ABSTRACT
BackgroundDuring the 2016/17 influenza season, influenza B/VIC lineage variant viruses emerged with two (K162N163) or three (K162N163D164) amino acid (aa) deletions in the haemagglutinin (HA) protein. There are currently five antigenically distinct HA proteins expressed by co-circulating influenza B viruses: B/YAM, B/VIC V1A (no deletion), B/VIC V1A-2DEL (2 aa deletion) and two antigenically distinguishable groups of B/VIC V1A-3DEL (3 aa deletion). The prevalence of these viruses differs across geographical regions, making it critical to have a sensitive, rapid diagnostic assay that detects and distinguishes these influenza B variant viruses during surveillance.AimOur objective was to develop a real-time RT-PCR (rRT-PCR) assay for detection and discrimination of influenza B/VIC lineage variant viruses.MethodsWe designed a diagnostic assay with one pair of conserved primers and three probes specific to each genetic group. We used propagated influenza B/VIC variant viruses and clinical specimens to assess assay performance.ResultsThis rRT-PCR assay detects and distinguishes the influenza B/VIC V1A, B/VIC V1A-2DEL, and B/VIC V1A-3DEL variant viruses, with no cross-reactivity. This assay can be run as a multiplex reaction, allowing for increased testing efficiency and reduced cost.ConclusionCoupling this assay with the Centers for Disease Control and Prevention's Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza B Lineage Genotyping Kit results in rapid detection and characterisation of circulating influenza B viruses. Detailed surveillance information on these distinct influenza B variant viruses will provide insight into their prevalence and geographical distribution and could aid in vaccine recommendations.
Subject(s)
Influenza B virus/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza B virus/classification , Influenza B virus/isolation & purification , Influenza, Human/epidemiology , Molecular Epidemiology/methodsABSTRACT
From June 2009 through December 2009, Haiti conducted sentinel surveillance for influenza. 499 samples were collected and tested using real-time RT-PCR. 197 (39.5%) were positive for influenza, including 95 (48%) pandemic (H1N1) 2009, 57 (29%) seasonal influenza A and 45 (23%) influenza B. The median age of pandemic (H1N1) 2009 cases was 21.7; two-thirds of pandemic (H1N1) 2009 cases were in patients aged 6 years - 35 years. Pandemic activity peaked in September and co-circulated with other influenza subtypes. The age distribution and seasonality of pandemic (H1N1) 2009 in Haiti were similar to other countries in the Caribbean region.
Subject(s)
Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human/epidemiology , Sentinel Surveillance , Adolescent , Adult , Aged , Child , Child, Preschool , Female , Haiti/epidemiology , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A virus/genetics , Influenza A virus/isolation & purification , Influenza B virus/genetics , Influenza B virus/isolation & purification , Influenza, Human/virology , Male , Middle Aged , Pandemics , Seasons , Young AdultABSTRACT
In the beginning of 2007-2008 Northern Hemisphere influenza season, the frequency of influenza A(H1N1) viruses bearing a previously defined oseltamivir resistance conferring amino acid change of Histidine to Tyrosine at position 274 (H274Y) of the neuraminidase (NA) increased dramatically. In order to rapidly detect such resistant viruses, an RT-PCR/restriction fragment length polymorphism (RT-PCR/RFLP) assay targeting amino acid 274 of the N1 NA molecule was developed to investigate the presence or absence of the H274Y mutation. The reverse primer was engineered to produce a BspHI site in the amplicon for oseltamivir-sensitive viruses with Histidine at position 274 (274H). A total of 50 influenza A(H1N1) specimens including 30 oseltamivir-sensitive and 20 oseltamivir-resistant ones submitted to the Centers for Disease Control and Prevention (CDC) during the 2007-2008 influenza season were successfully characterized by this assay. The assay was specific for grown A(H1N1) viruses and original clinical specimens, with a lower limit of detection of approximately 10 RNA transcript copies per reaction. Our RT-PCR/RFLP assay provides a simple, rapid and sensitive tool to monitor the emergence and spread of H274Y oseltamivir-resistant influenza A(H1N1) viruses.
Subject(s)
Antiviral Agents/therapeutic use , Drug Resistance, Viral , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human/virology , Mutation, Missense , Neuraminidase/genetics , Oseltamivir/therapeutic use , Reverse Transcriptase Polymerase Chain Reaction/methods , Viral Proteins/genetics , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/genetics , Influenza, Human/drug therapy , Polymorphism, Restriction Fragment LengthABSTRACT
Since its identification in April 2009, an A(H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The lack of similarity between the 2009 A(H1N1) virus and its nearest relatives indicates that its gene segments have been circulating undetected for an extended period. Its low genetic diversity suggests that the introduction into humans was a single event or multiple events of similar viruses. Molecular markers predictive of adaptation to humans are not currently present in 2009 A(H1N1) viruses, suggesting that previously unrecognized molecular determinants could be responsible for the transmission among humans. Antigenically the viruses are homogeneous and similar to North American swine A(H1N1) viruses but distinct from seasonal human A(H1N1).