Chicken UFL1 Restricts Avian Influenza Virus Replication by Disrupting the Viral Polymerase Complex and Facilitating Type I IFN Production.

During avian influenza virus (AIV) infection, host defensive proteins promote antiviral innate immunity or antagonize viral components to limit viral replication. UFM1-specific ligase 1 (UFL1) is involved in regulating innate immunity and DNA virus replication in mammals, but the molecular mechanism by which chicken (ch)UFL1 regulates AIV replication is unclear. In this study, we first identified chUFL1 as a negative regulator of AIV replication by enhancing innate immunity and disrupting the assembly of the viral polymerase complex. Mechanistically, chUFL1 interacted with chicken stimulator of IFN genes (chSTING) and contributed to chSTING dimerization and the formation of the STING-TBK1-IRF7 complex. We further demonstrated that chUFL1 promoted K63-linked polyubiquitination of chSTING at K308 to facilitate chSTING-mediated type I IFN production independent of UFMylation. Additionally, chUFL1 expression was upregulated in response to AIV infection. Importantly, chUFL1 also interacted with the AIV PA protein to inhibit viral polymerase activity. Furthermore, chUFL1 impeded the nuclear import of the AIV PA protein and the assembly of the viral polymerase complex to suppress AIV replication. Collectively, these findings demonstrate that chUFL1 restricts AIV replication by disrupting the viral polymerase complex and facilitating type I IFN production, which provides new insights into the regulation of AIV replication in chickens.

RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-ß expression during virus infection, the expression level of IFN-ß in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

Duck MARCH8 Negatively Regulates the RLR Signaling Pathway through K29-Linked Polyubiquitination of MAVS.

Mitochondrial antiviral signaling protein (MAVS) is a key adaptor in cellular innate immunity. Ubiquitination plays an important role in regulating MAVS-mediated innate immune responses; however, the molecular mechanisms underlying ubiquitination of MAVS have not been fully elucidated. In this study, we first identified the mitochondria-resident E3 ligase duck membrane-associated RING-CH 8 (duMARCH8) in ducks as a negative regulator of duck MAVS (duMAVS). Overexpression of duMARCH8 impaired the duMAVS-mediated signaling pathway, whereas knockdown of duMARCH8 resulted in the opposite effects. The suppression was due to duMARCH8 interacting with duMAVS and degrading it in a proteasome-dependent manner. We further found that duMARCH8 interacted with the 176-619 regions of duMAVS. Moreover, duMARCH8 catalyzed the K29-linked polyubiquitination of duMAVS at Lys 398 to inhibit the MAVS-mediated signaling pathway. Collectively, our findings reveal a new strategy involving MARCH8 that targets the retinoic acid-inducible gene-I-like receptor signaling pathway to regulate innate immune responses in ducks.

Interactive Multi-Stage Robotic Positioner for Intra-Operative MRI-Guided Stereotactic Neurosurgery.

Magnetic resonance imaging (MRI) demonstrates clear advantages over other imaging modalities in neurosurgery with its ability to delineate critical neurovascular structures and cancerous tissue in high-resolution 3D anatomical roadmaps. However, its application has been limited to interventions performed based on static pre/post-operative imaging, where errors accrue from stereotactic frame setup, image registration, and brain shift. To leverage the powerful intra-operative functions of MRI, e.g., instrument tracking, monitoring of physiological changes and tissue temperature in MRI-guided bilateral stereotactic neurosurgery, a multi-stage robotic positioner is proposed. The system positions cannula/needle instruments using a lightweight (203 g) and compact (Ø97 × 81 mm) skull-mounted structure that fits within most standard imaging head coils. With optimized design in soft robotics, the system operates in two stages: i) manual coarse adjustment performed interactively by the surgeon (workspace of ±30°), ii) automatic fine adjustment with precise (<0.2° orientation error), responsive (1.4 Hz bandwidth), and high-resolution (0.058°) soft robotic positioning. Orientation locking provides sufficient transmission stiffness (4.07 N/mm) for instrument advancement. The system's clinical workflow and accuracy is validated with lab-based (<0.8 mm) and MRI-based testing on skull phantoms (<1.7 mm) and a cadaver subject (<2.2 mm). Custom-made wireless omni-directional tracking markers facilitated robot registration under MRI.

Genetic characteristics of waterfowl-origin H5N6 highly pathogenic avian influenza viruses and their pathogenesis in ducks and chickens.

Waterfowl, such as ducks, are natural hosts for avian influenza viruses (AIVs) and act as a bridge for transmitting the virus to humans or susceptible chickens. Since 2013, chickens and ducks have been threatened by waterfowl-origin H5N6 subtype AIVs in China. Therefore, it is necessary to investigate the genetic evolution, transmission, and pathogenicity of these viruses. In this study, we determined the genetic characteristics, transmission, and pathogenicity of waterfowl-origin H5N6 viruses in southern China. The hemagglutinin (HA) genes of H5N6 viruses were classified into the MIX-like branch of clade 2.3.4.4h. The neuraminidase (NA) genes belonged to the Eurasian lineage. The PB1 genes were classified into MIX-like and VN 2014-like branches. The remaining five genes were clustered into the MIX-like branch. Therefore, these viruses belonged to different genotypes. The cleavage site of the HA proteins of these viruses was RERRRKR/G, a molecular characteristic of the H5 highly pathogenic AIV. The NA stalk of all H5N6 viruses contained 11 amino acid deletions at residues 58-68. All viruses contained 627E and 701D in the PB2 proteins, which were molecular characteristics of typical bird AIVs. Furthermore, this study showed that Q135 and S23 viruses could replicate systematically in chickens and ducks. They did not cause death in ducks but induced mild clinical signs in them. All the infected chickens showed severe clinical signs and died. These viruses were shed from the digestive and respiratory tracts and transmitted horizontally in chickens and ducks. Our results provide valuable information for preventing H5N6 avian influenza outbreaks.

Genetic characterization and pathogenicity of H7N9 highly pathogenic avian influenza viruses isolated from South China in 2017.

Since 2017, the new H7N9 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for more than 200,000 cases of chicken infection and more than 120,000 chicken deaths in China. Our previous study found that the Q26 was chicken-origin H7N9 HPAIV. In this study, we analyzed the genetic characterization of Q24, Q65, Q66, Q85, and Q102 H7N9 avian influenza viruses isolated from Guangdong, China in 2017. Our results showed that these viruses were highly pathogenic and belonged to two different genotypes, which suggested they occurred genetic reassortant. To investigate the pathogenicity, transmission, and host immune responses of H7N9 virus in chickens, we selected Q24 and Q26 viruses to inoculate chickens. The Q24 and Q26 viruses killed all inoculated chickens within 3 days and replicated effectively in all tested tissues. They were efficiently transmitted to contact chickens and killed them within 4 days through direct contact. Furthermore, we found that the expressions of several immune-related genes (e.g., TLR3, TLR7, MDA5, MAVS, IFN-ß, IL-6, IL-8, OAS, Mx1, MHC I, and MHC II) were upregulated obviously in the lungs and spleen of chickens inoculated with the two H7N9 viruses at 24 h post-inoculation (HPI). Among these, IL-6 and IFN-ß in lungs were the most upregulated (by 341.02-381.48-fold and 472.50-500.56-fold, respectively). These results suggest that the new H7N9 viruses isolated in 2017, can replicate and transmit effectively and trigger strong immune responses in chickens, which helps us understand the genetic and pathogenic variations of H7N9 HPAIVs in China.

Duck TRIM29 negatively regulates type I IFN production by targeting MAVS.

The innate immune response is a host defense mechanism that induces type I interferon and proinflammatory cytokines. Tripartite motif (TRIM) family proteins have recently emerged as pivotal regulators of type I interferon production in mammals. Here, we first identified duck TRIM29, which encodes 571 amino acids and shows high sequence homology with other bird TRIM29 proteins. DuTRIM29 inhibited IFN-ß and IRF7 promoter activation in a dose-dependent manner and downregulated the mRNA expression of IFN-ß, IRF7, Mx and IL-6 mediated by duRIG-I. Moreover, duTRIM29 interacted and colocalized with duMAVS in the cytoplasm. DuTRIM29 interacted with duMAVS via its C-terminal domains. In addition, duTRIM29 inhibited IFN-ß and IRF7 promoter activation and significantly downregulated IFN-ß and immune-related gene expression mediated by duMAVS in ducks. Furthermore, duTRIM29 induced K29-linked polyubiquitination and degradation of duMAVS to suppress the expression of IFN-ß. Overall, our results demonstrate that duTRIM29 negatively regulates type I IFN production by targeting duMAVS in ducks. This study will contribute to a better understanding of the molecular mechanism regulating the innate immune response by TRIM proteins in ducks.

Soft robotic manipulator for intraoperative MRI-guided transoral laser microsurgery.

Magnetic resonance (MR) imaging (MRI) provides compelling features for the guidance of interventional procedures, including high-contrast soft tissue imaging, detailed visualization of physiological changes, and thermometry. Laser-based tumor ablation stands to benefit greatly from MRI guidance because 3D resection margins alongside thermal distributions can be evaluated in real time to protect critical structures while ensuring adequate resection margins. However, few studies have investigated the use of projection-based lasers like those for transoral laser microsurgery, potentially because dexterous laser steering is required at the ablation site, raising substantial challenges in the confined MRI bore and its strong magnetic field. Here, we propose an MR-safe soft robotic system for MRI-guided transoral laser microsurgery. Owing to its miniature size (Ø12 × 100 mm), inherent compliance, and five degrees of freedom, the soft robot ensures zero electromagnetic interference with MRI and enables safe and dexterous operation within the confined oral and pharyngeal cavities. The laser manipulator is rapidly fabricated with hybrid soft and hard structures and is powered by microvolume (<0.004 milliter) fluid flow to enable laser steering with enhanced stiffness and lowered hysteresis. A learning-based controller accommodates the inherent nonlinear robot actuation, which was validated with laser path-following tests. Submillimeter laser steering accuracy was demonstrated with a mean error < 0.20 mm. MRI compatibility testing demonstrated zero observable image artifacts during robot operation. Ex vivo tissue ablation and a cadaveric head-and-neck trial were carried out under MRI, where we employed MR thermometry to monitor the tissue ablation margin and thermal diffusion intraoperatively.

Duck PIAS2 negatively regulates RIG-I mediated IFN-ß production by interacting with IRF7.

The protein inhibitor of activated STAT (PIAS) proteins are important signal transduction modulator family and regulate the innate immune signaling pathway induced by certain transcription factors, including NF-κB, IRF3, and JAK/STAT. The PIAS protein mechanism that regulates innate immune response in mammals has been well described in the literature; however, whether the PIAS gene exists in ducks as well as the role of PIAS in duck IFN-ß expression is still unclear. Here, we cloned duck PIAS (duPIAS), finding PIAS2 could repress IFN-ß production. DuPIAS2 contains SAP-PINIT-RLD-S/T characteristic domains, and its overexpression could inhibit virus-induced IFN-ß promoter activation. Moreover, duPIAS2 interacts with duck interferon regulatory factor 7 (IRF7) and inhibits IFN-ß promoter activation induced by duck IRF7. Additionally, its inhibitory function does not rely on its SUMO E3 ligase activity but rather its C-terminal portion. The above results demonstrate that duPIAS2 is a repressor of IFN-ß production induced by duck IRF7.

Duck TRIM32 Functions in IFN-ß Signaling Against the Infection of H5N6 Highly Pathogenic Avian Influenza Virus.

In mammals, tripartite motif 32 (TRIM32) is essential for regulating host innate immune responses to viral infections. However, the antiviral effect of TRIM32 in birds has not been reported. Here, we cloned the full-length duck TRIM32 (duTRIM32) cDNA from total spleen RNA of Peking duck. DuTRIM32 consists of 682 amino acids and has 95.5% similarity in amino acid sequences with chicken TRIM32 and 84.9% similarity with human TRIM32, respectively. DuTRIM32 mRNA was found to be ubiquitously expressed in all tested tissues from healthy ducks. Overexpression of duTRIM32 significantly activated the IFN-ß promoter and upregulated the mRNA levels of IFN-ß, IRF7, and Mx, which indicates that duTRIM32 is involved in the type I IFN pathway. Furthermore, duTRIM32 was found to directly interact with duck STING (duSTING) and to contribute to the expression of IFN-ß mediated by duSTING. The mRNA level of duTRIM32 was significantly upregulated in the lungs and spleens of H5N6 highly pathogenic avian influenza virus (HPAIV) infected ducks 3 days post-infection (DPI). Furthermore, overexpression of duTRIM32 could inhibit the replication of H5N6 HPAIV in duck embryo fibroblasts (DEFs). Therefore, these results indicate that duTRIM32 is involved in the type I IFN pathway and exhibit an antiviral effect against H5N6 HPAIV infection.

Pathogenicity of different H5N6 highly pathogenic avian influenza virus strains and host immune responses in chickens.

The H5N6 highly pathogenic avian influenza virus (HPAIV) has been circulating in China since 2013. In this report, we describe our recent chicken experimental studies investigating the pathogenicity and transmission of four H5N6 HPAIV field strains of different origins (GS39, CK44, DK47 and CK74) and the host immune responses. Four-week-old specific-pathogen-free chickens were inoculated intranasally with one of the four H5N6 HPAIV strains (one strain per group). Among the contact chickens, the GS39 and CK74 strains caused 100 % mortality, the CK44 strain caused 80 % mortality, and the DK47 strain caused 40 % mortality. The viruses were effectively replicated in multiple tissues of the inoculated chickens, in which high viral titers were detected in virus-infected tissues, and significantly upregulated expression of immune-related genes was found in the infected chickens at 24 hpi. The chicken serum antibody levels increased from 5log2 at 7 dpe to 7.67-8log2 at 14 dpe. The major histocompatibility complex molecules were upregulated 21.22- to 32.98-fold in lungs and 5.10- to 18.47-fold in spleens. In summary, H5N6 viruses can replicate within chickens and be effectively transmitted between chickens. Our study contributes to further understanding the pathogenesis of clade 2.3.4.4 H5N6 avian influenza viruses in chickens.

Duck PIAS2 Promotes H5N1 Avian Influenza Virus Replication Through Its SUMO E3 Ligase Activity.

The protein inhibitor of the activated STAT2 (PIAS2) has been implicated in many cellular processes and can also regulate viral replication in mammals. However, the role of PIAS2 in the highly pathogenic avian influenza virus (HPAIV) H5N1 replication in ducks is still unclear. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, we identified that duck PIAS2 (duPIAS2) was one protein that interacted with the nucleoprotein (NP) from the H5N1 HPAIV strain of DK212. Through confocal microscopy images and Co-IP assay, we confirmed NP could interact with duPIAS2. Overexpression of duPIAS2 in primary duck embryo fibroblast (DEF) cells was shown to promote DK212 replication, and knockdown of duPIAS2 could repress DK212 replication. We further found duPIAS2 could promote NP SUMOylation through duck SUMO1 (duSUMO1), and the potential SUMOylation sites of NP were at lysines 7, 48, and 87. Furthermore, duPIAS2 promoted the replication of DK212, here relying on the activity of its SUMO E3 ligase. Duck SENP1 (duSENP1), a deSUMOylation enzyme, could repress NP SUMOylation and also inhibit DK212 replication. Together, we identified duPIAS2 could interact with NP and that duPIAS2 promoted H5N1 HPAIV replication, which might be related to NP SUMOylation.

Comparative Pathogenicity and Transmissibility of the H7N9 Highly Pathogenic Avian Influenza Virus and the H7N9 Low Pathogenic Avian Influenza Virus in Chickens.

There were five outbreaks of H7N9 influenza virus in humans in China since it emerged in 2013, infecting >1000 people. The H7N9 low pathogenic influenza virus was inserted into four amino acids in the HA protein cleavage site to mutate into the H7N9 highly pathogenic virus. This emerging virus caused 15 outbreaks in chickens from the end of 2016 to date. Two H7N9 avian influenza virus (AIV) strains, A/chicken/Guangdong/A46/2013 (LPAIV) and A/chicken/Guangdong/Q29/2017 (HPAIV), were selected to compare the pathogenicity and transmissibility between H7N9 LPAIVs and HPAIVs in chickens. We inoculated 3- to 4-week-old specific-pathogen-free (SPF) chickens with 6 log10EID50/0.1 mL viruses via the ocular-nasal route and co-housed four chickens in each group. The inoculated chicken mortality rate in the A46 and Q29 groups was 1/5 and 5/5, respectively. Q29 virus replication was more efficient compared to the A46 virus in inoculated chickens. Infected chickens initiated viral shedding to naïve contact chickens through respiratory and digestive routes. Both viruses transmitted between chickens by naïve contact, but the Q29 virus had a higher pathogenicity in contact chickens than the A46 virus. Compared with early H7N9 LPAIVs, the pathogenicity and transmissibility of the emerging H7N9 HPAIV was stronger in chickens, indicating that H7N9 influenza virus may continue to threaten human and poultry health.

Phylogeny, Pathogenicity, Transmission, and Host Immune Responses of Four H5N6 Avian Influenza Viruses in Chickens and Mice.

H5Nx viruses have continuously emerged in the world, causing poultry industry losses and posing a potential public health risk. Here, we studied the phylogeny, pathogenicity, transmission, and immune response of four H5N6 avian influenza viruses in chickens and mice, which were isolated from waterfowl between 2013 and 2014. Their HA genes belong to Clade 2.3.4.4, circulated in China since 2008. Their NA genes fall into N6-like/Eurasian sublineage. Their internal genes originated from different H5N1 viruses. The results suggested that the four H5N6 viruses were reassortants of the H5N1 and H6N6 viruses. They cause lethal infection with high transmission capability in chickens. They also cause mild to severe pathogenicity in mice and can spread to the brain through the blood-brain barrier. During the infection, the viruses result in the up-regulation of PRRs and cytokine in brains and lungs of chickens and mice. Our results suggested that the high viral loads of several organs may result in disease severity in chickens and mice; there were varying levels of cytokines induced by the H5N6 viruses with different pathogenicity in chickens and mice.

Genetic characteristics, pathogenicity and transmission of H5N6 highly pathogenic avian influenza viruses in Southern China.

Since 2014, H5 highly pathogenic avian influenza viruses (HPAIVs) from clade 2.3.4.4 have been persistently circulating in Southern China. This has caused huge losses in the poultry industry. In this study, we analysed the genetic characteristics of seven H5N6 HPAIVs of clade 2.3.4.4 that infected birds in Southern China in 2016. Phylogenetic analysis grouped the HA, PB2, PA, M and NS genes as MIX-like, and the NA genes grouped into the Eurasian lineage. The PB1 genes of the GS24, GS25, CK46 and GS74 strains belonged to the VN 2014-like group and the others were grouped as MIX-like. The NP genes of GS24 and GS25 strains belonged to the ZJ-like group, but the others were MIX-like. Thus, these viruses came from different genotypes, and the GS24, GS25, CK46 and GS74 strains displayed genotype recombination. Additionally, our results showed that the mean death time of all chickens inoculated with 105 EID50 of CK46 or GS74 viruses was 3 and 3.38 days, respectively. The viruses replicated at high titers in all tested tissues of the inoculated chickens. They also replicated in all tested tissues of naive contact chickens, but their replication titers in some tissues were significantly different (p < 0.05). Thus, the viruses displayed high pathogenicity and variable transmission in chickens. Therefore, it is necessary to focus on the pathogenic variation and molecular evolution of H5N6 HPAIVs in order to prevent and control avian influenza in China.

Different Pathogenicity and Transmissibility of Goose-Origin H5N6 Avian Influenza Viruses in Chickens.

Highly pathogenic avian influenza H5N6 viruses have been circulating in poultry in Asia since 2013 and producing serious diseases in chickens. Here, we analyzed the genetic properties of 10 H5N6 subtypes AIVs from geese in 2015-2016 in Guangdong province. Phylogenic analysis showed that all HA genes of the 10 viruses belonged to clade 2.3.4.4, and their genes including HA, PA, PB1, M, NP, and NS all derived from Mix-like 1 (CH, VN, LS). Their PB2 genes come from Mix-like 2 (CH, VN, JP). The NA genes were classified into a Eurasian lineage. Therefore, the 10 viruses likely originate from the same ancestor and were all recombinant viruses between different genotypes. We selected A/Goose/Guangdong/GS144/2015(H5N6) (GS144) and A/Goose/Guangdong/GS148/2016(H5N6) (GS148) viruses to inoculate 5-week-old chickens intranasally with 104 EID50/0.1 mL dose intranasally to assess their pathogenicity and transmissibility. Inoculated chickens showed that the GS144 virus caused systematic infection with a lethality of 100%, but the lethality of GS148 virus was 0%. The two viruses were efficiently transmitted to contact chickens. The lethality of GS144 and GS148 virus in contact with chickens was 87.5% and 0%, respectively, which suggests that the transmissibility of GS144 virus was stronger than GS148 virus in chickens. Thus, different H5N6 viruses from the same waterfowl can show different pathogenicity and transmissibility in chickens. Continued surveillance and characteristic analysis of the H5N6 viruses will help us to keep abreast of evolution and variation in avian influenza viruses in the future.

Immune-Related Gene Expression in Ducks Infected With Waterfowl-Origin H5N6 Highly Pathogenic Avian Influenza Viruses.

Clade 2.3.4.4 H5 avian influenza viruses (AIVs) are widely prevalent and of significant concern to the poultry industry and public health in China. Nowadays, the clade 2.3.4.4 H5N6 virus has become a dominant AIV subtype among domestic ducks in southern China. We found that waterfowl-origin clade 2.3.4.4 H5N6 viruses (A/goose/Guangdong/16568/2016, GS16568 and A/duck/Guangdong/16873/2016, DK16873) isolated from southern China in 2016 could replicate in multiple organs of inoculated ducks. DK16873 virus caused mild infections and killed 2/5 of inoculated ducks, and GS16568 virus did not kill inoculated ducks. In addition, the two viruses could be transmitted via direct contact between ducks. DK16873 and GS16568 viruses killed 2/5 and 1/5 of contact ducks, respectively. Furthermore, ducks inoculated with the two H5N6 viruses exhibited different expressions of immune-related genes in their lungs. The expression of RIG-I, TLR3 and IL6 was significantly upregulated at 12 h post-inoculation (HPI) and most of the tested immune-related genes were significantly upregulated at 3 days post-inoculation (DPI). Notably, the expression of RIG-I and IL-6 in response to DK16873 virus was significantly higher than for GS16568 virus at 12 HPI and 3 DPI. Our research have provided helpful information about the pathogenicity, transmission and immune-related genes expression in ducks infected with new H5N6 AIVs.