Human Properdin Released By Infiltrating Neutrophils Can Modulate Influenza A Virus Infection

The complement system is designed to recognise and eliminate invading pathogens via activation of classical, alternative and lectin pathways. Human properdin stabilises the alternative pathway C3 convertase, resulting in an amplification loop that leads to the formation of C5 convertase, thereby acting as a positive regulator of the alternative pathway. It has been noted that human properdin on its own can operate as a pattern recognition receptor and exert immune functions outside its involvement in complement activation. Properdin can bind directly to microbial targets via DNA, sulfatides and glycosaminoglycans, apoptotic cells, nanoparticles, and well-known viral virulence factors. This study was aimed at investigating the complement-independent role of properdin against Influenza A virus infection. As one of the first immune cells to arrive at the site of IAV infection, we show here that IAV challenged neutrophils released properdin in a time-dependent manner. Properdin was found to directly interact with haemagglutinin, neuraminidase and matrix 1 protein Influenza A virus proteins in ELISA and western blot. Furthermore, modelling studies revealed that properdin could bind HA and NA of the H1N1 subtype with higher affinity compared to that of H3N2 due to the presence of an HA cleavage site in H1N1. In an infection assay using A549 cells, properdin suppressed viral replication in pH1N1 subtype while promoting replication of H3N2 subtype, as revealed by qPCR analysis of M1 transcripts. Properdin treatment triggered an anti-inflammatory response in H1N1-challenged A549 cells and a pro-inflammatory response in H3N2-infected cells, as evident from differential mRNA expression of TNF-α, NF-κB, IFN-α, IFN-β, IL-6, IL-12 and RANTES. Properdin treatment also reduced luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles; however, it was increased in the case of pseudotyped H3N2 particles. Collectively, we conclude that infiltrating neutrophils at the site of IAV infection can release properdin, which then acts as an entry inhibitor for pandemic H1N1 subtype while suppressing viral replication and inducing an anti-inflammatory response. H3N2 subtype can escape this immune restriction due to altered haemagglutinin and neuraminindase, leading to enhanced viral entry, replication and pro-inflammatory response. Thus, depending on the subtype, properdin can either limit or aggravate IAV infection in the host.

Rapid Differential Detection of H1 and H3 Subtype Swine Influenza Viruses by a TaqMan-MGB Based Duplex One-Step Real Time RT-PCR Assay

Swine influenza is not only an economically important respiratory disease in swine, but also constantly poses a threat to human health. Hence, developing a rapid, sensitive and efficient detection method of swine influenza virus (SIV) is highly essential. By aligning the HA gene sequences of SIV circulating in China in recent 10 years, a H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and Pandemic 2009 H1N1 (Pdm09 H1N1) lineages plus a H3 prime-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed, respectively. Further, a TaqMan-MGB based duplex one-step real time RT-PCR (RRT-PCR) assay was established and evaluated. The duplex RRT-PCR possessed the detection limit of 5 copies/μL HA plasmid for each of the EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs, and matched an overall detection sensitivity of 100% and specificity of 91.67% with traditional virus isolation through chicken embryo inoculation using experimentally infected mice lung samples. Besides, the method showed high repeatability both within-run and between-runs, and no cross-reactivity against some commonly circulated porcine viruses in China. Furthermore, the duplex RRT-PCR method revealed a relatively higher prevalent rate of H1 than H3 subtype SIV in 166 nasal swabs from pigs in some slaughterhouse during October ~ December, 2019. This developed assay could be very helpful for rapid differential detection and routine surveillance of EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs in China.

Adjuvanted Influenza Vaccines Elicits Higher Antibody Responses against the A(H3N2) Subtype than Non-Adjuvanted Vaccines

Background: vaccination is the best approach to prevent influenza infections so far. Serological studies on the effect of different vaccine types are important to address vaccination campaigns and protect our population. In our study, we compared the serological response against influenza A subtypes using the non-adjuvanted influenza vaccine (NAIV) in adults and the elderly and the adjuvanted influenza vaccine (AIV) in the elderly. Methods: We performed a retrospective analysis by hemagglutination inhibition assay (HI) of serum samples right before and 28 days after seasonal influenza vaccination during the 1996–2017 seasons. Conclusions: The AIV presents better performance against the A(H3N2) subtype in the elderly whereas the NAIV induces a better response against A(H1N1)pdm09 in the same group.

Antiviral activity of Siberian wild and cultivated plants

The article presents data on the antiviral activity of ethanol and aqueous extracts isolated from the herb plants Dracocephalum nutans, Glechoma hederacea, Melissa officinalis, Berteroa incana, Aegopodium podagraria, and Veronica longifolia against the A/Aichi/2/68 (H3N2) and A/chicken/ Kurgan/05/2005 (H5N1) subtypes of influenza A virus (IAV) in MDCK cell culture. It was found that the ethanol extracts of Glechoma hederacea, Berteroa incana, and Aegopodium podagraria have the antiviral activity in vitro in MDCK cell culture against H3N2 subtype of IAV. The aqueous extracts of Glechoma hederacea, Melissa officinalis, Aegopodium podagraria, and Veronica longifolia and ethanol extracts of Berteroa incana and Veronica longifolia are active against H5N1 subtype of IAV. None of the extracts of Dracocephalum nutans showed significant activity against the studied subtypes. The greatest efficiency was shown by the ethanol extract of Aegopodium podagraria against the H5N1 subtype and aqueous extracts from Glechoma hederacea and Melissa officinalis.against the H3N2 subtype. The ethanol extract of Berteroa incana has a high activity against both IAV subtypes, which makes this sample the most promising for creating new drugs for the prevention and treatment of influenza virus.

1772. Factors that Influence Viral Load in Patients Infected with Influenza Over Multiple Seasons

Background The factors that influence influenza viral load are poorly understood, but may have important implications for viral transmission and disease severity. We explored the relationship between patient and virus factors on influenza viral load across 4 consecutive influenza seasons. Methods Adult influenza-positive patients presenting to emergency departments in Baltimore, MD and Taipei, Taiwan between 2014 and 2018 were consented and enrolled. Nasopharyngeal (NP) samples and detailed paired data on symptom duration, demographics, and vaccination were collected. Viral load was inferred using the cycle threshold (Ct) values from quantitative real-time RT–PCR assays for 299 samples and influenza subtype was determined. Bivariate and multivariate analyses were conducted. Results Viral load was impacted by both patient and virus characteristics. Older age and shorter duration of symptoms was associated with a higher viral load (age: Ct difference -0.04, P = 0.022. symptoms: each increasing day, Ct difference +1.02, P < 0.001). Seasonal variability was observed, with the highest viral load associated with the 2014–2015 predominant H3N2 subtype (Ct 21.69, P < 0.001), where there was also a vaccine mismatch. Across seasons, H1N1 was associated with a lower viral load than H3N2 (Ct value + 2.66, P < 0.001). There was not association between gender and immunosuppression on viral load. Conclusion Our study demonstrates that both host and virus factors that can affect Ct values and inferred viral loads. As anticipated, higher viral load, were found to be associated with older age and shorter duration of symptoms; interestingly infection with the H3N2 subtype, traditionally associated with more severe disease was also associated with higher viral loads. Future-focused studies are required to better characterize these relationships, and directly evaluate relevance to both transmission and disease severity. Disclosures All authors: No reported disclosures.

Influenza incidence prediction for the United States: an update for the 2018-2019 season

Introduction:Seasonal influenza causes a high disease burden every year in the United States and worldwide. Anticipating epidemic size ahead of season can contribute to preparedness and more targetted control and prevention of seasonal influenza.Methods:A recently developed process-based epidemiological model that incorporates evolutionary change of the virus and generates incidence forecasts for the H3N2 subtype ahead of the season, was previously validated by several statistical criteria, including an accurate real-time prediction for the 2016-2017 influenza season. With this model, a new forecast is generated here for the upcoming 2018-2019 season. The accuracy of predictions published for the 2017-2018 season is also retrospectively evaluated.Results:For 2017-2018, the model correctly predicted the dominance of the H3N2 subtype and its higher than average incidence. Based on surveillance and sequence data up to June 2018, the new forecast for the upcoming 2018-2019 season indicates low levels for H3N2, and suggests an H1N1 dominant season with low incidence of influenza B.Discussion:Real-time forecasts, those generated with a model that was parameterized based on data preceding the predicted season, allows valuable evaluation of the approach. Anticipating the dominant subtype and the size of the upcoming epidemic ahead of season informs disease control. Further studies are needed to promote more accurate ahead-of-season forecasts and extend the approach to multiple subtypes.

Isolation of a Reassortant H1N2 Swine Flu Strain of Type “Swine-Human-Avian” and Its Genetic Variability Analysis

We isolated an influenza strain named A/Swine/Fujian/F1/2010 (H1N2) from a pig suspected to be infected with swine flu. The results of electron microscopy, hemagglutination (HA) assay, hemagglutination inhibition (HI) assay, and whole genome sequencing analysis suggest that it was a reassortant virus of swine (H1N1 subtype), human (H3N2 subtype), and avian influenza viruses. To further study the genetic evolution of A/Swine/Fujian/F1/2010 (H1N2), we cloned its whole genome fragments using RT-PCR and performed phylogenetic analysis on the eight genes. As a result, the nucleotide sequences of HA, NA, PB1, PA, PB2, NP, M, and NS gene are similar to those of A/Swine/Shanghai/1/2007(H1N2) with identity of 98.9%, 98.9%, 99.0%, 98.6%, 99.0%, 98.9%, 99.3%, and 99.3%, respectively. Similar to A/Swine/Shanghai/1/2007(H1N2), we inferred that the HA, NP, M, and NS gene fragments of A/Swine/Fujian/F1/2010 (H1N2) strain were derived from classical swine influenza H3N2 subtype, NA and PB1 were derived from human swine influenza H3N2 subtype, and PB2 and PA genes were derived from avian influenza virus. This further validates the role of swine as a “mixer” for influenza viruses.