Epidemiology and etiological roles of two : human C and 68

Tohoku University Graduate School of Medicine Hitoshi Oshitani

Classification of picornaviridae

• Genus of ‘Enterovirus’ – Human enterovirus • Human enterovirus A • Human etnerovirus B • Human • Human enterovirus D – Human rhinovirus • Human rhinovirus A • Human rhinovirus B • Human rhinovirus C

ICTV Picornaviridae Study Group Human and • Common virological characteristics Human – Positive strand, non‐enveroped rhinoviruses – Same genomic structure • Pathogenesis and clinical diseases are different – Enterovirus: Human enteroviruses • Resistant to acid • Causing enteric / systemic , e.g. – Rhinovirus: • Sensitive to acid • Only respiratory

Simmonds P et. al. J Gen Virol 2010 vol. 91 no. 10 2409‐2419

Human enteroviruses and rhinoviruses

Respiratory infection Enteric / systemic infection

Human rhinovirus A

Human Enterovirus enteroviruses 68

Human rhinovirus B Human rhinovirus C ?? Background of the study

• Etiological study on pediatric – Eastern Visayas Regional Medical Tacloban Center, Taclobal City, Leyte Island in the Philippines – May 2009 –present – Part of J‐GRID (Japan Initiative for Global Research Network on Infectious Diseases) supported by the Japanese government

Pediatric Pneumonia Study

•Patients ‐ Aged between 7 days and 14 years ‐ Severe pneumonia by the definition of Integrated Management of Childhood Illness (IMCI) •Respiratory were detected by multiplex PCR Eastern Visayas Regional Medical Center (EVRMC)

Outcome of patients

Others, 30, HAMA- 4% Died, 70, 9% Improved, HAMA- 151, 18% Deteriorated , 18, 2%

May 2008 –May 2009 (n = 819) Discharged, 550, 67% HAMA: Home against medical advice Identified viruses

RSV-A

Negative RSV-B

Rhino-A

Rhino-B May 2008 –May Multiple Rhino-C 2009 (n = 819) hMPV WU/KI FluA Viruses were detected Corona FluB in 506 cases (61.2 %) Boca EV68 Adeno

Identified viruses

RSV-A

Negative RSV-B

Rhino-A

Rhino-B May 2008 –May Multiple Rhino-C 2009 (n = 819) hMPV WU/KI FluA Viruses were detected Corona FluB in 506 cases (61.2 %) Boca EV68 Adeno

1. Detected from hospitalized children with respiratory infection in 1962. Only sporadic cases have been reported 2. Acid sensitive, grows at lower optimal temperture EV68 HRV Other EV Acid sensitive Yes Yes No Optimum growth temp. 33℃ 33℃ 37℃

Respiratory Intestine tract (Mainly)

* HRV and EV68 shares phenotypic characterizations

2. Isolation is relatively difficult.

Classification of Human Enteroviruses

Coxsackievirus B1‐6, A9, Enterovirus 69, 73, 74, 75, 77, 78, Echovirus1‐ 7, 9,11‐21,24‐27,29‐33 Species Japan USA

Coxsackievirus A 3613(48%) 1520(3.3%) A1,11,13,15,17‐22,24 B 3949(52%) 44418(96.3%) C 7 (0.09%) 176(0.38%) D 14(0.18%) 26 (0.05%) (From enterovirus surveillance data)

Poliovirus 1‐3 • Only 3 serotypes in Human Enterovirus 68,70, 94 enterovirus D • Detection of human enterovirus D has been very rare

Coxsackievirus A2‐10, 12,14,16 & ,76 Methods

1) Amplify partial viral by RT‐PCR Screen samples by PCR targeting 5’NTR of HRV ⇢ Submit positive samples to PCR targeting VP1 of EV68

↓ Genetic analysis using Mega 3.1 software

2) Epidemiological analysis using collected patients’ information

• Enterovirus 68 was detected from 21 cases with severe pneumonia, from October 2008 and February 2009, 2 of them died

Babatngon Basey Eastern

Visayas region

Tacloban city

Palo N Imamura T et al. Emerg Infect Dis. Vol 17: Dulag 0 200km 0 5km 1430‐1435, 2011 (5’NTR)

EV68

5’NCR（232bp） Bootstrap x500

Phylogenetic tree (VP1)

EV68, Philippines

EV68, USA

EV68, France

VP1（296bp） Bootstrap x500 Enterovirus 68 detection in Japan

Infectious Agents Surveillance Report, NIID, Japan

Increased detection of enterovirus 68 in 2010 in Japan

Increased detection of Enterovirus 68

MMWR (in press)

Increased detection of enterovirus 68 in Philippines (2008‐2009), USA (2009‐10), Japan (2010) and Netherland (2010) Recent increase in Enterovirus 68 in different parts of the world

• Two possibilities – Increased detection • Most studies detected Enterovirus 68 by PCR targeting rhinovirus detection – Global epidemic • Due to emergence of different virus: antigenicity, virulence, transmissibility

Phylogenetic tree of recent enterovirus 68

Japan (Yamagata)

Japan (Sendai)

Japan (Osaka)

Philippines

Nederland Human rhinovirus C

• Newly identified species of human rhinovirus • No virus isolation technique established • Phonotypic characteristics and pathogenesis are largely unknown – Associated with more severe respiratory infections?

Renwick et al JID 2007; 196: 1754‐60

Monthly distribution of HRVs

HRVs 447 (28.9 %) 229 2008 2009 2010 2011 HRVA (14.3%) HRVB 32 (2.0%) HRVC 186 (11.6%) Total 1597 Phylogenetic trees of detected rhinoviruses

• Many different genotypes were seen for HRVA, 2008 HRVA HRVB and HRVC 2009 HRVB • No significant clustering 2010 HRVC

HRV HRVB HRVC A

10 % died

No death

Wheezing

Wheezing was more frequently 4 % seen in HRVC than HRVA died Detection of rhinovirus RNA in serum samples

2008.05-2009.05 Serum that were collected from whose nasopharyngeal samples were pos. for HRVs

Nasopharyngeal serum swabs (n=816)

Serum- positivity Positivity (%) Sample No. OR (95%CI) (%) HRVA 135 (16.5%) 135 4 (3%) * 0.22 (0.08-0.62) HRVB 25 (3.1%) 25 0** - HRVC 83 (10.2%) 83 26 (31.3%)* 6.84 (3.53-13.23) Total 243 243 30 (12.3%) Ref HRVs *p<0.01, **p<0.05

Serum positive rates: HRVC >> HRVA > HRVB HRVC may be associated with viremia more often than HRVA and HRVB Fuji N et al (submitted)

HRVA HRVB Number of cases of cases Number of cases Number 7.7% 3.6% 5.9%

Days after the onset HRVC Days after the onset

60.0%

36.8%

Number of cases of cases Number 25.0% 14.3% 14.3% 11.1% 20%

Days after the onset

Positive rate peaked 2 days after onset Fuji N et al (submitted) • Serum positive HRVC were distributed in almost all genotypes HRVC • Serum positive HRVA were found in certain HRV genotypes which are B close to HRVC in HRV 5’NCR sequence A

HRVC

Serum sample positive

Conclusion

• Enterovirus 68 (EV68) – EV 68 was detected from 21 cases with severe respiratory infections – Subsequently increased detection of EV 68 was reported from Japan, USA, and Europe – EV 68 can be an important cause of respiratory infection • Human Rhinovirus C (HRVC) – HRVC was detected from 11.6 % of cases with severe respiratory infections – HRVC causes viremia more often and may have different pathogenesis from HRVA and HRVB