[Analysis of epidemiological characteristics of respiratory pathogens in children with influenza-like illnesses in a children's hospital in Beijing from 2022 to 2023].

To investigate the status and epidemiological characteristics of respiratory pathogens infections in children with influenza-like illnesses (ILI) in Beijing Children's Hospital from 2022 to 2023. A dual amplification technique was used to detect nucleic acids of seven common respiratory pathogens, including influenza A virus (Flu A), influenza B virus (Flu B), mycoplasma pneumoniae (MP), respiratory syncytial virus (RSV), parainfluenza virus (PIV), adenovirus (ADV), and Chlamydia pneumoniae (CP), in outpatient and inpatient children (aged 0-18 years) with influenza-like symptoms who sought medical care at Beijing Children's Hospital, from January 2022 to March 2023. A total of 43 663 children were included in the study, of which 27 903 tested positive for respiratory pathogens with a total detection rate of 63.91%. Flu A had the highest detection rate of 69.93% (27 332/39 084), followed by MP about 13.22% (380/2 875). The total detection rate of RSV, PIV and ADV was 7.69% (131/1 704). Flu B had a detection rate of 0.16% (64/39 084). No CP was detected in this study. A total of 7 cases of dual infections were detected, with a detection rate of 0.41% (7/1 704). The Chi-square test was used to analyze the differences in detection rates of pathogens among different genders, age groups, and different seasons. Among the seven pathogens, only Flu A had statistically significant differences in gender (χ2=16.712, P<0.001). The detection rates of Flu A and MP showed an increasing trend with age (both P trend<0.001), while the detection rates of RSV and PIV showed a decreasing trend with age (both P trend<0.001). Flu A had its epidemic peak in winter and spring, with detection rates of 61.30% (3 907/6 374) and 77.47% (23 207/29 958) respectively; MP and PIV had higher detection rates in autumn (25.14% and 7.64% respectively); RSV showed a relatively higher detection rate in winter (8.69%); Flu B and ADV had lower detection rates throughout the study period (0.16% and 1.17% respectively). In conclusion, children with ILI in 2022-2023 were mainly infected with a single respiratory pathogen, and occasionally dual pathogen infections were observed. Among them, the detection rate of Flu A was the highest, and only Flu A showed a gender difference in detection rate. As the age of the children patients increased, the detection rate of Flu A and MP showed an increasing trend, while RSV and PIV showed a decreasing trend. The prevalence of Flu A, Flu B, MP, PIV, and RSV were seasonal.

Red knots in Europe: a dead end host species or a new niche for highly pathogenic avian influenza?

The 2020/2021 epidemic in Europe of highly pathogenic avian influenza virus (HPAIV) of subtype H5 surpassed all previously recorded European outbreaks in size, genotype constellations and reassortment frequency and continued into 2022 and 2023. The causative 2.3.4.4b viral lineage proved to be highly proficient with respect to reassortment with cocirculating low pathogenic avian influenza viruses and seems to establish an endemic status in northern Europe. A specific HPAIV reassortant of the subtype H5N3 was detected almost exclusively in red knots (Calidris canutus islandica) in December 2020. It caused systemic and rapidly fatal disease leading to a singular and self-limiting mass mortality affecting about 3500 birds in the German Wadden Sea, roughly 1 % of the entire flyway population of islandica red knots. Phylogenetic analyses revealed that the H5N3 reassortant very likely had formed in red knots and remained confined to this species. While mechanisms of virus circulation in potential reservoir species, dynamics of spill-over and reassortment events and the roles of environmental virus sources remain to be identified, the year-round infection pressure poses severe threats to endangered avian species and prompts adaptation of habitat and species conservation practices.

Binary and quaternary mixtures of perfluoroalkyl substances (PFAS) differentially affect the immune response to influenza A virus infection.

Per- and polyfluoroalkyl substances (PFAS) are anthropogenic organofluorine compounds that persist indefinitely in the environment and bioaccumulate throughout all trophic levels. Biomonitoring efforts have detected multiple PFAS in the serum of most people. Immune suppression has been among the most consistent effects of exposure to PFAS. PFAS often co-occur as mixtures in the environment, however, few studies have examined immunosuppression of PFAS mixtures or determined whether PFAS exposure affects immune function in the context of infection. In this study, mixtures containing two or four different PFAS and a mouse model of infection with influenza A virus (IAV) were used to assess immunotoxicity of PFAS mixtures. PFAS were administered via the drinking water as either a binary mixture of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) or quaternary mixture of PFOS, PFOA, perfluorohexane sulfonate (PFHxS), and perfluorononanoic acid (PFNA). The results indicated that the binary mixture affected the T-cell response, while the quaternary mixture affected the B-cell response to infection. These findings indicate that the immunomodulatory effects of PFAS mixtures are not simply additive, and that the sensitivity of immune responses to PFAS varies by cell type and mixture. The study also demonstrates the importance of studying adverse health effects of PFAS mixtures.

An overview of influenza A virus detection methods: from state-of-the-art of laboratories to point-of-care strategies.

Influenza A virus (IAV), a common respiratory infectious pathogen, poses a significant risk to personal health and public health safety due to rapid mutation and wide host range. To better prevent and treat IAV, comprehensive measures are needed for early and rapid screening and detection of IAV. Although traditional laboratory-based techniques are accurate, they are often time-consuming and not always feasible in emergency or resource-limited areas. In contrast, emerging point-of-care strategies provide faster results but may compromise sensitivity and specificity. Here, this review critically evaluates various detection methods for IAV from established laboratory-based procedures to innovative rapid diagnosis. By analyzing the recent research progress, we aim to address significant gaps in understanding the effectiveness, practicality, and applicability of these methods in different scenarios, which could provide information for healthcare strategies, guide public health response measures, and ultimately strengthen patient care in the face of the ongoing threat of IAV. Through a detailed comparison of diagnostic models, this review can provide a reliable reference for rapid, accurate and efficient detection of IAV, and to contribute to the diagnosis, treatment, prevention, and control of IAV.

FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

The ubiquitin-like modifier FAT10 is upregulated under pro-inflammatory conditions, targets its substrates for proteasomal degradation and functions as a negative regulator of the type-I IFN response. Influenza A virus infection upregulates the production of type-I IFN and the expression of the E3 ligase TRIM21, which regulates type-I IFN production in a positive feedback manner. In this study, we show that FAT10 becomes covalently conjugated to TRIM21 and that this targets TRIM21 for proteasomal degradation. We further show that the coiled-coil and PRYSPRY domains of TRIM21 and the C-terminal diglycine motif of FAT10 are important for the TRIM21-FAT10 interaction. Moreover, upon influenza A virus infection and in the presence of FAT10 the total ubiquitination of TRIM21 is reduced and our data reveal that the FAT10-mediated degradation of TRIM21 diminishes IFNß production. Overall, this study provides strong evidence that FAT10 down-regulates the antiviral type-I IFN production by modulating additional molecules of the RIG-I signaling pathway besides the already published OTUB1. In addition, we elucidate a novel mechanism of FAT10-mediated proteasomal degradation of TRIM21 that regulates its stability.

Influenza A viral burst size from thousands of infected single cells using droplet quantitative PCR (dqPCR).

An important aspect of how viruses spread and infect is the viral burst size, or the number of new viruses produced by each infected cell. Surprisingly, this value remains poorly characterized for influenza A virus (IAV), commonly known as the flu. In this study, we screened tens of thousands of cells using a microfluidic method called droplet quantitative PCR (dqPCR). The high-throughput capability of dqPCR enabled the measurement of a large population of infected cells producing progeny virus. By measuring the fully assembled and successfully released viruses from these infected cells, we discover that the viral burst sizes for both the seasonal H3N2 and the 2009 pandemic H1N1 strains vary significantly, with H3N2 ranging from 101 to 104 viruses per cell, and H1N1 ranging from 101 to 103 viruses per cell. Some infected cells produce average numbers of new viruses, while others generate extensive number of viruses. In fact, we find that only 10% of the single-cell infections are responsible for creating a significant portion of all the viruses. This small fraction produced approximately 60% of new viruses for H3N2 and 40% for H1N1. On average, each infected cell of the H3N2 flu strain produced 709 new viruses, whereas for H1N1, each infected cell produced 358 viruses. This novel method reveals insights into the flu virus and can lead to improved strategies for managing and preventing the spread of viruses.

Genomic diversity and evolutionary dynamics of Influenza A viruses in Colombian swine: implications for one health surveillance and control.

Influenza A viruses (IAV) impose significant respiratory disease burdens in both swine and humans worldwide, with frequent human-to-swine transmission driving viral evolution in pigs and highlighting the risk at the animal-human interface. Therefore, a comprehensive One Health approach (interconnection among human, animal, and environmental health) is needed for IAV prevention, control, and response. Animal influenza genomic surveillance remains limited in many Latin American countries, including Colombia. To address this gap, we genetically characterized 170 swine specimens from Colombia (2011-2017). Whole genome sequencing revealed a predominance of pandemic-like H1N1 lineage, with a minority belonging to H3N2 and H1N2 human seasonal-like lineage and H1N1 early classical swine lineages. Significantly, we have identified reassortant and recombinant viruses (H3N2, H1N1) not previously reported in Colombia. This suggests a broad genotypic viral diversity, likely resulting from reassortment between classical endemic viruses and new introductions established in Colombia's swine population (e.g. the 2009 H1N1 pandemic). Our study highlights the importance of a One Health approach in disease control, particularly in an ecosystem where humans are a main source of IAV to swine populations, and emphasizes the need for continued surveillance and enhanced biosecurity measures. The co-circulation of multiple subtypes in regions with high swine density facilitates viral exchange, underscoring the importance of monitoring viral evolution to inform vaccine selection and public health policies locally and globally.

Surveillance of respiratory viruses by aerosol screening in indoor air as an early warning system for epidemics.

The development of effective methods for the surveillance of seasonal respiratory viruses is required for the timely management of outbreaks. We aimed to survey Influenza-A, Influenza-B, RSV-A, Rhinovirus and SARS-CoV-2 surveillance in a tertiary hospital and a campus over 5 months. The effectiveness of air screening as an early warning system for respiratory viruses was evaluated in correlation with respiratory tract panel test results. The overall viral positivity was higher on the campus than in the hospital (55.0% vs. 38.0%). Influenza A was the most prevalent pathogen in both locations. There were two influenza peaks (42nd and 49th weeks) in the hospital air, and a delayed peak was detected on campus in the 1st-week of January. Panel tests indicated a high rate of Influenza A in late December. RSV-A-positivity was higher on the campus than the hospital (21.6% vs. 7.4%). Moreover, we detected two RSV-A peaks in the campus air (48th and 51st weeks) but only one peak in the hospital and panel tests (week 49). Although rhinovirus was the most common pathogen in panel tests, rhinovirus positivity was low in air samples. The air screening for Influenza-B and SARS-Cov-2 revealed comparable positivity rates with panel tests. Air screening can be integrated into surveillance programs to support infection control programs for potential epidemics of respiratory virus infections except for rhinoviruses.

Glycopolymer with Sulfated Fucose and 6'-Sialyllactose as a Dual-Targeted Inhibitor on Resistant Influenza A Virus Strains.

The frequent mutations of influenza A virus (IAV) have led to an urgent need for the development of innovative antiviral drugs. Glycopolymers offer significant advantages in biomedical applications owing to their biocompatibility and structural diversity. However, the primary challenge lies in the design and synthesis of well-defined glycopolymers to precisely control their biological functionalities. In this study, functional glycopolymers with sulfated fucose and 6'-sialyllactose were successfully synthesized through ring-opening metathesis polymerization and a postmodification strategy. The optimized heteropolymer exhibited simultaneous targeting of hemagglutinin and neuraminidase on the surface of IAV, as evidenced by MU-NANA assay and hemagglutination inhibition data. Antiviral experiments demonstrated that the glycopolymer displayed broad and efficient inhibitory activity against wild-type and mutant strains of H1N1 and H3N2 subtypes in vitro, thereby establishing its potential as a dual-targeted inhibitor for combating IAV resistance.

Exploring the Efficacy of Peptides and Mimics against Influenza A Virus, Adenovirus, and Murine Norovirus.

The ongoing battle against viral pandemics continues, with the possibility of future outbreaks. The search for effective antiviral compounds that can combat a diverse range of viruses continues to be a focal point of research. This study investigated the efficacy of two natural antimicrobial peptides (AMPs) (lactoferricin and LL-37), two synthetic AMPs (melimine and Mel4), and nine AMP mimics (758, 1091, 1096, 1083, 610, NAPL, 3-BIPL, 4-BIPL, and Sau-22) against influenza A virus strains H1N1 and H3N2, human adenovirus 5 (HAdV-5), and murine norovirus 1 (MNV-1). These compounds were tested using virus pre-treatment, cell pre-treatment, or post-cell entry treatment assays, electron microscopy, and circular dichroism (CD), alongside evaluations of cytotoxicity against the host cells. After virus pre-treatment, the AMP mimics 610 and Sau-22 had relatively low IC50 values for influenza strains H1N1 (2.35 and 6.93 µM, respectively) and H3N2 (3.7 and 5.34 µM, respectively). Conversely, natural and synthetic AMPs were not active against these strains. For the non-enveloped viruses, the AMP Mel4 and mimic 1083 had moderate activity against HAdV-5 (Mel4 IC50 = 47.4 µM; 1083 IC50 = 47.2 µM), whereas all AMPs, but none of the mimics, were active against norovirus (LL-37 IC50 = 4.2 µM; lactoferricin IC50 = 23.18 µM; melimine IC50 = 4.8 µM; Mel4 IC50 = 8.6 µM). Transmission electron microscopy demonstrated that the mimics targeted the outer envelope of influenza viruses, while the AMPs targeted the capsid of non-enveloped viruses. CD showed that Mel4 adopted an α-helical structure in a membrane mimetic environment, but mimic 758 remained unstructured. The diverse activity against different virus groups is probably influenced by charge, hydrophobicity, size, and, in the case of natural and synthetic AMPs, their secondary structure. These findings underscore the potential of peptides and mimics as promising candidates for antiviral therapeutics against both enveloped and non-enveloped viruses.

Beyond COVID-19: Wastewater-based epidemiology for multipathogen surveillance and normalization strategies.

Wastewater-based epidemiology (WBE) is a critical tool for monitoring community health. Although much attention has focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of coronavirus disease 2019 (COVID-19), other pathogens also pose significant health risks. This study quantified the presence of SARS-CoV-2, influenza A virus (Inf-A), and noroviruses of genogroups I (NoV-GI) and II (NoV-GII) in wastewater samples collected weekly (n = 170) from July 2023 to February 2024 from five wastewater treatment plants (WWTPs) in Yamanashi Prefecture, Japan, by quantitative PCR. Inf-A RNA exhibited localized prevalence with positive ratios of 59 %-82 % in different WWTPs, suggesting regional outbreaks within specific areas. NoV-GI (94 %, 160/170) and NoV-GII (100 %, 170/170) RNA were highly prevalent, with NoV-GII (6.1 ± 0.8 log10 copies/L) consistently exceeding NoV-GI (5.4 ± 0.7 log10 copies/L) RNA concentrations. SARS-CoV-2 RNA was detected in 100 % of the samples, with mean concentrations of 5.3 ± 0.5 log10 copies/L in WWTP E and 5.8 ± 0.4 log10 copies/L each in other WWTPs. Seasonal variability was evident, with higher concentrations of all pathogenic viruses during winter. Non-normalized and normalized virus concentrations by fecal indicator bacteria (Escherichia coli and total coliforms), an indicator virus (pepper mild mottle virus (PMMoV)), and turbidity revealed significant positive associations with the reported disease cases. Inf-A and NoV-GI + GII RNA concentrations showed strong correlations with influenza and acute gastroenteritis cases, particularly when normalized to E. coli (Spearman's ρ = 0.70-0.81) and total coliforms (ρ = 0.70-0.81), respectively. For SARS-CoV-2, non-normalized concentrations showed a correlation of 0.61, decreasing to 0.31 when normalized to PMMoV, suggesting that PMMoV is unsuitable. Turbidity normalization also yielded suboptimal results. This study underscored the importance of selecting suitable normalization parameters tailored to specific pathogens for accurate disease trend monitoring using WBE, demonstrating its utility beyond COVID-19 surveillance.

Loss of Lymphatic IKKα Disrupts Lung Immune Homeostasis, Drives BALT Formation, and Protects against Influenza.

IκB kinase (IKK)α controls noncanonical NF-κB signaling required for lymphoid organ development. We showed previously that lymph node formation is ablated in IkkαLyve-1 mice constitutively lacking IKKα in lymphatic endothelial cells (LECs). We now reveal that loss of IKKα in LECs leads to the formation of BALT in the lung. Tertiary lymphoid structures appear only in the lungs of IkkαLyve-1 mice and are not present in any other tissues, and these highly organized BALT structures form after birth and in the absence of inflammation. Additionally, we show that IkkαLyve-1 mice challenged with influenza A virus (IAV) exhibit markedly improved survival and reduced weight loss compared with littermate controls. Importantly, we determine that the improved morbidity and mortality of IkkαLyve-1 mice is independent of viral load and rate of clearance because both mice control and clear IAV infection similarly. Instead, we show that IFN-γ levels are decreased, and infiltration of CD8 T cells and monocytes into IkkαLyve-1 lungs is reduced. We conclude that ablating IKKα in LECs promotes BALT formation and reduces the susceptibility of IkkαLyve-1 mice to IAV infection through a decrease in proinflammatory stimuli.

Anti-Influenza A Potential of Tagetes erecta Linn. Extract Based on Bioinformatics Analysis and In Vitro Assays.

Tagetes erecta Linn. (TE) is traditionally used to treat cardiovascular, renal, and gastrointestinal diseases. In this study, we investigated the active compounds and targets of TE extract that may exert antiviral effects against influenza A. Active compounds and targets of TE extract were identified using the Traditional Chinese Medicine Systems Pharmacology database (TCSMP). The influenza A-related gene set was screened using GeneCards and the Kyoto Encyclopedia of Genes and Genomes (KEGG). A protein-protein interaction (PPI) network was built to establish the hub targets. Pathway and target studies were conducted using Gene Expression Omnibus (GEO). The interactions between active compounds and potential targets were assessed by molecular docking. An in vitro study was performed using antiviral and plaque reduction assays. From the compound and target search, we identified 6 active compounds and 95 potential targets. We retrieved 887 influenza-associated target genes and determined 14 intersecting core targets between TE and influenza. After constructing a compound-target network, we discovered lutein and beta-carotene to be the key compounds. Next, PPI network analysis identified the top three hub genes associated with influenza (IL-6, HIF1A, and IL-1ß). Similarly, GEO analysis revealed IL-6, TGFB1, and CXCL8 to be the top three target genes. In our docking study, we identified that lutein and IL-6 had the strongest bindings. Our in vitro experimental results revealed that the TE extract exhibited therapeutic rather than prophylactic effects on influenza disease. We identified lutein as a main active compound in TE extract, and IL-6 as an important target associated with influenza, by using data mining and bioinformatics. Our in vitro findings indicated that TE extract exerted protective properties against the influenza A virus. We speculated that lutein, as a key active component in TE extract, is largely responsible for its antiviral effects. Therefore, we suggest TE extract as an alternative in the treatment of influenza.

The Novel A-Type Proanthocyanidin-Rich Phytocomplex SP4™ Acts as a Broad-Spectrum Antiviral Agent against Human Respiratory Viruses.

The appearance of new respiratory virus infections in humans with epidemic or pandemic potential has underscored the urgent need for effective broad-spectrum antivirals (BSAs). Bioactive compounds derived from plants may provide a natural source of new BSA candidates. Here, we investigated the novel phytocomplex formulation SP4™ as a candidate direct-acting BSA against major current human respiratory viruses, including coronaviruses and influenza viruses. SP4™ inhibited the in vitro replication of SARS-CoV-2, hCoV-OC43, hCoV-229E, Influenza A and B viruses, and respiratory syncytial virus in the low-microgram range. Using hCoV-OC43 as a representative respiratory virus, most of the antiviral activity of SP4™ was observed to stem primarily from its dimeric A-type proanthocyanidin (PAC-A) component. Further investigations of the mechanistic mode of action showed SP4™ and its PAC-A-rich fraction to prevent hCoV-OC43 from attaching to target cells and exert virucidal activity. This occurred through their interaction with the spike protein of hCoV-OC43 and SARS-CoV-2, thereby interfering with spike functions and leading to the loss of virion infectivity. Overall, these findings support the further development of SP4™ as a candidate BSA of a natural origin for the prevention of human respiratory virus infections.

Opening another door on influenza entry.

In this issue of Cell Host & Microbe, Karakus et al. find that an influenza virus enters cells by exclusively binding to a protein instead of sugars.

Filling two needs with one deed: a combinatory mucosal vaccine against influenza A virus and respiratory syncytial virus.

Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFß or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.

MEK-inhibitor treatment reduces the induction of regulatory T cells in mice after influenza A virus infection.

Regulatory T cells (Tregs) play a crucial and complex role in balancing the immune response to viral infection. Primarily, they serve to regulate the immune response by limiting the expression of proinflammatory cytokines, reducing inflammation in infected tissue, and limiting virus-specific T cell responses. But excessive activity of Tregs can also be detrimental and hinder the ability to effectively clear viral infection, leading to prolonged disease and potential worsening of disease severity. Not much is known about the impact of Tregs during severe influenza. In the present study, we show that CD4+/CD25+FoxP3+ Tregs are strongly involved in disease progression during influenza A virus (IAV) infection in mice. By comparing sublethal with lethal dose infection in vivo, we found that not the viral load but an increased number of CD4+/CD25+FoxP3+ Tregs may impair the immune response by suppressing virus specific CD8+ T cells and favors disease progression. Moreover, the transfer of induced Tregs into mice with mild disease symptoms had a negative and prolonged effect on disease outcome, emphasizing their importance for pathogenesis. Furthermore, treatment with MEK-inhibitors resulted in a significant reduction of induced Tregs in vitro and in vivo and positively influenced the progression of the disease. Our results demonstrate that CD4+/CD25+FoxP3+ Tregs are involved in the pathogenesis of severe influenza and indicate the potential of the MEK-inhibitor zapnometinib to modulate CD4+/CD25+FoxP3+ Tregs. Thus, making MEK-inhibitors even more promising for the treatment of severe influenza virus infections.

Phylogenetic and mutational analysis of H10N3 avian influenza A virus in China: potential threats to human health.

In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. This study focuses on a patient infected with the H10N3 subtype of avian influenza virus, admitted to the Third People's Hospital of Kunming City on March 6, 2024. Metagenomic RNA sequencing and polymerase chain reaction (PCR) analysis were conducted on the patient's sputum, confirming the H10N3 infection. The patient presented severe pneumonia symptoms such as fever, expectoration, chest tightness, shortness of breath, and cough. Phylogenetic analysis of the Haemagglutinin (HA) and neuraminidase (NA) genes of the virus showed that the virus was most closely related to a case of human infection with the H10N3 subtype of avian influenza virus found in Zhejiang Province, China. Analysis of amino acid mutation sites identified four mutations potentially hazardous to human health. Consequently, this underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus, utilizing advanced genomic surveillance techniques.

Modified transport medium for improving influenza virus detection.

Background: Accurate detection of influenza virus in clinical samples requires correct execution of all aspects of the detection test. If the viral load in a sample is below the detection limit, a false negative result may be obtained. To overcome this issue, we developed a modified transport medium (MTM) for clinical sample transportation to increase viral detection sensitivity. Method: We first validated the MTM using laboratory-stocked influenza A viruses (IAVs: H1N1, H3N2, H7N3, H9N2) and influenza B viruses (IBVs: Yamagata, Victoria). We also tested clinical samples. A total of 110 patients were enrolled and a pair of samples were collected to determine the sensitivity of real-time polymerase chain reaction (RT-PCR) following MTM treatment. Result: After 24 h culturing in MTM, the viral loads were increased, represented by a 10-fold increase in detection sensitivity for H1N1, H9N2, and IBVs, a 100-fold increase for H3N2, and a 1,000-fold increase for H7N3. We further tested the effects of MTM on 19 IAV and 11 IBV stored clinical samples. The RT-PCR results showed that the positive detection rate of IAV samples increased from 63.16% (12/19) without MTM culturing to 78.95% (15/19) after 48 h culturing, and finally 89.47% (17/19) after 72 h culturing. MTM treatment of IBV clinical samples also increased the positive detection rate from 36.36% (4/11, 0 h) to 63.64% (7/11, 48 h) to 72.73% (8/11, 72 h). For clinical samples detected by RT-PCR, MTM outperformed other transport mediums in terms of viral detection rate (11.81% increase, P=0.007). Conclusion: Our results demonstrated that the use of MTM for clinical applications can increase detection sensitivity, thus facilitating the accurate diagnosis of influenza infection.

Evaluating storage conditions and enhancement strategies on viral biomarker recovery for WBE applications.

Wastewater-based epidemiology (WBE) is a valuable disease surveillance tool. However, little is known on how factors such as transportation, storage, and wastewater characteristics influence the accuracy of the quantification methods. Hence, this study investigated the impact of storage temperatures and physicochemical characteristics of wastewater on SARS-CoV-2 and influenza A stability using droplet digital PCR. Additionally, strategies to enhance viral recovery were explored. Municipal influent wastewater stored between ±25 and -80 °C was assessed for a period of 84 days to determine viral degradation. Degradation up to 94.1% of influenza A and SARS-CoV-2 was observed in all samples with the highest at ±25 °C. Viral degradation was correlated to the changes in wastewater physicochemical characteristics. The low degradation observed of SARS-CoV-2 in the spiked pellets were indicative of viral adhesion to wastewater solids, which correlated with changes in pH. Ultrasonication frequencies ranging from 4 to 16 kHz, increased SARS-CoV-2 concentrations in the supernatant between 3.30 and 35.65%, indicating viral RNA attachment to wastewater solids. These results highlight the importance of additional pretreatment methods for maximizing RNA recovery from wastewater samples. Based on these findings, it was deduced that wastewater preservation studies are essential, and pretreatment should be included in the WBE methodology.