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Next generation sequencing data analysis pipeline. Schematic representation of the analysis pipeline for in silico processing of next-generation sequencing data.
Source publication
Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. We compared the suitability of two benchtop next-generation sequencers for whole genome influenza A...
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... addition, high quality reads will lead to a higher accuracy of de novo sequence assembly. Therefore, we performed a quality control using the CLC Genomics Workbench software; we also propose a NGS data analysis pipeline that is generally applicable (Figure 3). First, we removed adaptor contamination and the low quality ends of the sequencing reads from the data generated by the two deep sequencing techniques. ...
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... set the forward/reverse balance between 0.25 and 0.75, mean- ing that the minimum ratio between the number of for- ward and reverse reads that support the surmised variant should be at least 0.25. In addition, a nucleotide variant should be counted at least 10 times independently and should have an average Phred score of at least 20 (based on [40]) (Figure 3). Applying these variant filters removed most of the false positive variant calls and retained one variant from the Illumina MiSeq and six or five variants from the Ion Torrent PGM data (Table 2). ...
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... viral RT-PCR products were purified and subjected to NGS on the Illumina MiSeq and the Ion Torrent PGM platforms. Before assembly, the reads were processed in silico as described above for the plasmid-derived sequences ( Figure 3). Afterwards, the sequencing reads were assembled de novo using de Bruijn graphs [44]. ...
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... mapping the reads to the reference genome, we called the variants using the optimal parameters described above ( Figure 3). Since we started with viral RNA, we increased the background threshold for variant calling to 0.5%, what we believe is the biologically relevant frequency threshold. ...
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... PR8mut virus contains the tracer mutations C354T and A645T. nucleotide variants in the influenza A virus (Figure 3). This analysis pipeline will help to standardize variant calling in small RNA genomes based on NGS data. ...
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... then applied the analysis pipeline outlined in Figure 3 to PR8 and PR8mut virus, which were gener- ated by a plasmid-based reverse genetics system and amplified in MDCK cells. In our opinion, variants in the influenza virus genome that appear with a frequency below 0.5% are very difficult to distinguish from the background noise that is cumulatively introduced by RT- PCR and the inherent variation due to the chemistry of currently available Illumina and Ion Torrent sequencers. ...
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In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laborato...
Purpose
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Next generation sequencing (NGS) has aided characterization of genomic variation. While whole genome sequencing may capture all possible mutations, whole exome sequencing remains cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-...
Next-generation sequencing generates large amounts of data affected by errors in the form of substitutions, insertions, or deletions of bases. Error correction based on the high-coverage information, typically improves de novo assembly. Most existing tools can correct substitution errors only; some support insertions and deletions, but accuracy is...
Citations
... DNA amplicons were deep sequenced using Illumina MiSeq technology performed by the St. Jude Children's Research Hospital Hartwell Center with DNA libraries prepared using Nextera XT DNA-Seq library prep kits (Illumina, cat#FC-131-1024) with 96 dual-index barcodes and sequenced on an Illumina MiSeq personal genome sequencer. SNVs relative to the reference sequence [CA/09 (H1N1)] were determined by mapping reads using the low-variant detection method in CLC Genomics Workbench 12 (41). To determine whether the variants identified have been previously detected in human surveillance samples, we used the protein sequence variance analysis for HA and NA at the Influenza Research Database (42). ...
Obesity has been epidemiologically and empirically linked with more severe diseases upon influenza infection. To ameliorate severe disease, treatment with antivirals, such as the neuraminidase inhibitor oseltamivir, is suggested to begin within days of infection especially in high-risk hosts. However, this treatment can be poorly effective and may generate resistance variants within the treated host. Here, we hypothesized that obesity would reduce oseltamivir treatment effectiveness in the genetically obese mouse model. We demonstrated that oseltamivir treatment does not improve viral clearance in obese mice. While no traditional variants associated with oseltamivir resistance emerged, we did note that drug treatment failed to quench the viral population and did lead to phenotypic drug resistance in vitro . Together, these studies suggest that the unique pathogenesis and immune responses in obese mice could have implications for pharmaceutical interventions and the within-host dynamics of the influenza virus population.
IMPORTANCE
Influenza virus infections, while typically resolving within days to weeks, can turn critical, especially in high-risk populations. Prompt antiviral administration is crucial to mitigating these severe sequalae, yet concerns remain if antiviral treatment is effective in hosts with obesity. Here, we show that oseltamivir does not improve viral clearance in genetically obese or type I interferon receptor-deficient mice. This suggests a blunted immune response may impair oseltamivir efficacy and render a host more susceptible to severe disease. This study furthers our understanding of oseltamivir treatment dynamics both systemically and in the lungs of obese mice, as well as the consequences of oseltamivir treatment for the within-host emergence of drug-resistant variants.
... We used the influenza virus cycle threshold (C T ) to estimate the viral load in clinical samples. Diverse C T value ranges (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)39) were used. ...
Whole-genome sequencing (WGS) of influenza A virus (IAV) is crucial for identifying diverse subtypes and newly evolved variants and for selecting vaccine strains. In developing countries, where facilities are often inadequate, WGS is challenging to perform using conventional next-generation sequencers. In this study, we established a culture-independent, high-throughput native barcode amplicon sequencing workflow that can sequence all influenza subtypes directly from a clinical specimen. All segments of IAV in 19 clinical specimens, irrespective of their subtypes, were amplified simultaneously using a two-step reverse transcriptase PCR (RT-PCR) system. First, the library was prepared using the ligation sequencing kit, barcoded individually using the native barcodes, and sequenced on the MinION MK 1C platform with real-time base-calling. Then, subsequent data analyses were performed with the appropriate tools. WGS of 19 IAV-positive clinical samples was carried out successfully with 100% coverage and 3,975-fold mean coverage for all segments. This easy-to-install and low-cost capacity-building protocol took only 24 h complete from extracting RNA to obtaining finished sequences. Overall, we developed a high-throughput portable sequencing workflow ideal for resource-limited clinical settings, aiding in real-time surveillance, outbreak investigation, and the detection of emerging viruses and genetic reassortment events. However, further evaluation is required to compare its accuracy with other high-throughput sequencing technologies to validate the widespread application of these findings, including WGS from environmental samples. IMPORTANCE The Nanopore MinION-based influenza sequencing approach we are proposing makes it possible to sequence the influenza A virus, irrespective of its diverse serotypes, directly from clinical and environmental swab samples, so that we are not limited to virus culture. This third-generation, portable, multiplexing, and real-time sequencing strategy is highly convenient for local sequencing, particularly in low- and middle-income countries like Bangladesh. Furthermore, the cost-efficient sequencing method could provide new opportunities to respond to the early phase of an influenza pandemic and enable the timely detection of the emerging subtypes in clinical samples. Here, we meticulously described the entire process that might help the researcher who will follow this methodology in the future. Our findings suggest that this proposed method is ideal for clinical and academic settings and will aid in real-time surveillance and in the detection of potential outbreak agents and newly evolved viruses.
... Full genome amplification was performed using universal IAV primers from Van den Hoecke et al. [34], targeting the conserved 5'-and 3 -non coding regions for each gene segment. This includes the common universal forward primer (F_CommonUni12; 5 -GCC GGA GCT CTG CAG ATA TCA GCA AAA GCA GG-3 ) and the common universal reverse primer (R_CommonUni13; 5 -GCC GGA GCT CTG CAG ATA TCA GTA GAA ACA AGG-3 ). ...
... Additional P-gene primers were designed and evaluated to ensure full internal coverage of these larger segments (Table S2). The RNA of influenza-positive samples was used for cDNA synthesis with the SuperScript ® IV Reverse Transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA), using an initial 13 µL primer-annealing reaction mixture containing 2.5 µM forward universal primer (F_CommonUni12) (Metabion, Planegg, Germany), 10 mM dNTPs from Thermo Fisher Scientific (USA), as well as 11 µL template RNA [34]. The Phusion High Fidelity DNA Polymerase kit (New England Biolabs, Ipswich, MA, USA) was used for full genome amplifications. ...
... The reaction mixture contained a final 1× concentration of Phusion High-Fidelity Buffer (New England Biolabs, Ipswich, MA, USA), 0.2 mM dNTPs (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 µM of the respective segment-specific forward and reverse primers (Metabion, Planegg, Germany), and 0.50 µL of 2 U Phusion High Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA). The assay conditions were adapted from Van Den Hoecke et al., [34]. They consisted of an initial denaturation cycle of 30 s at 98 • C, followed by 35 cycles of denaturation of 10 s at 98 • C and one annealing-extension step of 7.5 min at 72 • C, and was concluded with a final extension cycle of 10 min at 72 • C. The nested round of the RT-PCR assay utilized a similar 50 µL reaction mixture as described above with the segment-specific primers; however, the cycle conditions for the second round of amplification were optimized to include a separate annealing step at various temperatures, as determined by the primers and the size of the genome segments (Table S2). ...
In recent years, bats have been shown to host various novel bat-specific influenza viruses, including H17N10 and H18N11 in the Americas and the H9N2 subtype from Africa. Rousettus aegyptiacus (Egyptian Rousette bat) is recognized as a host species for diverse viral agents. This study focused on the molecular surveillance of a maternal colony in Limpopo, South Africa, between 2017–2018. A pan-influenza hemi-nested RT-PCR assay targeting the PB1 gene was established, and influenza A virus RNA was identified from one fecal sample out of 860 samples. Genome segments were recovered using segment-specific amplification combined with standard Sanger sequencing and Illumina unbiased sequencing. The identified influenza A virus was closely related to the H9N2 bat-influenza virus, confirming the circulation of this subtype among Egyptian fruit bat populations in Southern Africa. This bat H9N2 subtype contained amino acid residues associated with transmission and virulence in either mammalian or avian hosts, though it will likely require additional adaptations before spillover.
... These primers were first introduced by Watson and colleagues in 2013 to be used in next-generation, short-read Illumina sequencing to detect minor viral variants within a population [45]. Even though their amplification protocol used three individual RT-qPCR reactions, various adaptations to the protocol have been made over the years to deliver more cost-efficient IAV sequencing alternatives [23,46,47]. The protocol was shown to be compatible with third-generation MinION sequencing workflows in our work, as well as in other reports in the context of swIAV epidemiology and point-of-care testing [48][49][50][51]. ...
... Altogether, these differences in method 2 are thought to result in a superior performance in the generation of (near-)complete swIAV genomes from oral fluids. The actual primers will probably impact the sequencing success through better and subtype-wide segment targeting, though the exact impact of these primers should be verified in future research [23,46,47]. Additionally, applying a range of RT temperatures and ramping rates during PCR amplification is thought to increase the chance of on-target binding of these primers in the presence of (highly abundant) other nucleic acids (i.e., (r)RNA from contaminating organisms or host in oral fluids) [33,52]. ...
Influenza A virus (IAV) is a single-stranded, negative-sense RNA virus and a common cause of seasonal flu in humans. Its genome comprises eight RNA segments that facilitate reassortment, resulting in a great variety of IAV strains. To study these processes, the genetic code of each segment should be unraveled. Fortunately, new third-generation sequencing approaches allow for cost-efficient sequencing of IAV segments. Sequencing success depends on various factors, including
proper sample storage and processing. Hence, this work focused on the effect of storage of oral fluids and swIAV sequencing. Oral fluids (n = 13) from 2017 were stored at -22 C and later transferred to -80 C. Other samples (n = 21) were immediately stored at -80 C. A reverse transcription quantitative PCR (RT-qPCR) pre- and post-storage was conducted to assess IAV viral loads. Next, samples were subjected to two IAV long-read nanopore sequencing methods to evaluate success in this complex matrix. A significant storage-associated loss of swIAV loads was observed. Still, a total of 17 complete and 6 near-complete Polish swIAV genomes were obtained. Genotype T, (H1avN2, seven herds), P (H1N1pdm09, two herds), U (H1avN1, three herds), and A (H1avN1, 1 herd) were circulated on Polish farms. In conclusion, oral fluids can be used for long-read swIAV sequencing when considering appropriate storage and segment amplification protocols, which allows us to monitor swIAV in an animal-friendly and cost-efficient manner.
... Ion Torrent semiconductor sequencing technology with the Ion Proton and Ion S5 series sequencers which benefits from fast sequencing makes these sequencers particularly useful for targeted detection of viruses in clinical specimens, such as HIV [150], hepatitis B virus [151], HCV [152] and rapid genome sequencing of several viruses, including Tuscany virus [153], polyomavirus [154], porcine reproductive and respiratory syndrome virus [155], orthoreovirus [156], bluetongue virus [157], rotavirus [158], influenza virus [159]. This technology has been used to study the virome of skin [160], ticks [161], intestines in piglets [162] and seals [163]. ...
The COVID-19 pandemic and heightened perception of the risk of emerging viral infections have boosted the efforts to better understand the virome or complete repertoire of viruses in health and disease, with a focus on infectious respiratory diseases. Next-generation sequencing (NGS) is widely used to study microorganisms, allowing the elucidation of bacteria and viruses inhabiting different body systems and identifying new pathogens. However, NGS studies suffer from a lack of standardization, in particular, due to various methodological approaches and no single format for processing the results. Here, we review the main methodological approaches and key stages for studies of the human virome, with an emphasis on virome changes during acute respiratory viral infection, with applications for clinical diagnostics and epidemiologic analyses.
... Several recent studies have successfully identified genetic variation in viral quasispecies during clinical influenza infections using deep sequencing with HTS [24,[28][29][30][31]. Deep sequencing allows higher genome coverages, and consequently more reliable estimation of the diversity within the quasispecies population present at very low abundances [32]. Apart from the increased experimental costs associated with the use of HTS, many challenges remain to detect low-frequency variants (LFV, i.e. defined as nucleotides differing from the consensus sequence at low allelic frequency at a specific genomic position), including high-quality sequencing reads to ensure that insertions and deletions (indels), and single nucleotide variants (SNVs), can be called confidently. ...
... Concisely, RT-PCR was used to generate sequencing amplicons in a reaction volume of 50 µl. The used protocol is based on Van den Hoecke et al. [32] with optimized volumes and RT-PCR conditions. Primers included CommonA-Uni12G ( GCCG GAGC TCTG CAGA TATC AGCG AAAGCAGG), CommonA-Uni12 ( GCCA GAGC TCTG CAGA TATC AGCA AAAGCAGG) and CommonA-Uni13G ( GCCG GAGC TCTG CAGA TATC AGTA GAAA CAAGG) [32]. ...
... The used protocol is based on Van den Hoecke et al. [32] with optimized volumes and RT-PCR conditions. Primers included CommonA-Uni12G ( GCCG GAGC TCTG CAGA TATC AGCG AAAGCAGG), CommonA-Uni12 ( GCCA GAGC TCTG CAGA TATC AGCA AAAGCAGG) and CommonA-Uni13G ( GCCG GAGC TCTG CAGA TATC AGTA GAAA CAAGG) [32]. The reaction volumes included 25 µl RT-PCR buffer, 1 µl SuperScript III One-Step RT-PCR Platinum Taq HiFi DNA Polymerase (Invitrogen, USA), 17.375 µl dH 2 O, 0.375 µl After purifying the generated amplicons with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Germany) according to the manufacturers' instructions, the concentration of each purification product was quantified with the Qubit 4 Fluorometer (Invitrogen, USA) using the Qubit broad-range assay. ...
Influenza viruses exhibit considerable diversity between hosts. Additionally, different quasispecies can be found within the same host. High-throughput sequencing technologies can be used to sequence a patient-derived virus population at sufficient depths to identify low-frequency variants (LFV) present in a quasispecies, but many challenges remain for reliable LFV detection because of experimental errors introduced during sample preparation and sequencing. High genomic copy numbers and extensive sequencing depths are required to differentiate false positive from real LFV, especially at low allelic frequencies (AFs). This study proposes a general approach for identifying LFV in patient-derived samples obtained during routine surveillance. Firstly, validated thresholds were determined for LFV detection, whilst balancing both the cost and feasibility of reliable LFV detection in clinical samples. Using a genetically well-defined population of influenza A viruses, thresholds of at least 104 genomes per microlitre and AF of ≥5 % were established as detection limits. Secondly, a subset of 59 retained influenza A (H3N2) samples from the 2016-2017 Belgian influenza season was composed. Thirdly, as a proof of concept for the added value of LFV for routine influenza monitoring, potential associations between patient data and whole genome sequencing data were investigated. A significant association was found between a high prevalence of LFV and disease severity. This study provides a general methodology for influenza LFV detection, which can also be adopted by other national influenza reference centres and for other viruses such as SARS-CoV-2. Additionally, this study suggests that the current relevance of LFV for routine influenza surveillance programmes might be undervalued.
... For the calculation of transition/transversion and nonsynonymous mutations, we extended the threshold of f i to ≥ 1%, as detection limit for reliable recognition of variants in the viral [53] and the background error from PCR and sequencing. For transition/transversion definition, one SBS with f i ≥ 0.01 observed at one location was counted as one occurrence of transition (Ts) or transversion (Tv) basing on the consensus sequence. ...
Background
Reassortment between human and avian influenza viruses (AIV) may result in novel viruses with new characteristics that may threaten human health when causing the next flu pandemic. A particular risk may be posed by avian influenza viruses of subtype H9N2 that are currently massively circulating in domestic poultry in Asia and have been shown to infect humans. In this study, we investigate the characteristics and compatibility of a human H1N1 virus with avian H9N2 derived genes.
Methods
The polymerase activity of the viral ribonucleoprotein (RNP) complex as combinations of polymerase-related gene segments derived from different reassortment events was tested in luciferase reporter assays. Reassortant viruses were generated by reverse genetics. Gene segments of the human WSN-H1N1 virus (A/WSN/1933) were replaced by gene segments of the avian A2093-H9N2 virus (A/chicken/Jiangsu/A2093/2011), which were both the Hemagglutinin (HA) and Neuraminidase (NA) gene segments in combination with one of the genes involved in the RNP complex (either PB2, PB1, PA or NP). The growth kinetics and virulence of reassortant viruses were tested on cell lines and mice. The reassortant viruses were then passaged for five generations in MDCK cells and mice lungs. The HA gene of progeny viruses from different passaging paths was analyzed using Next-Generation Sequencing (NGS).
Results
We discovered that the avian PB1 gene of H9N2 increased the polymerase activity of the RNP complex in backbone of H1N1. Reassortant viruses were able to replicate in MDCK and DF1 cells and mice. Analysis of the NGS data showed a higher substitution rate for the PB1-reassortant virus. In particular, for the PB1-reassortant virus, increased virulence for mice was measured by increased body weight loss after infection in mice.
Conclusions
The higher polymerase activity and increased mutation frequency measured for the PB1-reassortant virus suggests that the avian PB1 gene of H9N2 may drive the evolution and adaptation of reassortant viruses to the human host. This study provides novel insights in the characteristics of viruses that may arise by reassortment of human and avian influenza viruses. Surveillance for infections with H9N2 viruses and the emergence of the reassortant viruses in humans is important for pandemic preparedness.
... In contrast, the errors that occurred on the Illumina MiSeq platform were mostly nucleotide substitutions. 103 The accuracy of MinION sequencing, when aligned to a reference genome, has been reported to be much lower than Figure 4. A graphical illustration of the trend of reporting sequencing approaches for either partial or complete genome sequencing of influenza A virus (IAV) from swine globally. ...
The rapidly evolving antigenic diversity of influenza A virus (IAV) genomes in swine makes it imperative to detect emerging novel strains and track their circulation. We analyzed in our review the sequencing technologies used for subtyping and characterizing swine IAV genomes. Google Scholar, PubMed, and International Nucleotide Sequence Database Collaboration (INSDC) database searches identified 216 studies that have utilized Sanger, second-, and third-generation sequencing techniques to subtype and characterize swine IAV genomes up to 31 March 2021. Sanger dideoxy sequencing was by far the most widely used sequencing technique for generating either full-length (43.0%) or partial (31.0%) IAV genomes in swine globally; however, in the last decade, other sequencing platforms such as Illumina have emerged as serious competitors for the generation of whole-genome sequences of swine IAVs. Although partial HA and NA gene sequences were sufficient to determine swine IAV subtypes, whole-genome sequences were critical for determining reassortments and identifying unusual or less frequently occurring IAV subtypes. The combination of Sanger and second-generation sequencing technologies also greatly improved swine IAV characterization. In addition, the rapidly evolving third-generation sequencing platform, MinION, appears promising for on-site, real-time sequencing of complete swine IAV genomes. With a higher raw read accuracy, the use of the MinION could enhance the scalability of swine IAV testing in the field and strengthen the swine IAV disease outbreak response.
... Although the outcome of most random mutations is detrimental or lethal, non-deleterious mutations may be preserved and subsequently amplified in the population if they confer a fitness advantage [18]. High mutation frequencies and within-host selective pressures create quasi-species [19][20][21][22], defined as a proliferating population of non-identical but closely related viral genomes as seen with most RNA viruses, including influenza viruses [23,24]. Some mutations can be positively selected in order for a virus to escape from host antibody neutralization or to replicate more efficiently, leading to virus variants becoming predominant in the population [25]. ...
Antigenic characterization of emerging and re-emerging viruses is necessary for the prevention of and response to outbreaks, evaluation of infection mechanisms, understanding of virus evolution, and selection of strains for vaccine development. Primary analytic methods, including enzyme-linked immunosorbent/lectin assays, hemagglutination inhibition, neuraminidase inhibition, micro-neutralization assays, and antigenic cartography, have been widely used in the field of influenza research. These techniques have been improved upon over time for increased analytical capacity, and some have been mobilized for the rapid characterization of the SARS-CoV-2 virus as well as its variants, facilitating the development of highly effective vaccines within 1 year of the initially reported outbreak. While great strides have been made for evaluating the antigenic properties of these viruses, multiple challenges prevent efficient vaccine strain selection and accurate assessment. For influenza, these barriers include the requirement for a large virus quantity to perform the assays, more than what can typically be provided by the clinical samples alone, cell- or egg-adapted mutations that can cause antigenic mismatch between the vaccine strain and circulating viruses, and up to a 6-month duration of vaccine development after vaccine strain selection, which allows viruses to continue evolving with potential for antigenic drift and, thus, antigenic mismatch between the vaccine strain and the emerging epidemic strain. SARS-CoV-2 characterization has faced similar challenges with the additional barrier of the need for facilities with high biosafety levels due to its infectious nature. In this study, we review the primary analytic methods used for antigenic characterization of influenza and SARS-CoV-2 and discuss the barriers of these methods and current developments for addressing these challenges.
... If CPE and HA were negative at 72 h after the third passage samples were considered negative for IAV isolation. Isolates obtained were partially sequenced by sanger sequencing service at an external facility (Macrogen, Korea) and identified using the BLAST tool for Hemaglutinin viral gene (Van den Hoecke et al., 2015). ...
Background: Influenza A virus (IAV) surveillance in swine is critical not only due to the direct impact of the disease in the pork industry but also because IAV are prone to interspecies transmission (from human to pigs and vice versa); therefore, its monitoring is fundamental from a public and animal health perspective. Several diagnostic techniques have been used to detect IAV infection from nasal samples in swine, while samples of oral fluids (OF) are in use as novel alternatives for pathogen detection. The OF allow for efficient and feasible low-cost disease detection at the herd level, with low risk of stress for the animals. Objective: To describe a surveillance strategy of IAV at the herd level during respiratory disease outbreaks in swine farms at tropical settings using porcine oral fluids. Methods: An active surveillance strategy was conducted in several farms with past records of respiratory disease. The IAV detection was conducted in five purposively selected swine farms from years 2014 to 2017. We investigated a total of 18 respiratory outbreaks of the disease. Swine OF were collected for IAV testing. An OF sample is described as a pen-based specimen collected from a group of >20 pigs per pen and/or per barn (stall-housed individually with close contact among them). The IAV infection was investigated in OF by rRT-PCR testing and confirmed by viral isolation in cell culture. Results: We found 107 (7.4%) positives to IAV by rRT-PCR from a total of 1,444 OF samples tested. Additionally, 9 IAV isolates were all further identified as H1 subtype. Conclusions: Our results demonstrate that OF can be easily implemented as a novel, user-friendly, welfare-friendly, accurate and cost-effective sampling method for active surveillance and monitoring of IAV infections in swine farms in tropical settings. © 2022 Universidad de Antioquia. Publicado por Universidad de Antioquia, Colombia.
