Pyrosequencing as a fast and reliable tool to determine clade affiliation for equine <i>Influenza A virus</i>

Bernardino, Pedro N.; Mapes, S.; Corbin, Rachel; Pusterla, Nicola

doi:10.1177/1040638716638123

Cited by 8 publications

(9 citation statements)

References 15 publications

(19 reference statements)

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“…Our results on clade affiliation for EIV strains collected from 341 horses with equine influenza showed that clade 1 has predominated in the United States during the last 6 y. This is in agreement with 2016 and 2018 studies, 1,14 and also with the 2017 annual report of the OIE expert surveillance panel (http://www.oie.int/our-scientific-expertise/ specific-information-and-recommendations/equine-influenza). A 2014 study has shown that the A/eq/Kentucky/91 cold-adapted intranasal EIV vaccine strain can be detected via rtPCR for up to 5 d in recently vaccinated horses.…”

Section: Contemporary Equine Influenza a Virus (Eiv; Speciessupporting

confidence: 89%

“…Eighty-five EIV rtPCR-positive samples had been used previously to establish and validate a pyrosequencing assay able to differentiate between clade 1 and clade 2 Florida sublineage EIV strains. 1 Further, to assess the efficiency of multiplex rtPCR assays for the 2 EIV clades, analyses in triplicate were performed using Florida sublineage clade 1 (A/eq/ Kentucky/1/2012, A/eq/New Hampshire/1/2013, A/eq/ Ohio/1/2013, A/eq/Kentucky/1/2014, A/eq/Ohio/1/2014, A/eq/Kentucky/3/2015, A/eq/Minnesota/1/2015, A/eq/ Illinois/1/2016, A/eq/New York/1/2016) and clade 2 (A/eq/Newmarket/5/2003, A/eq/Richmond/2007, A/eq/ Shropshire/2010, A/eq/Devon/2011, A/eq/East Renfrewshire/2011), and one Eurasian lineage (A/eq/Aboyne/ 1/2005) EIV prototype strains.…”

Section: Contemporary Equine Influenza a Virus (Eiv; Speciesmentioning

confidence: 99%

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Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates

et al. 2019

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We validated 2 multiplex real-time PCR (rtPCR) assays based on single nucleotide polymorphisms (SNPs) of the hemagglutinin-1 (HA1) gene of H3N8 equine influenza A virus (EIV) to determine clade affiliation of prototype and field isolates. Initial validation of the 2 multiplex rtPCR assays (SNP1 and SNP2) was performed using nucleic acid from 14 EIV Florida sublineage clade 1 and 2 prototype strains. We included in our study previously banked EIV rtPCR-positive nasal secretions from 341 horses collected across the United States in 2012-2017 to determine their clade affiliation. All 14 EIV prototype strains were identified correctly as either Florida sublineage clade 1 or clade 2 using the 2 SNP target positions. Of 341 EIV rtPCR-positive samples, 337 (98.8%) and 4 (1.2%) isolates were classified as belonging to clade 1 and 2 Florida sublineage EIV, respectively. All clade 1 Florida sublineage EIV strains were detected in domestic horses, three clade 2 Florida sublineage EIV strains originated from horses recently imported into the United States, and one clade 2 Florida sublineage EIV strain originated from a healthy horse recently vaccinated with a modified-live intranasal EIV vaccine containing the American lineage strain A/eq/Kentucky/1991. EIV Florida sublineage clade differentiation using a fast and reliable multiplex rtPCR platform will help monitor the introduction of clade 2 Florida sublineage EIV strains into North America via international transportation.

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Section: Contemporary Equine Influenza a Virus (Eiv; Speciessupporting

confidence: 89%

Section: Contemporary Equine Influenza a Virus (Eiv; Speciesmentioning

confidence: 99%

Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates

et al. 2019

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“…Serological techniques, due to the high degree of antigenic and genetic variability of AMDV, are not always adequate [6] because they produce a certain percentage of false negative [7]. For this reason, molecular biology techniques such as PCR and its variants, isothermal amplification methods, and techniques based on pyrosequencing are increasingly used to identify AMDV [8]. The sensitivity and specificity of PCR techniques is higher than that of immunochromatographic analysis [9].…”

Section: Introductionmentioning

confidence: 99%

A comparative molecular characterization of AMDV strains isolated from cases of clinical and subclinical infection

et al. 2018

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The Aleutian mink disease virus (AMDV) is one of the most serious threats to modern mink breeding. The disease can have various courses, from progressive to subclinical infections. The objective of the study was to provide a comparative molecular characterization of isolates of AMDV from farms with a clinical and subclinical course of the disease. The qPCR analysis showed a difference of two orders of magnitude between the number of copies of the viral DNA on the farm with the clinical course of the disease (10) and the farm with the subclinical course (10). The sequencing results confirm a high level of homogeneity within each farm and variation between them. The phylogenetic analysis indicates that the variants belonging to different farms are closely related and occupy different branches of the same clade. The in silico analysis of the effect of differences in the sequence encoding the VP2 protein between the farms revealed no effect of the polymorphism on its functionality. The close phylogenetic relationship between the isolates from the two farms, the synonymous nature of most of the polymorphisms and the potentially minor effect on the functionality of the protein indicate that the differences in the clinical picture may be due not only to polymorphisms in the nucleotide and amino acid sequences, but also to the stage of infection on the farm and the degree of stabilization of the pathogen-host relationship.

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“…In the early 2000s, the Florida sub-lineage diverged into clades 1 (FC1) and 2 (FC2), and has been the dominant circulating sub-lineage ever since. Although not fully restricted geographically, FC2 viruses have predominately been isolated in Europe and Asia, whilst the majority of FC1 viruses have been isolated in North America [8,9,15,21,22,23,24,25,26]. …”

Section: Introductionmentioning

confidence: 99%

Evolution and Divergence of H3N8 Equine Influenza Viruses Circulating in the United Kingdom from 2013 to 2015

et al. 2017

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Equine influenza viruses (EIV) are a major cause of acute respiratory disease in horses worldwide and occasionally also affect vaccinated animals. Like other influenza A viruses, they undergo antigenic drift, highlighting the importance of both surveillance and virus characterisation in order for vaccine strains to be kept up to date. The aim of the work reported here was to monitor the genetic and antigenic changes occurring in EIV circulating in the UK from 2013 to 2015 and to identify any evidence of vaccine breakdown in the field. Virus isolation, reverse transcription polymerase chain reaction (RT-PCR) and sequencing were performed on EIV-positive nasopharyngeal swab samples submitted to the Diagnostic Laboratory Services at the Animal Health Trust (AHT). Phylogenetic analyses were completed for the haemagglutinin-1 (HA1) and neuraminidase (NA) genes using PhyML and amino acid sequences compared against the current World Organisation for Animal Health (OIE)-recommended Florida clade 2 vaccine strain. Substitutions between the new isolates and the vaccine strain were mapped onto the three-dimensional structure protein structures using PyMol. Antigenic analyses were carried out by haemagglutination inhibition assay using a panel of post-infection ferret antisera. Sixty-nine outbreaks of equine influenza in the UK were reported by the AHT between January 2013 and December 2015. Forty-seven viruses were successfully isolated in eggs from 41 of the outbreaks. Only three cases of vaccine breakdown were identified and in each case the vaccine used contained a virus antigen not currently recommended for equine influenza vaccines. Nucleotide sequencing of the HA and NA genes revealed that all of the viruses belonged to the Florida clade 2 sub-lineage of H3N8 EIV. Phylogenetic and sequence analyses showed that the two sub-populations, previously identified within clade 2, continued to circulate and had accrued further amino acid substitutions. Antigenic characterisation using post-infection ferret antisera in haemagglutination inhibition assays however, failed to detect any marked antigenic differences between the isolates. These findings show that Florida clade 2 EIV continue to circulate in the UK and support the current OIE recommendation to include an example of Florida clade 2 in vaccines.

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Pyrosequencing as a fast and reliable tool to determine clade affiliation for equine Influenza A virus

Cited by 8 publications

References 15 publications

Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates

Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates

A comparative molecular characterization of AMDV strains isolated from cases of clinical and subclinical infection

Evolution and Divergence of H3N8 Equine Influenza Viruses Circulating in the United Kingdom from 2013 to 2015

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