Construction and characterization of a full-length infectious clone of an emerging senecavirus A strain.

Senecavirus A (SVA) is an emerging virus that causes vesicular disease in pigs. Construction of a full-length SVA cDNA clone is crucial for understanding its replication and pathogenesis. Here, we successfully constructed a CMV-promoter-driven infectious cDNA clone of the SVA isolate SVA/GX/CH/2018, which we named rSVA GX01. Sequence comparison between the pSVA GX01 and the parental isolate (SVA/GX/CH/2018) revealed three single-nucleotide differences. Four-week-old piglets were experimentally infected with either the parental virus or the cloned virus. The results showed that the cloned rSVA GX01 displayed weak pathogenicity in 4-week-old pigs compared to the parental virus SVA CH-GX-01-2018. The infectious clone of SVA will serve as a valuable tool for studying the viral replication cycle and for functional analysis of the viral genome.

Construction of and evaluation of the immune response to two recombinant pseudorabies viruses expressing the B119L and EP364R proteins of African swine fever virus.

African swine fever (ASF) is an infectious disease caused by ASF virus (ASFV), which is characterized by high infectivity, rapid onset of disease, and a high mortality rate. Outbreaks of ASFV have caused great economic losses to the global pig industry, and there is a need to develop safe and effective vaccines. In this study, two recombinant pseudorabies virus (PRV) strains, rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L, expressing the EP364R and B119L protein, respectively, of ASFV, were constructed by homologous recombination technology. Western blotting and immunofluorescence analysis showed that these foreign proteins were expressed in cells infected with the recombinant strains. The strains showed good genetic stability and proliferative characteristics for 20 passages in BHK-21 cells. Both of these strains were immunogenic in mice, inducing the production of specific antibodies against the expressed ASFV proteins while providing protection against lethal challenge with PRV. Thus, the recombinant strains rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L could be used as candidate vaccines for both ASFV and PRV. In addition, our study identifies two potential target genes for the development of safe and efficient ASFV vaccines, provides a reference for the construction of bivalent ASFV and PRV vaccines, and demonstrates the feasibility of developing a live ASFV vector vaccine.

Molecular epidemiological and genetic characterization of pseudorabies virus in Guangxi, China.

Pseudorabies virus (PRV) is an important pathogen that can cause harm to the pig population. Since 2011, there have been a number of large-scale outbreaks of pseudorabies on Chinese farms where animals had been vaccinated with the Bartha-K61 vaccine. In order to understand the epidemiological trend and genetic variations of PRV in Guangxi province, China, 819 tissue samples were collected from swine farms where PRV infection was suspected from 2013 to 2019, and these were tested for infectious wild strains of PRV. The results showed a positive rate of PRV in Guangxi province of 28.21% (231/819). Thirty-six wild-type PRV strains were successfully isolated from PRV-positive tissue samples, and a genetic evolutionary analysis was performed based on the gB, gC, gD, gE, and TK genes. Thirty of the PRV strains were found to be closely related to the Chinese variant strains HeN1-China-2012 and HLJ8-China-2013. In addition, five PRV strains were genetically related to Chinese classical strains, and one isolate was a recombinant of the PRV variant and the vaccine strain Bartha-K61. Amino acid sequence analysis showed that all 36 PRV strains had characteristic variant sites in the amino acid sequences of the gB, gC, gD, and gE proteins. Pathogenicity analysis showed that, compared to classical PRV strains, the PRV variant strains were more pathogenic in mice and had a lower LD50. Taken together, our results show that wild-type PRV infections are common on pig farms in Guangxi province of China and that the dominant prevalent strains were those of the PRV variants. The PRV variant strains also had increased pathogenicity in mice. Our data will provide a useful reference for understanding the prevalence and genetic evolution of PRV in China.

Generation of a porcine reproductive and respiratory syndrome virus expressing a marker gene inserted between ORF4 and ORF5a.

In recent years, the availability of reverse genetics systems for porcine reproductive and respiratory syndrome virus (PRRSV) has created new perspectives for the use of recombinant viruses as expression vectors. Most of these recombinant PRRSV vectors express foreign genes through either an independent transcription unit inserted in ORF1b and ORF2, or in ORF7 and the 3' UTR. The aim of this study was to find an alternative site for foreign gene insertion into the PRRSV genome. Here, we constructed an infectious cDNA clone for a cell-adapted PRRSV strain, GXNN1396-P96. This cDNA-clone-derived recombinant virus (rGXAM) was comparable in its growth kinetics in MARC-145 cells to the parental virus, GX1396-P96. Using the infectious cDNA-clone, we inserted an independent transcription unit in ORF4 and ORF5a to generate a novel PRRSV-based recombinant virus expressing the green fluorescent protein (GFP) gene. Biological characterization of the recombinant virus, rGX45BSTRS-GFP, showed that it maintained similar growth characteristics but produced fewer infectious virions than the parental PRRSV. These data demonstrate that the ORF4 and ORF5a site is able to tolerate the insertion of foreign genes.

Genetic analysis of porcine circovirus type 2 (PCV2) strains between 2002 and 2016 reveals PCV2 mutant predominating in porcine population in Guangxi, China.

BACKGROUND: Porcine circovirus 2-associated disease (PCVAD) is acknowledged as one of the most economically important diseases for the swine industry worldwide. The aim of this study was to characterize and determine the genetic diversity of PCV2 in the porcine population of Guangxi, China. METHODS: The full length genome and open reading frame 2 (ORF2) of 95 PCV2 strains collected from the tissues and sera of pigs that had either died as a result of PCVAD or did not exhibit disease symptoms were analyzed. RESULTS: The results of multiple sequence alignments showed that there is considerable diversity among the PCV2 ORF2 sequences. Phylogenetic analyses based on the complete genome showed that current PCV2 strains in this study could be divided into PCV2a (1/95), PCV2b (39/95), PCV2d (43/95), PCV2e (10/95) and PCV2h (2/95). Among the 5 sub-genotypes, PCV2b was dominant in the porcine population from 2002 to 2008. The newly identified sub-genotype, PCV2d, was seen from 2003 and has increased every year. PCV2b and PCV2d formed two predominant genetic groups circulating in southern China between 2009 and 2013 and the sub-genotype PCV2d has become the dominant virus in China since 2014. CONCLUSIONS: This study reveals the complex genetic diversity of PCV2 and improves our understanding regarding the epidemiological trends of PCV2 sub-genotypes in China.

Molecular epidemiology and viremia of porcine astrovirus in pigs from Guangxi province of China.

BACKGROUND: Porcine astroviruses (PAstVs) are common in pigs worldwide. There are five distinct lineages with each lineage representing a different ancestral origin. Recently, multiple reports have demonstrated the evidence of extra-intestinal infection of PAstVs, but little is known about viremia. RESULTS: In this study, a total of 532 fecal samples and 120 serum samples from healthy pigs were collected and tested from 2013 to 2015 in Guangxi province, China; of these 300/532 (56.4%) and 7/120 (5.8%) of fecal samples tested positive for PAstVs, respectively. Our study revealed that there was wide genetic diversity and high prevalence of the virus in the pig population. All five of the known PAstVs genotypes (1-5) prevailed in the pig population of Guangxi province and were distributed in all age groups of pigs, from suckling piglets to sows, with PAstV2 (47.7%), PAstV1 (26.2%) and PAstV5 (21.5%) seen predominantly. Phylogenetic analysis of partial ORF1b and partial capsid sequences from fecal and serum samples revealed that they were divided into the five lineages. Among these genotypes, based on partial ORF2 genes sequencing 23 strains were grouped as PAstV1, including 6 serum-derived strains, and were regarded as the causative agents of viremia in pigs. CONCLUSIONS: Due to the information regarding the types of PAstV in blood is limit. This is the first report for the presence of PAstV1 in blood and PAstV3 in the feces of nursery pigs of China. This study provides a reference for understanding the prevalence and genetic evolution of PAstVs in pigs in Guangxi province, China. It also provides a new perspective for understanding of the extra-intestinal infection of PAstVs in pigs.

Genetic analysis of porcine productive and respiratory syndrome virus between 2013 and 2014 in Southern parts of China: identification of several novel strains with amino acid deletions or insertions in nsp2.

BACKGROUND: Porcine respiratory and reproductive syndrome virus (PRRSV) is one of the most economically significant pathogens in the Chinese swine industry. ORF5 and nsp2 are highly variable regions of the PRRSV genome. Therefore, nsp2 and GP5 are often selected for investigation of variations and phylogenetic analyses for their genetic diversities. Knowledge of the molecular evolution of PRRSV field strains may contribute to the control of PRRS in China. RESULTS: The results of multiple sequence alignments of GP5 showed that there is 84.5-100% aa identity among the 56 strains in this study. These strains shared 84.5-99.0% aa identity with the prototypical type 2 PRRSV VR-2332 and 56.6-59.2% with strain LV, prototypical type 1 PRRSV. Phylogenetic analysis showed there is considerable diversity among PRRSV ORF5 and the existence of two lineages (5 and 8). Most of the strains were classified into lineage 8 with multiple sub-lineages (3, 4 and 6). Moreover, PRRSV strains with 5 novel patterns of deletions or insertions in the nsp2 region were found. CONCLUSIONS: Phylogenetic analysis based on ORF5 sequences indicated the diversity of PRRSV in southern parts of China and the strains with 30 aa deletion in nsp2 are dominant in the porcine population. Also, new PRRSV strains with different patterns of deletions or insertions in nsp2 are emerging. The data presented here constitute a useful basis for further epidemiological studies regarding the heterogeneity of PRRSV strains in China and provide a basis for the prevention of PRRS in southern parts of China.

Effect of an 88-amino-acid deletion in nsp2 of porcine reproductive and respiratory syndrome virus on virus replication and cytokine responses in vitro.

Previously, a spontaneous 88-amino-acid (aa) deletion in nsp2 was associated with cell-adaptation of porcine reproductive and respiratory syndrome virus (PRRSV) strain JXM100, which arose during passaging of the highly pathogenic PRRSV (HP-PRRSV) strain JX143 in MARC-145 cells. Here, to elucidate the biological role of this deletion, we specifically deleted the region of a cDNA clone of HP-PRRSV strain JX143 (pJX143) corresponding to these 88 amino acids. The effect of the deletion on virus replication in cultured cells and transcriptional activation of inflammatory cytokines and chemokines in pulmonary alveolar macrophages (PAMs) was examined. Mutant virus with the 88-aa deletion in nsp2 (rJX143-D88) had faster growth kinetics and produced larger plaques in MARC-145 cells than the parental virus (rJX143), suggesting that the deletion enhanced virus replication in MARC-145 cells. In contrast, the overall yield of rJX143 was almost 1 log higher than that of rJX143-D88, suggesting that the 88-aa deletion in nsp2 decreased the production of infectious viruses in PAMs. Infection with the mutant virus with the 88-aa deletion resulted in increased mRNA expression of type I interferon (IFN-α and IFN-ß) and chemokines genes. In addition, the mRNA expression of antiviral genes (ISG15, ISG54 and PKR) regulated by the IFN response was upregulated in PAMs infected with the mutant virus rJX143-D88. Our results demonstrate that virus-specific host immunity can be enhanced by modifying certain nsp2 epitope regions. These findings provide important insights for understanding virus pathogenesis and development of future vaccines.

Construction of a reverse genetic system for porcine astrovirus.

In order to construct a full-length infectious cDNA clone of porcine astrovirus, three fragments covering the complete genome of PAstV1-GX1 strain were amplified by RT-PCR. All three PCR-amplified fragments were cloned into T-Vector pMD19 (Simple), and subsequently assembled into a full-length cDNA clone by subcloning. A silent nucleotide change creating a PstI site was engineered into the full-length cDNA clone to distinguish the rescued virus from the parental virus. Upon transfection of BHK-21 cells with the in vitro transcripts of both the original and constructed cDNAs, typical cytopathic effects were observed on PK-15 cells after serial passaging of the cell supernatant. The construction and recovery of the infectious cDNA clone of porcine astrovirus will provide a valuable experimental system to study the genome function and pathogenesis of astroviruses.

Phylogeographic analysis of porcine reproductive and respiratory syndrome virus 1 in Guangdong province, Southern China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered an important economic pathogen for the international swine industry. At present, both PRRSV-1 and PRRSV-2 have been confirmed to be co-circulating in China. However, there is little available information about the prevalence or distribution of PRRSV-1 in Guangdong province, southern China. In this study, we performed molecular detection of PRRSV-1 in 750 samples collected from 50 farms in 15 major pig farming regions in this province. After RT-PCR testing, 64% (32/50) of farms were confirmed as PRRSV-1-positive. Surprisingly, PRRSV-1 was circulating on at least one pig farm in all 15 regions; of the 750 samples, 186 samples (24.8%) were positive for PRRSV-1. Furthermore, 15 representative PRRSV-1 ORF5 sequences (606 bp) (n = 1 per region) were obtained from those PRRSV-1-positive regions. Sequence alignment analysis indicated that they shared 81.8% ~ 100% nucleotide and 81.2% ~ 100% amino acid similarity with each other. Although all current PRRSV-1 sequences were divided into pandemic subtype 1, most of them had unique glycoprotein-5 amino acid sequences that are significantly different from other known PRRSV-1 isolates. To conclude, the present findings revealed wide geographical distribution of PRRSV-1 in Guangdong province, southern China. This study further extends the epidemiological significance of PRRSV-1 in China.

Detection and genetic characterization of canine astroviruses in pet dogs in Guangxi, China.

BACKGROUND: Astroviruses (AstVs) have been reported to infect and cause gastroenteritis in most animal species. Human AstVs were regarded the causative agent of viral diarrhea in children. In dogs, little is known about the epidemiology and clinical significance of AstV infection. FINDINGS: In this study, we collected and tested 253 rectal swabs from pet dogs; of which 64 samples (25.3%) tested positive for AstVs with diarrhea and 15 more samples (5.9%) also was identified as AstVs, however without any clinical signs. Phylogenetic analysis of 39 partial ORF1b sequences from these samples revealed that they are similar to AstVs, which can be subdivided into three lineages. Interestingly, out of the 39 isolates sequenced, 16 isolates are shown to be in the Mamastrovirus 5/canine astrovirus (CAstV) lineage and the remaining 23 isolates displayed higher similarities with known porcine astrovirus (PoAstV) 5 and 2. Further, analysis of 13 capsid sequences from these isolates showed that they are closely clustered with Chinese or Italy CAstV isolates. CONCLUSIONS: The findings indicate that CAstVs commonly circulate in pet dogs, and our sequencing results have shown the genomic diversity of CAstVs leading to increasing number of clusters.

Emergence of human-like H3N2 influenza viruses in pet dogs in Guangxi, China.

BACKGROUND: After the 1968 H3N2 pandemic emerged in humans, H3N2 influenza viruses continuously circulated and evolved in nature. An H3N2 variant was circulating in humans in the 1990s and subsequently introduced into the pig population in the 2000s. This virus gradually became the main subtype of swine influenza virus worldwide. However, there were no reports of infections in dogs with this virus. FINDINGS: In 2013, 35 nasal swabs from pet dogs were positive for Influenza A virus by RT-PCR. Two viruses were isolated and genetically characterized. In the phylogenetic trees of all gene segments, two H3N2 canine isolates clustered with Moscow/10/99 and most H3N2 swine influenza viruses. These results indicated that two H3N2 CIVs possessed high homology with human/swine influenza viruses, which at the same time exhibited some amino acid substitutions in NA, polymerase basic protein 1 (PB1), and nucleoprotein (NP), which probably were related to the interspecies transmission. CONCLUSIONS: These two viruses share the highest homology with swine H3N2, Moscow/99-like viruses, which indicated that these viruses might originate from swine viruses.

Genetic manipulation of a transcription-regulating sequence of porcine reproductive and respiratory syndrome virus reveals key nucleotides determining its activity.

The factors that determine the transcription-regulating sequence (TRS) activity of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unclear. In this study, the effect of mutagenesis of conserved C nucleotides at positions 5 and 6 in the leader TRS (TRS-L) and/or canonical body TRS7 (TRS-B7) on the synthesis of subgenomic (sg) mRNA and virus infectivity was investigated in the context of a type 2 PRRSV infectious cDNA clone. The results showed that a double C mutation in the leader TRS completely abolished sg mRNAs synthesis and virus infectivity, but a single C mutation did not. A single C or double C mutation in TRS-B7.1 or/and TRS-B7.2 impaired or abolished the corresponding sg mRNA synthesis. Introduction of identical mutations in the leader and body TRSs partially restored sg mRNA7.1 and/or sg mRNA7.2 transcription, indicating that the base-pairing interaction between sense TRS-L and cTRS-B is a crucial factor influencing sg mRNA synthesis. Analysis of the mRNA leader-body junctions of mutants provided evidence for a mechanism of discontinuous minus-strand transcription. This study also showed that mutational inactivation of TRS-B7.1 or TRS-B7.2 did not affect the production of infectious progeny virus, and the sg mRNA formed from each of them could express N protein. However, TRS-B7.1 plays more important roles than TRS-B7.2 in maintaining the growth characteristic of type 2 PRRSV. These results provide more insight into the molecular mechanism of genome expression and subgenomic mRNA transcription of PRRSV.

The emergence, isolation, and phylogenetic analysis of a closely related human strain of parainfluenza virus 5 from a case of porcine reproductive and respiratory syndrome in China.

Reports of Parainfluenza virus 5 (PIV5) epidemics have been on a global upward trend, with an expanding host range across various animals. In 2020, we isolated a PIV5 strain from a PRRSV-positive serum sample. This strain was named GX2020. Genetic analysis revealed that GX2020 belongs to group A, represented by the AGS strain isolated from a human in the USA. Comparisons of amino acid identity in the coding regions showed that GX2020 had the highest amino acid identity (99.6%) with the AGS strain. The emergence of PIV5 strains genetically similar to human strains in pigs highlights its zoonotic potential and underscores the need for enhanced PIV5 surveillance in the future.

Isolation and characterization of Chlamydia felis and its pathogenesis in cats.

Feline upper respiratory tract disease (URTD) is a common but complicated disease that occurs in domestic cats, worldwide. 396 cats in Guangxi Province, China were screened for URTD-associated pathogens from March 2022 to August 2023. Mycoplasma felis was found to be the most prevalent infectious agent with a positivity rate of 24.75 %, followed by feline calicivirus (FCV), Chlamydia felis, feline herpesvirus 1 (FHV-1) and feline influenza A virus (FeIAV) with rates of 15.91, 11.62, 5.56 and 1.52 %, respectively. In particular, C. felis and M. felis were found in 13 of 55 co-infected cats. Of the 46 C. felis-positive samples, one strain, named as GXNN36, was successfully isolated using chicken embryos and it was characterized both in vivo and in vitro. For the cat studies, both high- and low-dose challenged groups showed severe conjunctivitis, accompanied by transient fever and respiratory symptoms. C. felis replicated well in turbinate, trachea and lung tissues with high copy numbers and the infection subsequently spread to the livers, spleens, pancreas, kidneys, hearts and intestines. These findings will help our understanding of the role of C. felis in feline URTD and provide a valuable model to evaluate the efficacy of vaccines and therapeutic remedies in the future.

Construction and characterization of recombinant senecavirus A expressing secreted luciferase for antiviral screening.

Senecavirus A (SVA) is a newly identified picornavirus associated with swine vesicular disease and neonatal mortality. The development of an SVA incorporating an exogenous reporter gene provides a powerful tool for viral research. In this study, we successfully constructed a recombinant SVA expressing Gaussia Luciferase (Gluc), termed rSVA-Gluc. The growth kinetics of rSVA-Gluc in BHK-21 cells were found to be comparable to those of the parental virus, and Gluc activity paralleled the virus growth curve. Genetic analysis revealed stable inheritance of the inserted reporter protein genes for at least six generations. We evaluated the utility of rSVA-Gluc in antiviral drug screening, and the results highlighted its potential as an effective tool for such purposes against SVA. DATA AVAILABILITY STATEMENT: The data that support the findings of this study are available on request from the corresponding author.

Development of a monoclonal antibody specifically recognizing a linear epitope on the capsid protein of the emerging Group III Getah virus.

Getah virus (GETV) is an emerging mosquito-borne alphavirus that can infect horses, pigs and other animals. Given the public health threat posed by GETV, research on its pathogenesis, diagnosis and prevention is urgently needed. In the current study, prokaryotic expression systems were used to express the capsid protein of GETV. This protein was then used to immunize BALB/c mice in order to generate monoclonal antibodies (mAbs). Subsequently, hybridoma cells secreting a mAb (2B11-4) against the capsid protein were obtained using the hybridoma technique. A B cell linear epitope, 18-PAYRPWR-24, located at the capsid protein's N-terminal region was identified using western blotting analysis with the produced mAb, 2B11-4. Sequence alignment indicated that this epitope was highly conserved in group III (GIII) strains of GETV, but varied among the other genotypes. Western blotting showed that mAb 2B11-4 could discriminate Group III GETVs from other genotypes. This study describes the preparation of a mAb against the GETV capsid protein and the identification of the specific localization of B-cell epitopes, and will contribute towards a better understanding of the biological importance of the GETV capsid protein. It will also pave the way for developing immunological detection methods and genotype diagnosis for GETVs.

Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus.

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.

A novel VP1-based enzyme-linked immunosorbent assay revealed widespread Enterovirus G infections in Guangxi, China.

Enterovirus G (EV-G) has recently been shown to affect weight gain and cause neurological symptoms in piglets. However, the serological investigation of EV-G is limited. In this study, we developed a novel serological detection method based on the structural protein, VP1 of EV-G. The intra-assay and inter-assay coefficient variations were 3.2-8.9% and 2.6-8.0%, respectively. There was no cross-reaction of the VP1-based enzyme-linked immunosorbent assay (ELISA) with antisera against the other known porcine viruses. In addition, a comparison was made with other methods including the developed indirect ELISAs based on VP2 and VP3 proteins and western blot (WB) analysis, which demonstrated the reliability of the novel method. Using the VP1-based ELISA, we carried out the first seroepidemiological survey of EV-G in China by testing 1041 serum samples collected from different pig farms in Guangxi from 2019 to 2021. Our results showed that 68.78% of the serum samples and 100% of the pig farms were positive for EV-G, with a relatively high incidence of seropositivity in pigs of different ages. This was specifically evident in fattening pigs and sows, which may suggest that the piglets have experienced an infection with EV-G during their growth process. Our data provide the first serological evidence of EV-G infections in pigs from China and reveal the widespread presence of EV-G infections in Guangxi, China.

Hunnivirus structural protein VP2 inhibits beta interferon production by targeting the IRF3 essential modulator.

Water buffalo Hunnivirus (BufHuV) belongs to the family Picornaviridae and is a newly discovered member of the Hunnivirus A genus. It causes intestinal diseases in cattle, mainly lead to subclinical infections, thereby seriously threatening the health of cattle herds. In addition, it can also bring about various clinical disease syndromes which results in severe economic losses to the cattle industry. To date, there have been no reports worldwide on the study of Hunnivirus virus infecting host cells and causing innate immune responses. In this study, we found that interferon treatment effectively blocked BufHuV replication and infection with the virus weakened the host antiviral responses. Inhibiting the transcription of IFN-ß and ISGs induced by either Sendai virus (SeV) or poly(I:C) in MDBK and HCT-8 cells, were dependent on the IRF3 or NF-κB signaling pathways, and this inhibited the activation of IFN-ß promoter by TBK1 and its upstream molecules, RIGI and MDA5. By constructing and screening five BufHuV proteins, we found that VP2, 2 C, 3 C and 3D inhibited the activation of IFN-ß promoter induced by SeV. Subsequently, we showed that VP2 inhibited the activation of IRF3 induced by SeV or poly (I:C), and it inhibited IRF3 activation by inhibiting its phosphorylation and nuclear translocation. In addition, we confirmed that VP2 inhibited the activation of IFNß induced by signaling molecules, MDA5 and TBKI. In summary, these findings provide new insights into the pathogenesis of Hunnivirus and its mechanisms involved in evading host immune responses.