Classification of picornaviridae

Epidemiology and etiological roles <strong>of</strong><br />

two picornaviruses:<br />

human rhinovirus C and enterovirus 68<br />

Tohoku University Graduate School <strong>of</strong> Medicine<br />

Hitoshi Oshitani<br />

<strong>Classification</strong> <strong>of</strong> <strong>picornaviridae</strong><br />

• Genus <strong>of</strong> ‘Enterovirus’<br />

– Human enterovirus<br />

• Human enterovirus A<br />

• Human etnerovirus B<br />

• Human enterovirus C<br />

• Human enterovirus D<br />

– Human rhinovirus<br />

• Human rhinovirus A<br />

• Human rhinovirus B<br />

• Human rhinovirus C<br />

ICTV Picornaviridae Study Group

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Human enteroviruses and rhinoviruses<br />

• Common virological<br />

characteristics<br />

– Positive strand, non‐enveroped<br />

virus<br />

– Same genomic structure<br />

• Pathogenesis and clinical<br />

diseases are different<br />

– Enterovirus:<br />

• Resistant to acid<br />

• Causing enteric / systemic<br />

infections, e.g. poliovirus<br />

– Rhinovirus:<br />

• Sensitive to acid<br />

• Only respiratory infection<br />

Human<br />

rhinoviruses<br />

Human<br />

enteroviruses<br />

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

Human enteroviruses and rhinoviruses<br />

Respiratory infection<br />

Human<br />

rhinovirus A<br />

Human<br />

rhinovirus B<br />

Enterovirus<br />

68<br />

Human<br />

rhinovirus C<br />

??<br />

Enteric / systemic<br />

infection<br />

Human<br />

enteroviruses

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Background <strong>of</strong> the study<br />

• Etiological study on pediatric<br />

pneumonia<br />

– Eastern Visayas Regional Medical<br />

Center, Taclobal City, Leyte Island in the<br />

Philippines<br />

– May 2009 –present<br />

– Part <strong>of</strong> J‐GRID (Japan Initiative for<br />

Global Research Network on Infectious<br />

Diseases) supported by the Japanese<br />

government<br />

Pediatric Pneumonia Study<br />

•Patients<br />

‐ Aged between 7 days and<br />

14 years<br />

‐ Severe pneumonia by the<br />

definition <strong>of</strong> Integrated<br />

Management <strong>of</strong> Childhood<br />

Illness (IMCI)<br />

•Respiratory viruses were<br />

detected by multiplex PCR<br />

Tacloban

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Eastern Visayas Regional Medical Center<br />

(EVRMC)<br />

May 2008 –May<br />

2009 (n = 819)<br />

Outcome <strong>of</strong> patients<br />

HAMA-<br />

Improved,<br />

151, 18%<br />

Others, 30,<br />

4%<br />

Died, 70, 9%<br />

HAMA-<br />

Deteriorated<br />

, 18, 2%<br />

Discharged,<br />

550, 67%<br />

HAMA: Home against medical advice

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Negative<br />

Negative<br />

Multiple<br />

Multiple<br />

Identified viruses<br />

WU/KI<br />

Corona<br />

Boca<br />

RSV-A<br />

RSV-B<br />

Rhino-A<br />

Rhino-B<br />

May 2008 –May<br />

2009 (n = 819)<br />

Rhino-C<br />

hMPV<br />

FluA Viruses were detected<br />

EV68<br />

FluB<br />

Adeno<br />

in 506 cases (61.2 %)<br />

Identified viruses<br />

WU/KI<br />

Corona<br />

Boca<br />

RSV-A<br />

RSV-B<br />

Rhino-A<br />

Rhino-B<br />

May 2008 –May<br />

2009 (n = 819)<br />

Rhino-C<br />

hMPV<br />

FluA Viruses were detected<br />

EV68<br />

FluB<br />

Adeno<br />

in 506 cases (61.2 %)

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Enterovirus 68<br />

1. Detected from hospitalized children with respiratory<br />

infection in 1962. Only sporadic cases have been reported<br />

2. Acid sensitive, grows at lower optimal temperture<br />

2. Isolation is relatively difficult.<br />

EV68 HRV Other EV<br />

Acid sensitive Yes Yes No<br />

Optimum growth temp. 33℃ 33℃ 37℃<br />

Respiratory<br />

tract<br />

Intestine<br />

(Mainly)<br />

* HRV and EV68 shares phenotypic characterizations<br />

<strong>Classification</strong> <strong>of</strong> Human Enteroviruses<br />

Coxsackievirus B1‐6, Coxsackievirus A9,<br />

Enterovirus 69, 73, 74, 75, 77, 78, Echovirus1‐<br />

7, 9,11‐21,24‐27,29‐33<br />

Coxsackievirus A2‐10, 12,14,16 & Enterovirus 71,76<br />

Coxsackievirus<br />

A1,11,13,15,17‐22,24<br />

Enterovirus 68,70, 94<br />

Poliovirus 1‐3<br />

Species Japan USA<br />

A 3613(48%) 1520(3.3%)<br />

B 3949(52%) 44418(96.3%)<br />

C 7 (0.09%) 176(0.38%)<br />

D 14(0.18%) 26 (0.05%)<br />

(From enterovirus surveillance data)<br />

• Only 3 serotypes in Human<br />

enterovirus D<br />

• Detection <strong>of</strong> human enterovirus<br />

D has been very rare

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Methods<br />

1) Amplify partial viral genome by RT‐PCR<br />

Screen samples by PCR targeting 5’NTR <strong>of</strong> HRV<br />

⇢ Submit positive samples to PCR targeting VP1 <strong>of</strong> EV68<br />

↓<br />

Genetic analysis using Mega 3.1 s<strong>of</strong>tware<br />

2) Epidemiological analysis using collected patients’ information<br />

Eastern<br />

Visayas<br />

region<br />

Babatngon<br />

0 200km<br />

0 5km<br />

Tacloban city<br />

Palo<br />

Dulag<br />

Basey<br />

N<br />

• Enterovirus 68 was detected<br />

from 21 cases with severe<br />

pneumonia, from October<br />

2008 and February 2009, 2<br />

<strong>of</strong> them died<br />

Imamura T et al. Emerg Infect Dis. Vol 17:<br />

1430‐1435, 2011

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Phylogenetic tree (5’NTR)<br />

5’NCR(232bp)<br />

Bootstrap x500<br />

Phylogenetic tree (VP1)<br />

VP1(296bp)<br />

Bootstrap x500<br />

EV68<br />

EV68, Philippines<br />

EV68, USA<br />

EV68, France

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Enterovirus 68 detection in Japan<br />

Increased detection <strong>of</strong> enterovirus 68 in 2010 in Japan<br />

Infectious Agents Surveillance Report, NIID, Japan<br />

Increased detection <strong>of</strong> Enterovirus 68<br />

MMWR (in press)<br />

Increased detection <strong>of</strong> enterovirus 68 in Philippines (2008‐2009), USA<br />

(2009‐10), Japan (2010) and Netherland (2010)

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Recent increase in Enterovirus 68 in<br />

different parts <strong>of</strong> the world<br />

• Two possibilities<br />

– Increased detection<br />

• Most studies detected Enterovirus 68 by PCR targeting<br />

rhinovirus detection<br />

– Global epidemic<br />

• Due to emergence <strong>of</strong> different virus: antigenicity,<br />

virulence, transmissibility<br />

Phylogenetic tree <strong>of</strong> recent enterovirus 68<br />

Japan (Yamagata)<br />

Japan (Sendai)<br />

Japan (Osaka)<br />

Philippines<br />

Nederland

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Human rhinovirus C<br />

• Newly identified species <strong>of</strong><br />

human rhinovirus<br />

• No virus isolation technique<br />

established<br />

• Phonotypic characteristics<br />

and pathogenesis are largely<br />

unknown<br />

– Associated with more severe<br />

respiratory infections?<br />

Renwick et al JID 2007; 196: 1754‐60<br />

Monthly distribution <strong>of</strong> HRVs<br />

2008 2009 2010 2011<br />

HRVs 447<br />

(28.9 %)<br />

229<br />

HRVA (14.3%)<br />

HRVB 32<br />

(2.0%)<br />

HRVC 186<br />

(11.6%)<br />

Total 1597

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10 %<br />

died<br />

2008<br />

2009<br />

2010<br />

No death<br />

4 %<br />

died<br />

Phylogenetic trees <strong>of</strong> detected<br />

rhinoviruses<br />

HRVA<br />

HRVB<br />

HRVC<br />

• Many different genotypes were seen for HRVA,<br />

HRVB and HRVC<br />

• No significant clustering<br />

HRV<br />

A<br />

HRVB<br />

Wheezing<br />

HRVC<br />

Wheezing was more frequently<br />

seen in HRVC than HRVA

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Number <strong>of</strong> cases<br />

Detection <strong>of</strong> rhinovirus RNA in serum samples<br />

2008.05-2009.05<br />

Serum that were collected from whose nasopharyngeal samples were pos. for HRVs<br />

Nasopharyngeal<br />

swabs (n=816)<br />

serum<br />

Positivity (%) Sample No.<br />

Serum- positivity<br />

(%)<br />

OR (95%CI)<br />

HRVA 135 (16.5%) 135 4 (3%) * 0.22 (0.08-0.62)<br />

HRVB 25 (3.1%) 25 0** -<br />

HRVC<br />

Total<br />

HRVs<br />

83 (10.2%)<br />

243<br />

83<br />

243<br />

26 (31.3%)*<br />

30 (12.3%)<br />

6.84 (3.53-13.23)<br />

Ref<br />

*p HRVA > HRVB<br />

HRVC may be associated with viremia more <strong>of</strong>ten than HRVA and HRVB<br />

7.7% 3.6% 5.9%<br />

HRVA HRVB<br />

Days after the onset HRVC<br />

Days after the onset<br />

Number <strong>of</strong> cases<br />

25.0%<br />

60.0%<br />

Positive rate peaked 2 days after onset<br />

Number <strong>of</strong> cases<br />

36.8%<br />

14.3%<br />

14.3%<br />

11.1% 20%<br />

Days after the onset<br />

Fuji N et al (submitted)<br />

Fuji N et al (submitted)

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HRV<br />

B<br />

HRVC<br />

HRVC<br />

HRV<br />

A<br />

Conclusion<br />

• Serum positive HRVC<br />

were distributed in<br />

almost all genotypes<br />

• Serum positive HRVA<br />

were found in certain<br />

genotypes which are<br />

close to HRVC in<br />

5’NCR sequence<br />

Serum sample<br />

positive<br />

• Enterovirus 68 (EV68)<br />

– EV 68 was detected from 21 cases with severe respiratory<br />

infections<br />

– Subsequently increased detection <strong>of</strong> EV 68 was reported from<br />

Japan, USA, and Europe<br />

– EV 68 can be an important cause <strong>of</strong> respiratory infection<br />

• Human Rhinovirus C (HRVC)<br />

– HRVC was detected from 11.6 % <strong>of</strong> cases with severe<br />

respiratory infections<br />

– HRVC causes viremia more <strong>of</strong>ten and may have different<br />

pathogenesis from HRVA and HRVB