Nanoparticles have gained attention as potential antiviral agents, but the effects of graphene oxide nanoparticles (GONPs) on influenza virus remain unclear. In this study, we evaluated the antiviral activity of GONPs against influenza virus strain A/Hunan-Lengshuitan/11197/2013(H9N2). Our results show that GONPs with a diameter of 4 nm exerted an antiviral effect, whereas those with a diameter of 400 nm had no effect. Treatment with 4-nm GONPs reduced viral titers by more than 99% and inhibited viral nucleoprotein expression in a dose-dependent manner. We also confirmed that 4-nm GONPs inhibited the infectivity of H9N2 in MDCK cells. A transmission electron microscopic analysis revealed morphological abnormalities in the GONP-treated virus, including the destruction of the envelope glycoprotein spikes and an irregular shape, suggesting that GONPs cause the destruction of the viral coat proteins. Our results highlight the potential utility of GONPs in the prevention and treatment of viral infections, especially those of emerging and re-emerging viruses.

Low pathogenic avian influenza (LPAI) subtype H9N2 is a causative agent that has raised increasing concern about its impact on poultry and potential public health threats. Even though H9N2 is endemic in Peninsular Malaysia, it was first reported in Sabah in August 2022, after an outbreak associated with high mortality in broiler chickens. In the present study, based on the hemagglutinin (HA) gene, we report the genetic variations and phylogenetic analysis of a H9N2 virus isolated from broiler chickens in Sabah. The sequence analysis of the HA gene revealed a 98% similarity to the H9N2 virus recently isolated from China in 2018. The amino acids in the HA cleavage site displayed a characteristic LPAI motif (PARSSR/ GLF). Notably, at position 226, the isolate had amino acid Leucine (L) demonstrating its ability to bind to the receptor of mammals, resulting in the potential risk of transmission to humans. In addition, the H9N2 isolate harboured seven potential N-glycosylation sites. The phylogenetic analysis revealed that the isolate belonged to clade h9.4.2.5 in the Y280 lineage, similar to previously reported in Malaysia. However, we observed that the isolate in this study falls in a different cluster compared with previous Malaysian isolates, suggesting different source of H9N2 introduction into the country. This prompts us to propose continuous and thorough surveillance of poultry across the country and the necessity of implementing farm biosecurity to minimize economic losses and potential threats to public health.

Sensitive, convenient and rapid detection and subtyping of influenza viruses are crucial for timely treatment and management of infected people. Compared with antigen detection, nucleic acid detection has higher specificity and can shorten the detection window. Hence, in this work, we improved the lateral flow assay (LFA, one of the most promising user-friendly and on-site methods) to achieve detection and subtyping of H1N1, H3N2 and H9N2 influenza virus nucleic acids. Firstly, the antigen-antibody recognition mode was transformed into a nucleic acid hybridization reaction. Secondly, Fe3O4-Au heterodimer nanoparticles were prepared to replace frequently used Au nanoparticles to obtain better coloration. Thirdly, four lines were arranged on the LFA strip, which were three test (T) lines and one control (C) line. Three T lines were respectively sprayed by the DNA sequences complementary to one end of H1N1, H3N2 and H9N2 influenza virus nucleic acids, while Fe3O4-Au nanoparticles were respectively coupled with the DNA sequences complementary to the other end of H1N1, H3N2 and H9N2 nucleic acids to construct three kinds of probes. The C line was sprayed by the complementary sequences to the DNAs on all three kinds of probes. In the detection, by hybridization reaction, the probes were combined with their target nucleic acids which were captured by the corresponding T lines to form color bands. Finally, according to the position of the color bands and their grey intensity, simultaneous qualitative and semi-quantitative detection of the three influenza virus nucleic acids was realized. The detection results showed that this multi-channel LFA had good specificity, and there was no significant cross reactivity among the three subtypes of influenza viruses. The simultaneous detection achieved comparable detection limits with individual detections. Therefore, this multi-channel LFA had good application potential for sensitive and rapid detection and subtyping of influenza viruses.

Influenza A/H9 viruses circulate worldwide in wild and domestic avian species, continuing to evolve and posing a zoonotic risk. A substantial increase in human infections with A/H9N2 subtype avian influenza viruses (AIVs) and the emergence of novel reassortants carrying A/H9N2-origin internal genes has occurred in recent years. Different names have been used to describe the circulating and emerging A/H9 lineages. To address this issue, an international group of experts from animal and public health laboratories, endorsed by the WOAH/FAO Network of Expertise on Animal Influenza, has created a practical lineage classification and nomenclature system based on the analysis of 10,638 hemagglutinin sequences from A/H9 AIVs sampled worldwide. This system incorporates phylogenetic relationships and epidemiologic characteristics designed to trace emerging and circulating lineages and clades. To aid in lineage and clade assignment, an online tool has been created. This proposed classification enables rapid comprehension of the global spread and evolution of A/H9 AIVs.

The H9N2 subtype of the avian influenza virus (AIV) is widely prevalent in birds, threatening the poultry industry and providing genetic material for emerging human pathogens. The prevalence and genetic characteristics of H9N2 in Yunnan Province, China, are largely unknown. Samples were collected from live poultry markets (LPMs) and breeding farms in Yunnan Province. H9N2-positive samples were identified by polymerase chain reaction (PCR), with a high positivity rate of 42.86% in tissue samples. The positivity rate of swab samples in the LPMs in Kunming was 3.97% (17/564), but no AIV was detected in samples from poultry farms in Lijiang, Wenshan, and Yuxi. Evolutionary analysis and genotyping were performed for the 17 strains of isolated H9N2 virus. Phylogenetic analysis revealed that all H9N2 viral genes had 91.6%-100% nucleotide homology, belonged to the G57 genotype, and had high homology with H9N2 viruses isolated from Guangdong and Guangxi, suggesting that the H9N2 viruses in Yunnan Province may have been imported by chicks. Using a nucleotide divergence cutoff of 95%, we identified ten distinct H9N2 genotypes that continued to evolve. The surface genes of the H9N2 isolates displayed substantial genetic diversity, highlighting the genetic diversity and complexity of the H9N2-subtype AIVs in Yunnan. Molecular analysis demonstrated that all 17 strains of H9N2 isolates had mutations at H183N, Q226L, L31P, and I268V in hemagglutinin; S31N in matrix protein 2; and no replacements at positions 274 and 292 of the neuraminidase protein. Sixteen strains had the A558V mutation and one strain had the E627V mutation in polymerase basic protein 2. Analysis of these amino acid sites suggests that H9N2 influenza viruses in Yunnan continue to mutate and adapt to mammals and are sensitive to neuraminidase inhibitors but resistant to adamantanes. It is necessary to strengthen surveillance of AIV H9N2 subtypes in poultry and LPMs in Yunnan to further understand their genetic diversity.

H9N2 avian influenza virus (AIV), one of the predominant subtypes circulating in the poultry industry, inflicts substantial economic damage. Mutations in the hemagglutinin (HA) and neuraminidase (NA) proteins of H9N2 frequently alter viral antigenicity and replication. In this paper, we analyzed the HA genetic sequences and antigenic properties of 26 H9N2 isolates obtained from chickens in China between 2012 and 2019. The results showed that these H9N2 viruses all belonged to h9.4.2.5, and were divided into two clades. We assessed the impact of amino acid substitutions at HA sites 145, 149, 153, 164, 167, 168, and 200 on antigenicity, and found that a mutation at site 164 significantly modified antigenic characteristics. Amino acid variations at sites 145, 153, 164 and 200 affected virus\'s hemagglutination and the growth kinetics in mammalian cells. These results underscore the critical need for ongoing surveillance of the H9N2 virus and provide valuable insights for vaccine development.

Although molecular imprinting technology has been widely used in the construction of virus sensors, it is still a great challenge to identify subtypes viruses specifically because of their high similarity in morphology, size and structure. Here, a monoclonal molecular imprinted polymers (MIPs) sensor for recognition of H5N1 is constructed to permit the accurate distinguishing of H5N1 from other influenza A virus (IAV) subtypes. Firstly, H5N1 are immobilized on magnetic microspheres to produce H5N1-MagNPs, then the high affinity nanogel H5N1-MIPs is prepared by solid phase imprinting technique. When H5N1-MIPs is combined with MagNP-H5N1, different concentrations of H5N1 are added for competitive substitution. The quantitative detection of H5N1 is realized by the change of fluorescence intensity of supernatant. As expected, the constructed sensor shows satisfactory selectivity, and can identify the target virus from highly similar IAV subtypes, such as H1N1, H7N9 and H9N2. The sensor was highly sensitive, with a detection limit of 0.58 fM, and a selectivity factor that is comparable to that of other small MIPs sensors is achieved. In addition, the proposed sensor is cheap, with a cost of only RMB 0.08 yuan. The proposed monoclonal sensor provides a new method for the specific recognition of designated virus subtype, which is expected to be used for large-scale screening and accurate treatment of infected people.

Avian influenza outbreaks, including ones caused by highly pathogenic A(H5N1) clade 2.3.4.4b viruses, have devastated animal populations and remain a threat to humans. Risk elements assessed for emerging influenza viruses include their susceptibility to approved antivirals. Here, we screened >20,000 neuraminidase (NA) or polymerase acidic (PA) protein sequences of potentially pandemic A(H5Nx), A(H7Nx), and A(H9N2) viruses that circulated globally in 2010-2023. The frequencies of NA or PA substitutions associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NA inhibitors (NAIs) (oseltamivir, zanamivir) or a cap-dependent endonuclease inhibitor (baloxavir) were low: 0.60% (137/22,713) and 0.62% (126/20,347), respectively. All tested subtypes were susceptible to NAIs and baloxavir at sub-nanomolar concentrations. A(H9N2) viruses were the most susceptible to oseltamivir, with IC50s 3- to 4-fold lower than for other subtypes (median IC50: 0.18 nM; n = 22). NA-I222M conferred RI of A(H5N1) viruses by oseltamivir (with a 26-fold IC50 increase), but NA-S246N did not reduce inhibition. PA-E23G, PA-K34R, PA-I38M/T, and the previously unreported PA-A36T caused RI by baloxavir in all subtypes tested. Avian A(H9N2) viruses endemic in Egyptian poultry predominantly acquired PA-I38V, which causes only a <3-fold decrease in the baloxavir EC50 and fails to meet the RI criteria. PA-E199A/D in A(H7Nx) and A(H9N2) viruses caused a 2- to 4-fold decrease in EC50 (close to the borderline for RI) and should be closely monitored. Our data indicate antiviral susceptibility is high among avian influenza A viruses with pandemic potential and present novel markers of resistance to existing antiviral interventions.

OBJECTIVE: This review aims to discuss the current state of human infections with Avian Influenza A (H5) and (H9) viruses, to support awareness of the global epidemiology among clinicians and public health professionals interested in emerging respiratory infections.
RESULTS: Among increasing numbers of detections in avian species of Avian Influenza A(H5N1) clade 2.3.4.4b globally, reported human cases of severe infection have been rare.Enhanced surveillance of persons exposed to avian species infected with Influenza A (H5N1) clade 2.3.4.4b in different countries has identified small numbers of asymptomatic individuals with Avian Influenza A (H5N1) detected by PCR from the upper respiratory tract; some of these instances have been considered to represent contamination rather than infection.There have also been recent sporadic human cases of Avian Influenza A(H9N2) internationally, including in China and Cambodia.
CONCLUSIONS: Human infections with Avian Influenza A(H5) and (H9) viruses remain of interest as an emerging infection both to clinicians and public health professionals. While maintaining effective surveillance is essential, one health strategies to control infection in avian species will be key to mitigating these risks.

Human cases of avian influenza virus (AIV) infections are associated with an age-specific disease burden. As the influenza virus N2 neuraminidase (NA) gene was introduced from avian sources during the 1957 pandemic, we investigate the reactivity of N2 antibodies against A(H9N2) AIVs. Serosurvey of healthy individuals reveal the highest rates of AIV N2 antibodies in individuals aged ≥65 years. Exposure to the 1968 pandemic N2, but not recent N2, protected against A(H9N2) AIV challenge in female mice. In some older adults, infection with contemporary A(H3N2) virus could recall cross-reactive AIV NA antibodies, showing discernable human- or avian-NA type reactivity. Individuals born before 1957 have higher anti-AIV N2 titers compared to those born between 1957 and 1968. The anti-AIV N2 antibodies titers correlate with antibody titers to the 1957 N2, suggesting that exposure to the A(H2N2) virus contribute to this reactivity. These findings underscore the critical role of neuraminidase immunity in zoonotic and pandemic influenza risk assessment.