Asymptomatic infection with clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) in carnivore pets, Italy, April 2023.

In April 2023, an outbreak of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses carrying the T271A mammalian adaptive mutation in the PB2 protein was detected in a backyard poultry farm in Italy. Five domestic dogs and one cat living on the premises had seroconverted in the absence of clinical signs. Virological and serological monitoring of individuals exposed to the virus proved the absence of human transmission, however, asymptomatic influenza A(H5N1) infections in mammalian pets may have important public health implications.

Autophagy mediates avian influenza H5N1 pseudotyped particle-induced lung inflammation through NF-κB and p38 MAPK signaling pathways.

Since avian influenza virus H5N1-induced hypercytokemia plays a key role in acute lung injury, understanding its molecular mechanism is highly desirable for discovering therapeutic targets against H5N1 infection. In the present study, we investigated the role of autophagy in H5N1-induced lung inflammation by using H5N1 pseudotyped viral particles (H5N1pps). The results showed that H5N1pps significantly induced autophagy both in A549 human lung epithelial cells and in mouse lung tissues, which was primarily due to hemagglutinin (HA) of H5N1 virus. Blocking autophagy with 3-methyladenine (an autophagy inhibitor) or siRNA knockdown of autophagy-related genes (beclin1 and atg5) dramatically attenuated H5N1pp-induced proinflammatory cytokines and chemokines, such as IL-1ß, TNF-α, IL-6, CCL2, and CCL5, both in vitro and in vivo. Autophagy-mediated inflammatory responses involved the activation of NF-κB and p38 MAPK signaling pathways, which required the presence of clathrin but did not rely on p62 or autophagosome-lysosome fusion. On the other hand, the activation of NF-κB also promoted H5N1pp-induced autophagosome formation. These data indicated a positive feedback loop between autophagy and NF-κB signaling cascade, which could exacerbate H5N1pp-induced lung inflammation. Our data demonstrated an essential role of autophagy in H5N1pp-triggered inflammatory responses, and targeting the autophagic pathway could be a promising strategy to treat H5N1 virus-caused lung inflammation.

The protective effect of a combination of human intracellular and extracellular antibodies against the highly pathogenic avian influenza H5N1 virus.

BACKGROUND: The highly pathogenic avian influenza H5N1 virus poses a serious threat to humans. Due to its antiviral activity, antibody-based therapy is one of the possible effective countermeasures. Here, a combination of intracellular and extracellular human antibodies was investigated and showed an improved protective effect. METHODS: The scFv4F5-based intracellular antibody vectors and IgG1 extracellular antibody were constructed and expressed, respectively, and the sensitivity, specificity, and affinity of these antibodies were determined in vitro. In vivo, the protective effect of IgG1 and the combination of antibodies were tested respectively. Furthermore, the dynamics of viral replication, the related cytokines and apoptosis-related proteins were detected. RESULTS: In vitro, the expressed intracellular antibody inhibited H5N1 virus propagation and the IgG1 exhibited high specificity, sensitivity, and affinity against the H5N1 virus. In vivo, the extracellular antibody could inhibit viral propagation in a dose-dependent manner. The protective effect of IgG1 was good in a mouse model, and the survival was 100% at a dose of 15 mg/kg under infection with 100 TCID50 virus. When the intracellular antibody was pre-transfected in combination with IgG1, it had a better protective effect. The survival was 16.67% under treatment with IgG1 alone and up to 83.33% under treatment with the combination of antibodies when challenge of 500 TCID 50 virus. Furthermore, the levels of cytokines IFN-γ, IL-6, IL-10 and some apoptosis-related proteins increased. CONCLUSIONS: This antibody combination technique could be used as an appropriate and powerful alternative to antiviral therapy.

T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects.

BACKGROUND: Protection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising. METHODS: This study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection. RESULTS: IFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry. CONCLUSIONS: This study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

Characterization of thermostable Newcastle disease virus recombinants expressing the hemagglutinin of H5N1 avian influenza virus as bivalent vaccine candidates.

Newcastle disease virus (NDV) has been used as a vector in the development of vaccines and gene delivery. In the present study, we generated the thermostable recombinant NDV (rNDV) expressing the different forms of hemagglutinin (HA) of highly pathogenic avian influenza virus (HPAIV) H5N1 based on the full-length cDNA clone of thermostable TS09-C strain. The recombinant thermostable Newcastle disease viruses, rTS-HA, rTS-HA1 and rTS-tPAs/HA1, expressed the HA, HA1 or modified HA1 protein with the tissue plasminogen activator signal sequence (tPAs), respectively. The rNDVs displayed similar thermostability, growth kinetics and pathogenicity compared with the parental TS09-C virus. The tPAs facilitated the expression and secretion of HA1 protein in cells infected with rNDV. Animal studies demonstrated that immunization with rNDVs elicited effective H5N1- and NDV-specific antibody responses and conferred immune protection against lethal H5N1 and NDV challenges in chickens and mice. Importantly, vaccination of rTS-tPAs/HA1 resulted in enhanced protective immunity in chickens and mice. Our study thus provides a novel thermostable NDV-vectored vaccine candidate expressing a soluble form of a heterologous viral protein, which will greatly aid the poultry industry in developing countries.

An epidemiological overview of human infections with HxNy avian influenza in the Western Pacific Region, 2003–2022

@#Avian influenza subtype A(HxNy) viruses are zoonotic and may occasionally infect humans through direct or indirect contact, resulting in mild to severe illness and death. Member States in the Western Pacific Region (WPR) communicate and notify the World Health Organization of any human cases of A(HxNy) through the International Health Regulations (IHR 2005) mechanism. This report includes all notifications in the WPR with illness onset dates from 1 November 2003 to 31 July 2022. During this period, there were 1972 human infections with nine different A(HxNy) subtypes notified in the WPR. Since the last report, an additional 134 human avian influenza infections were notified from 1 October 2017 to 31 July 2022. In recent years there has been a change in the primary subtypes and frequency of reports of human A(HxNy) in the region, with a reduction of A(H7N9) and A(H5N1), and conversely an increase of A(H5N6) and A(H9N2). Furthermore, three new subtypes A(H7N4), A(H10N3) and A(H3N8) notified from the People’s Republic of China were the first ever recorded globally. The public health risk from known A(HxNy) viruses remains low as there is no evidence of person-to-person transmission. However, the observed changes in A(HxNy) trends reinforce the need for effective and rapid identification to mitigate the threat of a pandemic from avian influenza if person-to-person transmission were to occur.