Abstract
Table 1. Primers used to amplify and sequence the HN region to differentiate between HPIV4a and HPIV4b Primer Sequence (5?-3?) Position (AB543336) Para4-HN-F1-710 CAACAATCCAGARRGACGTCACA 7409-7431 Para4-HN-F2-711 AGATTAYCCCAATTTTATTCCAACTG 7995-8220 Para4-HN-F3-712 AGGAGCAAAAGAYTCATACACAATAACT 8583-8610 Para4-HN-R2-715 CATTCTTTTGGRCAAGCCTTA 8930-8910 Para4-HN-R3-717 GGTTCCGCTTGATGGACTGT 9508-9489 Para4-HN-R4-718 GGCCGATGGTCCTTCGTATA 9567-9548 Para4-HN-F4-719 CCATCATTYTCCYTARGTCAAA 8049-8071 Para4-HN-F5-720 TGCAGAGGGTCGMYTATATAAYATTG 8721-8746 Para4-HN-R3-721 CGATTCYTCCCRTCATTTAAAT 8228-8207 Para4-HN-R5-723 TGATTCTATADAGGCTYGGATADGG 8812-8788 Position (AB543337) Para4-HN-R1-714 TTTGTTATTGATGGGTGATGTTTCAC 9467-9443 76
Abstract
Jpn.

Human parainfluenza virus type 4 (HPIV4) belongs to genus Rubulavirus, subfamily Paramyxovirinae of family Paramyxoviridae (1). Although serological surveys indicate that infection may be common, and even universal, reports and descriptions of HPIV4-associated diseases are limited (1) (2) (3) (4) . Thus, although mild and severe respiratory illness associated with HPIV4 infections are reported, correlations between HPIV4 infections and specific clinical syndromes, age of the child, and time of the year, or seasonal patterns have not been established thus far (1) (2) (3) (4) (5) (6) (7) (8) . Due to the difficulties in isolating HPIV4, such as slow growth, trypsin concentration, and subtle cytopathic effect (4), its diagnosis has probably been underestimated in the past. However, even though previously undetected cases can now be identified with more sensitive methods such as real-time reverse transcription-PCR (RT-PCR) (1, 2) , the number of HPIV4 infections reported is still quite low. For example, only 88 cases, including our 28 cases from Yamagata in 2011, were reported between 1982 and 2011 in Japan, according to the National Epidemiologi-cal Surveillance of Infectious Diseases (NESID) system (9) . In this study, we describe an outbreak of HPIV4 infections during the 2011-2012 winter season in Yamagata, Japan.

We carried out a longitudinal epidemiological study of acute respiratory infections (ARIs) among children in Yamagata, Japan, primarily based on virus isolation using a microplate method (10) . Six cell lines, namely HEF, HEp-2, Vero or VeroE6, MDCK, RD-18S, and GMK, have been used since 2001. In 2008, we added the HMV-II cell line, primarily for the isolation of HPIVs (10) (11) (12) . Then, when we accidentally succeeded in isolating one HPIV4 strain from a nasopharyngeal sample collected in October 2011, we added the LLC-MK2 cell line, which is reportedly sensitive for the isolation of HPIV4 (13) . Further, we performed RT-PCR screening for HPIV4 in fresh and stocked frozen samples. In the latter effort, cases found to be positive using the influenza virus test kit were excluded. Finally, we included 1,499 nasopharyngeal swab specimens obtained from patients with ARIs at pediatric clinics between January 77 2011 and April 2012. We screened the polymerase L gene coding region using the previously reported AVU-RUB-F1, AVU-RUB-F2, and AVU-RUB-R primers (14) , and we further amplified the hemagglutininneuraminidase (HN) glycoprotein region in HPIV4positive specimens to differentiate between HPIV4a and HPIV4b using our original primers shown in Table 1 .

We registered sequence data with GenBank (accession nos. AB753462-AB753479). When we found syncytia formation on the HMV-II and/or LLC-MK2 cell lines, we also carried out immunofluorescent staining to identify HPIV4, using two commercially available antibodies: mouse anti-parainfluenza 4 MAB8780 (Millipore, Temecula, Calif., USA) and FITC-conjugated rabbit anti-mouse immunoglobulins (DAKO, Glostrup, Denmark). Among the 1,499 clinical specimens, all 43 HPIV4positive cases were detected by RT-PCR, and the virus was isolated on the HMV-II and/or LLC-MK2 cell lines in 12 of the 43 cases (Fig. 1) . The monthly distribution of HPIV4 detection during the study period is shown in Fig. 2 . Further, 37 (86z) HPIV4-positive cases were found between September 2011 and February 2012. In other months, only 0-1 cases were detected. Sequence data were analyzed with CLUSTAL W version 1.83, and a phylogenetic tree was constructed via the neighbor-joining method (Fig. 3) . Among the 43 HPIV4-positive cases, 7 were identified as HPIV4a, 11 as HPIV4b, while the others remained unidentified because the HN region was not amplified enough for the analysis.

Among the 43 HPIV4-positive cases, 4 patients (9.3z) were aged º1 years, 10 (23.2z) were aged 1 year, 2 (4.7z) were aged 2 years, 3 (7z) were aged 3 years, 5 (11.6z) were aged 4 years, 6 (14z) were aged 5 years, 6 (14z) were aged 6-9 years, and 7 (16.2z) were aged 10-15 years. We detected plural viruses, including rhinovirus, human metapneumovirus, enterovirus, cytomegalovirus, and adenovirus in 11 cases. Among the 32 HPIV4-only cases, 25 (78.1z) were diagnosed as nasopharyngitis, 5 (15.6z) as bronchitis, and others as laryngitis and viral exanthema.

A number of HPIV1-3 strains have been isolated with our surveillance system using the microplate method (12); however, HPIV4 strains have been rarely isolated. A local outbreak of HPIV4 was observed for the first time during the winter season of 2011-2012, as described here. Since the number of reported cases of HPIV4 in Japan is quite limited (9), few HPIV4-associated outbreaks have been observed apart from that presentated by Watanabe et al. (13) . Even worldwide, the number of HPIV4 cases reported is quite limited. Laurichesse et al. and Fry et al. reported that the number of HPIV4-positive cases was markedly smaller than that of HPIV1-3-positive cases (5, 15) . Lau et al. reported 41 HPIV4 cases during an outbreak in a developmental disability unit and 35 cases (1.2z) among 2,912 nasopharyngeal aspirates in a 1-year study (16, 17) ; Ren et al. described 25 (10.2z) HPIV4 cases among 246 HPIV-positive cases over a 3-year period (6) . Finally, Scott et al. described 13 HPIV4-positive cases, most of which were severe cases, during their 6-year study (7) . Thus, our study experience together with the limited data, indicates the difficulties associated with the isolation and detection of HPIV4. To clarify the etiology and epidemiology of HPIV4 infections, HPIV4 infections should be recognized as common diseases that require active surveillance using sensitive cell lines such as LLC-MK2, as well as combining molecular method such as RT-PCR.

Watanabe et al. conducted an epidemiological study of HPIV4 among children with ARIs in Sendai and Yamagata. They isolated HPIV4 from 129 (0.8z) of 16,187 clinical specimens using the LLC-MK2 cell line between 2003 and 2009, and they commonly isolated HPIV4 during autumn (13) . Based on their study, we believe that a number of HPIV4 infections in Yamagata were missed, especially those during the autumn season. Vachon et al. suggested that HPIV4 infections can be relatively common during the autum and winter seasons (18); Billaud et al. reported that they detected 18 of 20 HPIV4-positive cases during the autumn and winter (8) . Now that we have succeeded in isolating and detecting HPIV4, we hope to determine if HPIV4 outbreaks commonly occur during autumn. Watanabe et al. also stated that approximately two-thirds of HPIV4-positive patients were diagnosed with lower respiratory infections such as pneumonia, bronchiolitis, and bronchitis (13) . However, we found only 5 cases of bronchitis, and most of the patients in our study were diagnosed with nasopharyngitis. Thus, the clinical picture of HPIV4 remains controversial.

In conclusion, epidemiological data on HPIV4 are still quite limited and further studies are needed to clarify the etiology and epidemiology of HPIV4 infections.

{'paper_id': '42140483', 'title': 'An outbreak of parainfluenza virus type 4 infections among children with acute respiratory infections during the 2011-2012 winter season in Yamagata, Japan.', 's2_url': 'https://api.semanticscholar.org/CorpusID:42140483', 'source': 'S2ORC'}