Evaluation of two multiplex RT-PCR assays for detection and subtype differentiation of Brazilian swine influenza viruses

Abstract

Influenza A virus (IAV) subtypes H1N1, H1N2, and H3N2 are endemic in swine herds in most pork producing countries; however, the viruses circulating in different geographic regions are antigenically and genetically distinct. In this sense, the availability of a rapid diagnostic assay to detect locally adapted IAVs and discriminate the virus subtype in clinical samples from swine is extremely important for monitoring and control of the disease. This study describes the development and validation of a multiplex RT-PCR assay for detection and subtyping of IAV from pigs. The analytical and diagnostic specificity of the assays was 100% (94.3-100.0, CI 95%), and the limit of detection was 10 −3 TCID 50 /mL. A total of 100 samples (IAV isolates and clinical specimens) were tested, and the virus subtype was determined for 80 samples (80%; 71.1-86.7, CI 95%). From these, 50% were H1N1, 22.5% were H1N2, and 7.5% were H3N2. Partial subtyping was determined for 8.75% samples (H1pdmNx and HxN2). Additionally, mixed infections with two virus subtypes (H1N2 + H3N2 and H1N1pdm + H1pdmN2; 2.5%) and reassortant viruses (H1pdmN2, 6.25%; and H1N1hu, 2.5%) were detected by the assay. A rapid detection of the most prevalent IAV subtypes and lineages in swine is provided by the assays developed here, improving the IAV diagnosis in Brazilian laboratories, and contributing to the IAV monitoring.

Swine influenza A virus subtypes circulating in Brazilian commercial pig herds from 2012 to 2019

“…Compared to previous years, in 2017–2018, serological and PCR subtyping data from clinical samples revealed a significant increase in the occurrence of H3N2, as well as in H1N1pdm + H3N2, and a decrease in H1N1pdm. The results of molecular analysis corroborate with data previously published demonstrating a higher detection of H3N2 in clinical samples in this period [ 24 ]. The increasing occurrence of H3N2 in swine may be related to the occurrence of this IAV subtype in the human population.…”

“…Some studies using multiplex real-time RT-PCR (RT-qPCR) for the detection of H3 and different H1 lineages circulating in Europe, subtyped between 89 and 94% of SIAV-positive clinical samples [ 15 , 22 , 23 ]. Another study using a one-step multiplex RT-qPCR assay for the identification of different subtypes and lineages of SIAV circulating in Brazil demonstrated high sensitivity and specificity when applied for the diagnostic of IAV in clinical samples [ 24 ]. However, real-time PCR requires a greater investment and qualified technicians, which could be limitations for diagnostic labs and customers.…”

Influenza A virus circulation in pig nurseries in the state of Santa Catarina, Brazil

“…agropec. bras., Brasília, v.57, e02816, 2022 DOI: 10.1590/S1678-3921.pab2022.v57.02816 introduction in the country in 2009 (Schaefer et al, 2011;Rajão et al, 2013) until 2015(Ciacci-Zanella et al, 2015Rech et al, 2018;Haach et al, 2020;Fraiha et al, 2021). However, since 2015, IAV outbreaks caused by the H3N2 virus have been reported more frequently among Brazilian pig herds (Haach et al, 2020;Fraiha et al, 2021).…”

“…RT-qPCR positive samples were subjected to viral isolation in Madin-Darby Canine Kidney (MDCK) cells or in embryonated specific pathogen free (SPF) chicken eggs (ECE) as in Zhang & Gauger (2014). For IAV subtyping, three isolated viruses were selected from each nursery, totalizing 33 samples, which were also tested by RT-qPCR (Haach et al, 2020). The serum -individual samples per assay -was separated from the blood samples and tested by the InfA Multi commercial kit for ELISA (BioCheck: Smart Veterinary Diagnostics, Scarborough, ME, USA), following the manufacturer's instructions.…”

Introductions of Human-Origin Seasonal H3N2, H1N2 and Pre-2009 H1N1 Influenza Viruses to Swine in Brazil

“…Data from swine surveillance activities in Brazil have indicated previously unappreciated genetic diversity in swine IAV as well as the persistence of novel human-to-swine incursions [ 16 , 17 ]. Currently, H1N1pdm09 and human-like H1N2 and H3N2 viruses circulate in swine in several Brazilian states [ 15 , 18 , 19 , 20 ]. A comprehensive time-scaled phylogenetic analysis revealed the introduction and circulation of three lineages of human seasonal IAV in swine that had not been previously observed in any other country [ 17 ].…”