Amino Acid Variation at Hemagglutinin Position 193 Impacts the Properties of H9N2 Avian Influenza Virus.

Despite active control strategies, including the vaccination program in poultry, H9N2 avian influenza viruses possessing mutations in hemagglutinin (HA) were frequently isolated. In this study, we analyzed the substitutions at HA residue 193 (H3 numbering) of H9N2 and investigated the impact of these mutations on viral properties. Our study indicated that H9N2 circulating in the Chinese poultry have experienced frequent mutations at HA residue 193 since 2013, with viruses that carried asparagine (N) being replaced by those with alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), and serine (S), etc. Our results showed the N193G mutation impeded the multiple cycles of growth of H9N2, and although most of the variant HAs retained the preference for human-like receptors as did the wild-type N193 HA, the N193E mutation altered the preference for both human and avian-like receptors. Furthermore, these mutations substantially altered the antigenicity of H9N2 as measured by both monoclonal antibodies and antisera. In vivo studies further demonstrated that these mutations showed profound impact on viral replication and transmission of H9N2 in chicken. Viruses with D, E, or S at residue 193 acquired the ability to replicate in lungs of the infected chickens, whereas virus with G193 reduced its transmissibility in infected chickens to those in direct contact. Our findings demonstrated that variations at HA residue 193 altered various properties of H9N2, highlighting the significance of the continued surveillance of HA for better understanding of the etiology and effective control of H9N2 in poultry. IMPORTANCE H9N2 are widespread and have sporadically caused clinical diseases in humans. Extensive vaccinations in poultry helped constrain H9N2; however, they might have facilitated the evolution of the virus. It is therefore of importance to monitor the variation of the circulating H9N2 and evaluate its risk to both veterinary and public health. Here, we found substitutions at position 193 of HA from H9N2 circulated since 2013 and assessed the impact of several mutations on viral properties. Our data showed these mutations resulted in substantial antigenic change. N193E altered the binding preference of HA for human-like to both avian and human-like receptors. More importantly, N193G impaired the growth of H9N2 and its transmission in chickens, whereas mutations from N to D, E, and S enhanced the viral replication in lungs of chickens. Our study enriched the knowledge about H9N2 and may help implement an effective control strategy for H9N2.

A novel mAb broadly neutralizes SARS-CoV-2 VOCs in vitro and in vivo, including the Omicron variants.

Novel immune escape variants have emerged as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. Many of the variants cause breakthrough infections in vaccinated populations, posing great challenges to current antiviral strategies targeting the immunodominance of the receptor-binding domain within the spike protein. Here, we found that a novel broadly neutralizing monoclonal antibody (mAb), G5, provided efficient protection against SARS-CoV-2 variants of concern (VOCs) in vitro and in vivo. A single dose of mAb G5 could significantly inhibit the viral burden in mice challenged with the mouse-adapted SARS-CoV-2 or SARS-CoV-2 Omicron BA.1 variant, as well as the body weight loss and cytokine release induced by mouse-adapted SARS-CoV-2. The refined epitope recognized by mAb G5 was identified as 1148 FKEELDKYF1156 in the stem helix of subunit S2. In addition, a human-mouse chimeric mAb was generated based on the variable region of heavy chain and VL genes of mAb G5. Our study provides a broad antibody drug candidate against SARS-CoV-2 VOCs and reveals a novel target for developing pan-SARS-CoV-2 vaccines.

Identification of three novel B cell epitopes in ORF2 protein of the emerging goose astrovirus and their application.

The outbreak of goose gout disease caused by novel goose astrovirus type 1 (GAstV-1) has resulted in huge economic losses to the goose industry in China since 2017. However, little is known about the B cell epitopes in major antigen of GAstV-1 and the serological approach for detection of GAstV-1 is not available. In this study, three novel monoclonal antibodies (mAbs) against the ORF2 protein were first generated and designated as 3G6, 5H7, and 6C6, respectively. Epitope mapping revealed that mAb 3G6, 5H7, and 6C6 recognized 695AVRFEKGGHE704, 685EKALSAPQAG694, and 635DDDPLSDVTS644 in ORF2, respectively. Sequence alignments found that the three epitopes were highly conserved in GAstV-1 but not in other AAstV members. Moreover, a mAb-based sandwich ELISA for the detection of GAstV-1 was first developed using mAb 6C6. The sandwich ELISA only reacted with GAstV-1 but not with GAstV-2 and the other goose-associated viruses tested. The limit of the detection of the sandwich ELISA reaches 1.58 × 103 TCID50/mL of GAstV-1. Notably, mAb 6C6 could also efficiently react with the GAstV-1 in tissue frozen sections of the clinical infected goose through IFA. The mAbs generated in this study pave the way for further studying on the role of ORF2 in the infection and pathogenesis of GAstV, and the sandwich ELISA and the tissue frozen section-IFA approaches established here provide efficient and rapid serological diagnostic tools for detection of GAstV-1. KEY POINTS: • Three novel B cell epitopes were identified in ORF2 of GAstV-1. • mAb-based ELISA and IFA for detection of GAstV-1 were developed.

OASL Triggered by Novel Goose Astrovirus via ORF2 Restricts Its Replication.

Although astroviruses causes enteric diseases and encephalitis in humans and nephritis and hepatitis in poultry, astrovirus infection is thought to be self-limiting. However, little is known about its molecular mechanism. In this study, we found that a novel goose astrovirus (GAstV), GAstV-GD, and its open reading frame 2 (ORF2) could efficiently activate the innate immune response and induce a high level of OASL in vitro and in vivo The truncation assay for ORF2 further revealed that the P2 domain of ORF2 contributed to stimulating OASL, whereas the acidic C terminus of ORF2 attenuated such activation. Moreover, the overexpression and knockdown of OASL could efficiently restrict and promote the viral replication of GAstV-GD, respectively. Our data not only give novel insights for elucidating self-limiting infection by astrovirus but also provide virus and host targets for fighting against astroviruses.IMPORTANCE Astroviruses cause gastroenteritis and encephalitis in human, and nephritis, hepatitis, and gout disease in poultry. However, the host immune response activated by astrovirus is mostly unknown. Here, we found that a novel goose astrovirus, GAstV-GD, and its ORF2 protein could efficiently induce a high level of OASL in vitro and in vivo, which could feed back to restrict the replication of GAstV-GD, revealing novel innate molecules triggered by astroviruses and highlighting that the ORF2 of GAstV-GD and OASL can be potential antiviral targets for astroviruses.

Fiber-1, Not Fiber-2, Directly Mediates the Infection of the Pathogenic Serotype 4 Fowl Adenovirus via Its Shaft and Knob Domains.

Recently, the disease of hepatitis-hydropericardium syndrome (HPS) caused by serotype 4 fowl adenovirus (FAdV-4) has spread widely and resulted in huge economic losses to the poultry industry. Although the genome of FAdV-4 has two fiber genes (fiber-1 and fiber-2), the exact role of the genes in the infection of FAdV-4 is barely known. In this study, through superinfection resistance analysis and an interfering assay, we found that fiber-1, but not fiber-2, was the key factor for directly triggering the infection of FAdV-4. The truncation analysis further revealed that both of the shaft and knob domains of fiber-1 were required for the infection. Moreover, the sera against the knob domain were able to block FAdV-4 infection, and the knob-containing fusion protein provided efficient protection against the lethal challenge of FAdV-4 in chickens. All the data demonstrated the significant roles of fiber-1 and its knob domain in directly mediating the infection of FAdV-4, which established a foundation for identifying the receptor of FAdV-4 and developing efficient vaccines against FAdV-4.IMPORTANCE Among 12 serotypes of fowl adenovirus (FAdV), FAdV-1, FAdV-4, and FAdV-10 all carry two fiber genes (i.e., fiber-1 and fiber-2), whereas other serotypes have only one. As important viral surface proteins, the fibers play vital roles in the infection and pathogenesis of FAdV. However, the importance of the fibers to the infection and pathogenesis of FAdV may be different from each other. Recent studies reveal that fiber-2 is identified as a determinant of virulence, but which fiber triggers the infection of FAdV-4 remains unknown. In this study, fiber-1 was identified as a key factor for directly mediating the infection of FAdV-4 through its shaft and knob domains, whereas fiber-2 did not play a role in triggering FAdV-4 infection. The results suggest that fiber-1 and its knob domain may serve as a target for identifying the receptor of FAdV-4 and developing efficient drugs or vaccines against FAdV-4.

A novel fiber-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4 fowl adenovirus.

Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.

Alternative Strategy for a Quadrivalent Live Attenuated Influenza Virus Vaccine.

Influenza virus infections continue to pose a major public health threat worldwide associated with seasonal epidemics and sporadic pandemics. Vaccination is considered the first line of defense against influenza. Live attenuated influenza virus vaccines (LAIVs) may provide superior responses compared to inactivated vaccines because the former can better elicit a combination of humoral and cellular responses by mimicking a natural infection. Unfortunately, during the 2013-2014, 2014-2015, and 2015-2016 seasons, concerns emerged about the effectiveness of the only LAIV approved in the United States that prevented the Advisory Committee on Immunization Practices (ACIP) from recommending its use. Such drawbacks open up the opportunity for alternative LAIV strategies that could overcome such concerns. Previously, we developed a combined strategy of temperature-sensitive mutations in the PB2 and PB1 segments and an epitope tag in the C terminus of PB1 that effectively attenuates influenza A viruses of avian and mammalian origin. More recently, we adopted a similar strategy for influenza B viruses. The resulting attenuated (att) influenza A and B viruses were safe, immunogenic, and protective against lethal influenza virus challenge in a variety of animal models. In this report, we provide evidence of the potential use of our att strategy in a quadrivalent LAIV (QIV) formulation carrying H3N2 and H1N1 influenza A virus subtype viruses and two antigenic lineages of influenza B viruses. In naive DBA/2J mice, two doses of the QIV elicited hemagglutination inhibition (HI) responses with HI titers of ≥40 and effectively protected against lethal challenge with prototypical pandemic H1N1 influenza A and influenza B virus strains.IMPORTANCE Seasonal influenza viruses infect 1 billion people worldwide and are associated with ∼500,000 deaths annually. In addition, the never-ending emergence of zoonotic influenza viruses associated with lethal human infections and of pandemic concern calls for the development of better vaccines and/or vaccination strategies against influenza virus. Regardless of the strategy, novel influenza virus vaccines must aim at providing protection against both seasonal influenza A and B viruses. In this study, we tested an alternative quadrivalent live attenuated influenza virus vaccine (QIV) formulation whose individual components have been previously shown to provide protection. We demonstrate in proof-of principle studies in mice that the QIV provides effective protection against lethal challenge with either influenza A or B virus.

Two novel monoclonal antibodies against fiber-1 protein of FAdV-4 and their application in detection of FAdV-4/10.

BACKGROUND: Recently, serotype 4 fowl adenovirus (FAdV-4) has spread widely and caused huge economic loss to poultry industry. However, little is known about the molecular pathogenesis of FAdV-4. Fiber protein is thought to be vital for its infection and pathogenesis. RESULTS: Two novel monoclonal antibodies (mAbs) targeting the fiber-1 protein of FAdV-4 were generated, designated as mAb 3B5 and 6H9 respectively. Indirect immunofluorescence assay (IFA) showed that both mAbs only reacted with the FAdV-4 and FAdV-10, not with other serotypes including FAdV-1, FAdV-5, FAdV-6, FAdV-7, FAdV-8 and FAdV-9 tested. Although both mAbs did not recognize the linear epitopes, they could efficiently immunoprecipitate the fiber-1 protein in LMH cells either infected with FAdV-4 or transfected with pcDNA3.1-Fiber-1. Moreover, mAb 3B5 as a capture antibody and HRP-conjugated mAb 6H9 as a detection antibody, a novel sandwich ELISA for efficient detection of FAdV-4 was generated. The limit of detection of the ELISA could reach to 1000 TCID50/ml of FAdV-4 and the ELISA could be efficiently applied to detect FAdV-4 in the clinical samples. CONCLUSION: The two mAbs specific targeting fiber-1 generated here would pave the way for further studying on the role of fiber-1 in the infection and pathogenesis of FAdV-4, and the established mAb based sandwich ELISA would provide an efficient diagnostics tool for detection of FAdV-4/10.

Development of an Alternative Modified Live Influenza B Virus Vaccine.

Influenza B virus (IBV) is considered a major human pathogen, responsible for seasonal epidemics of acute respiratory illness. Two antigenically distinct IBV hemagglutinin (HA) lineages cocirculate worldwide with little cross-reactivity. Live attenuated influenza virus (LAIV) vaccines have been shown to provide better cross-protective immune responses than inactivated vaccines by eliciting local mucosal immunity and systemic B cell- and T cell-mediated memory responses. We have shown previously that incorporation of temperature-sensitive (ts) mutations into the PB1 and PB2 subunits along with a modified HA epitope tag in the C terminus of PB1 resulted in influenza A viruses (IAV) that are safe and effective as modified live attenuated (att) virus vaccines (IAV att). We explored whether analogous mutations in the IBV polymerase subunits would result in a stable virus with an att phenotype. The PB1 subunit of the influenza B/Brisbane/60/2008 strain was used to incorporate ts mutations and a C-terminal HA tag. Such modifications resulted in a B/Bris att strain with ts characteristics in vitro and an att phenotype in vivo Vaccination studies in mice showed that a single dose of the B/Bris att candidate stimulated sterilizing immunity against lethal homologous challenge and complete protection against heterologous challenge. These studies show the potential of an alternative LAIV platform for the development of IBV vaccines.IMPORTANCE A number of issues with regard to the effectiveness of the LAIV vaccine licensed in the United States (FluMist) have arisen over the past three seasons (2013-2014, 2014-2015, and 2015-2016). While the reasons for the limited robustness of the vaccine-elicited immune response remain controversial, this problem highlights the critical importance of continued investment in LAIV development and creates an opportunity to improve current strategies so as to develop more efficacious vaccines. Our laboratory has developed an alternative strategy, the incorporation of 2 amino acid mutations and a modified HA tag at the C terminus of PB1, which is sufficient to attenuate the IBV. As a LAIV, this novel vaccine provides complete protection against IBV strains. The availability of attenuated IAV and IBV backbones based on contemporary strains offers alternative platforms for the development of LAIVs that may overcome current limitations.

Novel avian leukosis viruses from domestic chicken breeds in mainland China.

Two novel avian leukosis viruses (ALVs) were isolated from 1380 whole blood samples taken from domestic chicken breeds in China. The two ALVs were uniquely different from the env (Envelope) genes of ALV A-J and carried an LTR (long terminal repeat) cluster from ALV-E. Large scale sequence analysis further showed that these ALVs (with different env and LTRs) were recently endemic in domestic chicken breeds in both China and Japan. The emergence of these novel ALVs is challenging the current ALV eradication program, and as such novel ALVs should be monitored in a timely and careful manner to stop their transmission and further recombination in the future.

Antigenic mapping of the hemagglutinin of an H9N2 avian influenza virus reveals novel critical amino acid positions in antigenic sites.

H9N2 influenza virus is undergoing extensive genetic and antigenic evolution, warranting detailed antigenic mapping of its hemagglutinin (HA). Through examining antibody escape mutants of an Asian avian H9N2 virus, we identified 9 critical amino acid positions in H9 antigenic sites. Five of these positions, 164, 167, 168, 196, and 207, have not been reported previously and, thus, represent novel molecular markers for monitoring the antigenic change of H9N2 virus.

The amino acid variation at hemagglutinin sites 145, 153, 164 and 200 modulate antigenicity andreplication of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes circulating in the poultry industry, inflicts substantial economic damage. Mutations in the hemagglutinin (HA) and neuraminidase (NA) proteins of H9N2 frequently alter viral antigenicity and replication. In this paper, we analyzed the HA genetic sequences and antigenic properties of 26 H9N2 isolates obtained from chickens in China between 2012 and 2019. The results showed that these H9N2 viruses all belonged to h9.4.2.5, and were divided into two clades. We assessed the impact of amino acid substitutions at HA sites 145, 149, 153, 164, 167, 168, and 200 on antigenicity, and found that a mutation at site 164 significantly modified antigenic characteristics. Amino acid variations at sites 145, 153, 164 and 200 affected virus's hemagglutination and the growth kinetics in mammalian cells. These results underscore the critical need for ongoing surveillance of the H9N2 virus and provide valuable insights for vaccine development.

Molecular epidemiology of infectious bronchitis virus in eastern and southern China during 2021-2023.

As a highly infectious and contagious pathogen in chickens, infectious bronchitis virus (IBV) is currently grouped into nine genotypes (GI to GIX). However, the classification of serotypes of IBV is still not clear. In this study, 270 field strains of IBV were isolated from dead or diseased chicken flocks in eastern and southern China during January 2021 to April 2023. These isolated IBV strains could be classified into 2 genotypes, GI (including 5 lineages GI-1, GI-13, GI-19, GI-22, and GI-28) and GVI based on the complete S1 sequence. Further analysis showed that the GI-19, GI-13, GI-22, GI-28, and GVI were the dominant genotypes with the proportions of 61.48, 8.89, 8.89, 7.78, and 8.89% respectively, and the homology of S1 protein of these isolates ranged from 86.85 to 100% in GI-19, 92.22 to 100% in GI-13, 83.1 to 100% in GI-22, 94.81 to 100% in GI-28 and 90.0 to 99.8% in GVI, respectively. Moreover, cross-neutralization test with sera revealed that these isolates in GI-19 lineage could be classified into at least 3 serotypes according to the antigenic relationship. In addition, structure assay using PyMOL indicated that one mutation such as S120 in receptor binding site (RBD) of GI-19 might alter the antigenicity and conformation of S protein of IBV. Overall, our data demonstrate that not only multiple genotypes, but also multiple serotypes in a single genotype or lineage have been co-circulated in eastern and southern China, providing novel insights into the molecular evolution of the antigenicity of IBV and highlighting the significance of the selection of the dominant isolate for vaccine development in IBV endemic region.

Isolation and genomic characterization of chicken infectious anemia virus in Jiangsu province of China during 2020-2022.

As an immunosuppressive disease virus, chicken infectious anemia virus (CIAV) mainly infects chickens, causing aplastic anemia and systemic lymphoid tissue atrophy. In recent years, the prevalence of CIAV in the poultry industry globally has caused huge economic losses. In this study, a total of 223 clinical samples, including anal swabs, tissues, blood, and vaccines, were collected from 19 broiler farms or breeding companies in Jiangsu province, with symptoms of significant anemia and immunosuppression during 2020-2022. Among them, 75 samples (75/223, 33.6%) were positive for CIAV in polymerase chain reaction (PCR) test, and 20 CIAV strains were successfully isolated. The phylogenetic trees of the 20 isolates and 42 CIAV strains deposited in GenBank formed four distinct groups (A-D). And the isolates mainly belonged to Group A but with high genetic diversity. Analysis for VP1 indicated that these isolates possess key characteristics of highly pathogenic strains. Meanwhile, VP2 and VP3 were much conserved with much fewer mutations compare to VP1. The above epidemiological study of CIAV provides novel insights into molecular characterization of CIAV and lays the foundation for developing efficient strategies for control of CIAV in China.

Research Note: A novel peptide-based ELISA for efficient detection of antibody against chicken infectious anemia virus.

Chicken infectious anemia virus (CIAV) is the pathogen of chicken infectious anemia. Currently, due to the lack of effective diagnostics technology and prevention approach, CIAV has spread globally and caused huge economic losses to poultry industry. In this study, a novel peptide-based ELISA (pELISA) for efficient detection of antibody against CIAV was developed. The peptide (25CRLRRRYKFRHRRRQRYRRRAF45) used in pELISA was highly conserved in VP1 protein of different CIAV isolates. The specificity and reproducibility showed that the pELISA only reacted with sera against CIAV, not with sera against other pathogens tested, and the CV of the intra-/inter-assay of the pELISA was 6.8 to 9.22%. Moreover, the comparison assay using 56 clinical samples showed that the positive rate of the pELISA and the commercial ELISA kit (IDEXX) was 85.7 and 80.4%, respectively. The pELISA generated here provides a rapid and efficient serological detection method for diagnosis of CIAV infection and evaluation of the efficacy of CIAV vaccination.

A novel monoclonal antibody efficiently blocks the infection of duck adenovirus 3 by targeting Fiber-2.

Duck adenovirus 3 (DAdV-3), identified as the causative agent of a disease characterized by swelling and hemorrhage of liver and kidney, has caused substantial economic losses to duck industry in China. However, the neutralizing epitopes and the infection mechanism of DAdV-3 have not been extensively elucidated. In this study, a novel monoclonal antibody (mAb) targeting Fiber-2 protein of DAdV-3 was generated and designated as mAb 3E7. Indirect immunofluorescence assay showed that mAb 3E7 specifically reacted with the Fiber-2 in LMH cells transfected with pcDNA3.1-Fiber-2 or infected with DAdV-3. Moreover, mAb 3E7 could immunoprecipitate the Fiber-2 and efficiently inhibit the infection of DAdV-3 in vitro. Further epitope mapping revealed mAb 3E7 recognized the epitope 108LALGDGLE115 in Fiber-2, which was highly conserved among DAdV-3 strains. These findings not only identified a novel neutralizing epitope in Fiber-2, but also paved the way for further elucidating the vital roles of Fiber-2 in the infection and pathogenesis of DAdV-3.

A peptide-based ELISA for detection of antibodies against novel goose astrovirus type 1.

Goose gout disease is a high morbidity and mortality disease caused by novel serotype 1 goose astrovirus (GAstV-1), which has resulted in huge economic loss to the goose industry of China. However, few diagnostic methods have been developed for serological surveillance of GAstV-1. In our previous study, several novel B cell epitopes were identified in the ORF2 protein of GAstV-1. In this study, one novel peptide of 627-646 aa in the ORF2 recognized by monoclonal antibody (mAb) 6C6 was used as an antigen to develop an efficient peptide-based ELISA (pELISA) for detection of antibodies against GAstV-1. Specificity analysis showed that the pELISA only reacted with sera against GAstV-1, but not with sera against other pathogens tested. The sensitivity of the pELISA in detecting positive sera was higher than that of the IFA (Indirect immunofluorescence assay). The coefficients of variation (CV) of the intra-assay and inter-assay were both < 10%, indicating that the reproducibility of pELISA was good. For detection of clinical samples, the pELISA had 87.5% concordance with the IFA. Our data demonstrate that the pELISA generated here provides an accurate, rapid, and economical method for the detection antibodies against GAstV-1 for serological surveillance.

An efficient double-fluorescence approach for generating fiber-2-edited recombinant serotype 4 fowl adenovirus expressing foreign gene.

Recently, the infection of serotype 4 fowl adenovirus (FAdV-4) in chicken flocks has become endemic in China, which greatly threatens the sustainable development of poultry industry. The development of recombinant FAdV-4 expressing foreign genes is an efficient strategy for controlling both FAdV-4 and other important poultry pathogens. Previous reverse genetic technique for generating the recombinant fowl adenovirus is generally inefficient. In this study, a recombinant FAdV-4 expressing enhanced green fluorescence protein (EGFP), FA4-EGFP, was used as a template virus and directly edited fiber-2 gene to develop an efficient double-fluorescence approach to generate recombinant FAdV-4 through CRISPR/Cas9 and Cre-Loxp system. Moreover, using this strategy, a recombinant virus FAdV4-HA(H9) stably expressing the HA gene of H9N2 influenza virus was generated. Chicken infection study revealed that the recombinant virus FAdV4-HA(H9) was attenuated, and could induce haemagglutination inhibition (HI) titer against H9N2 influenza virus at early time points and inhibit the viral replication in oropharynx. All these demonstrate that the novel strategy for constructing recombinant FAdV-4 expressing foreign genes developed here paves the way for rapidly developing attenuated FAdV-4-based recombinant vaccines for fighting the diseases caused by both FAdV-4 and other pathogens.

Identification of novel B cell epitopes in Fiber-2 protein of duck adenovirus 3 and their application.

Duck adenovirus 3 (DAdV-3), a newly emerged duck adenovirus, has resulted in significant economic losses to the duck industry across China since 2014. However, little is known about the B cell epitopes in major antigen of DAdV-3 and the serological approach for detection of DAdV-3 is not available. In this study, four monoclonal antibodies (mAbs) specific to Fiber-2 protein of DAdV-3 were first generated and designated as 2G10, 3D9, 5E6, and 6B12. Indirect immunofluorescence assay (IFA) showed that all of the mAbs reacted with the Fiber-2. Moreover, mAbs 2G10, 5E6, and 6B12 demonstrated good activity with Fiber-2 in Western blot. Notably, the Fiber-2 could be immunoprecipitated efficiently by mAb 3D9. Epitope mapping revealed that mAbs 2G10, 3D9, 5E6, and 6B12 recognized 397-429aa, 463-481aa, 67-99aa, and 1-66aa of Fiber-2, respectively. Besides, a novel sandwich ELISA for efficient detection of DAdV-3 was developed based on mAb 3D9 and horseradish peroxidase (HRP) conjugated mAb 3D9. The sandwich ELISA only reacted with DAdV-3 but not with other duck-associated viruses. The limit of detection of the ELISA was 6.25 × 103 TCID50/mL. Overall, the mAbs generated laid the foundation for elucidating the critical role of Fiber-2 in mediating infection and pathogenesis, and the sandwich ELISA approach established here provided efficient and rapid serological diagnostic tool for DAdV-3.

Efficient cross-protection against serotype 4/8a fowl adenoviruses (FAdVs): recombinant FAdV-4 with FAdV-8a Fiber.

IMPORTANCE: Epidemiological data reveal that FAdV-4 and FAdV-8a are the dominant serotypes of FAdVs in the poultry industry in China. Although three commercial inactivated vaccines against FAdV-4 have been licensed in China, the bivalent vaccine against both FAdV-4 and FAdV-8a is not available. Here, we used CRISPR-Cas9 and Cre-LoxP system to generate a recombinant virus FAdV4-F/8a-rF2 expressing the Fiber of FAdV-8a. Notably, FAdV4-F/8a-rF2 was highly attenuated and could provide efficient protection against both FAdV-4 and FAdV-8a in the chicken infection model, highlighting the applaudable application of FAdV4-F/8a-rF2 as a novel live-attenuated bivalent vaccine against the diseases caused by the infection of FAdV-4 and FAdV-8a.