HutZ is required for efficient heme utilization and contributes to the pathogenicity of Avibacterium paragallinarum.

Avibacterium paragallinarum is the pathogen that causes infectious coryza, a highly contagious respiratory disease that brings a serious threat to chickens. Heme utilization systems play an important role in bacterial adversity adaptation and pathogenicity, and our previous report found the presence of heme utilization (HutZ) in Av. paragallinarum. However, little is known about the function of HutZ in Av. paragallinarum. In this study, the HutZ mutant strain of Av. paragallinarum was successfully developed and identified by PCR and western blot analysis. Mutation of HutZ significantly retards bacterial growth under reduced iron conditions, indicating the regulatory role of HutZ on growth and iron acquisition. Notably, the HutZ mutant strain had slower growth than the wild-type strain when heme was provided as the sole source of iron; thus, HutZ is crucial for heme utilization in Av. paragallinarum. Moreover, the HutZ mutant strain exhibited a markedly compromised tolerance to acid stress compared to the wild-type strain. Pathogenicity analysis showed that mutation of HutZ significantly weakened the ability of bacteria to invade and reproduce in host macrophage cells in vitro. Furthermore, the HutZ mutation could significantly decrease the bacterial virulence in chickens, which displayed lower morbidity and milder clinical symptoms. Hence, this is the first study to demonstrate in-depth the essential roles of HutZ on iron homeostasis and pathogenesis of Av. paragallinarum, which provides novel insight into advances of new prophylactic vaccines against this kind of bacteria.ImportanceHeme utilization (HutZ) protein has been characterized as an important heme-degrading enzyme that is critical for the cleavage of heme to biliverdin via verdoheme and can release iron to be used by bacteria. The interaction between HutZ and Av. paragallinarum is still unknown. Here, we unraveled the role of HutZ on the growth, iron acquisition, heme utilization, and resistance to acidic stress in Av. paragallinarum. We also uncovered the importance of HutZ for the success of Av. paragallinarum infection and provided new clues to the pathogenesis strategies of this organism. This work constitutes a relevant step toward an understanding of the role of HutZ protein as a master virulence factor. Therefore, this study is of great importance for understanding the mechanisms underlying Av. paragallinarum virulence and may contribute to therapeutic applications.

Melatonin alleviates lung injury in H1N1-infected mice by mast cell inactivation and cytokine storm suppression.

Influenza A virus (IAV) H1N1 infection is a constant threat to human health and it remains so due to the lack of an effective treatment. Since melatonin is a potent antioxidant and anti-inflammatory molecule with anti-viral action, in the present study we used melatonin to protect against H1N1 infection under in vitro and in vivo conditions. The death rate of the H1N1-infected mice was negatively associated with the nose and lung tissue local melatonin levels but not with serum melatonin concentrations. The H1N1-infected AANAT-/- melatonin-deficient mice had a significantly higher death rate than that of the WT mice and melatonin administration significantly reduced the death rate. All evidence confirmed the protective effects of melatonin against H1N1 infection. Further study identified that the mast cells were the primary targets of melatonin action, i.e., melatonin suppresses the mast cell activation caused by H1N1 infection. The molecular mechanisms involved melatonin down-regulation of gene expression for the HIF-1 pathway and inhibition of proinflammatory cytokine release from mast cells; this resulted in a reduction in the migration and activation of the macrophages and neutrophils in the lung tissue. This pathway was mediated by melatonin receptor 2 (MT2) since the MT2 specific antagonist 4P-PDOT significantly blocked the effects of melatonin on mast cell activation. Via targeting mast cells, melatonin suppressed apoptosis of alveolar epithelial cells and the lung injury caused by H1N1 infection. The findings provide a novel mechanism to protect against the H1N1-induced pulmonary injury, which may better facilitate the progress of new strategies to fight H1N1 infection or other IAV viral infections.

Specificity of DNA Vaccines against the Genogroup J and U Infectious Hematopoietic Necrosis Virus Strains Prevalent in China.

Infectious hematopoietic necrosis virus (IHNV) is the most important pathogen threatening the aquaculture of salmonid fish in China. In addition to the common genogroup J IHNV, genogroup U has been newly discovered in China. However, there is no effective DNA vaccine to fight against this emerging genogroup U IHNV in China. In this study, DNA vaccines encoding the IHNV viral glycoprotein (G) gene of the GS2014 (genogroup J) and BjLL (genogroup U) strains isolated from northern China were successfully developed, which were identified by restriction analysis and IFA. The expression of the Mx-1 gene and G gene in the spleens and muscles of the injection site as well as the titers of the serum antibodies were measured to evaluate the vaccine efficacy by RT-qPCR and ELISA. We found that DNA vaccine immunization could activate Mx1 gene expression and upregulate G gene expression, and the mRNA levels of the Mx1 gene in the muscles were significantly higher than those in the spleens. Notably, DNA vaccine immunization might not promote the serum antibody in fish at the early stage of immunization. Furthermore, the efficacy of the constructed vaccines was tested in intra- and cross-genogroup challenges by a viral challenge in vivo. It seemed that the DNA vaccines were able to provide great immune protection against IHNV infection. In addition, the genogroup J IHNV-G DNA vaccine showed better immune efficacy than the genogroup U IHNV-G or divalent vaccine, which could provide cross-immune protection against the genogroup U IHNV challenge. Therefore, this is the first study to construct an IHNV DNA vaccine using the G gene from an emerging genogroup U IHNV strain in China. The results provide great insight into the advances of new prophylactic strategies to fight both the genogroup J and U IHNV in China.

The Impacts of Iron Overload and Ferroptosis on Intestinal Mucosal Homeostasis and Inflammation.

Intestinal homeostasis is maintained through the interplay of the intestinal mucosa, local and systemic immune factors, and the microbial content of the gut. Iron is a trace mineral in most organisms, including humans, which is essential for growth, systemic metabolism and immune response. Paradoxically, excessive iron intake and/or high iron status can be detrimental to iron metabolism in the intestine and lead to iron overload and ferroptosis-programmed cell death mediated by iron-dependent lipid peroxidation within cell membranes, which contributes to several intestinal diseases. In this review, we comprehensively review recent findings on the impacts of iron overload and ferroptosis on intestinal mucosal homeostasis and inflammation and then present the progress of iron overload and ferroptosis-targeting therapy in intestinal diseases. Understanding the involved mechanisms can provide a new understanding of intestinal disease pathogenesis and facilitate advanced preventive and therapeutic strategies for intestinal dysfunction and diseases.

Transcriptomic Profiling of Mouse Mast Cells upon Pathogenic Avian H5N1 and Pandemic H1N1 Influenza a Virus Infection.

Mast cells, widely residing in connective tissues and on mucosal surfaces, play significant roles in battling against influenza A viruses. To gain further insights into the host cellular responses of mouse mast cells with influenza A virus infection, such as the highly pathogenic avian influenza A virus H5N1 and the human pandemic influenza A H1N1, we employed high-throughput RNA sequencing to identify differentially expressed genes (DEGs) and related signaling pathways. Our data revealed that H1N1-infected mouse mast P815 cells presented more up- and down-regulated genes compared with H5N1-infected cells. Gene ontology analysis showed that the up-regulated genes in H1N1 infection were enriched for more degranulation-related cellular component terms and immune recognition-related molecular functions terms, while the up-regulated genes in H5N1 infection were enriched for more immune-response-related biological processes. Network enrichment of the KEGG pathway analysis showed that DEGs in H1N1 infection were specifically enriched for the FoxO and autophagy pathways. In contrast, DEGs in H5N1 infection were specifically enriched for the NF-κB and necroptosis pathways. Interestingly, we found that Nbeal2 could be preferentially activated in H5N1-infected P815 cells, where the level of Nbeal2 increased dramatically but decreased in HIN1-infected P815 cells. Nbeal2 knockdown facilitated inflammatory cytokine release in both H1N1- and H5N1-infected P815 cells and aggravated the apoptosis of pulmonary epithelial cells. In summary, our data described a transcriptomic profile and bioinformatic characterization of H1N-1 or H5N1-infected mast cells and, for the first time, established the crucial role of Nbeal2 during influenza A virus infection.

First isolation and pathogenicity analysis of a genogroup U strain of infectious hematopoietic necrosis virus from rainbow trout in China.

Infectious hematopoietic necrosis virus (IHNV) is a major fish viral pathogen causing acute clinical disease and death in a variety of salmonids. IHNV isolates have been classified into five major genogroups according to the phylogenetic analysis of partial G gene fragments or the complete G gene sequence: U, M, E, L and J. Genogroup U strains have been reported in North America and Japan prior to 1982, and genogroup J is the only genogroup that has been reported in China. Here, one of IHNV strain (BjLL) was isolated from a local farm in China and were characterized in this study. The homogenate tissues of infected fry induced IHNV-positive cytopathic effects in epithelioma papulosum cyprinid (EPC) cells that were confirmed by RT-PCR and sequencing. The complete genome sequence of BjLL comprised 11,129 nucleotides, which had been submitted to GenBank (accession no. MF509592). By the sequence comparison and phylogenetic analysis for the G gene sequence of BjLL with 51 reference sequences in GenBank, we confirmed that this Chinese isolate belonged to genogroup U. Furthermore, virus exposure experiments with juvenile rainbow trout were conducted to assess the virulence and pathogenicity of BjLL. Compared with GS-2014 of genogroup J, BjLL was an obviously less virulent strain that could result in lower mortality. Besides, typical clinical symptoms and pathological damages could be seen in fish following infection of BjLL. The present study is the first report of genogroup U IHNV infection in China and will provide essential information for future studies on pathogenesis of IHNV BjLL and development of efficient control strategies.

A Novel Lateral Flow Assay for Rapid and Sensitive Nucleic Acid Detection of Avibacterium paragallinarum.

Avibacterium paragallinarum, the pathogen of infectious coryza, caused a highly contagious respiratory disease that poses a serious threat to chickens. Hence, it is necessary to do diagnostic screening for Av. paragallinarum. Existing technologies have been used for Av. paragallinarum testing, which, however, have some drawbacks such as time consuming and expensive that require well-trained personnel and sophisticated infrastructure, especially when they are limitedly feasible in some places for lack of resources. Nucleic acid hybridization-based lateral flow assay (LFA) is capable of dealing with these drawbacks, which is attributed to the advantages, such low cost, rapid, and simple. However, nucleic acid determination of Av. paragallinarum through LFA method has not been reported so far. In this study, we developed a novel LFA method that employed gold nanoparticle probes to detect amplified Av. paragallinarum dsDNA. Compared with agarose gel electrophoresis, this LFA strip was inexpensive, simple- to- use, and time- saving, which displayed the visual results within 5-8 min. This LFA strip had higher sensitivity that achieved the detection limit of 101 CFU/ml compared with 102 CFU/ml in agarose gel electrophoresis. Besides, great sensitivity was also shown in the LFA strip, and no cross reaction existed for other bacteria. Furthermore, Av. paragallinarum in clinical chickens with infectious coryza were perfectly detected by our established LFA strip. Our study is the first to develop the LFA integrated with amplification and sample preparation techniques for better nucleic acid detection of Av. paragallinarum, which holds great potential for rapid, accurate, and on-site determination methods for early diagnosis of Av. paragallinarum to control further spreading.

The Transcriptomic and Bioinformatic Characterizations of Iron Acquisition and Heme Utilization in Avibacterium paragallinarum in Response to Iron-Starvation.

Avibacterium paragallinarum is the pathogen of infectious coryza, which is a highly contagious respiratory disease of chickens that brings a potentially serious threat to poultry husbandry. Iron is an important nutrient for bacteria and can be obtained from surroundings such as siderophores and hemophores. To date, the mechanisms of iron acquisition and heme utilization as well as detailed regulation in A. paragallinarum have been poorly understood. In this study, we investigated the transcriptomic profiles in detail and the changes of transcriptomes induced by iron restriction in A. paragallinarum using RNA-seq. Compared with the iron-sufficiency control group, many more differentially expressed genes (DEGs) and cellular functions as well as signaling pathways were verified in the iron-restriction group. Among these DEGs, the majority of genes showed decreased expression and some were found to be uniquely present in the iron-restriction group. With an in-depth study of bioinformatic analyses, we demonstrated the crucial roles of the Hut protein and DUF domain-containing proteins, which were preferentially activated in bacteria following iron restriction and contributed to the iron acquisition and heme utilization. Consequently, RT-qPCR results further verified the iron-related DEGs and were consistent with the RNA-seq data. In addition, several novel sRNAs were present in A. paragallinarum and had potential regulatory roles in iron homeostasis, especially in the regulation of Fic protein to ensure stable expression. This is the first report of the molecular mechanism of iron acquisition and heme utilization in A. paragallinarum from the perspective of transcriptomic profiles. The study will contribute to a better understanding of the transcriptomic response of A. paragallinarum to iron starvation and also provide novel insight into the development of new antigens for potential vaccines against infectious coryza by focusing on these iron-related genes.

Safety, Immunogenicity, and Effectiveness of Defective Viral Particles Arising in Mast Cells Against Influenza in Mice.

Mast cells play pivotal roles in the pathogenesis of influenza A virus (IAV) infections. Defective viral particles (DPs) often arise during IAV replication, which can interfere with the replication of infectious viruses and stimulate the antiviral response of host cells. Therefore, DPs are expected to have immune-protective functions in clinic. However, the potent immunogenicity and effectiveness of DPs arising in mast cells during IAV replication have not been reported. In the present study, we showed that DPs generated in the human mastocytoma cell line HMC-1 following H1N1 infection were safe to mice after vaccination. Compared with lung adenocarcinoma cells, A549, DPs generated in infected mast cells had much better immunostimulatory activity, enhancing both humoral and cellular immunity of hosts. Notably, they could significantly increase the expression of immune-associated cytokines, especially the IFN-γ. Due to the robust immunogenicity, thus DPs generated in infected mast cells could stimulate the robust protective immune reaction effectively to fight against lethal IAV re-challenge after vaccination, which result in the high survival, decreased lung injury as well as inhibition of viral replication and inflammatory response in lungs. This study is the first to illustrate and explore the safety, immunogenicity, and effectiveness of DPs arising in mast cells against influenza as favorable potential vaccination. The results provide insight into the advances of new prophylactic strategies to fight influenza by focusing on DPs generated in mast cells.

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.

Isolation and pathogenicity testing of avian reticuloendotheliosis virus from layer chickens in China.

Reticuloendotheliosis virus (REV) can cause runting, immunosuppression, acute reticulum cell neoplasia, and chronic lymphoid tumors in a variety of domestic and wild birds. We diagnosed a case of reticuloendotheliosis with obvious tumors in liver and kidney. We isolated and sequenced the virus and performed pathogenicity testing of the REV strain. Immunohistochemistry and PCR confirmed that the diseased layer chickens were infected with REV. The strain, named BJ1503, was successfully isolated from the case by inoculation of tissue homogenates onto chicken embryo fibroblasts. The length of the proviral REV genome is 8,293 nucleotides. The isolate had 99.7% identity with REV-HA9901 (AY842951.1), which was isolated from Jiangsu, China, in 1999. The chickens infected with REV-BJ1503 had depressed weight gain and lymphoid atrophy. Our findings suggest that REV isolate BJ1503 was phylogenetically close to the earlier strain found in China, with minor variations, and the virus was associated with severe production problems.

Pre-Treatment with Zirconia Nanoparticles Reduces Inflammation Induced by the Pathogenic H5N1 Influenza Virus.

BACKGROUND: New approaches are urgently needed to fight influenza viral infection. Previous research has shown that zirconia nanoparticles can be used as anticancer materials, but their antiviral activity has not been reported. Here, we investigated the antiviral effect of zirconia (ZrO2) nanoparticles (NPs) against a highly pathogenic avian influenza virus. MATERIALS AND METHODS: In this study, the antiviral effects of ZrO2 on H5N1 virus were assessed in vivo, and the molecular mechanism responsible for this protection was investigated. RESULTS: Mice treated with 200 nm positively-charged NPs at a dose of 100 mg/kg showed higher survival rates and smaller reductions in weight. 200 nm ZrO2 activated mature dendritic cells and initially promoted the expression of cytokines associated with the antiviral response and innate immunity. In the lungs of H5N1-infected mice, ZrO2 treatment led to less pathological lung injury, significant reduction in influenza A virus replication, and overexpression of pro-inflammatory cytokines. CONCLUSION: This antiviral study using zirconia NPs shows protection of mice against highly pathogenic avian influenza virus and suggests strong application potential for this method, introducing a new tool against a wide range of microbial infections.

p-STAT1 regulates the influenza A virus replication and inflammatory response in vitro and vivo.

Influenza A virus infection activates various intracellular signaling pathways, which is mediated by the transcription factors. Here, a quantitative phosphoproteomic analysis of A549 cells after infection with influenza A virus (H5N1) was performed and we found that the transcription factor STAT1 was highly activated. Unexpectedly, upon inhibition of p-STAT1, titers of progeny virus and viral protein synthesis were both reduced. The STAT1 inhibitor Fludarabine (FLUD) inhibited an early progeny step in viral infection and reduced the levels of influenza virus genomic RNA (vRNA). Concomitantly, there was reduced expression of inflammatory cytokines in p-STAT1 inhibited cells. In vivo, suppression of p-STAT1 improved the survival of H5N1 virus-infected mice, reduced the pulmonary inflammatory response and viral burden. Thus, our data demonstrated a critical role for p-STAT1 in influenza virus replication and inflammatory responses. We speculate that STAT1 is an example of a putative antiviral signaling component to support effective replication.

Chronic heat stress negatively affects the immune functions of both spleens and intestinal mucosal system in pigs through the inhibition of apoptosis.

With the globe warming, chronic heat stress (CHS) has been considered to be a common hazard that could negatively affect pig's growth and reproduction performance. However, the effects of CHS on the immune functions of pigs were seldom reported, especially the cellular immune functions of intestinal mucosal system. In order to resolve this problem, a pig CHS model was built firstly and the effects of CHS on numbers of T cells in spleen and small intestines were observed. Exposure to a temperature of 39 °C, 4 h/d for 10d, the expression of heat stress protein 70 (HSP70) was increased dramatically. Under CHS condition, the numbers of CD3+ T cells were increased dramatically in both spleens and small intestines. Besides, the numbers of CD4+T cells and the value of CD4+/CD8+T cells in spleens were also significantly increased. The results highly revealed that CHS made the equilibrium state of immune function destroyed. Furthermore, CHS mainly promoted the expression of anti-apoptosis factor B cell lymphoma-2 (Bcl-2) and thus inhibited the apoptosis of lymphocytes in spleens and intestinal mucosa. This study demonstrates for the first time that CHS negatively affects the immune functions of both spleens and intestinal mucosal system in pigs through the inhibition of apoptosis. Our study can richer the data for study of mechanism of CHS and provide new knowledge for reference of making new strategy to control the disease induced by CHS.

Genomic and Bioinformatic Characterization of Mouse Mast Cells (P815) Upon Different Influenza A Virus (H1N1, H5N1, and H7N2) Infections.

Influenza A virus (IAV) is a segmented negative-stranded RNA virus that brings a potentially serious threat to public health and animal husbandry. Mast cells play an important role in both the inherent and adaptive immune response. Previous studies have indicated that mast cells support the productive replication of H1N1, H5N1, and H7N2. To date, the distinct molecular mechanism behind the pathogenesis in mast cells among the three different viruses has been poorly understood. In this study, we investigated the genomic profiles in detail and the dynamic change of genomes regulated by different subtypes of IAV in mouse mast cells using microassays. Compared with any two of the three IAV-infected groups, many more differentially expressed genes (DEGs), cellular functions, and signaling pathways were confirmed in H1N1 or H7N2 group, with the H7N2 group showing the highest levels. However, few DEGs were detected and various cellular functions and signaling pathways were dramatically suppressed in the H5N1 group. With an in-depth study on the H1N1 and H7N2 groups, we demonstrated the essential role of the 5-HT signaling pathway and the cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathway, which were preferentially activated in P815 cells infected by H1N1, and the crucial role of the HIF-1 signaling pathway that was preferentially activated in P815 cells infected by the H7N2 virus. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) results showed significantly increased mRNA levels of 5-HT and PKG in H1N1-infected P815 cells and increased HIF-1 in H7N2-infected P815 cells. In addition, exosomes were preferentially secreted from H1N1-infected or H7N2-infected P815 cells and are potentially pivotal in innate immunity to fight IAV infection. This study provides novel information and insight into the distinct molecular mechanism of H1N1, H5N1, and H7N2 viruses in mast cells from the perspective of genomic profiles.

iTRAQ-based proteomic and bioinformatic characterization of human mast cells upon infection by the influenza A virus strains H1N1 and H5N1.

Mast cells can support the replication of influenza A virus, although how this occurs is poorly understood. In the present study, using quantitative MS, we analyzed the proteome of human mast cells infected with different influenza A virus strains at 12 h post-infection. Forty-one differentially expressed proteins were identified in human mast cells upon infection by the virulent H5N1 (A/Chicken/Henan/1/04) virus compared to the seasonal H1N1 (A/WSN/33) virus. Bioinformatic analyses confirmed that H1N1 significantly regulates the RNA degradation pathway via up-regulation of CCR4-NOT transcription complex subunit 4, whereas apoptosis could be suppressed by H5N1 via down-regulation of the tumor protein p53 signaling pathway with P ≤ 0.05 at 12 h post-infection. The hypoxia-inducible factor-1 signaling pathway of human mast cells is more susceptible to infection by H5N1 than by H1N1 virus.

H5N1 Influenza a Virus Replicates Productively in Pancreatic Cells and Induces Apoptosis and Pro-Inflammatory Cytokine Response.

The inflammatory response and apoptosis have been proved to have a crucial role in the pathogenesis of the influenza A virus (IAV). Previous studies indicated that while IAV commonly causes pancreatitis and pancreatic damage in naturally and experimentally infected animals, the molecular mechanisms of the pathogenesis of IAV infection are less reported. In the present study, we showed for the first time that both avian-like (α-2,3-linked) and human-like (α-2,6-linked) sialic acid (SA) receptors were expressed by the mouse pancreatic cancer cell line PAN02 and the human pancreatic cancer cell line PANC-1. Using growth kinetics experiments, we also showed that PAN02 and PANC-1 cells supported the productive replication of the H5N1 highly pathogenic avian influenza while exhibited the limited replication of IAV subtypes H1N1 and H7N2 in vitro. The in vivo infection of H5N1 in pancreatic cells was confirmed by the histopathological and immunohistochemical staining of pancreas tissue from mice. Other than H1N1 and H7N2, severe damage and extensive positive signals were observed in pancreas of H5N1 infected mice. All three virus subtypes induced apoptosis but also triggered the infected PAN02 and PANC-1 cells to release pro-inflammatory cytokines and chemokines including interferon (IFN)-α, IFN-ß, IFN-γ, chemokine (C-C motif) ligand 2 (CCL2), tumor necrosis factor (TNF)-α, and interleukin (IL)-6. Notably, the subtypes of H5N1 could significantly upregulate these cytokines and chemokines in both two cells when compared with H1N1 and H7N2. The present data provide further understanding of the pathogenesis of H5N1 IAV in pancreatic cells derived from humans and mammals and may also benefit the development of new treatment against H5N1 influenza virus infection.

Lethal influenza A virus preferentially activates TLR3 and triggers a severe inflammatory response.

The "cytokine storm" and excessive inflammation triggered by lethal avian influenza virus (IAV) are responsible for its high virulence and mortality. However, the molecular mechanism behind these effects is unclear. In this study, we used LA795 cells and a mouse model to assess the crucial role of TLR3 during infection with lethal avian influenza A virus and subsequent inflammation. The results showed that IAVs could replicate and proliferate well in LA795 cells and that the replication of H5N1 was more efficient than human H1N1 and lowly pathogenic avian H7N2 viruses. The TLR3 signaling pathways were activated preferentially in vitro and in vivo and a range of pro-inflammatory cytokines were released following H5N1 infection. RNAi and TLR3 knockout mice were used to validate the results. These results are the first to provide insight into the preferential involvement of TLR3 in lethal avian influenza A virus infection and inflammation compared with others such as human or lowly pathogenic avian influenza A viruses. The data will increase understanding of the pathogenesis of lethal avian influenza A virus infection and may help facilitate the development of novel therapeutic aids targeting TLR3 signaling pathways.

Antiviral effects of Shuanghuanglian injection powder against influenza A virus H5N1 in vitro and in vivo.

The current study was to identify a protective role of Shuanghuanglian (SHL) injection powder in vitro and in vivo after H5N1 viral infection. Immunofluorescent staining was used to determine the susceptibility of rat intestinal mucosa microvascular endothelial cells (RIM-MVECs) to the H5N1 virus. Viral replication of RIM-MVECs was measured by transmission electron microscopy (TEM) a hemagglutination assay and real-time quantitative PCR. H5N1 virally infected RIM-MVECs, and BALB/c mice were treated with SHL to investigate its therapeutic effect. Animal survival and the weight of H5N1 virally infected BALB/c mice after SHL treatment was noted, and histology and real-time PCR applied to mouse lungs were used to confirm the anti-H5N1 viral effects of SHL. RIM-MVECs supported replication of the H5N1 virus in vitro. SHL treatment reduced viral titers in H5N1 virally infected RIM-MVECs and mouse lungs. SHL -treated mice survived compared to controls. Mild pathological changes, reduced inflammatory cell infiltration and fewer viral antigens were observed in the lungs of SHL-treated mice at days 3 and 6 post-infection. In conclusion, SHL may have the antiviral activity against the H5N1 virus infection by inhibiting viral replication and alleviating lung injury.

Pathogenicity differences between QX-like and Mass-type infectious bronchitis viruses.

Infectious bronchitis is a highly contagious, acute viral respiratory disease of chickens, caused by infectious bronchitis virus (IBV). In recent years, the isolation rate of QX-like IBV has increased in the world. To clarify this phenomenon and better understand the pathogenicity of QX-like IBV, we examined differences in pathogenicity between two IBV strains, SD and M41, which belong to QX-like and Mass-type IBV, respectively. SD strain was more virulent in 3-week-old specific-pathogen-free chickens than M41 strain causing higher mortality with severe renal lesions. The tissue distribution of the two virus strains was tested by real-time RT-PCR. The results showed that the viral genome copy numbers in the tissues of chickens inoculated with SD strain were higher than those in chickens inoculated with M41 strain, with the exception of the trachea and lung. This study indicates that there are tremendous differences in pathogenicity and tissue tropism between the QX-like strain and Mass-type strain. These findings may benefit the prevention of infectious bronchitis in the poultry industry in China.