H5N1 Avian Influenza Virus Infection Research Articles

Type IIb Heat Labile Enterotoxin B Subunit as a Mucosal Adjuvant to Enhance Protective Immunity against H5N1 Avian Influenza Viruses.

Human infections with highly pathogenic avian influenza H5N1 viruses persist as a major global health concern. Vaccination remains the primary protective strategy against H5N1 and other novel avian influenza virus infections. We investigated the use of E. coli type IIb heat labile enterotoxin B subunit (LTIIb-B5) as a mucosal adjuvant for intranasal immunizations with recombinant HA proteins against H5N1 avian influenza viruses. Use of LTIIb-B5 adjuvant elicited more potent IgG, IgA, and neutralizing antibody titers in both sera and bronchoalveolar lavage fluids, thus increasing protection against lethal virus challenges. LTIIb-B5 mucosal adjuvanticity was found to trigger stronger Th17 cellular response in spleen lymphocytes and cervical lymph nodes. Studies of anti-IL-17A monoclonal antibody depletion and IL-17A knockout mice also suggest the contribution from Th17 cellular response to anti-H5N1 protective immunity. Our results indicate a link between improved protection against H5N1 live virus challenges and increased Th17 response due to the use of LTIIb-B5 mucosal adjuvant with HA subunit proteins.

Defective Viral Particles Produced in Mast Cells Can Effectively Fight Against Lethal Influenza A Virus.

Mast cells play an important role in the pathogenesis of highly pathogenic H5N1 avian influenza virus (H5N1-HPAIV) infection. Defective viral particles (DPs) can interfere with the replication of infectious viruses and stimulate the innate immune response of host cells. However, DPs arising from mast cells during HPAIV replication and their potent antiviral actions has not been reported. Here, we showed that the human mastocytoma cell line, HMC-1, allowed for the productive replication of the H5N1-HPAIV. Compared with alveolar cell line A549, DPs were propagated preferentially and abundantly in mast cells following IAV infection, which can be attributed to the wide existence of Argonaute 2 (AGO2) in HMC-1 cells. In addition, DPs generated in H5N1-infected cells could provide great therapeutic protection on mice to fight against various influenza A viruses, which included not only homologous H5N1-HPAIV, but also heterologous H1N1, H3N2, H7N2, and H9N2. Importantly, DPs generated in H5N1-infected HMC-1 cells could diminish viral virulence in vivo and in vitro by triggering a robust antiviral response through type II interferon signaling pathways. This study is the first to illustrate the arising of DPs in H5N1-HPAIV infected mast cells and explore their favorable ability to protect mice from influenza A viruses infection, which provides a novel insight and valuable information for the progress of new strategies to fight influenza A viruses infection, especially highly pathogenic avian influenza virus infection by focusing on the DPs generated in mast cells.

Nitrate Is Crucial for the Proliferation of Gut Escherichia coli Caused by H9N2 AIV Infection and Effective Regulation by Chinese Herbal Medicine Ageratum-Liquid.

H9N2 avian influenza virus (AIV) infection in chickens is often accompanied by secondary bacterial infection, but the mechanism is unclear. The aim of the present study was to reveal that mechanism and explore non-antibiotic treatment. 16s rRNA sequencing and metabonomics were performed in the intestinal contents of chickens infected with H9N2 AIV or H9N2 AIV and fed with ageratum-liquid (AL) to reveal the metabolite that promote intestinal Escherichia coli (E. coli) proliferation caused by H9N2 AIV, as well as to determine the regulatory effect of AL. It was found that H9N2 AIV infection led E. coli to become the dominant gut microbe and promoted E. coli translocation from the intestinal tract to the visceral tissue through the damaged intestinal barrier. H9N2 AIV infection induces inflammation in the intestinal mucosa and promotes the secretion and release of nitrate from the host intestinal epithelium. In addition, nitrate promoted E. coli proliferation in the inflamed intestinal tract following H9N2 AIV infection. Furthermore, Chinese herbal medicine AL can restore intestinal homeostasis, inhibit the production of nitrate in the intestinal epithelium and effectively prevent the proliferation and translocation of E. coli in the intestines. This is the first report on the mechanism of E. coli secondary infection induced by H9N2 AIV, where herbal medicine AL was shown to have a good preventive effect on the secondary infection.

Analysis of a highly pathogenic avian influenza (H5N1) virus causing the first outbreak in domestic poultry in Bulgaria in January 2015

This study documents the clinical signs, necropsy findings and viral antigen distribution of the highly pathogenic avian influenza (HPAI) H5N1 virus infection in domestic poultry (a backyard farm) and the phylogenetic analysis of the virus. On January 29, 2015, an outbreak of HPAI H5N1 in domestic poultry was reported on a backyard farm in Bulgaria. Out of the twenty-two chickens with clinical signs, twenty died while the remaining two were destroyed. The morbidity was 100%, whereas the overall mortality and lethality were 90.91%. The clinical observations made were sudden death, high mortality, weakness, and recumbency. Although multisystemic lesions were observed occasionally, the main pathologic findings were observed in the nervous, circulatory, respiratory, and gastrointestinal systems. An influenza virus nucleocapsid protein was identified by an immunohistochemical analysis in all the analysed organs: brain 3/3, trachea 3/3, lung 3/3, intestine 3/3, heart 3/3, which confirmed the systemic infection. The phylogenetic analyses of the virus showed a close genetic relationship with the H5N1 viruses of Asian origin, isolated in 2012 and 2013, belonging to the clade 2.3.2.1c. The HA-gene genetically clusters with HPAI H5N1 viruses isolated from wild pelicans in Romania and Bulgaria, thereby demonstrating the link between wild and domestic birds in the epidemiology of avian influenza. The contact between the affected chickens and migrating water birds over Bulgaria’s territory was suspected as a reason for the outbreak in the backyard farm. In addition, the detection of the virus in wild bird populations in Bulgaria three days earlier strongly supports the hypothesis of migrating wild birds spreading HPAI H5N1.

Co-infection of Salmonella enteritidis with H9N2 avian influenza virus in chickens

ABSTRACT Salmonella and avian influenza virus are important pathogens affecting the poultry industry and human health worldwide. In this experimental study, we evaluated the consequences of co-infection of Salmonella enteritidis (SE) with H9N2 avian influenza virus (H9N2-AIV) in chickens. Four groups were included: control group, H9N2-AIV group, H9N2-AIV + SE group, and SE group. Infected chickens were intranasally inoculated with H9N2-AIV at 21 days of age and then orally administered SE on the same day. The birds were monitored for clinical signs, mortality rates, and alterations in body weight. Sera, intestinal fluids, oropharyngeal, and cloacal swabs, and tissue samples were collected at 2, 6, 10, and 14 days post-infection (dpi). Significant increases in clinical signs and mortality rates were observed in the H9N2-AIV + SE group. Moreover, chickens with co-infection showed a significant change in body weight. SE faecal shedding and organ colonization were significantly higher in the H9N2-AIV + SE group than in the SE group. H9N2-AIV infection compromised the systemic and mucosal immunity against SE, as evidenced by a significant decrease in lymphoid organ indices as well as systemic antibody and intestinal immunoglobulin A (IgA) responses to SE and a significant increase in splenic and bursal lesion scores. Moreover, SE infection significantly increased shedding titres and duration of H9N2-AIV. In conclusion, this is the first report of co-infection of SE with H9N2-AIV in chickens, which leads to increased pathogenicity, SE faecal shedding and organ colonization, and H9N2-AIV shedding titre and duration, resulting in substantial economic losses and environmental contamination, ultimately leading to increased zoonoses.

Induction of cross-group broadly reactive antibody response by natural H7N9 avian influenza virus infection and immunization with inactivated H7N9 vaccine in chickens.

Pre-existing immunity against the conserved haemagglutinin (HA) stalk underlies the elicitation of cross-group antibody induced by natural H7N9 virus infection and immunization in humans. However, whether broadly reactive antibodies can be induced by H7N9 infection and immunization in the absence of pre-existing stalk-specific immunity is unclear. In this study, antibody response induced by H7N9 virus infection and immunization with inactivated and viral-vectored H7N9 vaccines in naïve chickens was analysed. The results showed that H7N9 infection and immunization with inactivated vaccine resulted in potent induction of haemagglutination-inhibition (HI), virus neutralization (VN) and HA-binding antibodies, whereas Newcastle disease virus (NDV)-vectored H7N9 vaccine induced marginal HI and VN titres but high levels of HA-binding antibody. In addition, H7N9 infection and immunization induced stalk-specific antibodies in naïve chickens and these antibodies recognized different epitopes in the stalk. Virus infection and immunization with inactivated vaccine elicited antibodies cross-reactive with both group 1 and group 2 HAs, while antibodies induced by NDV-H7N9 vaccination showed a narrower cross-reactivity within group 2. Moreover, only homologous neutralizing activity of the sera against H7N9 virus was observed, and cross-binding antibodies did not show heterosubtypic neutralizing activity. Our results indicated that cross-group binding but non-neutralizing antibodies primarily targeting the stalk can be induced by natural H7N9 infection and immunization with inactivated vaccine in naïve chickens. This suggests that at least in a naïve chicken model, pre-existing stalk-specific immunity is not required for induction of broadly reactive antibodies. Additionally, H7N9-based immunogens may be explored as vaccine candidates or as a prime component to induce broadly protective influenza immunity.

Highly pathogenic H7N9 avian influenza virus infection associated with up-regulation of PD-1/PD-Ls pathway-related molecules

To investigate the main transcriptional and biological changes of human host during low and highly pathogenic avian H7N9 influenza virus infection and to analyze the possible causes of escalated virulence and the systematic progression of H7N9 virus infection, we utilized whole transcriptome sequencing (RNA-chip and RNA-seq) and other biomolecular methods to analyze and verify remarkable changes of host cells during these two subtypes of H7N9 influenza viruses infection. Whole transcriptome analysis showed the global profiles of differentially expressed genes (DEGs) and identified 458 DEGs associated with major changes in biological processes of the host cells after infection with 2017 HPAI H7N9 virus versus 2013 LPAI H7N9 virus, mainly including drastically increased defense responses to viruses (e.g. negative regulation of viral gene replication), IFNs related pathways, immune response/native immune response, and inflammatory response. Genes of programmed cell death 1 (PD-1) pathways were found changed remarkably and several highly correlated non-coding RNAs were identified. The results suggested that HPAI H7N9 virus induces stronger immune response and suppressing response than LPAI H7N9. Meanwhile, PD-1/PD-Ls signaling pathways work together in regulating host responses including antiviral defense, lethal inflammation caused by the virus and immune response, thus contribute to the high pathogenicity of 2017H7N9 virus that can be regulated by non-coding RNAs. The present study represents a comprehensive understanding and good reference of regulation of pathogenicity of H7N9 virus even other fatal viruses and correlated host immune responses.

Dendrigraft poly-L-lysines delivery of DNA vaccine effectively enhances the immunogenic responses against H9N2 avian influenza virus infection in chickens

Biodegradable nanomaterials can protect antigens from degradation, promote cellular absorption, and enhance immune responses. We constructed a eukaryotic plasmid [pCAGGS-opti441-hemagglutinin (HA)] by inserting the optimized HA gene fragment of H9N2 AIV into the pCAGGS vector. The pCAGGS-opti441-HA/DGL was developed through packaging the pCAGGS-opti441-HA with dendrigraft poly-l-lysines (DGLs). DGL not only protected the pCAGGS-opti441-HA from degradation, but also exhibited high transfection efficiency. Strong cellular immune responses were induced in chickens immunized with the pCAGGS-opti441-HA/DGL. The levels of IFN-γ and IL-2, and lymphocyte transformation rate of the vaccinated chickens increased at the third week post the immunization. For the vaccinated chickens, T lymphocytes were activated and proliferated, the numbers of CD3+CD4+ and CD4+/CD8+ increased, and the chickens were protected completely against H9N2 AIV challenge. This study provides a method for the development of novel AIV vaccines, and a theoretical basis for the development of safe and efficient gene delivery carriers.

Antiviral activity of water extracts of some medicinal and nutritive plants from the Apiaceae family

During the past two decades, several human infections with avian influenza H5N1 virus have been reported. An increase in the recorded cases of human viral infections led to more public health concern, because of their potential pandemic proportions in the human's society. Moreover, an increase in the cases of drug-resistant influenza A virus has brought the urgent need for alternative anti-influenza drugs. In the present study, water extracts from eight commonly available medicinal and nutritive plants from the Apiaceae family including; Dill, Celery, Caraway, Coriander, Cumin, Fennel, Anise, and Parsley were prepared. The cytotoxicity of each of extract was individually determined in the Madin–Darby canine kidney (MDCK) cells. Thereafter, these extracts were investigated for their in vitro antiviral activities against the avian influenza H5N1 virus infection. Current results revealed that water extracts of the eight plants showed antiviral inhibitory activities with percentages ranging from 0- 71%. Among the tested plants, only anise plant (Pimpeniella anisum) had significant antiviral activity against the avian influenza H5N1 virus. Thus, the mode of action of this effective anise extract was investigated against the same virus. It was found that water extract of the anise plant induced virucidal effect, as well as direct effect on replication of the avian influenza H5N1 virus. The aim of the present study was to shed light on searching for alternative therapeutic sources for future treatment of the H5N1 influenza virus infection.

Estimating Transmission Potential of H5N1 Viruses Among Humans in Egypt Using Phylogeny, Genetic Distance and Sampling Time Interval.

In 2014 and 2015, the number of human cases of H5N1 avian influenza virus infections had increased dramatically in Egypt. This increase might be related to increase in the transmission potential of the virus among humans. To clarify the cause of the increase in H5N1 human cases, we investigate the transmissibility of H5N1 viruses among humans via estimating the basic reproduction number R0 using nucleotide sequences and sampling dates of viruses. To this end, full-length hemagglutinin gene sequences of human and avian H5N1 influenza viruses isolated from 2006 to 2016 in Egypt were obtained from the NCBI influenza virus resource. Taking into account the phylogeny, genetic distance, sampling time difference among viruses, R0 was estimated to be 0.05 (95% CI: 0.01, 0.13) assuming that human-to-human transmissions occurred within a city, 0.23(95% CI: 0.14, 0.35) assuming human-to-human transmissions among cities. Our results indicate that human-to-human transmission of H5N1 viruses in Egypt is limited, and the large increase in human cases is likely attributed to other factor than increase in human-to-human transmission potential.

Tandem mass tag-based quantitative proteomic analysis of lycorine treatment in highly pathogenic avian influenza H5N1 virus infection.

Highly pathogenic H5N1 influenza viruses (HPAIV) cause rapid systemic illness and death in susceptible animals, leading to a disease with high morbidity and mortality rates. Although vaccines and drugs are the best solution to prevent this threat, a more effective treatment for H5 strains of influenza has yet to be developed. Therefore, the development of therapeutics/drugs that combat H5N1 influenza virus infection is becoming increasingly important. Lycorine, the major component of Amaryllidaceae alkaloids, exhibits better protective effects against A/CK/GD/178/04 (H5N1) (GD178) viruses than the commercial neuraminidase (NA) inhibitor oseltamivir in our prior study. Lycorine demonstrates outstanding antiviral activity because of its inhibitory activity against the export of viral ribonucleoprotein complexes (vRNPs) from the nucleus. However, how lycorine affects the proteome of AIV infected cells is unknown. Therefore, we performed a comparative proteomic analysis to identify changes in protein expression in AIV-infected Madin-Darby Canine Kidney cells treated with lycorine. Three groups were designed: mock infection group (M), virus infection group (V), and virus infection and lycorine-treated after virus infection group (L). The multiplexed tandem mass tag (TMT) approach was employed to analyze protein level in this study. In total, 5,786 proteins were identified from the three groups of cells by using TMT proteomic analysis. In the V/M group, 1,101 proteins were identified, of which 340 differentially expressed proteins (DEPs) were determined during HPAIV infection; among the 1,059 proteins identified from the lycorine-treated group, 258 proteins presented significant change. Here, 71 proteins showed significant upregulation or downregulation of expression in the virus-infected/mock and virus-infected/lycorine-treated comparisons, and the proteins in each fraction were functionally classified further. Interestingly, lycorine treatment decreased the levels of the nuclear pore complex protein 93 (Nup93, E2RSV7), which is associated with nuclear–cytoplasmic transport. In addition, Western blot experiments confirmed that the expression of Nup93 was significantly downregulated in lycorine treatment but induced after viral infection. Our results may provide new insights into how lycorine may trap vRNPs in the nucleus and suggest new potential therapeutic targets for influenza virus.

NP and NS1 proteins of H5N1 virus significantly upregulated IFITM1, IFITM2, and IFITM3 in A549 cells.

Avian influence virus H5N1 causes serious public health concern with significant morbidity and mortality from poultry to humans. Interferon-induced transmembrane (IFITM) proteins usually protect cells from many virus infections by viral entry and replication. The purpose of this study was to investigate whether H5N1 viral proteins involved in regulation IFITM1, IFITM2, and IFITM3 following H5N1 infection. NS1, M1, NP, PB2, HA and NA genes of H5N1 virus were generated by PCR and cloned into pcDNA3.1/myc-His (+) A vector for genes over-expression experiments. Gene expression levels was performed using Real-time PCR. Research displayed that NS1, M1, NP, and PB2 proteins of H5N1 virus increased IFITM1, IFITM2, and IFITM3 expression in A549 cells, only IFITM1 was upregulated by M1 in HEK293T cells. However, our study did not find that HA and NA of H5N1 virus affected IFITM genes family or interferon genes expression. Taken together, our data suggested that IFITM1, IFITM2, and IFITM3 might be directly upregulated via NS1, M1, NP, and PB2 proteins during H5N1 avian influenza virus infection. This study provided new insights into the influence of NS1 and NP proteins on regulation of IFITM1, IFITM2, and IFITM3 expression following H5N1 infection.

Prior exposure to immunogenic peptides found in human influenza A viruses may influence the age distribution of cases with avian influenza H5N1 and H7N9 virus infections.

The epidemiology of H5N1 and H7N9 avian viruses of humans infected in China differs despite both viruses being avian reassortants that have inherited six internal genes from a common ancestor, H9N2. The median age of infected populations is substantially younger for H5N1 virus (26 years) compared with H7N9 virus (63 years). Population susceptibility to infection with seasonal influenza is understood to be influenced by cross-reactive CD8+ T cells directed towards immunogenic peptides derived from internal viral proteins which may provide some level of protection against further influenza infection. Prior exposure to seasonal influenza peptides may influence the age-related infection patterns observed for H5N1 and H7N9 viruses. A comparison of relatedness of immunogenic peptides between historical human strains and the two avian emerged viruses was undertaken for a possible explanation in the differences in age incidence observed. There appeared to be some relationship between past exposure to related peptides and the lower number of H5N1 virus cases in older populations, however the relationship between prior exposure and older populations among H7N9 virus patients was less clear.

Highly Pathogenic H5N1 Influenza A Virus Spreads Efficiently in Human Primary Monocyte-Derived Macrophages and Dendritic Cells.

Influenza A viruses cause recurrent epidemics and occasional global pandemics. Wild birds are the natural reservoir of influenza A virus from where the virus can be transmitted to poultry or to mammals including humans. Mortality among humans in the highly pathogenic avian influenza H5N1 virus infection is even 60%. Despite intense research, there are still open questions in the pathogenicity of the H5N1 virus in humans. To characterize the H5N1 virus infection in human monocyte-derived macrophages (Mɸs) and dendritic cells (DCs), we used human isolates of highly pathogenic H5N1/2004 and H5N1/1997 and low pathogenic H7N9/2013 avian influenza viruses in comparison with a seasonal H3N2/1989 virus. We noticed that the H5N1 viruses have an overwhelming ability to replicate and spread in primary human immune cell cultures, and even the addition of trypsin did not equalize the infectivity of H7N9 or H3N2 viruses to the level seen with H5N1 virus. H5N1 virus stocks contained more often propagation-competent viruses than the H7N9 or H3N2 viruses. The data also showed that human DCs and Mɸs maintain 1,000- and 10,000-fold increase in the production of infectious H5N1 virus, respectively. Both analyzed highly pathogenic H5N1 viruses showed multi-cycle infection in primary human DCs and Mɸs, whereas the H3N2 and H7N9 viruses were incapable of spreading in immune cells. Interestingly, H5N1 virus was able to spread extremely efficiently despite the strong induction of antiviral interferon gene expression, which may in part explain the high pathogenicity of H5N1 virus infection in humans.

The PA-interacting host protein nucleolin acts as an antiviral factor during highly pathogenic H5N1 avian influenza virus infection

Polymerase acidic (PA) protein is a multifunctional regulator of influenza A virus (IAV) replication and pathogenesis. In a previous study, we reported that nucleolin (NCL) is a novel PA-interacting host protein. In this study, we further explored the role of NCL during highly pathogenic H5N1 avian influenza virus infection. We found that depletion of endogenous NCL in mammalian cells by siRNA targeting during H5N1 infection resulted in significantly increased viral polymerase activity, elevated viral mRNA, cRNA and vRNA synthesis, accelerated viral replication, and enhanced apoptosis and necrosis. Moreover, siRNA silencing of NCL significantly exacerbated the inflammatory response, resulting in increased secretion of IL-6, TNF-α, TNF-β, CCL-4, CCL-8, IFN-α, IFN-β and IFN-γ. Conversely, overexpression of NCL significantly decreased IAV replication. Collectively, these data show that NCL acts as a novel potential antiviral factor during H5N1 infection. Further studies exploring the antiviral mechanisms of NCL may accelerate the development of new anti-influenza drugs.

Down-regulation of cellular protein heme oxygenase-1 inhibits proliferation of avian influenza virus H9N2 in chicken oviduct epithelial cells.

The pathogenesis of H9N2 subtype avian influenza virus (AIV) infection in hens is often related to oviduct tissue damage. Our previous study suggested that H9N2 AIV induces cellular apoptosis by activating reactive oxygen species (ROS) accumulation and mitochondria-mediated apoptotic signalling in chicken oviduct epithelial cells (COECs). Heme oxygenase-1 (HO-1) is an inducible enzyme that exerts protective effects against oxidative stress and activated HO-1 was recently shown to have antiviral activity. To study the potential involvement of HO-1 in H9N2 AIV proliferation, the role of its expression in H9N2-infected COECs was further investigated. Our results revealed that H9N2 AIV infection significantly up-regulated the expression of HO-1 and that HO-1 down-regulation by ZnPP, a classical inhibitor of HO-1, could inhibit H9N2 AIV replication in COECs. Similarly, the small interfering RNA (siRNA)-mediated knockdown of HO-1 also markedly decreased the virus production in H9N2-infected COECs. In contrast, adenoviral-mediated over-expression of HO-1 concomitantly promoted H9N2 AIV replication. Taken together, our study demonstrated the involvement of HO-1 in AIV H9N2 proliferation, and these findings suggested that HO-1 is a potential target for inhibition of AIV H9N2 replication.

Mycophenolic mofetil, an alternative antiviral and immunomodulator for the highly pathogenic avian influenza H5N1 virus infection

Infection with the highly pathogenic avian influenza H5N1 virus results in a high incidence of mortality in humans. Severe complications from infection are often associated with hypercytokinemia. However, current neuraminidase inhibitors (NAIs) have several limitations including the appearance of oseltamivir-resistant H5N1 virus and the inability to completely ameliorate hyper-immune responses. To overcome these limitations, we evaluated the anti-viral activity of mycophenolic mofetil (MMF) against A/Vietnam/1194/2004 (H5N1) virus infection using MDCK cells and mice. The IC50 of MMF (0.94 μM) was comparable to that of zanamivir (0.87 μM) in H5N1 virus-infected MDCK cells based on ELISA. Time-course assays demonstrated that MMF completely inhibited H5N1 viral mRNA replication and protein expression for approximately 8 h after the initiation of treatment. In addition, MMF treatment protected 100% of mice, and lung viral titers were substantially reduced. The anti-viral mechanism of MMF against H5N1 virus infection was further confirmed to depend on the inhibition of cellular inosine monophosphate dehydrogenase (IMPDH) by exogenous guanosine, which inhibits viral mRNA and protein expression. Moreover, IL-1β, IFN-β, IL-6, and IP-10 mRNA expression levels were significantly downregulated in MDCK cells with MMF treatment. These results indicated that MMF could represent a novel inhibitor of viral replication and a potent immunomodulator for the treatment of H5N1 virus infection.

Construction of a highly efficient CRISPR/Cas9-mediated duck enteritis virus-based vaccine against H5N1 avian influenza virus and duck Tembusu virus infection

Duck enteritis virus (DEV), duck tembusu virus (DTMUV), and highly pathogenic avian influenza virus (HPAIV) H5N1 are the most important viral pathogens in ducks, as they cause significant economic losses in the duck industry. Development of a novel vaccine simultaneously effective against these three viruses is the most economical method for reducing losses. In the present study, by utilizing a clustered regularly interspaced short palindromic repeats (CRISPR)/associated 9 (Cas9)-mediated gene editing strategy, we efficiently generated DEV recombinants (C-KCE-HA/PrM-E) that simultaneously encode the hemagglutinin (HA) gene of HPAIV H5N1 and pre-membrane proteins (PrM), as well as the envelope glycoprotein (E) gene of DTMUV, and its potential as a trivalent vaccine was also evaluated. Ducks immunized with C-KCE-HA/PrM-E enhanced both humoral and cell-mediated immune responses to H5N1 and DTMUV. Importantly, a single-dose of C-KCE-HA/PrM-E conferred solid protection against virulent H5N1, DTMUV, and DEV challenges. In conclusion, these results demonstrated for the first time that the CRISPR/Cas9 system can be applied for modification of the DEV genome rapidly and efficiently, and that recombinant C-KCE-HA/PrM-E can serve as a potential candidate trivalent vaccine to prevent H5N1, DTMUV, and DEV infections in ducks.

Protection of chickens against H9N2 avian influenza virus challenge with recombinant Lactobacillus plantarum expressing conserved antigens

Avian influenza virus (AIV) is spreading worldwide and is a serious threat to the health of poultry and humans. In many countries, low pathogenic AIVs, such as H9N2, have become an enormous economic burden on the commercial poultry industry because they cause mild respiratory disease and decrease egg production. A recombinant Lactobacillus plantarum NC8 strain expressing NP-M1-DCpep from H9N2 AIV has been studied in a mouse model. However, it remains unknown whether this L. plantarum strain can induce an immune response and provide protection against H9N2 AIV in chickens. In this study, chickens that were orally vaccinated with NC8-pSIP409-NP-M1-DCpep exhibited significantly increased T cell-mediated immune responses and mucosal sIgA and IgG levels, which provided protection against H9N2 AIV challenge. More importantly, compared with oral administration of NC8-pSIP409-NP-M1-DCpep, intranasal administration induced stronger immune responses and provided effective protection against challenge with the H9N2 virus by reducing body weight loss, lung virus titers, and throat pathology. Taken together, these findings suggest that L. plantarum expressing NP-M1-DCpep has potential as a vaccine to combat H9N2 AIV infection.

Inhibition of H9N2 Virus Invasion into Dendritic Cells by the S-Layer Protein from L. acidophilus ATCC 4356.

Probiotics are essential for the prevention of virus invasion and the maintenance of the immune balance. However, the mechanism of competition between probiotics and virus are unknown. The objectives of this study were to isolate the surface layer (S-layer) protein from L. acidophilus ATCC 4356 as a new antiviral material, to evaluate the stimulatory effects of the S-layer protein on mouse dendritic cells (DCs) and to verify its ability to inhibit the invasion of H9N2 avian influenza virus (AIV) in DCs. We found that the S-layer protein induced DCs activation and up-regulated the IL-10 secretion. The invasion and replication of the H9N2 virus in mouse DCs was successfully demonstrated. However, the invasion of H9N2 virus into DCs could be inhibited by treatment with the S-layer protein prior to infection, which was verified by the reduced hemagglutinin (HA) and neuraminidase (NA) mRNA expression, and nucleoprotein (NP) protein expression in the DCs. Furthermore, treatment with the S-layer protein increases the Mx1, Isg15, and Ddx58 mRNA expressions, and remits the inflammatory process to inhibit H9N2 AIV infection. In conclusion, the S-layer protein stimulates the activation of mouse DCs, inhibits H9N2 virus invasion of DCs, and stimulates the IFN-I signaling pathway. Thus, the S-layer protein from Lactobacillus is a promising biological antiviral material for AIV prevention.