Virus Subtype Research Articles

Drug Resistance Mutations (DRMs) for Long-Acting Injectable Cabotegravir and Rilpivirine (CAB/RPV LAI) in the HIV-1 Subtype A6 Epidemic in Poland

HIV subtype A6 with the L74I polymorphism, which increases the risk of cabotegravir/rilpivirine treatment failure, causes more and more infections in Poland. In this multicenter, observational, cross-sectional study (2023–2024), we analyzed viral subtypes and drug-resistance mutations to drugs used for long-acting injectable antiretroviral treatment and pre-exposure prophylaxis. Among 357 people with HIV, 247 (69%) were Polish nationals, and 102 (29%) were from former Soviet Union countries. Of the 357 people included, 159 (45%) had subtype B, and 177 (50%) had subtype A6 infections, with 165 (87%) of the latter characterized by the L74I polymorphism. Subtype A6 was more frequent in women (66% vs. 46% in men, p < 0.05) and among people from former Soviet countries (77% vs. 39% in Polish nationals, p < 0.05). About 40% of people had either drug-resistance mutations for cabotegravir/rilpivirine or HIV A6 subtype with the L74I polymorphism; 4.5% had both of these conditions. Compared to subtype B infections, subtype A6 infections were characterized by more frequent major transmitted drug-resistance mutations for non-nucleoside reverse transcriptase inhibitors (8.5% vs. 1.9%, p = 0.007) and rilpivirine (5.1% vs. 0.6%, p = 0.016). Due to the frequent occurrence of the L74I polymorphism and drug-resistance mutations in HIV A6 subtype infection, about 40% of people with HIV in Poland may be at risk of long-acting injectable treatment failure.

HIV-1 resistance mutations and genetic diversity among children failing antiretroviral treatment in five healthcare facilities in Benin, West Africa.

Antiretroviral treatment increases the risk of accumulation of resistance mutations that negatively impact the possibilities of future treatment. This study aimed to present the frequency of HIV-1 antiretroviral resistance mutations and the genetic diversity among children with virological failure in five pediatric care facilities in Benin. A cross-sectional study was carried out from November 20, 2020, to November 30, 2022, in children under 15 years of age who failed ongoing antiretroviral treatment at five facilities care in Benin (VL > 3log10 on two consecutive realizations three months apart). Viral loads were measured using the m2000 RealTime Abbott platform. Genotyping was carried out with the commercial Viroseq kit. Sequences were read on the ABI 3500 sequencer and then edited with ViroSeqHIVv3.0 software. The HIV drug resistance database at Stanford University was used to identify mutations and viral subtypes were assigned by phylogenetic analyses. The HIV-1 pol gene was sequenced in 47 participants with virological failure of antiretroviral treatment. The median age was 120 [Interquartile Range 90-144] months. The prevalent treatment was EFV base regimen (22/47; 46.8%). Median viral load was 4.39 log10 [IQR 3.81-4.86 log10] respectively. Resistance testing was successful among (37/47; 78.72%) children, resistance mutations were detected in (32/37; 86.48%) children, and (29/32; 90.62%) had at least one surveillance drug resistance mutation. Respectively (25/32; 78.12%), (28/32; 87.5%), (4/32; 12.90%), (22/32; 68.75%) had at least one resistance mutation associated with NRTIs, NNRTIs, PIs and NNRTIs+NRTIs. (12/32; 37.5%) of children carried mutations related to TAMs. the most frequently NRTIs identified were M184V (21/62; 33.9%) followed by TAMs (20/62; 32.2%) and T69G/D (2/62; 3.2%)s. Among mutations associated with NNRTIs K103N represented (18/64; 28.1%) followed by P225H (7/64; 10.9%). The I54V (3/6; 50%) mutation is the major PI observed. Genetic diversity is characterized by a preponderance of CRF02_AG (72%, 23/32), followed by unique recombinant forms (URFs) (25%, 8/32) and one subtype G. A high rate of mutations has been observed in children. These data underline the importance of implementing routine genotypic testing in the biological monitoring of infected children to anticipate the accumulation of resistance mutations and thus compromise the treatment options available in Benin.

Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

Background: Current influenza A vaccines primarily induce neutralizing antibodies targeting the variable hemagglutinin (HA) head domain, limiting their effectiveness against diverse or emerging influenza A virus (IAV) subtypes. The conserved HA stem domain, particularly the long α-helix (LAH) epitope, is a focus of universal vaccine research due to its cross-protective potential. Additionally, Fc-mediated functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are recognized as important protective immune mechanisms. This study evaluated IgG responses to the HA head, stem, and LAH regions and assessed cross-reactive potential through neutralization, ADCC, and ADCP assays. Methods: IgG responses to the HA head, stem, and LAH regions were measured in vaccinated individuals. Functional assays were conducted for neutralization, ADCC, and ADCP to evaluate the association between antibody levels and immune function. Results: The results showed that HA head-specific IgG increased significantly after vaccination in 50 individuals, whereas stem-specific IgG increased by 72% and LAH-specific IgG by 12–14%. Among the induced antibody subclasses, IgG1 was predominantly increased. Neutralization titers were detected in viruses of the same strain as the vaccine strain, but not in classical or pandemic strains (H5N1, H7N9). HA stem-specific IgG1 antibody titers showed a significant correlation with ADCC/ADCP activity breadth, but no correlation was observed with neutralization breadth. Conclusion: These findings suggest that although current influenza vaccines can induce HA stem-targeted cross-reactive antibodies, their quantity may be insufficient for broad cross-protection, underscoring the need for improved vaccine strategies.

Isolation and Characterization of H1 Subtype Swine Influenza Viruses Recently Circulating in China

Pigs serve as a mixing vessel for influenza viruses and can independently promote the emergence of pandemic strains in humans. During our surveillance of pig populations from 2021 to 2023 in China, 11 H1 subtype swine influenza viruses (SIVs) were isolated. All viruses were reassortants, possessing internal genes of identical origins (PB2, PB1, PA, NP, M: pdm09/H1N1 origin, NS: North American triple reassortant origin). The H1N1 isolates were all the dominant G4 EA H1N1 viruses in China. Two H1N2 isolates carried early human pdm09/H1N1 HA genes, suggesting a possible pig-to-human transmission route. Mutations that dictate host range specificity were identified in all isolates, a phenomenon which may enhance the affinity to human receptors. These H1 subtype viruses effectively replicated both in vivo and in vitro without prior adaptation and exhibited different pathogenicity and growth characteristics. Some of the H1 viruses were even found to cause lethal infections in mice. Taken together, our study indicates that the H1 subtype SIVs recently circulating in China pose a potential threat to human health and emphasizes the importance of continuing to closely monitor their evolution and spread.

Novel H16N3 avian influenza viruses isolated from migratory gulls in China in 2023

As a rare subtype of avian influenza virus, H16 viruses are predominant in gulls but rarely found in domestic birds. The low prevalence of H16 viruses has limited our understanding of their epidemiology and evolutionary dynamics. In this study, we isolated three novel H16N3 viruses from migratory gulls in East Asian–Australasian Flyway in eastern China in 2023, which are significantly different from previously identified isolates. To fully understand the epidemiology and genetics characteristics of the global H16 viruses, we compared the host divergence of several rare subtypes and determined that the H13 and H16 subtypes were predominantly pooled into different species of gulls by sharing their internal genes, whereas the waterfowl of Anatidae served as the primary natural reservoirs of the H8, H11, H12, H14, and H15 subtypes. Detailed phylogenetic analysis revealed the evolutionary divergence of globally circulating H16 viruses and their frequent gene reassortment. Furthermore, the gull origin H13 and H16 viruses collectively served as gene donors for the newly emerged highly pathogenic clade 2.3.4.4b H5N1 viruses because the H13/H16-like PA, NP, and NS genes have been introduced into circulating H5N1 viruses since May 2022 in Europe. To date, the H5N1 reassortants containing the H13/H16-like gene segments have been detected in wild and domestic birds and resulted in mammal and human infections. These results improve our knowledge of the ecology and genetics of H16 viruses and emphasize the need for surveillance to monitor the emergence of novel avian influenza viruses in migratory birds.

Topologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution.

The widespread and evolution of RNA viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the importance of fast identification of virus subtypes, particularly in non-laboratory settings. Rapid and inexpensive at-home testing of viral nucleic acids with single-base resolution remains a challenge. Topologically constrained DNA ring is engineered as substrates for the trans-cleavage of Cas13a to yield an accelerated post isothermal amplification. The capacity of CRISPR/Cas13a for discriminating single nucleotide variant (SNV) in viral genome is leveraged by designing synthetic mismatches and hairpin structure in CRISPR RNA (crRNA), enabling robust discrimination of different SARS-CoV-2 variants. Via optimisation of CasTDR3pot to be one-pot assay, CasTDR1pot can detect Omicron and its subvariants, with only a few copies in clinical samples in less than 30min without pre-amplification. The detection system boasts high sensitivity (0.1 aM), single-base specificity, and the advantage of a rapid "sample-to-answer" process, which takes only 30min. In the detection of SARS-CoV-2 clinical samples and their variant strains, CasTDR1pot has achieved 100% accuracy. Furthermore, the design of a portable signal-reading device facilitates user-friendly result interpretation. For the detection needs of different RNA viruses, the system can be adapted simply by designing the corresponding crRNA. Our study provides a rapid and accurate molecular diagnostic tool for point-of-care testing, epidemiological screening, and the detection of diseases associated with other RNA biomarkers with excellent single nucleotide differentiation, high sensitivity, and simplicity. National Key Research and Development Program of China (No. 2023YFB3208302), National Natural Science Foundation of China (No. 22377110, 22034004, 82402749, 82073787, 22122409), National Key Research and Development Program of China (No. 2021YFA1200104), Henan Province Fund for Cultivating Advantageous Disciplines (No. 222301420019).

Highly pathogenic avian influenza virus (H5N5) detected in an Atlantic walrus (Odobenus rosmarus rosmarus) in the Svalbard Archipelago, Norway, 2023

We present the first documented case of highly pathogenic avian influenza virus (HPAIV) subtype H5N5 in an Atlantic walrus (Odobenus rosmarus rosmarus). The animal was found dead in Svalbard, Norway, in 2023. Sequence analysis revealed the highest genetic similarity with virus isolates from different avian hosts.

Isoleucine at position 137 of haemagglutinin acts as a mammalian adaptation marker of H9N2 avian influenza virus

ABSTRACT The H9N2 subtype of avian influenza virus (AIV) is widely distributed among poultry and wild birds and is also a threat to humans. During AIV active surveillance in Liaoning province from 2015 to 2016, we identified 10 H9N2 strains exhibiting different lethality to chick embryos. Two representative strains, A/chicken/China/LN07/2016 (CKLN/07) and A/chicken/China/LN17/2016 (CKLN/17), with similar genomic background but different chick embryo lethality, were chosen to evaluate the molecular basis for this difference. A series of reassortants between CKLN/07 and CKLN/17 were generated and their chick embryo lethality was assessed. We found that the isoleucine (I) residue at position 137 (H3 numbering) in the haemagglutinin (HA) was responsible for the chick embryo lethality of the H9N2 virus. Further studies revealed that the threonine (T) to I mutation at HA position 137 enhanced viral replication in vitro and in vivo. Moreover, the HA-T137I substitution in H9N2 avian influenza virus increased the guinea pig transmission efficiency. We also found that the HA-T137I substitution was critical for α2,6-linked sialic acid binding preference and HA activation and stability of H9N2 virus. Our findings demonstrated that HA-137I is a key molecular marker for mammalian adaptation of H9N2 AIV.

Rapid and specific on-site H5Nx avian influenza diagnosis via RPA and PAM-independent CRISPR-Cas12a assay combined with anti-NP antibody-based viral RNA purification.

Rapid and accurate detection of H5Nx avian influenza viruses is critical for effective surveillance and control measures. Currently, RT-qPCR with spin column RNA extraction is the gold standard for HPAIV surveillance, but its long reaction time and need for specialized equipment limit its effectiveness for rapid response. In this study, we introduce a centrifuge-free, rapid detection method for on-site detection of H5Nx viruses that combines magnetic bead-based ribonucleoprotein (RNP) purification and concentration with a CRISPR-Cas12a system that is independent of the protospacer adjacent motif (PAM) sequence. Our approach employs anti-NP monoclonal antibodies for the targeted isolation of RNA bound to RNPs, facilitating a quick and specific RNA extraction process that negates the need for centrifugation. Additionally, by denaturing the RT-RPA amplicon using 60% DMSO, we activate the trans-ssDNA cleavage activity of the Cas12a protein without the need for a specific PAM (5'-TTTV-3') sequence. This strategy increases flexibility in CRISPR RNA design, providing a significant advantage when targeting genes with high variability. We validated the efficacy of our magnetic RNP purification and concentration method in combined with an RT-RPA/PAM-independent Cas12a assay for detecting the H5 gene. The assay achieved a sensitivity threshold of 101 EID50 with fluorescent detection and 102 EID50 using lateral flow strips. It also exhibited high specificity, yielding positive results solely for H5Nx viruses among various influenza A virus subtypes. Furthermore, in clinical samples, the assay demonstrated 80% sensitivity and 100% specificity. These results highlight the advantages of using NP-specific antibodies for RNP purification and CRISPR-Cas12a with viral gene-specific crRNA to achieve exceptional diagnostic specificity.

The Development of a Novel Broad-Spectrum Influenza Polypeptide Vaccine Based on Multi-Epitope Tandem Sequences.

Polypeptide vaccines have the potential to improve immune responses by targeting conserved and weakly immunogenic regions in antigens. This study aimed to identify and evaluate the efficacy of a novel influenza universal vaccine candidate consisting of multiple polypeptides derived from highly conserved regions of influenza virus proteins hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2). Immunoinformatics tools were used to screen conserved epitopes from different influenza virus subtypes (H1N1, H3N2, H5N1, H7N9, H9N2, and IBV). A polypeptide vaccine, P125-H, was constructed by linking multiple epitopes using Ii-Key technology. The immunogenicity of P125-H was assessed in mice using MF59-adjuvanted P125-H via intraperitoneal injection. Hemagglutination inhibition (HI) and neutralizing antibody responses were measured, along with IFN-γ levels in spleen lymphocytes. Protective efficacy was evaluated using viral challenge with lethal doses of H1N1 and H7N9. Mice immunized with P125-H generated high levels of HI and neutralizing antibodies against multiple influenza strains. IFN-γ production was significantly elevated in spleen lymphocytes upon stimulation with the vaccine. P125-H protected mice from influenza infection, reducing weight loss and the viral load in the lungs, mitigating lung pathology, and decreasing mortality. The P125-H vaccine induced broad cross-protection against multiple influenza strains and elicited robust immune responses. It demonstrates strong potential as a candidate for a universal influenza vaccine.

Neurotropic Tick-Borne Flavivirus in Alpine Chamois (Rupicapra rupicapra rupicapra), Austria, 2017, Italy, 2023.

The European subtype of tick-borne encephalitis virus (TBEV-Eur; species Orthoflavivirus encephalitidis, family Flaviviridae) was the only tick-borne flavivirus present in central Europe known to cause neurologic disease in humans and several animal species. Here, we report a tick-borne flavivirus isolated from Alpine chamois (Rupicapra rupicapra rupicapra) with encephalitis and attached ticks, present over a wide area in the Alps. Cases were detected in 2017 in Salzburg, Austria, and 2023 in Lombardy and Piedmont, Italy. The virus strains exhibit 94.8-97.3% nucleotide identities to each other and are more closely related to Louping ill viruses (LIV; Orthoflavivirus loupingi; 90-92% identities) than to TBEV-Eur (less than 88%). The chamois-derived virus strains, tentatively termed "Alpine chamois encephalitis virus", form a well-supported independent genetic clade with Spanish goat encephalitis virus, clearly separated from other LIV. This supports its designation as a new virus subtype with the proposed shared taxonomic name "Spanish goat and Alpine chamois encephalitis virus subtype" within the species Orthoflavivirus loupingi. The zoonotic potential of this newly identified virus subtype as well as its host range in other animal species including farm animals needs to be further investigated.

A lineage 1 branch porcine reproductive and respiratory syndrome virus live vaccine candidate provides broad cross-protection against HP-like PRRSV in piglets

ABSTRACT Multiple porcine reproductive and respiratory syndrome virus (PRRSV) subtypes coinfect numerous pig farms in China, and commercial PRRSV vaccines offer limited cross-protection against heterologous strains. Our previous research confirmed that a PRRSV lineage 1 branch attenuated live vaccine (SD-R) provides cross-protection against HP-PRRSV, NADC30-like PRRSV and NADC34-like PRRSV. HP-PRRSV has undergone significant genetic variation following nearly two decades of evolution and has transformed into a subtype referred to as HP-like PRRSV, which also exhibits high pathogenicity. The effectiveness of immunising piglets with the SD-R strain to provide protection against infection with HP-like PRRSV remains uncertain. In the present study, we evaluated the protective effects of SD-R vaccine strains on DLF-challenged piglets. The results revealed that piglets challenged with DLF presented clinical symptoms such as continuous high fever and an obvious decrease in daily weight gain. Importantly, the piglets immunised with SD-R exhibited notable reductions in pathological damage, especially of decreases in DLF-induced thymic atrophy. Moreover, the serum of SD-R-immunised piglets strongly neutralised DLF, and the number of SD-R-vaccinated piglets demonstrating viraemia was greatly reduced. These results suggest that the PRRSV lineage 1 branch live vaccine candidate provides broad cross-protection against HP-like PRRSV in piglets.

Development of avian influenza A(H5) virus datasets for Nextclade enables rapid and accurate clade assignment.

The ongoing panzootic of highly pathogenic avian influenza (HPAI) A(H5) viruses is the largest in history, with unprecedented transmission to multiple mammalian species. Avian influenza A viruses of the H5 subtype circulate globally among birds and are classified into distinct clades based on their hemagglutinin (HA) genetic sequences. Thus, the ability to accurately and rapidly assign clades to newly sequenced isolates is key to surveillance and outbreak response. Co-circulation of endemic, low pathogenic avian influenza (LPAI) A(H5) lineages in North American and European wild birds necessitates the ability to rapidly and accurately distinguish between infections arising from these lineages and epizootic HPAI A(H5) viruses. However, currently available clade assignment tools are limited and often require command line expertise, hindering their utility for public health surveillance labs. To address this gap, we have developed datasets to enable A(H5) clade assignments with Nextclade, a drag-and-drop tool originally developed for SARS-CoV-2 genetic clade classification. Using annotated reference datasets for all historical A(H5) clades, clade 2.3.2.1 descendants, and clade 2.3.4.4 descendants provided by the Food and Agriculture Organization/World Health Organization/World Organisation for Animal Health (FAO/WHO/WOAH) H5 Working Group, we identified clade-defining mutations for every established clade to enable tree-based clade assignment. We then created three Nextclade datasets which can be used to assign clades to A(H5) HA sequences and call mutations relative to reference strains through a drag-and-drop interface. Nextclade assignments were benchmarked with 19,834 unique sequences not in the reference set using a pre-released version of LABEL, a well-validated and widely used command line software. Prospective assignment of new sequences with Nextclade and LABEL produced very well-matched assignments (match rates of 97.8% and 99.1% for the 2.3.2.1 and 2.3.4.4 datasets, respectively). The all-clades dataset also performed well (94.8% match rate) and correctly distinguished between all HPAI and LPAI strains. This tool additionally allows for the identification of polybasic cleavage site sequences and potential N-linked glycosylation sites. These datasets therefore provide an alternative, rapid method to accurately assign clades to new A(H5) HA sequences, with the benefit of an easy-to-use browser interface.

Influenza vaccine outcomes: a meta-analysis revealing morbidity benefits amid low infection prevention.

The morbidity and mortality associated with influenza viruses are a significant public health challenge. Annual vaccination against circulating influenza strains reduces hospitalisations and increases survival rates but requires a yearly redesign of vaccines against prevalent subtypes. The complex genetics of influenza viruses with high antigenic drift create an ongoing challenge in vaccine development to address dynamic influenza epidemiology. Understanding the evolution of influenza viruses and the vaccine's effectiveness against different types and subtypes is pivotal to designing public health measures against influenza. We conducted a systematic review and meta-analysis of 192 705 patients, collecting information on the incidence and severity of the disease. The results of this meta-analysis were further validated using data from 6 594 765 patients from TriNetX. We analysed the prevalence of the most common influenza A virus (IAV) subtypes (H1N1 and H3N2) and influenza B virus (IBV), as well as vaccination effectiveness against them in three age groups, given that age is associated with influenza disease severity. Our analysis reflects that overall vaccination against H1N1 IAV and IBV is effective in reducing infection and influenza-related complications in children aged <5 years old, individuals between 5 and 65 years old and older adults aged >65 years old. By contrast, while vaccination against H3N2 IAV is effective in protecting against infection in infants <5 years old, it provides reduced protection against infection in older individuals. Despite higher infection rates, vaccination against H3N2 remains as highly effective as vaccination against H1N1 and IBV in reducing influenza-related morbidity and mortality in all age groups. Detailing vaccine effectiveness in terms of infection protection and disease burden across different age groups is necessary for understanding vaccine impacts in terms of other outcomes, e.g. hospitalisations, mortality and disease severity; for improving vaccine formulations and public awareness; and for enhancing vaccination campaigns to improve coverage and public acceptance.

Rapid, flexible fabrication of a microfluidic electrochemical chip nucleic acid target for selective, label-free detection of influenza virus DNA using catalytic redox-recycling.

H5N1 flu is a highly virulent and variable subtype of influenza with significant epidemic and pandemic potential. In this study, we introduce a novel, maskless, and rapid manufacturing process for a microfluidic chip integrated with electrodes for the quantitative detection of H5N1-DNA sequences. This detection leverages a catalytic redox-recycling signal via a novel Fe₃O₄@TMU-8 nanocomposite, which facilitates the turnover of the oxidation state of [Ru(NH₃)₆]³⁺, thereby amplifying the electrochemical signal output. The positively charged [Ru(NH₃)₆]³⁺ molecule associates with the phosphate backbone of the nucleic acids in H5N1-DNA. Changes in the aptasensor's redox-recycling signal, due to the hybridization of DNA sequences with [Ru(NH₃)₆]³⁺, were used as the electrochemical sensing response. Under optimal conditions, the signal exhibited a linear relationship with H5N1-DNA concentration, ranging from 1fM to 1nM, with a detection limit of 0.16fM. This report details the fabrication of the microfluidic device using Poly(methyl methacrylate) (PMMA) sheet substrates. A laser system was employed to generate microfluidic patterns directly on the PMMA sheet. This biosensing device demonstrated long-term stability and good reproducibility, making it suitable for the quantitative assay of H5N1-DNA sequences. The results from food sample analyses further confirmed the applicability and effectiveness of the resulting biosensor.

The PA-X host shutoff site 100 V exerts a contrary effect on viral fitness of the highly pathogenic H7N9 influenza A virus in mice and chickens

ABSTRACT Several viruses, including influenza A virus (IAV), encode viral factors to hijack cellular RNA biogenesis processes to direct the degradation of host mRNAs, termed “host shutoff.” Host shutoff enables viruses to simultaneously reduce antiviral responses and provides preferential access for viral mRNAs to cellular translation machinery. IAV PA-X is one of these factors that selectively shuts off the global host genes. However, the specific role of PA-X host shutoff activity in viral fitness of IAV remains poorly understood. Herein, we successfully mapped PA-X 100 V as a novel site important for host shutoff of the H7N9 and H5N1 viruses. By analysing the polymorphism of this residue in various subtype viruses, we found that PA-X 100 was highly variable in H7N9 viruses. Structural analysis revealed that 100 V was generally close to the PA-X endonuclease active site, which may account for its host shutoff activity. By generating the corresponding mutant viruses derived from the parental H7N9 virus and the PA-X-deficient H7N9 virus, we determined that PA-X 100 V significantly enhanced viral fitness in mice while diminishing viral virulence in chickens. Mechanistically, PA-X 100 V significantly increased viral polymerase activity and viral replication in mammalian cells. Furthermore, PA-X 100 V highly blunted the global host response in 293T cells, particularly restraining genes involved in energy metabolism and inflammatory response. Collectively, our data provided information about the intricate role of the PA-X host shutoff site in regulating the viral fitness of the H7N9 influenza virus, which furthers our understanding of the complicated pathogenesis of the influenza A virus.

Evidence of an emerging triple-reassortant H3N3 avian influenza virus in China

The H3 subtype of avian influenza virus (AIV) stands out as one of the most prevalent subtypes, posing a significant threat to public health. In this study, a novel triple-reassortant H3N3 AIV designated A/chicken/China/16/2023 (H3N3), was isolated from a sick chicken in northern China. The complete genome of the isolate was determined using next-generation sequencing, and the AIV-like particles were confirmed via transmission electron microscopy. Subsequent phylogenetic analyses revealed that HA and NA genes of the H3N3 isolate clustered within the Eurasian lineage of AIVs, exhibiting the closest genetic relationship with other H3N3 AIVs identified in China during 2023. Interestingly, the HA and NA genes of the nove H3N3 isolate were originated from H3N8 and H10N3 AIVs, respectively, and the six internal genes originated from prevalent H9N2 AIVs. These findings indicated the novel H3N3 isolate possesses a complex genetic constellation, likely arising from multiple reassortment events involving H3N8, H9N2, and H10N3 subtype influenza viruses. Additionally, the presence of Q226 and T228 in the HA protein suggests the H3N3 virus preferentially binds to α-2,3-linked sialic acid receptors. The HA cleavage site motif (PEKQTR/GIF) and the absence of E627K and D701N mutations in PB2 protein classify the virus as a characteristic low pathogenicity AIV. However, several mutations in internal genes raise concerns about potential increases in viral resistance, virulence, and transmission in mammalian hosts. Overall, this study provides valuable insights into the molecular and genetic characterization of the emerging triple-reassortant H3N3 AIVs, and continued surveillance of domestic poultry is essential for monitoring the H3N3 subtype evolution and potential spread.

Intranasal Immunization with DNA Vaccine HA-CCL19/Polyethylenimine/Chitosan Composite Provides Immune Protection Against H7N9 Infection.

The H7N9 avian influenza virus (AIV) constitutes a novel subtype of influenza virus that has emerged within the past decade. Empirical studies have demonstrated that H7N9 AIV holds the potential to trigger a human pandemic. Vaccines constitute the sole armament available to humanity in combating influenza epidemics. DNA vaccines present numerous merits; however, substantial conundrums persist regarding how to augment their immunogenicity and implement their delivery through mucosal immunization. In this study; BALB/c mice were utilized as a model to investigate the effect of CCL19 as a molecular adjuvant and to determine the immune response elicited by polyethylene imine (PEI) and chitosan (CS) as adjuvants during the delivery of a DNA vaccine through the nasal mucosal route. Our results revealed that the CCL19 molecular adjuvant exerts a substantial immunomodulatory enhancement effect on the H7N9-HA DNA vaccine, inducing more pronounced cellular and humoral immunity. Additionally, our results indicated that the composite formed by the HA-CCL19 DNA in combination with PEI and CS effectively activates local mucosal immunity as well as systemic humoral and cellular immunity, offering 100% protection against lethal doses of homologous virus challenges. CCL19 conspicuously augments the immunogenicity of the influenza virus HA DNA and conserves the integrity of the vaccine antigen. Simultaneously, CS and PEI proficiently facilitate the mucosal delivery of DNA, thereby eliciting mucosal immunity related to DNA vaccines. This study investigated the feasibility of utilizing nasal mucosa for DNA vaccine immunization, which holds significant implications for the advancement and application of DNA vaccines in public health.

Targets of influenza human T-cell response are mostly conserved in H5N1.

Frequent recent spillovers of subtype H5N1 clade 2.3.4.4b highly pathogenic avian influenza (HPAI) virus into poultry and mammals, especially dairy cattle, including several human cases, increased concerns over a possible future pandemic. Here, we performed an analysis of epitope data curated in the Immune Epitope Database (IEDB). We found that the patterns of immunodominance of seasonal influenza viruses circulating in humans and H5N1 are similar. We further conclude that a significant fraction of the T-cell epitopes is conserved at a level associated with cross-reactivity between avian and seasonal sequences, and we further experimentally demonstrate extensive cross-reactivity in the most dominant T-cell epitopes curated in the IEDB. Based on these observations, and the overall similarity of the neuraminidase (NA) N1 subtype encoded in both HPAI and seasonal H1N1 influenza virus as well as cross-reactive group 1 HA stalk-reactive antibodies, we expect that a degree of pre-existing immunity is present in the general human population that could blunt the severity of human H5N1 infections.IMPORTANCEInfluenza A viruses (IAVs) cause pandemics that can result in millions of deaths. The highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype is presently among the top viruses of pandemic concern, according to the WHO and the National Institute of Allergy and Infectious Diseases (NIAID). Previous exposure by infection and/or vaccination to a given IAV subtype or clade influences immune responses to a different subtype or clade. Analysis of human CD4 and CD8 T-cell epitope conservation between HPAI H5N1 and seasonal IAV sequences revealed levels of identity and conservation conducive to T cell cross-reactivity, suggesting that pre-existing T cell immune memory should, to a large extent, cross-recognize avian influenza viruses. This observation was experimentally verified by testing responses from human T cells to non-avian IAV and their HPAI H5N1 counterparts. Accordingly, should a more widespread HPAI H5N1 outbreak occur, we hypothesize that cross-reactive T-cell responses might be able to limit disease severity.

Viral Transcript and Tumor Immune Microenvironment-Based Transcriptomic Profiling of HPV-Associated Head and Neck Squamous Cell Carcinoma Identifies Subtypes Associated with Prognosis.

Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HPV-positive HNSCC) has distinct biological characteristics from HPV-negative HNSCC. Using an AI-based analytical platform on meta cohorts, we profiled expression patterns of viral transcripts and HPV viral genome integration, and classified the tumor microenvironment (TME). Unsupervised clustering analysis revealed five distinct and novel TME subtypes across patients (immune-enriched, highly immune and B-cell enriched, fibrotic, immune-desert, and immune-enriched luminal). These TME subtypes were highly correlated with patient prognosis. In order to understand specific factors associated with prognosis, we used the unsupervised clustering of an HPV-positive HNSCC cohort from The Cancer Genome Atlas (TCGA) (n = 53) based on HPV transcript expression, and identified four HPV-related subtypes (E2/E5, E6/E7, E1/E4 and L1/L2). Utilizing both viral transcript and TME subtypes, we found that the E2/E5 HPV subtype was associated with an immune-enriched TME and had a higher overall survival rate compared to other subtypes. The E2/E5 subtype was also enriched for samples without HPV-genome integration, suggesting that the episomal HPV status and E2/E5 expression pattern may be associated with an inflamed microenvironment and improved prognosis. In contrast, E6/E7 subtype samples were associated with the fibrotic and immune-desert TME subtypes, with lower values of T-cell and B-cell gene expression signatures and lower overall survival. Both E1/E4 and L1/L2 subtypes were associated with the immune-enriched luminal subtype. Our results suggest that HPV-transcript expression patterns may drive the modulation of the TME, and thereby impact prognosis.