Research Note: Rapid generation of a novel fowl adenovirus serotype 11 reverse genetic platform.

Fowl adenovirus serotype 11 (FAdV-11) is one of the main causative agents of inclusion body hepatitis (IBH) in broilers. Outbreaks of FAdV-11-related IBH have been increasingly reported in China and many other geographical areas worldwide. However, the critical virulence factors of FAdV-11 remain uncertain due to the lack of technical platforms for efficient manipulation of FAdV-11 genome. Here, we reported the establishment of a FAdV-11 reverse genetic system based on a novel FAdV-11 Chinese isolate FJSW/2021 using the exonuclease combined with RecET (ExoCET), Redαß recombineering and ccdB counter-selection techniques for the first time. A recombinant FAdV-11 was rescued efficiently by using the established reverse genetic platform through swapping the ORF11 gene of the FAdV-11 FJSW/2021 with the ZsGreen fluorescent protein expression cassette. This study provides an effective technical platform for identifying virulence factors of FAdV-11 and developing recombinant FAdV-11-vectored vaccine candidates.

Genomic characterization and pathogenicity of a novel fowl adenovirus serotype 11 isolated from chickens with inclusion body hepatitis in China.

Fowl adenovirus serotype 11 (FAdV-11) is one of the primary causative agents of inclusion body hepatitis (IBH), which causes substantial economic losses in the world poultry industry. In this study, we characterized the genome of the fowl adenovirus serotype 11 (FAdV-11) isolate FJSW/2021. The full genome of FJSW/2021 was 44, 154 base pairs (bp) in length and had a similar organization to that of previously reported FAdV-11 isolates. Notably, compared with those of other reported FAdV-11 strains, the preterminal protein (pTP) of FAdV-11 FJSW/2021 has six amino acid (aa) insertions (S-L-R-I-I-C) between 470 and 475 and one aa mutation of L476F; moreover, the tandem repeat (TR) regions of TR1 and TR2 were 33 bp (1 repeat) and 1,080 bp (8 repeats) shorter than those of the Canadian nonpathogenic isolate ON NP2, respectively. The pathogenicity of FJSW/2021 was studied in 10-day-old specific pathogen-free chicken embryos following allantoic cavity inoculation and in 1-day-old, 1-wk-old and 2-wk-old SPF chickens following intramuscular inoculation with 107 TCID50 of the virus. The results showed that FJSW/2021 can induce typical severe IBH in chicks less than 2 wk old. These findings highlighted the genetic differences between the pathogenic and non-pathogenic FAdV-11 isolates. The data will provide guidance for identifying the virulence factors of FAdV-11 strains. The animal challenge model developed in our study will allow precise evaluation of the efficacy of potential FAdV-11 vaccine candidates.

An inactivated novel chimeric FAdV-4 containing fiber of FAdV-8b provides full protection against hepatitis-hydropericardium syndrome and inclusion body hepatitis.

Fowl adenovirus serotype 4 (FAdV-4) and FAdV-8b are causative agents of hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH), respectively. HHS and IBH co-infections were often reported in clinical, yet there are no commercially available bivalent vaccines for prevention and control of both FAdV-4 and -8b. In the present study, a chimeric FAdV-4 was firstly generated by substituting fiber-1 of FAdV-4 with fiber of FAdV-8b. The chimeric virus, rFAdV-4-fiber/8b, exhibited similar replication ability in vitro and pathogenicity in vivo to the parental wild type FAdV-4. A single dosage of vaccination with the inactivated rFAdV-4-fiber/8b induced high antibody titers against fiber-2 of FAdV-4 and fiber of FAdV-8b and provided full protection against FAdV-4 and -8b challenge. These results demonstrated that fiber of FAdV-8b could replace the role of fiber-1 of FAdV-4 in the process of viral infection, and rFAdV-4-fiber/8b could be used to make a potential bivalent vaccine for the control and prevention of HHS and IBH.

The Role of Hexon Amino Acid 188 Varies in Fowl Adenovirus Serotype 4 Strains with Different Virulence.

Hepatitis-hydropericardium syndrome (HHS) induced by fowl adenovirus serotype 4 (FAdV-4) has caused huge economic losses to poultry industries. The key genes responsible for different virulence of FAdV-4 strains are not fully elucidated. Previous studies indicated that hexon of pathogenic FAdV-4 has a conserved arginine (R) at position 188, and a conserved isoleucine (I) is present at this position in reported nonpathogenic FAdV-4. Recently, it was reported that R188 of hexon is the determinant site for pathogenicity of the emerging Chinese FAdV-4 strain. However, the role of hexon amino acid 188 (aa188) has not been examined in the nonpathogenic FAdV-4 strain. In this study, three recombinant FAdV-4 viruses, H/H/R188I, O/O/I188R, and H/O/I188R, were constructed by mutating hexon aa188 of FAdV-4 pathogenic strain CH/HNJZ/2015 (H) and nonpathogenic strain ON1 (O), and pathogenicity was assessed in specific-pathogen-free (SPF) chickens. Consistent with previous findings, H/O/I188R exhibited pathogenicity similar to that of CH/HNJZ/2015, yet H/H/R188I induced no mortality. Unexpectedly, all chickens infected with O/O/I188R survived. Postmortem examination of O/O/I188R-infected chickens showed typical lesions of inclusion body hepatitis rather than HHS. Expression of proinflammatory cytokines in CH/HNJZ/2015- and H/O/I188R-infected chickens was significantly higher than that in H/H/R188I-, ON1-, and O/O/I188R-infected chickens. Analysis of predicted hexon protein structures indicated that aa188 mutation leads to conformational changes in the L1 loop of HNJZ-hexon but not in ON1-hexon. In summary, the present study demonstrated that the role of hexon aa188 in the virulence of FAdV-4 varies between different strains. Induction of HHS requires factors aside from hexon aa188 in the emerging Chinese FAdV-4 strain. IMPORTANCE HHS induced by FAdV-4 has caused huge economic losses to the poultry industry. The key determinants for the different virulence of FAdV-4 have not been fully elucidated. Here, we investigated the role of hexon aa188 in FAdV-4 strains with different virulence and showed that the role of hexon aa188 varies in FAdV-4 strains with different genetic contents. The hexon R188 may be the key amino acid for causing inclusion body hepatitis by the pathogenic FAdV-4 strain, and induction of HHS by FAdV-4 may need other viral cofactors. Moreover, the hexon R188I mutation greatly affected the expression of proinflammatory cytokines induced by the pathogenic strain CH/HNJZ/2015, but no significant difference was observed between the nonpathogenic strain ON1 and ON1 with hexon I188R mutation. We found that hexon aa188 mutation induced conformational changes to hexon protein in CH/HNJZ/2015 but not in ON1, which might be the underlying reason for the changing virulence.

Hypervirulent FAdV-4 infection induces activation of the NLRP3 inflammasome in chicken macrophages.

Fowl adenovirus serotype 4 (FAdV-4) is the primary causative agent of hepatitis-hydropericardium syndrome (HHS) causing great economic losses to the world poultry industry. The exact factors responsible for the pathogenesis of hypervirulent FAdV-4 have not been completely elucidated. Hypervirulent FAdV-4 infection induces inflammatory damages in accompany with a high level of proinflammatory interleukin-1 beta (IL-1ß) secretion in a variety of organs. Investigation of the mechanisms underlying hypervirulent FAdV-4-induced IL-1ß secretion would contribute to understanding of the pathogenesis of FAdV-4. Here, we investigated whether FAdV-4 infection activates NLRP3 inflammasome in chicken macrophage cell line HD11. The results showed that stimulation of HD11 with hypervirulent FAdV-4 induced NLRP3- and Caspase-1-dependent secretion of IL-1ß. Genetic knockdown of NLRP3 or Caspase-1 expression, a critical component of inflammasome, significantly downregulated IL-1ß expression, indicating that activation of the NLRP3 inflammasome contributed to the FAdV-4-induced IL-1ß secretion. Moreover, ATP signaling and potassium efflux were involved in the process of NLRP3 inflammasome activation. Our data indicated that hypervirulent FAdV-4 infection induces the activation of NLRP3 inflammasome and followed by massive secretion of IL-1ß of macrophages, which thereby contribute to the inflamed lesion of tissues.

Evaluation of immunogenicity and protective efficacy of a novel Senecavirus A strain-based inactivated vaccine in mice.

Senecavirus A (SVA) is an emerging picornavirus associated with porcine idiopathic vesicular disease (PIVD), which is clinically indistinguishable from foot-and-mouth disease and other vesicular diseases in pigs. In recent years, the wide spread of SVA has caused huge economic losses to the world's pig industry. However, there are no vaccines currently available to prevent and control the infection of SVA due to the extensive diversity of SVA isolates and high cost of the pig model for vaccine evaluation. In the present study, a novel SVA CH-HNCY-2019 strain with unique amino-acid mutations in VP1, VP3 and 3C was isolated from the central part of China. A mouse model was proposed to for evaluation of the immunogenicity and protective efficacy of the inactivated CH-HNCY-2019 vaccine. The results indicated that one dose immunization of 107TCID50 inactivated CH-HNCY-2019 vaccine in mice induced a high titer of neutralizing antibody and complete protection. After challenging with the homologous virus, no viral RNA or histopathological damages were detected in the heart, liver, spleen, lung, kidney, intestine and brain tissues of the immunized mice. However, viral RNA and different degrees of histopathological damages were observed in all corresponding tissues of the unimmunized mice. In summary, the present study proved that mouse is a candidate animal model for the primary evaluation of the immunogenicity and protection efficacy of SVA vaccines for the first time. In addition, the inactivated SVA CH-HNCY-2019 vaccine was immunogenic and could protect mice against homologous viral challenges.

Single-Dose Vaccination of Recombinant Chimeric Newcastle Disease Virus (NDV) LaSota Vaccine Strain Expressing Infectious Bursal Disease Virus (IBDV) VP2 Gene Provides Full Protection against Genotype VII NDV and IBDV Challenge.

Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) are the two most important and widespread viruses causing huge economic losses in the global poultry industry. Outbreaks of genotype VII NDV and IBDV variants in vaccinated poultry flocks call for genetically matched vaccines. In the present study, a genetic matched chimeric NDV LaSota vaccine strain expressing VP2 gene of IBDV variant, rLaS-VIIF/HN-VP2 was generated for the first time, in which both the F and HN genes of LaSota were replaced with those of the genotype VII NDV strain FJSW. The cleavage site of the FJSW strain F protein in the rLaS-VIIF/HN-VP2 genome was mutated to the avirulent motif found in LaSota. Expression of IBDV VP2 protein was confirmed by western blot. The rLaS-VIIF/HN-VP2 maintained the efficient replication ability in embryonated eggs, low pathogenicity and genetic stability comparable to that of parental LaSota virus. One dose oculonasal vaccination of one-week-old SPF chickens with rLaS-VIIF/HN-VP2 induced full protection against genotype VII NDV and IBDV lethal challenge. These results indicate that the rLaS-VIIF/HN-VP2 is a promising bivalent vaccine to prevent infections of IBDV and genotype VII NDV.