An Updated Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of H5N1 Avian Influenza Viruses

Abstract Background In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. Methods We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). Results We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). Conclusions Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.

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Simultaneous subtyping and pathotyping of avian influenza viruses in chickens in Taiwan using reverse transcription loop-mediated isothermal amplification and microarray

Journal of Veterinary Medical Science ◽

10.1292/jvms.15-0602 ◽

2016 ◽

Vol 78 (8) ◽

pp. 1223-1228 ◽

Cited By ~ 2

Author(s):

Lih-Chiann WANG ◽

Dean HUANG ◽

Hui-Wen CHEN

Keyword(s):

Avian Influenza ◽

Reverse Transcription ◽

Influenza Viruses ◽

Isothermal Amplification ◽

Avian Influenza Viruses ◽

Loop Mediated Isothermal Amplification

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Subpopulation Primers Essential for Exhaustive Detection of Diverse Hemagglutinin Genes of H5 Subtype Avian Influenza Viruses by Loop-Mediated Isothermal Amplification Method

Journal of Clinical Microbiology ◽

10.1128/jcm.00985-18 ◽

2018 ◽

Vol 56 (9) ◽

Author(s):

Y. Furuyama ◽

Y. Takahashi ◽

K. Noguchi ◽

H. Murakami ◽

M. Sakaguchi ◽

...

Keyword(s):

Avian Influenza ◽

Screening Test ◽

Influenza Viruses ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Amplification Method ◽

Ha Gene ◽

Field Samples ◽

Base Mismatch ◽

H5 Subtype

ABSTRACT Loop-mediated isothermal amplification (LAMP) is a potential screening test for avian influenza (AI), but its narrow detection spectrum limits its applications. To improve this narrow detection spectrum, 3 types of primers were compared for detection of diverse H5 subtype hemagglutinin (HA) genes. Four and 6 genes, of 10 genetically different H5 HA genes tested, were detected with S primers specific for A/duck/Tsukuba/9/2005 (H5N2) and with M primers (which contained mixed bases), respectively. In contrast, all 10 HA genes became positive with population primers (P primers) (a mixture of primers designed for each subpopulation of 2,202 HA genes). Our study indicated that the P primers for the forward inner primer (FIP) and backward inner primer (BIP) sites were essential for exhaustive detection, whereas those for the F3, forward loop (FL), backward loop (BL), and B3 sites were exchangeable with M primers. A base mismatch experiment demonstrated that HA genes with ≤2 base mismatches per primer site and ≤10 base mismatches per HA gene were amplifiable. Reverse transcription-LAMP was broadly reactive, specific for H5 subtype HA genes, and applicable to field samples, with the sensitivity of real-time PCR. The in silico analysis suggested that most H5 HA genes (2,586 positive genes/2,588 genes tested) registered in the GenBank database might be amplifiable. These results indicate that the use of subpopulation primers in LAMP allows exhaustive detection of diverse HA genes and H5 LAMP can be used as a reliable AI screening test in general diagnostic laboratories.

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Faculty Opinions recommendation of Evolution of H5N1 avian influenza viruses in Asia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1028090.335985 ◽

2005 ◽

Author(s):

Angela McLean

Keyword(s):

Avian Influenza ◽

Influenza Viruses ◽

Avian Influenza Viruses ◽

H5n1 Avian Influenza

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Glycine at Position 622 in PB1 Contributes to the Virulence of H5N1 Avian Influenza Virus in Mice

Journal of Virology ◽

10.1128/jvi.02387-15 ◽

2015 ◽

Vol 90 (4) ◽

pp. 1872-1879 ◽

Cited By ~ 12

Author(s):

Xiaoxiao Feng ◽

Zeng Wang ◽

Jianzhong Shi ◽

Guohua Deng ◽

Huihui Kong ◽

...

Keyword(s):

Avian Influenza ◽

Aspartic Acid ◽

H5n1 Virus ◽

Genetic Basis ◽

Vaccine Development ◽

Influenza Viruses ◽

Avian Influenza Viruses ◽

Live Attenuated Vaccine ◽

H5n1 Influenza ◽

H5n1 Avian Influenza

ABSTRACTWe isolated two H5N1 viruses, A/duck/Hunan/S4020/2008 (DK/08) and A/chicken/Guangxi/S2039/2009 (CK/09), from live-bird markets during routine surveillance and found that these two viruses are genetically similar but differ in their replication and virulence in mice. The CK/09 virus is lethal for mice with a 50% mouse lethal dose (MLD50) of 1.6 log1050% egg infectious doses (EID50), whereas the DK/08 virus is nonpathogenic for mice with an MLD50value of 6.2 log10EID50. We explored the genetic basis of the virulence difference of these two viruses by generating a series of reassortant viruses and mutants in the lethal virus CK/09 background and evaluating their virulence in mice. We found that the PB1 gene of the DK/08 virus dramatically attenuated the virulence of the CK/09 virus and that the amino acid at position 622 in PB1 made an important contribution. We further demonstrated that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impaired the binding of PB1 to viral RNA, thereby dramatically decreasing the polymerase activity and attenuating H5N1 virus virulence in mice. Our results identify a novel virulence-related marker of H5N1 influenza viruses and provide a new target for live attenuated vaccine development.IMPORTANCEH5N1 avian influenza viruses have caused the deaths of nearly 60% of the humans that they have infected since 1997 and clearly represent a threat to public health. A thorough understanding of the genetic basis of virulence determinants will provide important insights for antiviral drug and live attenuated vaccine development. Several virulence-related markers in the PB2, PA, M1, and NS1 proteins of H5N1 viruses have been identified. In this study, we isolated two H5N1 avian influenza viruses that are genetically similar but differ in their virulence in mice, and we identified a new virulence-related marker in the PB1 gene. We found that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impairs the binding of PB1 to viral RNA, thereby attenuating H5N1 virus virulence in mice. This newly identified virulence-related marker could be applied to the development of live attenuated vaccines against H5N1 influenza.

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