐ and E. muris‐infected cells detected IgG against E. muris in only a single positive sample. This study demonstrates the presence of several potentially important tick‐borne pathogens in Shimane Prefecture and suggests the need for further study on the causative agents of FUOs.

A Clustering Outbreak of Hand, Foot, and Mouth Disease Caused by Coxsackie Virus A10

A clustering outbreak of hand, foot, and mouth disease (HFMD) occurred from July, 1981 to January, 1982 in Matsue City and Gotsu City, Shimane Prefecture. Thirty‐seven patients with clinical HFMD were virologically and serologically examined, and Coxsackie virus A10 (CA10) was isolated in 18 patients from vesicles (7/16), throat‐swabs (9/31) and feces (6/7). During the period, no CA16 or enterovirus 71 were isolated from HFMD patients or from other diseases such as pharyngitis, febrile diseases, and aseptic meningitis. Serological diagnosis was performed employing an African green monkey kidney cell (AG‐1)‐adapted CA10 which demonstrated cytopathogenic effects on the cells. Paired sera from seven patients including three cases in which isolation failed showed a significant increase of neutralizing antibody titer against CA10. Finally, an etiological diagnosis was made in 21 out of 37 patients with clinical HFMD. This is the first report of a clustering outbreak of HFMD caused by CA10 in Japan.

Oligonucleotide Fingerprint Analysis of Coxsackievirus A10 Isolated in Japan

Eight coxsackievirus A10 strains isolated in 1978 and in 1981 and 1982 from patients with hand, foot-and-mouth disease and with herpangina at a dispensary in Matsue city were compared by RNA fingerprinting techniques. The oligonucleotide maps of the four 1978 isolates were related to each other by 85 to 93% with respect to their large T1 oligonucleotides. In contrast, the oligonucleotide maps of the four 1981 and 1982 isolates were very different from each other. Co-electrophoresis experiments revealed that the 1981 and 1982 strains shared only 17 to 34% of their large oligonucleotides. In addition, some large oligonucleotides were found in most of the fingerprint maps of isolates from 1978 to 1982, suggesting that there are regions in the genome of coxsackievirus A10 which are not subject to mutational changes.

Properties of Virus Isolated from an Epidemic of Hand-Foot-and-Mouth Disease in 1973 in the City of Matsue

The virus strains isolated from clinical cases in an epidemic of hand‐foot‐and‐mouth disease in Matsue in 1973 were characterized and its properties were compared with those of the Coxsackievirus group A type 16 (CA 16) prototype strain. The virus isolated in 1973 was similar to CA16 prototype virus with respect to morphology in electron microscopy, resistance to ether and capability to replicate in medium containing fluorodeoxyuridine. Cross neutralization tests using guinea‐pig and horse antisera revealed that there was little or no detectable common antigen between the two viruses. The two viruses also differed in heat stability of virion infectivity: the 1973‐viruses were much more resistant to heat than the prototype virus. Under one‐step growth conditions in Vero cell cultures, growth rate and virus yield of the 1973‐viruses were lower than those of CA16, but this property was independent of incubation temperatures, pH of culture medium and other culture conditions. Several other differences in property between the 2 strains are also described. It is concluded that the epidemic in 1973 was caused by a virus whose properties differed greatly from those of the CA16 prototype.

Isolation and characterization of a cold-sensitive strain of coxsackievirus A10

A coxsackievirus A10 strain, isolated from a clinical specimen from a patient with pharyngitis, was characterized with respect to its growth properties in different cultured cells and at different incubation temperatures. This virus multiplied within cultured cells and produced cytopathogenic effects, whereas a prototype strain of coxsackievirus A10 did not. The isolate multiplied efficiently in cultured cells at 37 degrees C but its replication was markedly restricted at 32 degrees C. Temperature shift experiments indicated that the cold-sensitive event affected the late function(s) of the virus.

Reduced replication capacity of influenza A(H1N1)pdm09 virus during the 2010–2011 winter season in Tottori, Japan

A novel swine‐origin influenza A(H1N1)pdm09 virus has been circulating in humans since March–April, 2009. The 2009–2010 epidemic involved predominantly a single subtype of A(H1N1)pdm09 (at 96%, 46/48) in the sentinel sites of this study. However, A(H1N1)pdm09 started to circulate together with other type/subtype (49%, 33/68) at the first peak in the next epidemic season in 2010–2011: A(H1N1)pdm09/A(H3N2) (9%, 6/68), A(H1N1)pdm09/B (35%, 24/68), and A(H1N1)pdm09/A(H3N2)/B (4%, 3/68). Single infection of A(H1N1)pdm09 became a rare event (8%, 5/65) at the second peak of the same season in 2010–2011 compared with that at the first peak (50%, 34/68). Concurrently with this decline, single infections of others, A(H3N2) or B, became evident (6%, 4/65; 14%, 9/65, respectively). Triple infections were more common (29%, 19/65) at the second peak than at the first peak (4%). The A(H1N1)pdm09 detected in 2010–2011 produced less virus upon 72 hr of incubation in vitro after the inoculations at 104 and 3,300 copies/ml (2.3 × 109 and 2.3 × 109 copies/ml on average) than that in 2009–2010 (3.7 × 109 and 1.3 × 1010 copies/ml on average; P < 0.05 by ANOVA test), respectively. As described above, the replication capacity of A(H1N1)pdm09 seems to have deteriorated in the 2010–2011 season presumably due to substantial herd immunity and allowed the existence of other type/subtype. These results suggest that assessment of replication capacity is indispensable for analysis of influenza epidemics. J Med. Virol. 85:1871–1877, 2013.