Effects of Nonpharmaceutical COVID-19 Interventions on Pediatric Hospitalizations for Other Respiratory Virus Infections, Hong Kong.

To determine the effects of nonpharmaceutical interventions (NPIs) for coronavirus disease on pediatric hospitalizations for infection with respiratory viruses other than severe acute respiratory syndrome coronavirus 2, we analyzed hospital data for 2017-2021. Compared with 2017-2019, age-specific hospitalization rates associated with respiratory viruses greatly decreased in 2020, when NPIs were in place. Also when NPIs were in place, rates of hospitalization decreased among children of all ages for infection with influenza A and B viruses, respiratory syncytial virus, adenovirus, parainfluenza viruses, human metapneumovirus, and rhinovirus/enterovirus. Regression models adjusted for age and seasonality indicated that hospitalization rates for acute febrile illness/respiratory symptoms of any cause were reduced by 76% and by 85%-99% for hospitalization for infection with these viruses. NPIs in Hong Kong were clearly associated with reduced pediatric hospitalizations for respiratory viruses; implementing NPIs and reopening schools were associated with only a small increase in hospitalizations for rhinovirus/enterovirus infections.

Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses.

BACKGROUND: Neutrophil is of the most abundant number in human immune system. During acute influenza virus infection, neutrophils are already active in the early phase of inflammation - a time in which clinical biopsy or autopsy material is not readily available. However, the role of neutrophil in virus infection is not well understood. Here, we studied the role of neutrophil in host defense during influenza A virus infection, specifically assessing if it contributes to the differential pathogenesis in H5N1 disease. METHODS: Neutrophils were freshly isolated from healthy volunteers and subjected to direct influenza H1N1 and H5N1 virus infection in vitro. The ability of the naïve neutrophils to infiltrate from the basolateral to the apical phase of the influenza virus infected alveolar epithelium was assessed. The viral replication, innate immune responses and Neutrophil extracellular trap (NET) formation of neutrophils upon influenza virus infection were evaluated. RESULTS: Our results demonstrated that influenza virus infected alveolar epithelium allowed neutrophil transmigration. Significantly more neutrophils migrated across the H5N1 influenza virus infected the epithelium than the counterpart infected by the seasonal influenza H1N1 virus infected. Neutrophils were equally susceptible to H5N1 and H1N1 virus infection with similar viral gene transcription. Productive replication was observed in H5N1 infected neutrophils. H5N1 induced higher cytokine and chemokine gene transcription than H1N1 infected neutrophils, including TNF-α, IFN-ß, CXCL10, MIP-1α and IL-8. This inferred a more intense inflammatory response posed by H5N1 than H1N1 virus. Strikingly, NADPH oxidase-independent NET formation was only observed in H1N1 infected neutrophils at 6 hpi while no NET formation was observed upon H5N1 infection. CONCLUSION: Our data is the first to demonstrate that NET formation is abrogated in H5N1 influenza virus infection and might contribute to the severity of H5N1 disease.

Tropism of influenza B viruses in human respiratory tract explants and airway organoids.

Despite causing regular seasonal epidemics with substantial morbidity, mortality and socioeconomic burden, there is still a lack of research into influenza B viruses (IBVs). In this study, we provide for the first time a systematic investigation on the tropism, replication kinetics and pathogenesis of IBVs in the human respiratory tract.Physiologically relevant ex vivo explant cultures of human bronchus and lung, human airway organoids, and in vitro cultures of differentiated primary human bronchial epithelial cells and type-I-like alveolar epithelial cells were used to study the cellular and tissue tropism, replication competence and induced innate immune response of 16 IBV strains isolated from 1940 to 2012 in comparison with human seasonal influenza A viruses (IAVs), H1N1 and H3N2. IBVs from the diverged Yamagata- and Victoria-like lineages and the earlier undiverged period were included.The majority of IBVs replicated productively in human bronchus and lung with similar competence to seasonal IAVs. IBVs infected a variety of cell types, including ciliated cells, club cells, goblet cells and basal cells, in human airway organoids. Like seasonal IAVs, IBVs are low inducers of pro-inflammatory cytokines and chemokines. Most results suggested a higher preference for the conducting airway than the lower lung and strain-specific rather than lineage-specific pathogenicity of IBVs.Our results highlighted the non-negligible virulence of IBVs which require more attention and further investigation to alleviate the disease burden, especially when treatment options are limited.

Therapeutic Implications of Human Umbilical Cord Mesenchymal Stromal Cells in Attenuating Influenza A(H5N1) Virus-Associated Acute Lung Injury.

Background: Highly pathogenic avian influenza viruses can cause severe forms of acute lung injury (ALI) in humans, where pulmonary flooding leads to respiratory failure. The therapeutic benefits of bone marrow mesenchymal stromal cells (MSCs) have been demonstrated in a model of ALI due to influenza A(H5N1) virus. However, clinical translation is impractical and limited by a decline in efficacy as the age of the donor increases. Umbilical cord MSCs (UC-MSCs) are easier to obtain by comparison, and their primitive source may offer more-potent therapeutic effects. Methods: Here we investigate the therapeutic efficacy of UC-MSCs on the mechanisms of pulmonary edema formation and alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs were also tested in a mouse model of influenza ALI. Results: We found that UC-MSCs were effective in restoring impaired alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs consistently outperformed bone marrow MSCs, partly because of greater growth factor secretion of angiopoietin 1 and hepatocyte growth factor. Conditioned UC-MSC medium and UC-MSC exosomes were also able to recapitulate these effects. However, UC-MSCs only slightly improved survival of A(H5N1)-infected mice. Conclusions: Our results suggest that UC-MSCs are effective in restoring alveolar fluid clearance and protein permeability in A(H5N1)-associated ALI and confer functional in addition to practical advantages over conventional bone marrow MSCs.

Tropism, replication competence, and innate immune responses of influenza virus: an analysis of human airway organoids and ex-vivo bronchus cultures.

BACKGROUND: Human airway organoids are three-dimensional cultures derived from stem cells, which self-organise in ex-vivo conditions to form so-called mini-airways. The cellular morphology of these cultures is physiologically similar to the human airway, with cilia, goblet cells, and club cells facing the inner lumen and basal cells situated at the outer layer. The aim of this study was to compare replication competence, tissue tropism, and host responses elicited by human and avian strains of influenza A virus in ex-vivo human bronchus and human airway organoids. METHODS: Between Sept 29, 2016, and Jan 4, 2017, we obtained ex-vivo cultures of the human bronchus and cultured human airway organoids from lung stem cells obtained from human lung tissues removed as part of the routine clinical care of patients undergoing surgical resection at the Department of Cardiothoracic Surgery, University of Hong Kong, Queen Mary Hospital, Hong Kong. We compared viral replication competence, tissue tropism, and cytokine and chemokine induction of avian influenza A viruses isolated from humans (Sh2/H7N9, H5N1/483, H5N6/39715), and human H1N1pdm/415742 in airway organoids and ex-vivo bronchus explant cultures. FINDINGS: Virus tropism and replication kinetics of human and avian influenza A viruses in human airway organoids mimicked those found in ex-vivo cultures of human bronchus explants. In both airway organoids and bronchus explants, influenza A H1N1 subtype (H1N1) and avian influenza A H7N9 viruses replicated to significantly higher titres than did the highly pathogenic avian influenza (HPAI) H5N1, whereas HPAI H5N6 replication was moderate. H1N1, H7N9, and H5N6 viruses infected ciliated cells and goblet cells, but not basal cells in both airway organoids and bronchus explants. The expression of cytokines, interleukin 6, and interferon ß, and the chemokine regulated-on-activation, normal T-cell expressed and secreted, was significantly higher in human airway organoids infected with HPAI H5N1 virus than H1N1pdm/415742, Sh2/H7N9, and H5N6/39715 viruses, and the expression of monocyte chemoattractant protein-1 was significantly higher in human organoids infected with HPAI H5N1 virus than H1N1pdm/415742 and Sh2/H7N9 viruses. INTERPRETATION: Human airway organoid cultures provided results that were comparable to those observed in human ex-vivo bronchus cultures, and thus provide an alternative physiologically relevant experimental model for investigating virus tropism and replication competence that could be used to assess the pandemic threat of animal influenza viruses. FUNDING: US National Institute of Allergy and Infectious Diseases, Research Grants Council of the Hong Kong Special Administrative Region.

Evaluation of the human adaptation of influenza A/H7N9 virus in PB2 protein using human and swine respiratory tract explant cultures.

Novel avian H7N9 virus emerged in China in 2013 resulting in a case fatality rate of around 39% and continues to pose zoonotic and pandemic risk. Amino acid substitutions in PB2 protein were shown to influence the pathogenicity and transmissibility of H7N9 following experimental infection of ferrets and mice. In this study, we evaluated the role of amino acid substitution PB2-627K or compensatory changes at PB2-591K and PB2-701N, on the tropism and replication competence of H7N9 viruses for human and swine respiratory tracts using ex vivo organ explant cultures. Recombinant viruses of A/Shanghai/2/2013 (rgH7N9) and its mutants with PB2-K627E, PB2-K627E + Q591K and PB2-K627E + D701N were generated by plasmid-based reverse genetics. PB2-E627K was essential for efficient replication of rgH7N9 in ex vivo cultures of human and swine respiratory tracts. Mutant rgPB2-K627E + D701N replicated better than rgPB2-K627E in human lung but not as well as rgH7N9 virus. The rgPB2-K627E mutant failed to replicate in human type I-like pneumocytes (ATI) and peripheral blood monocyte-derived macrophages (PMϕ) at 37 °C while the compensatory mutant rgPB2-K627E + Q591K and rgPB2-K627E + D701N had partly restored replication competence in PMϕ. Our results demonstrate that PB2-E627K was important for efficient replication of influenza H7N9 in both human and swine respiratory tracts.

Highly pathogenic avian influenza H5N1 virus delays apoptotic responses via activation of STAT3.

Highly pathogenic avian influenza (HPAI) H5N1 virus continues to pose pandemic threat, but there is a lack of understanding of its pathogenesis. We compared the apoptotic responses triggered by HPAI H5N1 and low pathogenic H1N1 viruses using physiologically relevant respiratory epithelial cells. We demonstrated that H5N1 viruses delayed apoptosis in primary human bronchial and alveolar epithelial cells (AECs) compared to H1N1 virus. Both caspase-8 and -9 were activated by H5N1 and H1N1 viruses in AECs, while H5N1 differentially up-regulated TRAIL. H5N1-induced apoptosis was reduced by TRAIL receptor silencing. More importantly, STAT3 knock-down increased apoptosis by H5N1 infection suggesting that H5N1 virus delays apoptosis through activation of STAT3. Taken together, we demonstrate that STAT3 is involved in H5N1-delayed apoptosis compared to H1N1. Since delay in apoptosis prolongs the duration of virus replication and production of pro-inflammatory cytokines and TRAIL from H5N1-infected cells, which contribute to orchestrate cytokine storm and tissue damage, our results suggest that STAT3 may play a previously unsuspected role in H5N1 pathogenesis.

Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo.

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

Impact of the 2009 H1N1 Pandemic on Age-Specific Epidemic Curves of Other Respiratory Viruses: A Comparison of Pre-Pandemic, Pandemic and Post-Pandemic Periods in a Subtropical City.

BACKGROUND: The 2009 H1N1 influenza pandemic caused offseason peaks in temperate regions but coincided with the summer epidemic of seasonal influenza and other common respiratory viruses in subtropical Hong Kong. This study was aimed to investigate the impact of the pandemic on age-specific epidemic curves of other respiratory viruses. METHODS: Weekly laboratory-confirmed cases of influenza A (subtypes seasonal A(H1N1), A(H3N2), pandemic virus A(H1N1)pdm09), influenza B, respiratory syncytial virus (RSV), adenovirus and parainfluenza were obtained from 2004 to 2013. Age-specific epidemic curves of viruses other than A(H1N1)pdm09 were compared between the pre-pandemic (May 2004-April 2009), pandemic (May 2009-April 2010) and post-pandemic periods (May 2010-April 2013). RESULTS: There were two peaks of A(H1N1)pdm09 in Hong Kong, the first in September 2009 and the second in February 2011. The infection rate was found highest in young children in both waves, but markedly fewer cases in school children were recorded in the second wave than in the first wave. Positive proportions of viruses other than A(H1N1)pdm09 markedly decreased in all age groups during the first pandemic wave. After the first wave of the pandemic, the positive proportion of A(H3N2) increased, but those of B and RSV remained slightly lower than their pre-pandemic proportions. Changes in seasonal pattern and epidemic peak time were also observed, but inconsistent across virus-age groups. CONCLUSION: Our findings provide some evidence that age distribution, seasonal pattern and peak time of other respiratory viruses have changed since the pandemic. These changes could be the result of immune interference and changing health seeking behavior, but the mechanism behind still needs further investigations.

Modulation of sterol biosynthesis regulates viral replication and cytokine production in influenza A virus infected human alveolar epithelial cells.

Highly pathogenic H5N1 viruses continue to transmit zoonotically, with mortality higher than 60%, and pose a pandemic threat. Antivirals remain the primary choice for treating H5N1 diseases and have their limitations. Encouraging findings highlight the beneficial effects of combined treatment of host targeting agents with immune-modulatory activities. This study evaluated the undefined roles of sterol metabolic pathway in viral replication and cytokine induction by H5N1 virus in human alveolar epithelial cells. The suppression of the sterol biosynthesis by Simvastatin in human alveolar epithelial cells led to reduction of virus replication and cytokine production by H5N1 virus. We further dissected the antiviral role of different regulators of the sterol metabolism, we showed that Zometa, FPT inhibitor III, but not GGTI-2133 had anti-viral activities against both H5N1 and H1N1 viruses. More importantly, FPT inhibitor III treatment significantly suppressed cytokine production by H5N1 virus infected alveolar epithelial cells. Since both viral replication itself and the effects of viral hyper-induction of cytokines contribute to the immunopathology of severe H5N1 disease, our findings highlights the therapeutic potential of FPT inhibitor III for severe human H5N1 diseases. Furthermore, our study is the first to dissect the roles of different steps in the sterol metabolic pathway in H5N1 virus replication and cytokine production.

Highly pathogenic avian influenza A H5N1 and pandemic H1N1 virus infections have different phenotypes in Toll-like receptor 3 knockout mice.

Toll-like receptors (TLRs) play an important role in innate immunity to virus infections. We investigated the role of TLR3 in the pathogenesis of H5N1 and pandemic H1N1 (pH1N1) influenza virus infections in mice. Wild-type mice and those defective in TLR3 were infected with influenza A/HK/486/97 (H5N1) or A/HK/415742/09 (pH1N1) virus. For comparison, mice defective in the gene for myeloid differential factor 88 (MyD88) were also infected with the viruses, because MyD88 signals through a TLR pathway different from TLR3. Survival and body weight loss were monitored for 14 days, and lung pathology, the lung immune-cell profile, viral load and cytokine responses were studied. H5N1-infected TLR3(-/-) mice had better survival than H5N1-infected WT mice, evident by significantly faster regain of body weight, lower viral titre in the lung and fewer pathological changes in the lung. However, this improved survival was not seen upon pH1N1 infection of TLR3(-/-) mice. In contrast, MyD88(-/-) mice had an increased viral titre and decreased leukocyte infiltration in the lungs after infection with H5N1 virus and poorer survival after pH1N1 infection. In conclusion, TLR3 worsens the pathogenesis of H5N1 infection but not of pH1N1 infection, highlighting the differences in the pathogenesis of these two viruses and the different roles of TLR3 in their pathogenesis.

Anti-inflammatory and antiviral effects of indirubin derivatives in influenza A (H5N1) virus infected primary human peripheral blood-derived macrophages and alveolar epithelial cells.

Human disease caused by highly pathogenic avian influenza A (HPAI) (H5N1) is associated with fulminant viral pneumonia and mortality rates in excess of 60%. Acute respiratory syndrome (ARDS) has been found to be the most severe form of acute lung injury caused by H5N1 virus infection while cytokine dysregulation and viral replication are thought to contribute to its pathogenesis. In this study, the antiviral and anti-inflammatory effects of two indirubin derivatives: indirubin-3'-oxime (IM) and E804 on primary human peripherial blood-derived macrophages and type-I like pneumocytes (human alveolar epithelial cells) during influenza A (H5N1) virus infection were investigated. We found that both of the indirubin derivatives strongly suppress the pro-inflammatory cytokines including IP-10 (CXCL10), one of the key factors which contribute to the lung inflammation during H5N1 virus infection. In addition, we also demonstrated that the indirubin derivative delays the virus replication in the primary cell culture models. Our results showed that indirubin derivatives have a potential to be used as an adjunct to antiviral therapy for the treatment of severe human H5N1 disease.

Toll-like receptor 10 is involved in induction of innate immune responses to influenza virus infection.

Toll-like receptors (TLRs) play key roles in innate immune recognition of pathogen-associated molecular patterns of invading microbes. Among the 10 TLR family members identified in humans, TLR10 remains an orphan receptor without known agonist or function. TLR10 is a pseudogene in mice and mouse models are noninformative in this regard. Using influenza virus infection in primary human peripheral blood monocyte-derived macrophages and a human monocytic cell line, we now provide previously unidentified evidence that TLR10 plays a role in innate immune responses following viral infection. Influenza virus infection increased TLR10 expression and TLR10 contributed to innate immune sensing of viral infection leading to cytokine induction, including proinflammatory cytokines and interferons. TLR10 induction is more pronounced following infection with highly pathogenic avian influenza H5N1 virus compared with a low pathogenic H1N1 virus. Induction of TLR10 by virus infection requires active virus replication and de novo protein synthesis. Culture supernatants of virus-infected cells modestly up-regulate TLR10 expression in nonvirus-infected cells. Signaling via TLR10 was activated by the functional RNA-protein complex of influenza virus leading to robust induction of cytokine expression. Taken together, our findings identify TLR10 as an important innate immune sensor of viral infection and its role in innate immune defense and immunopathology following viral and bacterial pathogens deserves attention.

Tropism of and innate immune responses to the novel human betacoronavirus lineage C virus in human ex vivo respiratory organ cultures.

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection.

Tropism and innate host responses of a novel avian influenza A H7N9 virus: an analysis of ex-vivo and in-vitro cultures of the human respiratory tract.

BACKGROUND: Since March, 2013, an avian-origin influenza A H7N9 virus has caused severe pneumonia in China. The aim of this study was to investigate the pathogenesis of this new virus in human beings. METHODS: We obtained ex-vivo cultures of the human bronchus, lung, nasopharynx, and tonsil and in-vitro cultures of primary human alveolar epithelial cells and peripheral blood monocyte-derived macrophages. We compared virus tropism and induction of proinflammatory cytokine responses of two human influenza A H7N9 virus isolates, A/Shanghai/1/2013 and A/Shanghai/2/2013; a highly pathogenic avian influenza H5N1 virus; the highly pathogenic avian influenza H7N7 virus that infected human beings in the Netherlands in 2003; the 2009 pandemic influenza H1N1 virus, and a low pathogenic duck H7N9 virus that was genetically different to the human disease causing A H7N9 viruses. FINDINGS: Both human H7N9 viruses replicated efficiently in human bronchus and lung ex-vivo cultures, whereas duck/H7N9 virus failed to replicate in either. Both human A H7N9 viruses infected both ciliated and non-ciliated human bronchial epithelial cells and replicated to higher titres than did H5N1 (p<0.0001 to 0.0046) and A/Shanghai/1/2013 replicated to higher titres than did H7N7 (p=0.0002-0.01). Both human A H7N9 viruses predominantly infected type II alveolar epithelial cells and alveolar macrophages in the human lung and replicated to higher titres than did H5N1 (p<0.0001 to 0.0078); A/Shanghai/1/2013 replicated to higher titres than did H1N1 (p=0.0052-0.05) and H7N7 (p=0.0031-0.0151). Human H7N9 viruses were less potent inducers of proinflammatory cytokines compared with H5N1 virus. INTERPRETATION: Collectively, the results suggest that the novel H7N9 viruses are better adapted to infect and replicate in the human conducting and lower airways than are other avian influenza viruses, including H5N1, and pose an important pandemic threat. FUNDING: Area of Excellence Scheme of the University Grants Committee (AoE/M-12/96), Hong Kong Special Administrative Region.

Proinflammatory cytokine response and viral replication in mouse bone marrow derived macrophages infected with influenza H1N1 and H5N1 viruses.

The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes.

Detection of highly pathogenic influenza and pandemic influenza virus in formalin fixed tissues by immunohistochemical methods.

Tissues infected with highly pathogenic avian influenza viruses such as H5N1 and H7N7 are normally required to be fixed in formalin or paraformaldehyde before examination in order to inactivate the virus. In this study commercially available monoclonal antibodies to the influenza nucleoprotein (NP) were evaluated in order to determine which antibodies would identify positive cells in tissues fixed in formalin or paraformaldehyde. An assessment of which antigen retrieval process would unmask antigens blocked by formalin fixation was also made. Of six commercially available monoclonal antibodies tested, only one (HB65, European Veterinary Laboratories) was able to identify all formalin fixed avian, swine and human influenza virus infected tissues, and this was after pronase induced epitope retrieval. This monoclonal antibody is recommended for routine diagnostic use for the detection of influenza A infected tissues that have been fixed in formalin or paraformaldehyde.

Type 1 responses of human Vγ9Vδ2 T cells to influenza A viruses.

γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection.