Mobile imaging platform for digital influenza virus counting

ABSTRACTViruses with approximately 50% homology to human influenza C virus (ICV) have recently been isolated from swine and cattle. The overall low homology to ICV, lack of antibody cross-reactivity to ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion assays, and inability to productively reassort with ICV led to the proposal that these viruses represented a new genus of influenza virus, influenzavirus D (IDV). To further our understanding of the epidemiology of IDV, real-time reverse transcription-PCR was performed on a set of 208 samples from bovines with respiratory disease. Ten samples (4.8%) were positive and six viruses were successfully isolatedin vitro. Phylogenetic analysis of full-genome sequences of these six new viruses and four previously reported viruses revealed two distinct cocirculating lineages represented by D/swine/Oklahoma/1334/2011 (D/OK) and D/bovine/Oklahoma/660/2013 (D/660), which frequently reassorted with one another. Antigenic analysis using the HI assay and lineage-representative D/OK and D/660 antiserum found up to an approximate 10-fold loss in cross-reactivity against heterologous clade antiserum. One isolate, D/bovine/Texas/3-13/2011 (D/3-13), clustered with the D/660 lineage, but also had high HI titers to heterologous (D/OK) clade antiserum. Molecular modeling of the hemagglutinin esterase fusion protein of D/3-13 identified a mutation at position 212 as a possible antigenic determinant responsible for the discrepant HI results. These results suggest that IDV is common in bovines with respiratory disease and that at least two genetic and antigenically distinct clades cocirculate.IMPORTANCEA novel bovine influenza virus was recently identified. Detailed genetic and antigenic studies led to the proposal that this virus represents a new genus of influenza, influenzavirus D (IDV). Here, we show that IDV is common in clinical samples of bovine respiratory disease complex (BRDC), with a prevalence similar to that of other established BRDC etiological agents. These results are in good agreement with the near-ubiquitous seroprevalence of IDV previously found. Phylogenetic analysis of complete genome sequences found evidence for two distinct cocirculating lineages of IDV which freely reassort. Significant antigenic differences, which generally agreed with the surface glycoprotein hemagglutinin esterase phylogeny, were observed between the two lineages. Based on these results, and on the ability of IDV to infect and transmit in multiple mammalian species, additional studies to determine the pathogenic potential of IDV are warranted.

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Characterization of H5N1 Influenza Virus Quasispecies with Adaptive Hemagglutinin Mutations from Single-Virus Infections of Human Airway Cells

Journal of Virology ◽

10.1128/jvi.02004-17 ◽

2018 ◽

Vol 92 (11) ◽

pp. e02004-17 ◽

Cited By ~ 9

Author(s):

Yohei Watanabe ◽

Yasuha Arai ◽

Norihito Kawashita ◽

Madiha S. Ibrahim ◽

Emad M. Elgendy ◽

...

Keyword(s):

Genetic Diversity ◽

Influenza Virus ◽

Virus Infection ◽

Clinical Samples ◽

Human Airway ◽

H5n1 Influenza ◽

Starting Point ◽

Virus Adaptation ◽

Quasispecies Diversity ◽

Airway Cells

ABSTRACTTransmission of avian influenza (AI) viruses to mammals involves phylogenetic bottlenecks that select small numbers of variants for transmission to new host species. However, little is known about the AI virus quasispecies diversity that produces variants for virus adaptation to humans. Here, we analyzed the hemagglutinin (HA) genetic diversity produced during AI H5N1 single-virus infection of primary human airway cells and characterized the phenotypes of these variants. During single-virus infection, HA variants emerged with increased fitness to infect human cells. These variants generally had decreased HA thermostability, an indicator of decreased transmissibility, that appeared to compensate for their increase in α2,6-linked sialic acid (α2,6 Sia) binding specificity and/or in the membrane fusion pH threshold, each of which is an advantageous mutational change for viral infection of human airway epithelia. An HA variant with increased HA thermostability also emerged but could not outcompete variants with less HA thermostability. These results provided data on HA quasispecies diversity in human airway cells.IMPORTANCEThe diversity of the influenza virus quasispecies that emerges from a single infection is the starting point for viral adaptation to new hosts. A few studies have investigated AI virus quasispecies diversity during human adaptation using clinical samples. However, those studies could be appreciably affected by individual variability and multifactorial respiratory factors, which complicate identification of quasispecies diversity produced by selective pressure for increased adaptation to infect human airway cells. Here, we found that detectable HA genetic diversity was produced by H5N1 single-virus infection of human airway cells. Most of the HA variants had increased fitness to infect human airway cells but incurred a fitness cost of less HA stability. To our knowledge, this is the first report to characterize the adaptive changes of AI virus quasispecies produced by infection of human airway cells. These results provide a better perspective on AI virus adaptation to infect humans.

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Detection of the influenza virus yesterday and now.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1865 ◽

2014 ◽

Vol 61 (3) ◽

Cited By ~ 6

Author(s):

Agnieszka Woźniak-Kosek ◽

Bogumiła Kempińska-Mirosławska ◽

Grażyna Hoser

Keyword(s):

Influenza Virus ◽

Virus Detection ◽

Diagnostic Methods ◽

Detection Methods ◽

Clinical Samples ◽

Serological Tests ◽

Human Influenza Virus ◽

Rapid Spread ◽

Influenza Virus Detection

Demographic changes and the development of transportation contribute to the rapid spread of influenza. Before an idea of a 'person to person' spread appeared, divergent theories were developed to explain influenza epidemics in the past. Intensified virological and serological tests became possible after isolation of the human influenza virus in 1933. The first influenza virus detection methods were based on its isolation in egg embryos or cell lines and on demonstration of the presence of the viral antigens. Molecular biology techniques associated with amplification of RNA improved the quality of tests as well as sensitivity of influenza virus detection in clinical samples. It became possible to detect mixed infections caused by influenza types A and B and to identify the strain of the virus. Development of reliable diagnostic methods enabled fast diagnosis of influenza which is important for choosing an appropriate medical treatment.

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Characterization of a Porcine Lung Epithelial Cell Line Suitable for Influenza Virus Studies

Journal of Virology ◽

10.1128/jvi.75.19.9517-9525.2001 ◽

2001 ◽

Vol 75 (19) ◽

pp. 9517-9525 ◽

Cited By ~ 41

Author(s):

Sang Heui Seo ◽

Olga Goloubeva ◽

Richard Webby ◽

Robert G. Webster

Keyword(s):

Influenza Virus ◽

Epithelial Cell ◽

Cell Line ◽

Mdck Cells ◽

Influenza Viruses ◽

Lung Epithelial Cell ◽

Clinical Samples ◽

Epithelial Cell Line ◽

Porcine Endogenous Retrovirus ◽

Lung Epithelial

ABSTRACT We established a porcine lung epithelial cell line designated St. Jude porcine lung cells (SJPL) and demonstrated that all tested influenza A and B viruses replicated in this cell line. The infectivity titers of most viruses in SJPL cells were comparable to or better than those in MDCK cells. The propagation of influenza viruses from clinical samples in SJPL cells did not lead to antigenic changes in the hemagglutinin molecule. The numbers of both Sia2-3Gal and Sia2-6Gal receptors on SJPL cells were greater than those on MDCK cells. Influenza virus infection of SJPL cells did not lead to apoptosis, as did infection of MDCK cells. No porcine endogenous retrovirus was detected in SJPL cells, and in contrast to MDCK cells, SJPL cells did not cause tumors in nude mice.

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Isolation of influenza virus in human lung embryonated fibroblast cells (MRC-5) from clinical samples.

Journal of Clinical Microbiology ◽

10.1128/jcm.33.7.1948-1949.1995 ◽

1995 ◽

Vol 33 (7) ◽

pp. 1948-1949 ◽

Cited By ~ 6

Author(s):

M de Oña ◽

S Melón ◽

P de la Iglesia ◽

F Hidalgo ◽

A F Verdugo

Keyword(s):

Influenza Virus ◽

Human Lung ◽

Clinical Samples ◽

Fibroblast Cells

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Primary Swine Respiratory Epithelial Cell Lines for the Efficient Isolation and Propagation of Influenza A Viruses

Journal of Virology ◽

10.1128/jvi.01091-20 ◽

2020 ◽

Vol 94 (24) ◽

Author(s):

Victoria Meliopoulos ◽

Sean Cherry ◽

Nicholas Wohlgemuth ◽

Rebekah Honce ◽

Karen Barnard ◽

...

Keyword(s):

Influenza Virus ◽

Epithelial Cells ◽

Cell Lines ◽

Influenza A ◽

Vaccine Development ◽

Mdck Cells ◽

Influenza Viruses ◽

Clinical Samples ◽

Respiratory Epithelial Cells ◽

Respiratory Epithelial

ABSTRACT Influenza virus isolation from clinical samples is critical for the identification and characterization of circulating and emerging viruses. Yet efficient isolation can be difficult. In these studies, we isolated primary swine nasal and tracheal respiratory epithelial cells and immortalized swine nasal epithelial cells (siNEC) and tracheal epithelial cells (siTEC) that retained the abilities to form tight junctions and cilia and to differentiate at the air-liquid interface like primary cells. Critically, both human and swine influenza viruses replicated in the immortalized cells, which generally yielded higher-titer viral isolates from human and swine nasal swabs, supported the replication of isolates that failed to grow in Madin-Darby canine kidney (MDCK) cells, and resulted in fewer dominating mutations during viral passaging than MDCK cells. IMPORTANCE Robust in vitro culture systems for influenza virus are critically needed. MDCK cells, the most widely used cell line for influenza isolation and propagation, do not adequately model the respiratory tract. Therefore, many clinical isolates, both animal and human, are unable to be isolated and characterized, limiting our understanding of currently circulating influenza viruses. We have developed immortalized swine respiratory epithelial cells that retain the ability to differentiate and can support influenza replication and isolation. These cell lines can be used as additional tools to enhance influenza research and vaccine development.

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DETECTION OF INFLUENZA VIRUS AND PATHOGENS OF ACUTE RESPIRATORY VIRAL INFECTIONS IN POPULATION OF KAZAKHSTAN DURING 2018-2019 EPIDEMIC SEASON

Russian Journal of Infection and Immunity ◽

10.15789/2220-7619-doi-1348 ◽

2019 ◽

Author(s):

N. G. Klivleyeva ◽

N. S. Ongarbayeva ◽

A. M. Baimukhametova ◽

N. T. Saktaganov ◽

G. V. Lukmanova ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Syncytial Virus ◽

Influenza A ◽

Viral Infections ◽

Influenza Viruses ◽

Clinical Samples ◽

Type B ◽

Epidemic Season ◽

Parainfluenza Viruses ◽

Syncytial Virus

Influenza and other acute respiratory viral infections are the most common contemporary infectious diseases resulting in prominent harm to human health and great economic damage. At least five groups of viruses including more than 300 subtypes are currently referred to ARVI pathogens. Such infectious agents are characterized by variability resulting in their altered antigenic characteristics, increased contagiousness, "evasion from immune response and resistance to antivirals. Relevance of influenza and other ARVIs is also accounted for by rapid development of bacteria-associated respiratory diseases. Continuous variability of influenza viruses and emergence of new ARVI pathogens pose a serious threat. In recent years, a simultaneous circulation of subtype A (H1N1) and A (H3N2) influenza viruses with a predominance of a pandemic strain as well as type B viruses have been observed. Among the causative agents of non-influenza ARVIs, respiratory syncytial virus, rhino- and adenoviruses, and I/III parainfluenza viruses are recorded most often. Here we present the data of virology and serological examination of clinical samples collected during the 2018 – 2019 epidemic season in the Republic of Kazakhstan. For this, 2794 clinical samples (2530 nasopharyngeal swabs and 264 blood serums) were collected from patients diagnosed with ARVI, ARI, bronchitis, and pneumonia. Analysis of nasopharyngeal swabs for detection of influenza by RT-PCR demonstrated that mixed etiology influenza viruses with predominance of A/H1N1pdm virus circulated in Kazakhstan. The genetic fingerprints of influenza virus were found in 511 swabs (20.20% of total examined samples). Influenza A virus RNA was detected in 508 biological samples: A/H1N1 – in 289, A/H3N2 – in 209, and unidentified virus subtype in 10 samples. Type B influenza virus was detected in 3 samples. Study of 264 serum samples by HAI assay and ELISA showed emergence of antibodies against influenza A/H1N1, A/H3N2, and B viruses in residents from various regions of Kazakhstan that indirectly confirmed co-circulation of these viruses. 42 influenza virus strains were isolated in chicken embryos, from which 28 were assigned to A/H1N1pdm virus, 13 to A/H3N2 virus, and one isolate was identified as influenza B virus. Laboratory diagnostics of clinical samples for ARVIs established that among identified non-influenza agents respiratory syncytial virus dominated, while rhinoviruses and adenoviruses were less common. Metapneumoviruses, bocaviruses, coronaviruses, and type I parainfluenza viruses were detected in few cases. Comparison of study data with those obtained after examining circulation of influenza viruses during the 2017 – 2018 epidemic season showed that in 2018 – 2019 in Kazakhstan similar to the previous epidemic season, influenza A and B viruses continued to circulate, with prevalence of A/H1N1pdm virus. Identification of non-influenza viruses causing respiratory infections in 2018 – 2019 showed predominance of respiratory syncytial virus, which correlated with data on the 2017 – 2018 epidemic season.

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Reverse Transcription Recombinase-aided Amplification Assay for H5 Subtype avian Influenza Virus

10.21203/rs.3.rs-1147624/v1 ◽

2021 ◽

Author(s):

Suchun Wang ◽

Yang Li ◽

fuyou zhang ◽

Nan Jiang ◽

Qingye Zhuang ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Reverse Transcription ◽

Clinical Sample ◽

Economic Losses ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Single Temperature ◽

H5 Subtype

Abstract Background: The H5 subtype avian influenza virus (AIV) has caused huge economic losses to the poultry industry and is a threat to human health. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks. Methods: In this study, we developed a reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of H5 subtype AIV. Assays were performed at a single temperature (39°C), and the results were obtained within 20 min. Results: The assay showed no cross-detection with Newcastle disease virus or infectious bronchitis virus. The analytical sensitivity was 103 RNA copies/μL at a 95% confidence interval according to probit regression analysis, with 100% specificity. Compared with published reverse transcription quantitative real-time polymerase chain reaction assays, the κ value of the RT-RAA assay in 420 avian clinical samples was 0.983 (p < 0.001). The sensitivity for avian clinical sample detection was 97.26% (95% CI, 89.56–99.52%), and the specificity was 100% (95% CI, 98.64–100%). Conclusions: These results indicated that our RT-RAA assay may be a valuable tool for detecting H5 subtype AIV.

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Faculty Opinions recommendation of Mobile imaging platform for digital influenza virus counting.

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

10.3410/f.736211511.793568631 ◽

2019 ◽

Author(s):

Fumihiko Kajiya

Keyword(s):

Influenza Virus ◽

Mobile Imaging

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H7N7 Avian Influenza Virus Mutation from Low to High Pathogenicity on a Layer Chicken Farm in the UK

Viruses ◽

10.3390/v13020259 ◽

2021 ◽

Vol 13 (2) ◽

pp. 259

Author(s):

Alexander M. P. Byrne ◽

Scott M. Reid ◽

Amanda H. Seekings ◽

Alejandro Núñez ◽

Ana B. Obeso Prieto ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Economic Losses ◽

Clinical Samples ◽

Mortality Data ◽

Molecular Evidence ◽

High Pathogenicity ◽

Clinical Protection ◽

The Uk

Avian influenza virus (AIV) subtypes H5 and H7 are capable of mutating from low to high pathogenicity strains, causing high mortality in poultry with significant economic losses globally. During 2015, two outbreaks of H7N7 low pathogenicity AIV (LPAIV) in Germany, and one each in the United Kingdom (UK) and The Netherlands occurred, as well as single outbreaks of H7N7 high pathogenicity AIV (HPAIV) in Germany and the UK. Both HPAIV outbreaks were linked to precursor H7N7 LPAIV outbreaks on the same or adjacent premises. Herein, we describe the clinical, epidemiological, and virological investigations for the H7N7 UK HPAIV outbreak on a farm with layer chickens in mixed free-range and caged units. H7N7 HPAIV was identified and isolated from clinical samples, as well as H7N7 LPAIV, which could not be isolated. Using serological and molecular evidence, we postulate how the viruses spread throughout the premises, indicating potential points of incursion and possible locations for the mutation event. Serological and mortality data suggested that the LPAIV infection preceded the HPAIV infection and afforded some clinical protection against the HPAIV. These results document the identification of a LPAIV to HPAIV mutation in nature, providing insights into factors that drive its manifestation during outbreaks.

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