New respiratory viruses

Dr Klara Pósfay Barbe

Hôpital des Enfants Geneva, Switzerland

2nd Pediatric Infectious Disease Training Course

Respiratory tract infections § Leading cause worldwide of morbidity and

mortality (5 Mio in kids < 5yo/y from resp virus) § Etiologic agent often unknown

– E.i.: CAP cultures detected agents in only 46% § Most LRTI due to viruses: max 40% viruses

identified even with PCR § In children: RSV, parainfluenza viruses,

influenza = major causes of bronchiolitis and LRTI

§ Ease of spread; children 2­3x more infected than adults

Ruiz M et al; Am J Respir Crit Care Med 1999; 160:397

Until recently

RSV parainfluenza influenza rhinovirus …

Etiologies of LRTI & URTI in children

???

History § Since 2001, many new human viruses

discovered thanks to molecular techniques – Amplification by random PCR

(a procedure using a generic primer sequence with a random 3' end)

– Cloning of the PCR products – Large­scale sequencing of the clones – Automated editing and database

searches of the sequencing results

Rhinovirus

Influenza

RSV

Parainfluenza

Hum. metapn.

Coronavirus

Enterovirus

Adenovirus

Bocavirus

1956, 2007 “new HRVs”

1933

1956

1956

2001

1965, SARS, NL63, HKU1 2003­05

1940s ­ 50s

1953

2005

Panel of > 20 RT­PCR assays

Lecture outline

§ New respiratory viruses: why are they important? § Human metapneumovirus (hMPV) § Coronavirus NL63 (HCoV) § Bocavirus (HBoV)

Respiratory tract infections : why are they important?

§ Cohort of 197 newborns in Bern § Weekly standardized interview § First acute respiratory infection with

cough or wheeze § Nasopharyngeal swab: baseline & at

week 3

Kaiser L et al PIDJ 2006; Regamey N et al. PIDJ 2007

Respiratory tract infections during the first year of life

0.00 0.25 0.50 0.75 1.00 0

25

50

75

100

age (years)

% of infants with

ARI

16% virus negative

84% virus positive

112 episodes in 197 newborns (67%)

Regamey N et al. PIDJ 2007

Visit to general practitioners

HRV HCoV PIV RSV HMPV Others Mixed No virus 0

10

20

30

40

50

60

70

% of cases with

visit to GP

Regamey N et al. PIDJ 2007

Severity of symptoms

HRV HCoV PIV RSV Others Mixed No virus

0

1

2

3

4

5

6

7

8

9

Total sym

ptom

severity

score

„Common­cold“ viruses

„Classical“ viruses

p=0.57 p=0.002

p=0.01

HRV HCoV PIV RSV Others Mixed No virus

0

1

2

3

4

5

6

7

8

9

Total sym

ptom

severity

score

„Common­cold“ viruses

„Classical“ viruses

p=0.57 p=0.002

p=0.01

Regamey N et al. PIDJ 2007

Viral distribution

38%

Virus n (%)

RSV A – B 17 (15.2) Para 1 – 3 19 (17.0) Flu A – B 4 (3.6) Adenovirus 3 (2.7)

38

Virus n (%)

RSV A – B 17 (15.2) Para 1 – 3 19 (17.0) Flu A – B 4 (3.6) Adenovirus 3 (2.7)

48%

Rhinovirus 26 (23.2)

Coronavirus OC43 7 (6.3) Coronavirus E229 3 (2.7) Coronavirus NL63 9 (8.0) Coronavirus HKU1 1 (0.9)

Metapneumovirus 2 (1.8)

43%

Rhinovirus 26 (23.2)

Coronavirus OC43 7 (6.3) Coronavirus E229 3 (2.7) Coronavirus NL63 9 (8.0) Coronavirus HKU1 1 (0.9)

Metapneumovirus 2 (1.8)

Dual or triple infections in 9 cases

Virus negative 16%

Regamey N et al. PIDJ 2007

Conclusion

§ These “unknown” viruses might have an important

– Clinical impact – Socioeconomic impact – Longterm impact (asthma, bronchiectasis,

etc.) – Infection control issues

Lecture outline

§ New respiratory viruses: why are they important? § Human metapneumovirus (hMPV) § Coronavirus NL63 (HCoV) § Bocavirus (HBoV) § Others?

Human metapneumovirus (hMPV)

§ First described in 2001 § Has been circulating for > 50 years

(banked serologies) § By the age of 5 yo, ~100% have

serological evidence of hMPV infection

van den Hoogen BG et al. Nat Med 2001; 7: 719 Leung J et al. J Clin Microbiol 2005; 43:1213

hMPV: the virus § Paramyxovirus (subfamily Pneumovirinae) § Enveloped, single­stranded RNA virus of negative

polarity (genome must be transcribed to mRNA to be translated into protein)

§ Different from RSV: lacks 2 non­structural proteins and has a slightly different gene order

§ Two main lineages: A (A1 & A2) and B (B1 & B2) § Nucleotide sequence variation between A & B:

11.8­47.7% § Different genotypes may co­circulate during the

same year

Mackay Im et al. J Infect Dis 2004; 190:1913 Principi N et al. Clin Microbiol Infect 2006; 12: 301

Most frequent? More severe?

hMPV: local changes § Increased inflammatory cell infiltrates

(mononuclear mostly) in the lung interstitium § Increased myofibroblast thickening

near airway epithelium § Cell degeneration/necrosis § Can persist several weeks in the lungs

despite immune response (strategy to overcome host defense?)

Alvarez R et al. J Virol 2004; 78:6927

Day 0 Day 2

Day 4 Day 7

Day 10 Day 14

Alvarez R et al. J Virol 2004; 78:6927

Histopathology of HMPV infection in mice. Interstitial inflammatory cell infiltrates were examined in the lung

hMPV: epidemiology

§ Worldwide distribution § Season: overlap with RSV & influenza § Mostly found in infants, young children,

elderly and immunocompromised § Uncommon asymptomatic infection

Williams JV et al. NEJM 2004; 350:443

hMPV: clinical manifestations

§ Causes URI and LRTI (pneumonia) § Often bronchiolitis § Associated with

– Febrile seizures – Rash – Diarrhea – Enlarged liver – Altered liver function tests

Peiris JS et al. Emerg Infect Dis 2003; 9:628

hMPV: diagnosis

§ Poor replication in cell culture § Cytopathic effect in certain cell lines

only § Confirmation by RT­PCR § New assays ≤ 2 hours § Serologies by ELISA? § Stays detectable by PCR for 1 month in

NP aspirate at room temperature Bonroy C et al. Clin Microbiol Infect 2007; 13: 504

Difference between hMPV & RSV & influenza viruses

§ 1505 children: RT­PCR of NP swabs – 2.8% hMPV; 9.5%* RSV; 15.3%* influenza viruses – 16.7% dual infection (1x RSV, 6x flu) – hMPV+: all acute respiratory infection – Fever* if hMPV + & influenza + § Families more sick, more medical visits, more

antipyretics than RSV+ è socioeconomic impact – Wheezing* if hMPV+& RSV+

Bosis S et al. J Med Virol 2005; 75(1): 101 Principi N et al. Clin Microbiol Infect 2006; 12: 301

hMPV & RSV co­infection § 45 infants ventilated for RSV bronchiolitis:

bronchoalveolar lavage ; RT­PCR for hMPV § BAL cytokines & chemokine by ELISA

§ 68% also hMPV positive (demographics, symptoms° and PICU data similar if RSV+ / hMPV+ or ­)

° except for length of time between symptoms and intubation (longer if dual)

§ No difference in cellularity, cytokines (TNF, IFNγ, IL­ 4,­9,­10,­13) and chemokines (CXCL8 &10, CCL2, 3,5,11): timing? different production sites?

McNamara PS et al. Pediatr Pulmonol 2007 Mejias A et al. ICAAC 2007; V­1258

§ Other studies – If PICU for RSV+ bronchiolitis, 70% co­

infected with hMPV – 10x increase RR of admission in PICU for

mechanical ventilation if RSV+ & hMPV+, especially if < 3 years old

In contrast: hMPV & RSV co­ infection

Greensill J et al. Emerg Infect Dis 2003; 9:372 Semple MG et al. JID 2005; 191:382 Konig B et al. J Clin Microbiol 2004; 42:4632

è Influences the severity of RSV infection?

hMPV & other viruses

§ 185 non­influenza clinical samples (NP, sputum, throat, tracheal) in pediatrics § 22.2% hMPV positive § Co­infection with adenovirus or SARS

possible § Cause of underestimation of burden of

disease : only other virus­negative tested for hMPV?

Kaida A et al. Microbiol Immunol 2007

hMPV & bacteria

§ Co­infection with S. pneumoniae plays a role in inducing lower respiratory tract infection § Other bacteria coinfection:

– S. aureus, Stenotrophomonas maltophilia: clinical link not determined

Madhi SA et al. JID 2006 Boivin G et al. JID 2002

hMPV in Switzerland

§ 1’500 NP samples (PCR for hMPV) in children with respiratory illness § 5% + hMPV (vs 19.5% RSV) § hMPV:

– mean age 32 mo* (vs 16 mo) – 45% admitted (vs 54%) – 18% intensive care (vs 8%)[70% dual infection] – 77% LRTI

Baer G et al; Eur J Pediatr 2007

Treatment for hMPV

§ No treatment available § Monoclonal antibody (mAb 338)

against the fusion protein of hMPV in mouse model – Decreased viral titers – Decreased airway obstruction – Less severe perivascular, alveolar and

interstitial inflammation on day 5 & 42

Hamelin M et al; ICAAC 2007; V­1259

Treatment for hMPV § hMPV fusion protein (F) is most

important target of protective immunity

§ Production of a fully human monoclonal antibody fragment (Fab DS7)

§ Fab intranasally 3 days post hMPV infection

§ 24 hours later: – > 1500­fold reduction in viral titers

in lungs if treated – 4­fold reduction in nasal tissues – Dose­response

Williams JV et al. J Virol 2007; 81(15): 8315

Nose

Lung

Coronavirus

The common cold virus

Coronaviruses: HCoV

§ SARS­CoV most aggressive human CoV probably originated in wild animal reservoir

Masters PS. Adv Virus Res. 2006;66:193

Other theory

Coronaviruses § 1960s: HCoV­OC43 & ­229E: common colds § 2003: SARS­CoV § 2003: HCoV­NL63 § 2004: HCoV­HKU1

§ Worldwide distribution § Positive­stranded RNA viruses (largest viral genome

among RNA viruses); spiked membrane proteins (crown­like structure!)

Not tested in humans, No animal models, No cultures possible for HKU

Masters PS. Adv Virus Res. 2006;66:193

HCoV­NL63 § Closely related to HCoV­229E (65% sequence identity)

§ 1240 NP specimen (negative for other known viruses) in children with ARI – 2.1% positive for HCoV­NL63 – Mostly February­March (none August­November)

– 7 days ­ 9.5 years (mostly < 1yo) – Male: female= 2: 1

§ Shares with SARS­CoV receptor for target cell entry (treatment ??)

Bastien N et al. J Clin Microbiol 2005; 43: 4567 Esper F et al. JID 2005; 191:492 Van der Hoek L et al. PLoS Med 2005; 2:e240

In literature: 1.3­8.8%

Except in Hong­Kong: Spring­summer

HCoV: clinical presentation § Severe LRTI, pneumonia, bronchiolitis, but usually not

lethal § URTI (fever, cough , rhinorrhea)

– up to 40° ; mean duration 2.6 days +/­1.2 days (≠RSV or Influenza A)

§ Febrile seizures: – 38% HCoV­HKU1 – 6% HCoV­OC43

§ Typical of CoV­NL63: croup (6.6 x more likely if +)

§ Mostly in children, adults with underlying disease or elderly

§ More often found in outpatients Van der Hoek L. Antiviral Therapy 2007; 12: 651 Hayden FG. Curr Opin Infect Dis 2006; 19:169

P< 0.05

HCoV­NL63

§ Co­infection frequent >50% (influenza, RSV,…)

§ Nosocomial acquisition? § Lifelong immunity? Adults + maternal

antibodies not efficient? § Viral load high in NP during first 1­2 days,

than decreases (=influenza, ≠ SARS)

Chiu SS et al. CID 2005; 40:1721 Wu PS et al. Eur J Pediatr 2007; Feb Van der Hoek L et al. PLoS Med 2005

HCoV in Switzerland

Birth cohort § 7% HCoV­NL63 § 9% other coronaviruses § ½ cases were negative by PCR 3­

weeks after onset of illness § But…½ cases were positive by PCR 3

weeks after onset, even if symptom­ free

Kaiser L et al. PIDJ; 2005; 24: 1015

HCoV­NL63: importance § 2’060 children < 15 yo

in ER: NP aspirates (RT­PCR) § HCoV: 79 cases(3.8%)

– 72.2% only HCoV – 13 cases HCoV­NL63 – Mild disease – Few secondary cases

§ 1890 patients with pneumonia (RT­PCR) § HCoV: 64 cases (3.3%)

– 69.5% only HCoV – 8 cases HCoV­NL63 – Association between

HCoV­NL63 & pneumonia = protective!

Esposito S et al. J Med Virol 2006; 78:1609 Dare RK et al. JID 2007; 196: 1321

Limited clinical and socioeconomic impact of HCoV­NL63

HCoV: treatment

§ IVIG seems to be a HCoV­NL63 inhibitor § Inhibition of viral replication via peptides

derived from HR2 domain § Inhibition of viral replication through RNA

interference § Transcriptional level: pyrimidine analogues § Protease inhibitors (post­translational

processing): for ex. N3

Van der Hoek L et al. FEMS Microbiol Rev 2006; 30:760

Human Bocavirus (HBoV)

HBoV § 2005: described by Allander et al § Close to Parvovirus § Worldwide distribution § Mainly in children > 6 mo (=hMPV, ≠RSV) &

less than 24 mo § Few reports in adults § Detection by RT­PCR § Transmission? Nosocomial? § Incubation time?

Allander T et al. Proc Natl Acad Sci USA 2005; 102:12891 Völz S et al. J Clin Virol 2007; 40: 229

no culture, no animal model

HBoV: infection

§ In animals, similar viruses cause diarrhea (Bovine parvovirus; Canine minute virus)

§ Doesn’t (yet?) fulfill Koch’s postulates – Frequency increased if symptomatic

Koch’s postulates

1. The microorganism must be found in all organisms suffering from the disease (but not in healthy organisms)

2. The microorganism must be isolated from a diseased organism and grown in pure culture

3. The cultured microorganism should cause disease when introduced into a healthy organism

4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent

HBoV: infection

§ Associated with resp. tract infections – Cough, rhinorrhea, fever up to 39.5°

§ Associated with gastrointestinal disease (~25%?) – Vomiting, diarrhea – Fecal excretion

§ Seasonality: late winter­early spring? Canada: no preferred season

Arnold JC et al. CID 2006; 43:283 Vicente D et al; Emerg Infect Dis 2007; 13: 636 Bastien N et al. J Clin Microbiol 2006; 12: 848

Völz S et al. 2007

HBoV: prevalence § Range (0) 1.5­19% § In Switzerland (healthy birth cohort):

– 4.5% – 80% associated with other virus – Rapidly cleared (< 3 weeks, except for 1

patient) § Probably life­long immunity (same

strain for RTI and diarrhea; lack of variation in the surface protein)

Regamey N et al. 2007; PIDJ; 26: 147

95 1.6 yo 19 259 Allander T 2007

Same season RTI

& fecal

33 56

< 5 years 5 / 6.9; 2.1

400/1200; 1435 fecal

Lau SKP 2007

< 5 years 0.8 962 with gi sympt

Lee JI 2007

71 0­adults 13.8; 43!!!; 0.8

225 symp kids; 100 asympto;

126 adults with COPD or

pneumonia

Longtin J 2007 (ICAAC)

Found in stool and

urine

60 < 14 yo 13.4 917 Pozo 2007

Co­infection= modifyer of

disease?

36 9 mo (3­17)

2.8 389 Völz 2007

Co­inf %

Age (range)

+HBoV in %

N

HBoV: Co­infections

§ RSV § Adenovirus § Rhinovirus § Norovirus § Other respiratory viruses § Rotavirus, astrovirus, … § Several viruses

Pozo F et al. J Clin Virol 2007; 40: 224 Völz S et al 2007

HBoV: to think about….

§ 16 patients with Kawasaki disease § 31.2% + for HBoV

§ 259 children admitted for wheezing: – PCR, Cultures, Ag detection, serologies

(16 viruses): 95% + results (19% HBoV) – High viral loads if HBoV alone & wheezing – Found also in serum: systemic infection

Catalano­Pons C et al. Clin Microbiol Infect 2007; 13: 1220 Allander T et al. CID 2007; 44: 904

In summary: hMPV, HCoV& HBoV

§ In common

– Worldwide distribution – Season: end of winter – Children (elderly) – URTI & LRTI – Co­infections frequent – No treatment….yet

In summary: hMPV, HCoV& HBoV

~not in adults? 2:1 male: female

Two strains: co­ circulation

positive

Kawasaki? Mild illness Influences severity of RSV?

Also gastrointestinal

disease

Croup Bronchiolitis

6­24 mo <1 yo Age: 2­3 yo

HBoV HCoV­NL63 hMPV

Are we done?

Preview…

§ Characterisation of a newly identified human rhinovirus, HRV­QPM, discovered in infants with bronchiolitis § Identification of a third polyomavirus § Identification of a Novel Human

Polyomavirus from patients with ARTI (= KI & WU polyomaviruses)

P. McErlean et al. J Clin Virology; 2007; 39: 67 Allander T et al. J Virol; 2007 Gaynor AM PLoS Pathogens, in press

Papers to read

hMPV Principi N et al. Human Metapneumovirus in paediatric patients.

Clin Microbiol Infect 2006; 12: 301­308

HCoV (­NL63) Van der Hoek L. Human coronaviruses: what do they cause?

Antiviral Therapy 2007; 12: 651­658 Pyrc K et al. The novel Human Coronavirus NL63 and HKU1. J Virol

2007; 81(7): 3051­7

HBoV Völz S et al; Prospective study of HBoV infection in pediatric

university hospital in Germany 2005/2006. J Clin Virol 2007; 40:229­235