Dogs are considered mixing vessels for influenza viruses, posing a pandemic potential via viral reassortment. Our previous studies indicated that the avian-origin H3N2 canine influenza virus (A/canine/Zhejiang/1/2010, abbreviated C1) is virulent in canine and mice. Furthermore, we found that the HA and NA genes of C1 share a close genetic relationship with an H3N2 avian influenza virus (A/duck/Shanghai/06/2009, abbreviated D6), but they exhibit distinct pathogenicity. However, the understanding mechanisms remain unclear. In the present study, we explored the genetic determinants that contribute to the different pathogenicity between the C1 and D6. By using the reverse genetics approaches, we rescued several single-gene and position-substituted reassortant viruses based on the C1. The replication in Madin-Darby canine kidney cells and pathogenic trial in mice showed that the neuraminidase (NA) gene played a critical role in C1 virulence. Further analysis demonstrated that the K54E and S154P mutations in NA significantly reduced NA enzymatic activity, impairing viral release from infected cells. Consequently, these mutant viruses lost their ability to infect mice. Overall, our findings identify two novel virulence determinants in NA and elucidate the mechanisms behind the distinct pathogenicity between the C1 and D6 in mice. These results may provide some new targets for H3N2 influenza virus vaccines and antiviral drug development.

Chicken infectious anemia virus (CIAV) is a major immunosuppressive pathogen that causes huge economic losses in the poultry industry. The virus persists in the host for an extended period, making it difficult to eliminate and highlighting the need for rapid and accurate detection methods. In this study, we developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) kit for on-site detection of CIAV. Optimal primers and probes were designed and screened based on conserved regions of the full-gene sequences of 251 CIAV strains, ensuring high specificity, with no cross-reactions with 12 other common avian viruses. It demonstrated high sensitivity with a minimum detection limit of 10 copies of CIAV DNA, achieving efficient detection in just 15 min. The kit also showed good repeatability and stability, with 100 % concordance with qPCR in detecting 50 field samples. The developed CIAV RPA-LFD kit provides a practical tool for the rapid detection of CIAV, suitable for both on-site and laboratory use.

West Nile virus (WNV) is a zoonotic, mosquito-borne flavivirus with birds as its primary reservoir host. Infection of domestic ducks leads to disease, decreased egg production, and mortality, highlighting the need for an effective WNV vaccine for ducks. In this study, several live-attenuated chimeric vaccine candidates were engineered with WNV pre-Membrane (prM) and Envelope (E) genes in the backbone of the duck Tembusu virus (TMUV) vaccine strain FX2010-180P. Two mutations in E (D120N and S156P) were introduced to alter glycosylation and reduce virulence. In mice, the vaccine candidates 180P/WNV-prM-E, 180P/WNV-prM-ED120N and 180P/WNV-prM-ES156P exhibited high neurovirulence via intracranial inoculation, but were safe when administered intramuscularly with a dose of 104 TCID50. Neutralizing antibody levels after a single intramuscular dose were modest. In ducks, low viraemia was detected in all groups of intramuscularly inoculated ducks at 2 dpi but the virus was cleared at 3 dpi. Low virus titers were detected in the spleen of 2 out of 3 ducks in the 180P/WNV-prM-E group and 1out of 3 ducks in the 180P/WNV-prM-ES156P group, but not in the 180P/WNV-prM-ED120N group. All vaccinated ducks developed strong neutralizing antibody responses by day 14, with 180P/WNV-prM-E inducing the highest neutralization levels. This study supports the use of the TMUV vaccine strain as a platform for developing live-attenuated, chimeric flavivirus vaccines against WNV in ducks.

Since 2015, outbreaks of goose Astrovirus type 2 (GAstV-2) have caused significant economic losses in the goose industry by inducing severe visceral and articular gout in goslings, with mortality rates reaching up to 50 %. The capsid (Cap) protein of GAstV-2 is the primary structural protein that elicits immune responses, but it has not been extensively studied for neutralizing epitopes or infection mechanisms. In this study, two novel neutralizing monoclonal antibodies (mAbs) 4A7 and 8H3 were generated, which specifically target the Cap protein and effectively inhibit GAstV-2 infection in vitro. Epitope mapping using alanine-scanning mutagenesis revealed that the epitopes recognized by mAbs 4A7 and 8H3 are located within the amino acids sequences 443ESCSFLVF450 and 425QVTPSLVYNF434 of the Cap protein, respectively. These two epitopes are highly conserved among GAstV-2 strains, but show substantial divergence from GAstV-1 strains. Structural analysis indicated that these epitopes are situated on the surface of the Cap protein, within the P2 domain, which is critical for virus-receptor interactions and immune recognition. The findings not only identify novel neutralizing epitopes in GAstV-2 but also highlight the potential of mAbs 4A7 and 8H3 for developing epitope-based vaccines and diagnostic assays.

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in chickens and remains a major concern for poultry production. Although live-attenuated vaccines are widely used, residual virulence and latency raise safety concerns. The UL50 gene encodes a conserved deoxyuridine triphosphatase (dUTPase) and has been implicated in herpesvirus virulence, but its value as a vaccine attenuation target in ILTV remains uncertain. Here, we evaluated the effects of complete UL50 ORF deletion on the pathogenicity and immunogenicity of ILTV in chickens to assess its potential as a target for developing safer live-attenuated ILTV vaccines. Four-day-old chickens were inoculated via the eye-drop or intranasal route to evaluate pathogenicity and protective efficacy against challenge with the virulent SH2016 strain. The rGD2018-ΔUL50 exhibited further attenuation, inducing only minimal clinical signs and showing no detectable viral shedding or viral replication in the larynx and trachea. In contrast, the parental low-virulence GD2018 strain caused slightly more evident mild clinical signs. Despite its marked attenuation, the UL50-deleted strain conferred partial protection against challenge, whereas GD2018-inoculated chickens were fully protected. These results indicate that UL50 plays an important role in ILTV virulence, and that its complete deletion leads to over-attenuation, compromising protective efficacy. Optimization of UL50-based attenuation strategies, such as partial modification or combination with other genetic approaches, may facilitate the development of safer and effective next-generation ILTV live vaccines.

Duck hepatitis A virus type 3 (DHAV-3), an avian-specific pathogen primarily impacting ducklings, poses a significant threat to the duck farming industry by causing high mortality rates. A DHAV-3 strain SH52 was isolated from the diseased ducks and phylogenetic analysis of the whole genome revealed that the DHAV-3 SH52 belongs to the prevalent strains in China. The DHAV-3 SH52 strain replicates at high levels in various organs of 8-day-old specific pathogen-free (SPF) shelducks, causing pathological damage and leading to high lethality in 8-day-olds following intramuscular infection. The findings of this study provide an ideal animal challenge model for evaluating the efficacy of DHAV-3 vaccines in the future.

In 2014, Duck Adenovirus type 3 (DAdV-3) emerged in Muscovy ducks and has since spread rapidly across China, causing significant economic losses to the duck industry. Given this situation, the development of reliable diagnostic tools is crucial for effective disease control. In this study, a neutralizing monoclonal antibody (mAb) 2F12 specific to DAdV-3 was generated, which showed a blocking rate of over 70% and a neutralization titer of up to 1:794. A blocking enzyme-linked immunosorbent assay (b-ELISA) was further developed based on mAb 2F12 to efficiently detect neutralizing antibodies against DAdV-3. The cut-off values of percent inhibition (PI) were set based on testing 84 negative duck serum samples, with a value below 16.79% (mean (X¯) + 2 standard deviations (SD)) for negative sera and over 21.62% (X¯ + 3SD) for positive sera. The b-ELISA exhibited a high specificity, reacting exclusively with DAdV-3 positive serum and showing no cross-reactivity with other representative positive sera tested. Additionally, the b-ELISA showed significantly higher sensitivity than the serum neutralization test (SNT), detecting antibodies 16-fold greater than the endpoint dilution of the SNT. The established b-ELISA, validated with 90 field serum samples from six duck farms, was well-suited for clinical detection of DAdV-3 antibodies and for monitoring post-vaccination antibody levels, representing a significant advancement in DAdV-3 detection and prevention.

A duck adenovirus type 3 strain, SD2019, was isolated from sick Muscovy ducks in our laboratory in 2019. To study the biological properties of the virus, an infectious clone of the SD2019 strain was successfully established. The plasmid containing the whole genome of DAdV-3 was digested with Pac I and the linearized DNAs were electortransfected into LMH cells; the cells showed cytopathic effects (CPEs) at 96 h post transfection and the rescued virus (rSD2019) was identified by PCR and indirect immunofluorescence assays (IFAs). The biological characteristics of strain rSD2019 were studied in vitro and in vivo and the results show that rSD2019 grew to similar titers as compared with the wild-type SD2019 strain in LMH cells, as well as showing similar replication and virulence in Muscovy ducks. The establishment of a reliable reverse genetics system for DAdV-3 provides a foundation for future studies of DAdV-3.

Duck enteritis virus (DEV), an epornitic pathogen, causes substantial economic losses in the commercial duck industry and poses persistent risks to wild and migratory waterfowl populations. However, due to the large genomic capacity of the DEV, the understanding of the virulence-associated genes of DEV is still limited. In previous studies, we developed an attenuated strain E74 by serial passage of a virulent strain E1 on primary chicken embryo fibroblasts (CEFs). The bird experiment showed that the mortality rate of E1 on ducks reached 100%, and high-titered viruses were detected in all tested tissue samples. In contrast, the E74 virus has lost its pathogenicity in ducks and can only be detected at a relatively low viral load in the spleen. Furthermore, the E74 stimulated a significant increase in antibodies in the ducks at 7 days post-inoculation. To further investigate the molecular basis of the attenuation of DEV in ducks, the complete genomes of E74 and E1 were sequenced and analyzed. Compared with E1, E74 had a 5152 bp deletion in the UL region, which resulted in the lack of the hypothetical protein, LORF5, UL55 and LORF4 genes. To test the influence of the deletion on the viral pathogenicity, a rescued virus rE1-Δ5152 with the 5152 bp deletion in the UL region was generated on the E1 backbone. Animal experiments showed that the lethality of rE1-Δ5152 in ducks had disappeared. Those findings suggest that the hypothetical protein, LORF5, UL55, and LORF4 genes of DEV are associated with virus virulence, and the flexibility of this region provided excellent insertion sites for exogenous genes when DEV is used as a recombinant vaccine vector.

Avian viral arthritis (AVA), caused by avian reovirus (ARV), is a viral disease in chickens that has led to significant economic losses in the poultry industry. Recent studies have shown that traditional ARV vaccines based on the S1133 strain fail to protect against emerging ARV variants. In this study, we isolated and characterized three ARV strains (G4, YV, WF) from immunized chicken flocks with respiratory and arthritic symptoms. Genomic analysis revealed that the σC genes of G4, YV, and WF shared only 55.5%, 55.7%, and 58.7% sequence homology, respectively, with the S1133 strain. Phylogenetic analysis placed them in different branches, indicating they are variant strains. YV and WF belong to genotype III, and G4 falls into genotype VI. Whole genome analysis revealed gene segment reassortment among the variants. Pathogenicity testing in three-week-old SPF chickens showed that G4 (genotype VI) caused swelling of footpads, whereas WF (genotype III) did not. G4-infected chickens exhibited significantly higher viral loads in the thymus, lungs, spleen, and bursa of Fabricius than those in the WF-infected chickens, indicating viruses from different genotypes showed various pathogenesis. These results suggested an urgent need for new updates of vaccines against the variant ARVs, especially the genotype VI virus.

Duck egg-reducing syndrome virus (DERSV) is a novel Avihepatovirus and is responsible for a gradual decline in the laying rate of ducks, decreasing from a peak of 90% to 50%. The development of a rapid detection method for DERSV is crucial for the identification and control of virus infections. In this study, we developed a quantitative reverse transcription PCR (RT-qPCR) assay for detecting DERSV. Specific primers and a probe were designed to target a conserved region of the 3D gene. The assay demonstrated high specificity, with no cross-reactivity to other non-target duck viruses. It had a detection limit of 102 copies and a linear range from 102 to 109 copies per reaction. The assay’s efficiency was 92.59%, with a regression coefficient (R2) of 0.999. The coefficient of variation for both intra-and inter-assays was less than 2.00%. Among the 153 clinical samples collected from 2016 to 2023, the RT-qPCR detected a DERSV positive ratio of 47.06% (72/153). In conclusion, the utilization of the real-time RT-qPCR assay holds potential for the detection of DERSV in epidemiological and pathogenesis studies.

Background: Maternal-derived antibody (MDA) interferes with immune responses, leading to the failure of H9N2 avian influenza vaccinations in poultry. So far, none of the commercially available H9N2 avian influenza vaccines used in poultry have been able to overcome MDA interference. Methods: To develop a vaccine that can overcome MDA interference, one or multiple copies of the minimum-binding domain (P29) from the complement protein C3d were inserted in between the signal peptide and the head domain of the hemagglutinin (HA) protein on a H9N2 avian influenza virus (A/Chicken/Shanghai/H514/2017, named H514). Results: The HA proteins containing P29 stimulated stronger type I interferences than wild-type HA proteins in vitro. The modified viruses with the HA proteins containing one copy of P29 (rH514-P29.1) and two copies of P29.2 (rH514-P29.2) were successfully rescued using reverse genetics. The inactivated vaccines developed with rH514-P29.1 or rH514-P29.2 induced higher and faster humoral immune responses than the vaccine developed with rH514 in specific pathogen-free (SPF) chickens. To evaluate the vaccines’ efficacy in the presence of MDA and to ensure a uniform level of MDA, passively transferred antibody (PTA) was used as a model to mimic MDA in 1-day-old SPF chickens. Our results showed that the rH514-P29.2 inactivated vaccine induced significantly higher HI titers than the rH514 inactivated vaccine in the presence of PTA. More importantly, it reduced viral shedding after being challenged with H514 in the presence of PTA. Conclusions: Our results suggest that vaccine antigens fused with two copies of P29 can decrease the interference of MDA on immunity in chickens. Overall, our results provide a new strategy for overcoming MDA interference.

During the life cycle of the influenza virus, viral RNPs (vRNPs) are transported to the nucleus for replication. Given that a large number of progeny viral RNA occupies the nucleus, whether there is any host protein located in the nucleus that recognizes the viral RNA and inhibits the viral replication remains largely unknown. In this study, to explore the role of hnRNPH1 in influenza virus infection, we knocked down and over-expressed the hnRNPH1 proteins in 293T cells, then infected the cells with the influenza virus. The results showed that the host hnRNPH1 inhibits the replication of H1N1 and H9N2 influenza viruses by restraining the polymerase activity of viruses. hnRNPH1 contains two RNA recognition motifs (RRM1) and RRM2. Further studies indicated that hnRNPH1 specifically binds to the viral RNA of the PB1, PA, and NP genes. Mutation of the key residues tryptophan and tyrosine in RRM1 and RRM2 abolished the binding affinity to viral RNA and the suppression of polymerase activity of the influenza virus. All the results suggested that hnRNPH1 suppresses polymerase activity and replication of the influenza virus by binding viral RNA.

Tembusu virus (TMUV) is an emerging mosquito-borne flavivirus, primarily transmitted by Culex spp. mosquitoes. The 2010 outbreak of TMUV in ducks revealed that the virus had acquired direct contact and aerosol transmission routes, enabling its rapid spread in duck farms. Recently, cluster 3 TMUV has increasingly been isolated from chickens, ducks and geese. In this study, we examined the pathogenicity and transmission routes of the cluster 3 TMUV Shandong 2021 (SD) strain in ducks and chickens. Our results show that TMUV SD can infect both species, but only in ducks could TMUV be detected in throat and cloacal swabs. In ducks, the virus can spread without mosquito involvement to co-housed naïve birds, demonstrating direct transmission capability. Conversely, no virus shedding and direct transmission were observed in chickens, suggesting that mosquitoes are required for virus transmission between chickens. Indeed, Culex pipiens mosquitoes could become infected by biting chickens infected with the TMUV SD and transmit the virus to naïve chickens. Our results, for the first time, provide direct evidence that TMUV can be transmitted by mosquitoes in a laboratory setting. Furthermore, our findings indicate that viral excretion through the respiratory tract and/or digestive tract is essential for direct contact transmission of TMUV, which is a critical factor in the epidemic spread of TMUV. These insights into the transmission dynamics of a cluster 3 TMUV emphasize the importance of effective vector control and biosecurity measures in managing and preventing outbreaks.

Pigs serve as a mixing vessel for influenza viruses and can independently promote the emergence of pandemic strains in humans. During our surveillance of pig populations from 2021 to 2023 in China, 11 H1 subtype swine influenza viruses (SIVs) were isolated. All viruses were reassortants, possessing internal genes of identical origins (PB2, PB1, PA, NP, M: pdm09/H1N1 origin, NS: North American triple reassortant origin). The H1N1 isolates were all the dominant G4 EA H1N1 viruses in China. Two H1N2 isolates carried early human pdm09/H1N1 HA genes, suggesting a possible pig-to-human transmission route. Mutations that dictate host range specificity were identified in all isolates, a phenomenon which may enhance the affinity to human receptors. These H1 subtype viruses effectively replicated both in vivo and in vitro without prior adaptation and exhibited different pathogenicity and growth characteristics. Some of the H1 viruses were even found to cause lethal infections in mice. Taken together, our study indicates that the H1 subtype SIVs recently circulating in China pose a potential threat to human health and emphasizes the importance of continuing to closely monitor their evolution and spread.

Maternal-derived antibodies (MDAs) interfere with immune responses, leading to the failure of H9N2 avian influenza vaccination in poultry. So far, none of commercial H9N2 avian influenza vaccines used in poultry can overcome MDAs interference. To develop a vaccine overcoming MDAs interference, different copies of minimum-binding domain (P29) of complement protein C3d were inserted between the signal peptide and the head domain of hemagglutinin (HA) of a H9N2 avian influenza virus (A/Chicken/Shanghai/H514/2017, named H514). The HA proteins containing P29 stimulated stronger typeⅠinterfere than pure HA proteins in vitro. The modified viruses with HA containing one copy of P29 (rH514-P29.1) and two copies of P29.2 (rH514-P29.2) were successfully rescued by using reverse genetics. The inactivated H9N2 vaccines developed with rH514-P29.1 or rH514-P29.2 induced higher and faster adaptive immune responses than the vaccine developed with rH514 in chickens without MDAs. Importantly, in the chickens with MDAs, the inactivated rH514-P29.2 vaccine induced significantly higher HI titers than the vaccine without P29, and reduced viral shedding after challenged with H514, which suggests that vaccine antigens fused two copies of P29 can decrease the interference of MDAs to immunity in chickens. Overall, our results provide a new strategy to overcome MDAs interference.

ABSTRACT Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity. IMPORTANCE The H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

Maternal-derived antibodies (MDAs) are one of reasons why vaccination with the H9N2 inactivated whole virus (IWV) vaccine failed in poultry. Unmethylated CpG motif-containing oligodeoxynucleotides (CpG ODN) shows great potential to overcome MDAs interference in mammals, but whether it has similar characteristics in poultry is still unknown. In the present study, different classes and various copies of CpG ODN motifs were cloned into two different plasmids (pCDNA3.1 or T vector). Immunomodulatory activities and immunoadjuvant efficacy of these CpG ODN plasmids were tested in vitro and in vivo in the presence of passively transferred antibodies (PTAs) that were used to mimic MDAs. Results showed that the T vector enriched with 30 copies of CpG-A ODN and 20 copies of CpG-B ODN (T-CpG-AB) significantly up-regulated mRNA expression of chicken-interferon-α (ch-IFN-α), chicken-interferon-β (ch-IFN-β) and chicken-interleukin-12 protein 40 (ch-IL-12p40). When administered as adjuvant of the H9N2 IWV vaccine, the minimal dose of T-CpG-AB plasmid was 30 µg per one-day-old chicken, which could induce strong humoral immune responses in the presence of PTAs. Furthermore, T-CpG-AB plasmid-based vaccine triggered both strong humoral immune responses and cytokines expression in the presence of PTAs in chickens. Overall, our findings suggest that T-CpG-AB plasmid can be an excellent adjuvant candidate for the H9N2 IWV vaccine to overcome MDAs interference in chickens.

ABSTRACT Replication of influenza A virus (IAV) is highly reliant on host cell function, and to identify the key host factor required in the influenza A virus life cycle, a genome-wide CRISPR/Cas9 knockout (KO) screen was conducted in A549 cells infected by H1N1 influenza virus. The results showed that glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) plays a crucial role in the replication of influenza A virus, and knockout of the GNE significantly reduced the replication of multiple subtype influenza A viruses in A549 cells, and restoration of the GNE expression in the GNE-knockout cells partially recovered IAV infection. Additionally, overexpression of the GNE in wild-type (WT) cells promoted IAV infection to a certain extent. Further study showed that the GNE is involved in α-2,3- and α-2,6-linked sialic acid (Sia) synthesis, which is the major receptor of the influenza A virus, and the GNE knockout downregulated the α-2,3- and α-2,6-linked sialic acid expression. In summary, the knockout of the GNE inhibits adsorption and endocytosis of IAV. This study provides a new approach to elucidate the replication mechanism of IAV and identifies GNE as a potential target for anti-influenza drug development. IMPORTANCE Influenza A virus infection requires the assistance of the host proteins. Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) was identified as an important host factor in influenza A virus infection by genome-wide CRISPR screening. GNE knockout (KO) extensively inhibited the replication of multiple subtype influenza A viruses. It indirectly participated in IAV adsorption and endocytosis by regulating the expression of sialic acid (Sia), the main receptor of IAV. This finding provides novel insights into the replication mechanism of IAV.

Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses to the poultry industry in China since 2010. However, the potential threat of TMUV to mammals has not been well studied. In this study, a TMUV HB strain isolated from diseased ducks showed high virulence in BALB/c mice inoculated intranasally compared with the reference duck TMUV strain. Further studies revealed that the olfactory epithelium is one pathway for the TMUV HB strain to invade the central nervous system of mice. Genetic analysis revealed that the TMUV HB virus contains two unique residues in E and NS3 proteins (326K and 519T) compared with duck TMUV reference strains. K326E substitution weakens the neuroinvasiveness and neurovirulence of TMUV HB in mice. Remarkably, the TMUV HB strain induced significantly higher levels of IL-1β, IL-6, IL-8, and interferon (IFN)-α/β than mutant virus with K326E substitution in the brain tissue of the infected mice, which suggested that TMUV HB caused more severe inflammation in the mouse brains. Moreover, application of IFN-β to infected mouse brain exacerbated the disease, indicating that overstimulated IFN response in the brain is harmful to mice upon TMUV infection. Further studies showed that TMUV HB upregulated RIG-I and IRF7 more significantly than mutant virus containing the K326E mutation in mouse brain, which suggested that HB stimulated the IFN response through the RIG-I-IRF7 pathway. Our findings provide insights into the pathogenesis and potential risk of TMUV to mammals.