An update on the laboratory detection and epidemiology of astrovirus, adenovirus, sapovirus, and enterovirus in gastrointestinal disease
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1 An update on the laboratory detection and epidemiology of astrovirus, adenovirus, sapovirus, and enterovirus in gastrointestinal disease Christopher McIver, Principal Hospital Scientist, Microbiology Department (New South Wales Health Pathology), St George Hospital
2 Outline Astrovirus Literature review since 2000 Adenovirus Emergence of novel strains Enterovirus Sapovirus
3 Outline Astrovirus Literature review since 2000 Adenovirus Emergence of novel strains Enterovirus Sapovirus Established molecular methods need to be updated to ensure coverage
4
5 McIver et al Pathology, 33: 353 N = 412 < 6 years of age Parasites: <3%
6 McIver et al Pathology, 33: 353 N = 412 < 6 years of age Parasites: <3%
7 McIver et al Pathology, 33: 353 N = 412 < 6 years of age Parasites: <3%
8 McIver et al Pathology, 33: 353
9 McIver et al Pathology, 33: 353
10 28.2% McIver et al Pathology, 33: 353
11 McIver et al Pathology, 33: 353
12 RT-PCR Norwalk-like virus 10/172 (5.8%) McIver et al Pathology, 33: 353
13
14 Enteric Rotavirus A Enteric Rotavirus B Detection rate = 2.5% Detection rate = 0.2% Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb 0 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Enteric Norovirus Genogroup I Enteric Norovirus Genogroup II Detection rate = 0.4% Detection rate = 4.7% Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb 0 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Source: Microbiology Department (SEALS), Prince of Wales Hospital, Randwick
15 Enteric Rotavirus A Enteric Rotavirus B Detection rate = 2.5% Detection rate = 0.2% Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb 0 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Enteric Norovirus Genogroup I Enteric Norovirus Genogroup II Detection rate = 0.4% Detection rate = 4.7% Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb 0 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Source: Microbiology Department (SEALS), Prince of Wales Hospital, Randwick
16 Enteric Rotavirus A Enteric Rotavirus B Detection rate = 2.5% Detection rate = 0.2% Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb 0 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Enteric Norovirus Genogroup I Enteric Norovirus Genogroup II Detection rate = 0.4% Detection rate = 4.7% Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb 0 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Source: Microbiology Department (SEALS), Prince of Wales Hospital, Randwick
17 Enteric Adenovirus 40/ Enteric Astrovirus Detection rate = 1.8% Detection rate = 1.7% Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb 0 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Enterovirus Detection rate = 0.8% Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Source: Microbiology Department (SEALS), Prince of Wales Hospital, Randwick
18 Enteric Adenovirus 40/ Enteric Astrovirus Detection rate = 1.8% Detection rate = 1.7% Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb 0 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Enterovirus Detection rate = 0.8% Detection rate = unsure Calicivirus: Sapovirus 50 0 Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Source: Microbiology Department (SEALS), Prince of Wales Hospital, Randwick
19 Common characteristics of select enteric viruses Mild diarrhea (short duration and without dehydration) Persist in the convalescence Epidemics uncommon Non-encapsulated structures Environmentally resilient Resistant to disinfectants Adenovirus DNA Astrovirus, Enterovirus and Sapovirus: ssrna
20 Common characteristics of select enteric viruses Mild diarrhea (short duration of diarrhea and no dehydration) Persist in the convalesence Epidemics uncommon Non-encapsulated structures Environmentally resilient Resistant to disinfectants Adenovirus DNA Astrovirus, Enterovirus and Sapovirus: ssrna
21 Common characteristics of select enteric viruses Mild diarrhea (short duration of diarrhea and no dehydration) Persist in the convalescence Epidemics uncommon Non-encapsulated structures Environmentally resilient Resistant to disinfectants Adenovirus DNA Astrovirus, Enterovirus and Sapovirus: ssrna
22 Enteric viruses have a high predisposition to mutations and recombination New strains are always emerging
23 Astrovirus Appleton and Higgins (1975) cause of diarrhea outbreak in a maternity unit, UK Clinical and serological studies (Kurtz et al. 1979) 8 serotypes associated with human infections Subsequently been isolated in the faeces of animals Testing (EIA and RT-PCR) have established association with diarrhea and vomiting in infants and elderly
24 Astrovirus Appleton and Higgins (1975) cause of diarrhea outbreak in a maternity unit, UK Clinical and serological studies (Kurtz et al. 1979) 8 serotypes associated with human infections Subsequently been isolated in the faeces of animals Testing (EIA and RT-PCR) have established association with diarrhea and vomiting in infants and elderly
25 Astrovirus Appleton and Higgins (1975) cause of diarrhea outbreak in a maternity unit, UK Clinical and serological studies (Kurtz et al. 1979) 8 serotypes associated with human infections Subsequently been isolated in the faeces of animals Testing (EIA and RT-PCR) have established association with diarrhea and vomiting in infants and elderly
26 Children presenting in outpatients (n = 238) and hospital (n = 176) Detection rate: 4.6% McIver et al J Virol. Meth. 84:
27 Children presenting in outpatients (n = 238) and hospital (n = 176) Detection rate: 4.6% McIver et al J Virol. Meth. 84:
28 Children presenting in outpatients (n = 238) and hospital (n = 176) Detection rate: 4.6% McIver et al J Virol. Meth. 84:
29 2014 Source: Bosch et al Clin. Microbiol Rev 2014, 27 (4):1048.
30 2014 Source: Bosch et al Clin. Microbiol Rev 2014, 27 (4):1048.
31 2014 Source: Bosch et al Clin. Microbiol Rev 2014, 27 (4):1048.
32 Genomic structure of Astrovirus Source: Bosch et al Clin. Microbiol Rev 2014, 27 (4):1048.
33 Genomic structure of Astrovirus Non-structural proteins Structural proteins Source: Bosch et al Clin. Microbiol Rev 2014, 27 (4):1048.
34 Micro-mass sequencing to systematically identify viruses present in stools Finkbeiner et al PLoS Pathogens. 4:e
35 ORF1a ORF1b
36 ORF1a ORF1b Phylogenetic analysis of highly divergent astrovirus-like sequence reads
37 ORF1a ORF1b Phylogenetic analysis of highly divergent astrovirus-like sequence reads
38 Phylogenetic analysis of open reading frames ORF1a Serine protease ORF1b polymerase ORF2 capsid Finkbeiner et al Virol J. 5:117
39 Phylogenetic analysis of open reading frames ORF1a Serine protease ORF1b polymerase ORF2 capsid Finkbeiner et al Virol J. 5:117
40 Phylogenetic analysis of open reading frames ORF1a Serine protease ORF1b polymerase ORF2 capsid Finkbeiner et al Virol J. 5:117 Finkbeiner et al J Virol. 83: 10836
41 Phylogenetic analysis of open reading frames ORF1a Serine protease ORF1b polymerase ORF2 capsid Finkbeiner et al Virol J. 5:117 Finkbeiner et al J Virol. 83: 10836
42 Phylogenetic analysis of open reading frames ORF1a Serine protease ORF1b polymerase ORF2 capsid Finkbeiner et al Virol J. 5:117 Finkbeiner et al J Virol. 83: 10836
43 Phylogenetic analysis of open reading frames ORF1a Serine protease ORF1b polymerase ORF2 capsid Finkbeiner et al Virol J. 5:117 Finkbeiner et al J Virol. 83: 10836
44 Khamrin et al J Med Virol. 88:356
45 JAPAN: Finkbeiner (2009) target: Pan-astrovirus consensus primers targeting RdRp 54/330 (16.4%) stools from children with acute gastroenteritis Khamrin et al J Med Virol. 88:356
46 Phylogenetic analysis ORF1b: RdRp Khamrin et al J Med Virol. 88:356
47 Phylogenetic analysis ORF1b: RdRp 17/54 (31.5%) Khamrin et al J Med Virol. 88:356
48 Phylogenetic analysis ORF1b: RdRp 35/54 (64.8%) Khamrin et al J Med Virol. 88:356
49 Phylogenetic analysis ORF1b: RdRp 2/54 (3.7%) Khamrin et al J Med Virol. 88:356
50 Molecular detection of astroviruses 8 classic serotypes 5 conserved end of ORF2 (detection and sequencing) Noel et al J Clin Microbiol, 33:797
51 Molecular detection of astroviruses 8 classic serotypes 5 conserved end of ORF2 (detection and sequencing) Noel et al J Clin Microbiol, 33:797 Novel types ORF1b Chu et al J Virol, 82:9107 Kapoor et al Gen Virol, 90:2965 Finkbeiner et al Emerg Infect Dis, 15:441
52 Adenovirus Non-enveloped DNA viruses, nm Family: Adenoviridae Genus: Mastadenovirus 7 known species A-G: encompassing 68 types =adenovirus&client Protracted diarrhea (compared to rotavirus), lasting 7 8 days Detection rate is higher in developing countries than in developed.
53 Beijing, China /2,233 (9.8%) children Liu et al PLOS, 9:e88791
54 Beijing, China /2,233 (9.8%) children Community-acquired Liu et al PLOS, 9:e88791
55 Beijing, China /2,233 (9.8%) children Hospital-acquired Community-acquired Liu et al PLOS, 9:e88791
56 Beijing, China /2,233 (9.8%) children Hospital-acquired Community-acquired Liu et al PLOS, 9:e88791
57 Adenoviruses have broad tropisms Ghebremedhin Euro J Microbiol and Immunol, 4:26
58 Adenoviruses have broad tropisms Ghebremedhin Euro J Microbiol and Immunol, 4:26
59 Adenoviruses have broad tropisms Ghebremedhin Euro J Microbiol and Immunol, 4:26
60 Adenoviruses have broad tropisms Ghebremedhin Euro J Microbiol and Immunol, 4:26
61 A F G Complete genome sequencing C B D E Robinson et al Scientific Reports, 3:1812
62 A F G Compared to other species: Low mutation rate C Highly dependent on homologous recombination B D E Robinson et al Scientific Reports, 3:1812
63 Liu et al PLOS, 9:e88791
64 Hospitalized children with community-acquired Liu et al PLOS, 9:e88791
65 Hospitalized children with hospital-acquired Liu et al PLOS, 9:e88791
66 Outpatients- community acquired Liu et al PLOS, 9:e88791
67 Adenovirus type 41 (41.6%) Liu et al PLOS, 9:e88791
68 Adenovirus type 31 (19.7%) Liu et al PLOS, 9:e88791
69 Molecular detection of adenoviruses Established methods need to be updated to ensure coverage of newly identified types (based on implementation of genomic analyses) Liu et al PLOS, 9:e88791
70 Molecular detection of adenoviruses Established methods need to be updated to ensure coverage of newly identified types (based on implementation of genomic analyses) Liu et al PLOS, 9:e88791
71 Molecular detection of adenoviruses Established methods need to be updated to ensure coverage of newly identified types (based on implementation of genomic analyses) Liu et al PLOS, 9:e88791
72 Enterovirus Spread by the faecal-oral route Known to cause hand, foot and mouth disease and herpangina Complications including: Meningitis, encephalitis, acute flaccid paralysis, acute cardiopulmonary, respiratory infection, and myocardial injury
73 Enterovirus Diarrhea is less prominent low prevalence and self-limiting nature Enterovirus is not often included in a pathogen screen (before multiplexing PCR) Epidemiologic studies of gastroenteritis Have shown a clinical correlation with detection Most cases were mild without severe symptoms
74 Prevalence studies of enterovirus Japan, patients with gastroenteritis Echoviruses accounted for 625 (4.7%) of infections Infectious agents surveillance report in Japan, 1985 to 1999, Tokyo, Japan Administration of Health and Welfare
75 , India Children < 9 years (n = 2,330) Culture RT-PCR (degenerate primers) Rao et al Infect., Genetics and Evol. 17:153
76 , India Children < 9 years (n = 300) Culture RT-PCR (degenerate primers) Rotavirus 13.8% Rao et al Infect., Genetics and Evol. 17:153
77 , India Children < 9 years (n = 300) Culture RT-PCR (degenerate primers) Rotavirus 13.8% Enterovirus 16.3% Rao et al Infect., Genetics and Evol. 17:153
78 , India Children < 9 years (n = 300) Culture RT-PCR (degenerate primers) Rotavirus 13.8% Enterovirus 16.3% 77.4% confirmed by RT-PCR Broad specificity Rao et al Infect., Genetics and Evol. 17:153
79 , India Children < 9 years (n = 300) Culture RT-PCR (degenerate primers) Rotavirus 13.8% Enterovirus 16.3% Serotyped (genotyped) according to the VP1 sequence analysis Rao et al Infect., Genetics and Evol. 17:153
80 , India Rao et al Infect., Genetics and Evol. 17:153 N = 380
81 , India Rao et al Infect., Genetics and Evol. 17:153 Associated with serious diseases in children such as septic meningitis and acute flaccid paralysis Non-polio acute flaccid paralysis: 66 serotypes, EV71, E13 and CBV5 frequently detected In contrast, only 37 serotypes besides the 3 OPV types are detected in diarrheal children
82 , India Rao et al Infect., Genetics and Evol. 17:153 E11 Major cause of highly fatal perinatal/neonatal disseminated infections involving multiple organs causing serious severe systemic illness Non-polio acute flaccid paralysis: 66 serotypes, EV71, E13 and CBV5 frequently detected In contrast, only 37 serotypes besides the 3 OPV types are detected in diarrheal children
83 , India Enterovirus-positive diarrhea samples 0.64% co-infection with rotavirus Diarrheagenic E. coli: 2% Rao et al Infect., Genetics and Evol. 17:153
84 , India ROTAVIRUS Enterovirus-positive diarrhea samples 0.64% co-infection with rotavirus Diarrheagenic E. coli: 2% Rao et al Infect., Genetics and Evol. 17:153
85 , India Enterovirus Enterovirus-positive diarrhea samples 0.64% co-infection with rotavirus Diarrheagenic E. coli: 2% Rao et al Infect., Genetics and Evol. 17:153
86 , India Enterovirus-positive diarrhea samples 0.64% co-infection with rotavirus Diarrheagenic E. coli: 2% Rao et al Infect., Genetics and Evol. 17:153
87 Guangdong, China enterovirus-related hand, foot, mouth disease/ herpangia Serotypes identified in 763 cases (with and without diarrhea) Serotypes tend to reflect circulating strains No strong association of diarrhea with specific strains Zhou et al BMC Infect Dis, 16:128
88 Molecular detection of enteroviruses VP1 Target Study by Rao et al sets of species-specific degenerate primers Rao et al. 2013: Used VP1 region (above)and 5 UTR using 2 sets of species-specific degenerate primers: 3 degenerate primers Forward: Specific for 4 enterovirus species Reverse primer for species A and B Reverse primer for species D
89 Sapovirus First identified in outbreak in Sapporo, Japan in 1977 Responsible for gastroenteritis in all ages in both outbreaks and sporadic cases world-wide Clinical symptoms are indistinguishable from noroviruses Increasing reports of infections (particularly in SE Asia) suggest a rise in prevalence and virulence
90
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95 Genogroups Genotypes GI.1 GI.7 GIV.1 GV.1 and GV.2 GII.1 GII.7 Porcine Oka et al CMR, 28:32
96 Genogroups Genotypes GI.1 GI.7 GIV.1 GV.1 and GV.2 GII.1 GII.7 Porcine Oka et al CMR, 28:32
97 Genogroups Genotypes GI.1 GI.7 GIV.1 GV.1 and GV.2 GII.1 GII.7 Porcine Oka et al CMR, 28:32
98 Sapovirus: local prevalence study 95 stool samples < 18 years (Sydney Children s Hospital) Negative: Salmonella, Shigella and Campylobacter,Rotavirus, Adenovirus, Astrovirus and Norovirus Sapovirus detected in 7/95 (7.4%) stool samples 9mths - 7 years Other studies in Australia in 1998, 2001, and 2005: % of the total caliciviruses detected Hansman et al Emerg Infect Dis., 12:141
99 Sapovirus: local prevalence study Hansman et al Emerg Infect Dis., 12:141
100 Sapovirus: local prevalence study Hansman et al Emerg Infect Dis., 12:141
101 Molecular detection of sapovius Reverse transcription-pcr is a major and routine method for detection in clinical specimens Due to high diversity of sapoviruses, most assays include multiple or degenerate primers Currently, limited primer sets have demonstrated the ability to detect all genogroups of human sapoviruses *Oka et al CMR, 28:32
102 Molecular detection of sapovius Target Number of methods cited* Partial RdRP 11 Cross-react with norovirus, rotavirus and astrovirus RdRp-VP1 junction 11 Have the highest detection rate, recommended for clinical testing Partial VP1 9 Contains capsid coding region (preferred method, products can be sequenced for genotyping) *Oka et al CMR, 28:32
103 Concluding remarks Astrovirus, adenovirus, sapovirus, and enterovirus are common causes of gastroenteritis in Australia.
104 Concluding remarks Astrovirus, adenovirus, sapovirus, and enterovirus are common causes of gastroenteritis in Australia. These enteric viruses (exception of sapovirus)are now routinely screened in cases of gastroenteritis using capture enzyme immunoassays and molecular techniques including multiplex PCRs and allow epidemiologic surveillance of these putative agents.
105 Concluding remarks Astrovirus, adenovirus, sapovirus, and enterovirus are common causes of gastroenteritis in Australia. These enteric viruses (exception of sapovirus)are now routinely screened in cases of gastroenteritis using capture enzyme immunoassays and molecular techniques including multiplex PCRs and allow epidemiologic surveillance of these putative agents. In developing countries, these viruses have a higher prevalence and recent studies based on genomic analysis have revealed the emergence of newer strains due to homologous recombination and mutations.
106 Concluding remarks Astrovirus, adenovirus, sapovirus, and enterovirus are common causes of gastroenteritis in Australia. These enteric viruses (exception of sapovirus)are now routinely screened in cases of gastroenteritis using capture enzyme immunoassays and molecular techniques including multiplex PCRs and allow epidemiologic surveillance of these putative agents. In developing countries, these viruses have a higher prevalence and recent studies based on genomic analysis have revealed the emergence of newer strains due to homologous recombination and mutations. Established methods need to be updated periodically to ensure coverage of newly identified types.
107 Acknowledgements William Rawlinson, Grant Hansman Virology Research Laboratory, Microbiology Department, Prince of Wales Hospital Peter White, Elise Tu, Rowena Bull, Mi-Jurng Kim, John Sebastian Eden School of Biomolecular Sciences and Biotechnology, University of NSW Ruth Bishop, Enzo Palombo, Hussein Mustafa, Carl Kirkwood Department of Gastroenterology, Royal Children s Hospital, Melbourne Michael Catton, John Marshall, Jennifer Doultree Victorian Infectious Diseases Research Laboratories, North Melbourne
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