An Inactivated Novel Trivalent Vaccine Provides Complete Protection against FAdV-4 Causing Hepatitis-Hydropericardium Syndrome and FAdV-8b/-11 Causing Inclusion Body Hepatitis

Outbreaks of hepatitis-hydropericardium syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) and inclusion body hepatitis (IBH) related to FAdV-8b and FAdV-11 have been increased in chickens in China since 2015. Clinical concurrent infections of FAdV-4, FAdV-8b, and FAdV-11 are quite common, yet there are no commercially available trivalent vaccines against infection by these three serotypes. In our previous study, a bivalent vaccine based on a recombinant FAdV-4, of which fber -1 was replaced with the fber of FAdV-8b, has been developed. In this study, a novel recombinant rFAdV-4-fber/8b +11 was constructed by inserting FAdV-11 fber gene into the 1966-bp deletion region of rFAdV-4-fber/8b genome. Te in vitro replication ability of the rFAdV-4-fber/8b +11 was similar to the parental FAdV-4. One dose immunization with the inactivated rFAdV-4-fber/8b +11 vaccine generated robust immune responses against FAdV-4, FAdV-8b, and FAdV-11, and provided efcient clinical protection against FAdV-4, FAdV-8b, and FAdV-11 challenge. Tis study provides a novel strategy for developing potential trivalent vaccines for the prevention and control of HHS and IBH.


In vitro Growth Properties and Stability of the rFAdV-4-
Fiber/8b + 11.Te rescued rFAdV-4-fber/8b + 11 was serially passaged continuously in LMH cells.Te growth kinetics of the 15 th passage of rFAdV-4-fber/8b + 11 and wildtype FAdV-4 was assessed in LMH cells at a multiplicity of infection of 0.001, and the viruses were collected at 24, 48, 60, 72, 96, and 120 h postinfection (hpi), respectively.Te median tissue culture infective dose (TCID 50 ) of the collected viruses was determined in three technical replicates and calculated by the Reed-Muench method and presented as the mean ± standard error of mean.

Enzyme-Linked Immunosorbent Assay (ELISA).
Te antibody responses against FAdV-4, FAdV-8b, and FAdV-11 induced by the inactivated vaccine were evaluated by a FAdV-specifc indirect ELISA with some modifcations [33].Briefy, ELISA plates were coated with E. coli expressed Fiber-2 protein of FAdV-4, Fiber protein of FAdV-8b, or FAdV-11, respectively.After washing with PBS containing 0.1% Tween-20 (PBST), diluted sera were added and incubated at 37 °C for 1 h After repeated washing with PBST, the plate was incubated with rabbit antichicken IgY conjugated with horseradish peroxidase (Abcam, Cambridge, UK) at 37 °C for 1 h.Ten, the plate was washed again and incubated with TMB substrates (Solarbio, Beijing, China) for 10 min at 37 °C.Ten, 2 M sulphuric acid (Solarbio, Beijing, China) was added to each well to stop the reaction.Te OD450 nm was measured on the Multiskan GO spectrophotometer (Termo Fisher Scientifc, MA, USA).

Quantifcation of Viral Loads and Detection of Viral
Shedding.To quantify the viral loads in tissues and detect the viral shedding through cloaca, total DNA was extracted from tissue or cloacal swab samples using commercial DNA extraction kit (Tiangen, Beijing, China).Te viral loads of challenged viruses were determined by a previously described SYBR Green I quantitative real-time PCR (qRT-PCR) [34].Te fnal viral DNA concentration was calculated as copy numbers per milligram of the tissue sample.

Statistical Analysis.
All the data were presented as the means ± standard error of mean.Statistical analysis in this study was executed using the Unpaired t-test within the GraphPad 6 software.P < 0.05 was considered statistically signifcant.
To further evaluated the efcacy of the trivalent vaccine, the gross and microscopic lesions, viral DNA in diferent tissues and viral shedding through cloaca were examined.As indicated in Figure 5(a), After FAdV-4 challenge, all sick/ dead chickens in FAdV-4 challenge control group exhibited typical HHS-indicative gross lesions.In contrast, chickens immunized with the inactivated rFAdV4-fber/8b + 11 vaccine did not show any obvious lesions (Figure 5(a)).FAdV-8b or FAdV-11 challenge control chickens groups presented typical gross IBH-indicative lesions, characterized by swollen livers with white focal necrosis or petechial hemorrhages (Figures 5(b) and 5(c)).However, no obvious gross lesions in the kidney and heart were observed in unvaccinated and FAdV-8b or FAdV-11 challenged chickens (data not shown).
Te typical histopathological lesions presented by the sick/dead chickens in the FAdV-4 challenge control group included widened myocardial cell gap and myocardial fber rupture; severe degeneration and necrosis of hepatocytes; degeneration and necrosis of renal tubular epithelia; necrosis of mucosal epithelia in duodenums; and necrosis and depletion of lymphocytes in the bursa of Fabricius (Figure 6(a)).Comparing with the massive severe degeneration and necrosis of hepatocytes caused by FAdV-4, the primary histopathological lesions presented by the unvaccinated and FAdV-8b or FAdV-11 challenge groups included multifocal degeneration and necrosis of hepatocytes and mononuclear cell infltration (Figure 6(b)).However, the inactivated rFAdV4-fber/8b + 11 vaccine immunized chickens did not show HHS-or IBH-indicative histopathological lesions (Figures 6(a As indicated in Figure 7, viral DNA copy numbers in diferent tissues from vaccinated chickens were signifcantly lower than those of challenge control chickens.Only Transboundary and Emerging Diseases a background level of viral loads was detected in most tissues from vaccine immunized and uninfected control chickens.Te efcacy of inactivated rFAdV4-fber/8b + 11 vaccine protected chickens from shedding challenge virus was also assessed.As shown in Table 2, vaccinated chickens stopped excreting challenged FAdV-4, FAdV-8b, and FAdV-11 after 3, 4, and 5 dpc, respectively.All the challenge control chickens excreted the challenge viruses until the end of experiment.

Discussion
FAdV have been related with numerous disease conditions in chickens.FAdV-4 causing HHS and FAdV-8b or FAdV-11 causing IBH have spread worldwide and resulted in signifcant economic losses to the world poultry industry.
Vaccination has been demonstrated as one of the most efective and cost-efcient infectious disease interventions that there is.However, the control of HHS and IBH turns out to be more complicated by the involvement of multiple FAdV serotypes [3].Most HHS are caused by FAdV-4, whereas FAdV-2, FAdV-8a, FAdV-8b, and FAdV-11 are related to IBH.Te outbreaks of HHS and IBH have been increased since 2015 [13,26,35].Tere is an urgent need for developing multivalent vaccines against various FAdV serotypes causing HHS and IBH.
FAdV-4 possesses two fber genes that encode Fiber-1 and Fiber-2 proteins.Fiber-1 plays crucial role in mediating FAdV-4 infection and Fiber-2 plays important roles in inducing protective immune responses [37,43,44].FAdV-8b and FAdV-11 only have one fber gene in their genomes.Based on the above facts, we speculate that the Fiber of FAdV-8b or FAdV-11 plays dual roles in mediating viral infection and inducing efective antiviral immune responses.In our previous study, a recombinant FAdV-4, rFAdV-4fber/8b was successfully constructed by replacing fber-1 of FAdV-4 with the fber of FAdV-8b [29].Te recombinant rFAdV-4-fber/8b maintained efcient replication capacity and pathogenicity as the parent FAdV-4.Te inactivated bivalent vaccine based on the rFAdV-4-fber/8b also provided efcient protection against FAdV-4 causing HHS and FAdV-8b causing IBH.As the 1966-bp natural deletion region and more than 20 genes in the FAdV-4 genome have been identifed as potential insertion sites for foreign genes [19,45], thus using wild type or modifed FAdV-4 as a vector to express fber genes from other serotypes of FAdV provides another strategy for developing multivalent vaccines for simultaneous control of HHS and IBH.
Epidemiological data have indicated that FAdV-4, FAdV-8b, and FAdV-11 are the predominant serotypes in China [25,46,47].Clinical concurrent infections of various serotypes of FAdV are quite common, yet there are no trivalent vaccines for the prevention and control of HHS and IBH.In the present study, a novel recombinant rFAdV-4fber/8b + 11 coexpressing Fibers of FAdV-8b and FAdV-11 was constructed by inserting the fber gene of FAdV-11 into the 1966-bp deletion region of the rFAdV-4-fber/8b genome.Te rFAdV-4-fber/8b + 11 showed similar growth kinetics in vitro with the wild-type FAdV-4.A single immunization with the inactivated rFAdV-4-fber/8b + 11 vaccine generated specifc immune responses against FAdV-4, FAdV-8b, and FAdV-11.All immunized chickens developed full resistance to clinical disease and did not present HHS-and IBH-indicative gross and histopathological lesions.Compared with unvaccinated and challenged chickens, vaccinated chickens exhibited signifcantly lower viral loads in various tissues.Tis study reconfrmed Fiber as a critical immunogen for developing vaccines against diseases caused by various FAdV.Since both the recombinant rFAdV-4-fber/8b in our previous study and the recombinant rFAdV-4-fber/8b + 11 in the present study can replicate efciently as the parent FAdV-4, it implies that the Fiber of FAdV-8b and Fiber-1 of FAdV-4 might play the similar role in initiating viral infection of target cells.In order to   Transboundary and Emerging Diseases verify whether the Fiber of FAdV-11 may also play a similar role as Fiber-1 of FAdV-4, we are making another chimeric FAdV-4 in which fber-1 of FAdV-4 will be seamlessly replaced with the fber of FAdV-11.In summary, a novel chimeric FAdV-4 coexpressing Fibers of FAdV-8b and FAdV-11 was generated for the frst time.Te inactivated rFAdV-4-fber/8b + 11 could be used as a trivalent vaccine for simultaneous prevention of the concurrent infection of FAdV-4, FAdV-8b, and FAdV-11.Tis study provides a novel strategy for developing potential multivalent vaccines for the prevention of IBH and HHS.

Figure 5 :
Figure 5: Te representative gross lesion in the heart, liver, and kidney from chickens in each group after challenging with FAdV-4 (a), FAdV-8b (b), and FAdV-11 (c).(a) Chickens in the unvaccinated and FAdV-4 challenge group showed accumulation of clear straw-colored fuid in the pericardial sac, swollen and discolored liver, and enlarged kidneys with distended tubules.(b) and (c) Chickens in the FAdV-8b or FAdV-11 challenge control group exhibited IBH-indicative gross lesions, characterized by swollen livers with small white focal necrosis and petechial hemorrhages.

Figure 6 :Figure 7
Figure 6: Representative histopathological lesions in tissues from chickens in each group after challenging with FAdV-4, FAdV-8b, and FAdV-11.(a) Histopathological lesions including widened myocardial cell gap and myocardial fber rupture; severe degeneration and necrosis of hepatocytes; degeneration and necrosis of renal tubular epithelium; mucosal epithelial cell nuclear fragmentation and necrosis in duodenums; and severe depletion and necrosis of lymphocytes in the bursa of Fabricius were observed in the chickens from the unvaccinated and FAdV-4 challenge group.(b) Compared with the massive severe degeneration and necrosis of hepatocytes caused by FAdV-4, the primary histopathological lesions presented by the unvaccinated and FAdV-8b or FAdV-11 challenge groups included multifocal degeneration and necrosis of hepatocytes and mononuclear cell infltration.However, chickens from the inactivated rFAdV4-fber/8b + 11 vaccinated group did not present histopathological lesions of HHS and IBH mentioned above (HE staining, original magnifcation ×400).

Table 1 :
Primers used for generation of recombinant virus and detection of viral shedding.