Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR

Dovas, Chrysostomos Ι.; Papanastassopoulou, Maria; Georgiadis, Marios; Chatzinasiou, E.; Maliogka, Varvara I.; Georgiades, George

doi:10.1128/aem.01929-09

Cited by 42 publications

(27 citation statements)

References 43 publications

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“…Although viral particles may not be infectious anymore, their RNA is still protected from degradation in the matrix protein and the core and could consequently be detected by qRT‐PCR (HA5, NA1 and M genes) 3 . As already described in a previous study, 32 PCR inhibitors were detected in almost 30% of the mud samples, but even after serial dilutions, these specimens still tested negative by HA5 qRT‐PCR (data not shown).…”

Section: Discussionsupporting

confidence: 61%

Environment: a potential source of animal and human infection with influenza A (H5N1) virus

Horm

Gutiérrez

Sorn³

et al. 2012

Influenza Resp Viruses

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Please cite this paper as: Horm et al. (2012) Environment: a potential source of animal and human infection with influenza A (H5N1) virus. Influenza and Other Respiratory Viruses 6(6), 442–448. Background Very little is known regarding the persistence of highly pathogenic avian influenza H5N1 viruses in natural settings during outbreaks in tropical countries, although environmental factors may well play a role in the persistence and in the transmission of H5N1 virus. Objective To investigate various environmental compartments surrounding outbreak areas as potential sources for H5N1 virus transmission. Methods Environmental specimens were collected following outbreaks of avian influenza in Cambodia between April 2007 and February 2010. The methods used to concentrate H5N1 virus from water samples were based either on agglutination of the virus with chicken red blood cells or on adsorption on glass wool, followed by an elution‐concentration step. An elution‐concentration method was used for mud specimens. All samples that tested positive by real‐time RT‐PCRs (qRT‐PCRs) targeting the HA5, M and NA1 genes were inoculated into embryonated hen eggs for virus isolation. Results Of a total of 246 samples, 46 (19%) tested positive for H5N1 by qRT‐PCRs. Viral RNA was frequently detected in dust, mud and soil samples from the farms’ environment (respectively, 46%, 31% and 15%). Samples collected from ponds gave a lower proportion of positive samples (6%) as compared to those collected from the farms (24%). In only one sample, infectious virus particles were successfully isolated. Conclusion During H5N1 virus outbreaks, numerous environmental samples surrounding outbreak areas are contaminated by the virus and may act as potential sources for human and/or animal contamination.

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Section: Discussionsupporting

confidence: 61%

Environment: a potential source of animal and human infection with influenza A (H5N1) virus

Horm

Gutiérrez

Sorn³

et al. 2012

Influenza Resp Viruses

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“…In this study, the number of matrix (M) gene copies detected by qRT-PCR was one log 10 higher than the number of viral particles estimated by FFF-MALS. However, there is evidence that virus samples contain virus particle-associated nuclease-resistant RNA, which can artificially increase the number gene copies (Dovas et al, 2010; Wei et al, 2007). It is also possible that FFF-MALS quantitation may underestimate the number of virus particles counted, due to increased error when virus is eluted within the aggregate fraction (McEvoy et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles

Bousse

Shore

Goldsmith

et al. 2013

Journal of Virological Methods

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Summary Recent advances in instrumentation and data analysis in field flow fractionation and multi-angle light scattering (FFF-MALS) have enabled greater use of this technique to characterize and quantitate viruses. In this study, the FFF-MALS technique was applied to the characterization and quantitation of type A influenza virus particles to assess its usefulness for vaccine preparation. The use of FFF-MALS for quantitation and measurement of control particles provided data accurate to within 5% of known values, reproducible with a coefficient of variation of 1.9 %. The methods, sensitivity and limit of detection were established by analyzing different volumes of purified virus, which produced a linear regression with fitting value R2 of 0.99. FFF-MALS was further applied to detect and quantitate influenza virus in the supernatant of infected MDCK cells and allantoic fluids of infected eggs. FFF fractograms of the virus present in these different fluids revealed similar distribution of monomeric and oligomeric virions. However, the monomer fraction of cell grown virus has greater size variety. Notably, β-propialactone (BPL) inactivation of influenza viruses did not influence any of the FFF-MALS measurements. Quantitation analysis by FFF-MALS was compared to infectivity assays and real-time RT-PCR (qRT-PCR) and the limitations of each assay were discussed.

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“…Some reports investigated the persistence of influenza viruses in environmental media such as bird feces and river water as well as on various environmental surfaces (1,6,11,22,30,41,42,47,51). However, despite the profound mortality caused by highly pathogenic AI virus (H5N1) in a number of chickens and wild birds, its persistence in virus-contaminated organs or tissues, such as carcasses of infected birds, remains to be elucidated (37).…”

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confidence: 99%

Persistence of Avian Influenza Virus (H5N1) in Feathers Detached from Bodies of Infected Domestic Ducks

Yamamoto

Nakamura

Yamada

et al. 2010

Appl Environ Microbiol

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Asian lineage highly pathogenic avian influenza virus (H5N1) continues to cause mortality in poultry and wild bird populations at a panzootic scale. However, little is known about its persistence in contaminated tissues derived from infected birds. We investigated avian influenza virus (H5N1) persistence in feathers detached from bodies of infected ducks to evaluate their potential risk for environmental contamination. Four-week-old domestic ducks were inoculated with different clades of avian influenza virus (H5N1). Feathers, drinking water, and feces were collected on day 3 postinoculation and stored at 4°C or 20°C. Viral persistence in samples was investigated for 360 days by virus isolation and reverse transcription-PCR. Infectious viruses persisted for the longest period in feathers, compared with drinking water and feces, at both 4°C and 20°C. Viral infectivity persisted in the feathers for 160 days at 4°C and for 15 days at 20°C. Viral titers of 10 4.3 50% egg infectious doses/ml or greater were detected for 120 days in feathers stored at 4°C. Viral RNA in feathers was more stable than the infectivity. These results indicate that feathers detached from domestic ducks infected with highly pathogenic avian influenza virus (H5N1) can be a source of environmental contamination and may function as fomites with high viral loads in the environment.

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Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR

Cited by 42 publications

References 43 publications

Environment: a potential source of animal and human infection with influenza A (H5N1) virus

Environment: a potential source of animal and human infection with influenza A (H5N1) virus

Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles

Persistence of Avian Influenza Virus (H5N1) in Feathers Detached from Bodies of Infected Domestic Ducks

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