Molecular characterization of a virulent goose astrovirus genotype-2 with high mortality in vitro and in vivo.

Goose astrovirus (GAstV) is a newly identified viral pathogen threatening waterfowl, exhibiting a high prevalence across various regions in China. Notably, the Guanghan District of Deyang City, situated in Sichuan Province, has faced a outbreak of GAstV, resulting in significant mortality among goslings due to the induction of gout-like symptoms. In our research, we successfully isolated a GAstV strain known as GAstV SCG3. This strain exhibits efficient replication capabilities, proving virulent in goslings and goose embryos. Our study delved into the characteristics of GAstV SCG3 both in vitro and in vivo. Additionally, we examined tissue phagocytosis and the distribution of GAstV SCG3 in deceased goslings using H&E staining and IHC techniques. According to the classification established by the ICTV, GAstV SCG3 falls under the category of GAstV genotype-2. Notably, it demonstrates the highest homology with the published AHAU5 sequences, reaching an impressive 98%. Furthermore, our findings revealed that GAstV SCG3 exhibits efficient proliferation exclusively in goose embryos and in LMH cells, while not manifesting in seven other types of avian and mammalian cells. Significantly, the mortality of GAstV on goslings and goose embryos are 93.1 and 80%, respectively. Moreover, the viral load in the livers of infected goslings surpasses that in the kidneys when compared with the attenuated strain GAstV SCG2. The mortality of GAstV is usually between 20% and 50%, our study marks the first report of a virulent GAstV strain with such a high mortality.

A Pigeon-Derived Sub-Genotype XXI.1.2 Newcastle Disease Virus from Bangladesh Induces High Mortality in Chickens.

Newcastle disease virus (NDV) is a significant pathogen of poultry; however, variants also affect other species, including pigeons. While NDV is endemic in Bangladesh, and poultry isolates have been recently characterized, information about viruses infecting pigeons is limited. Worldwide, pigeon-derived isolates are commonly of low to moderate virulence for chickens. Here, we studied a pigeon-derived NDV isolated in Bangladesh in 2010. To molecularly characterize the isolate, we sequenced its complete fusion gene and performed a comprehensive phylogenetic analysis. We further studied the biological properties of the virus by estimating mean death time (MDT) and by experimentally infecting 5-week-old naïve Sonali chickens. The studied virus clustered in sub-genotype XXI.1.2 with NDV from pigeons from Pakistan isolated during 2014-2018. Deduced amino acid sequence analysis showed a polybasic fusion protein cleavage site motif, typical for virulent NDV. The performed in vivo pathogenicity testing showed a MDT of 40.8 h, and along with previously established intracerebral pathogenicity index of 1.51, these indicated a velogenic pathotype for chickens, which is not typical for pigeon-derived viruses. The experimental infection of chickens resulted in marked neurological signs and high mortality starting at 7 days post infection (dpi). Mild congestion in the thymus and necrosis in the spleen were observed at an advanced stage of infection. Microscopically, lymphoid depletion in the thymus, spleen, and bursa of Fabricius were found at 5 dpi, which progressed to severe in the following days. Mild to moderate proliferation of glial cells was noticed in the brain starting at 2 dpi, which gradually progressed with time, leading to focal nodular aggregation. This study reports the velogenic nature for domestic chickens of a pigeon-derived NDV isolate of sub-genotype XXI.1.2. Our findings show that not all pigeon-derived viruses are of low virulence for chickens and highlight the importance of biologically evaluating the pathogenicity of NDV isolated from pigeons.

Novel duck reovirus exhibits pathogenicity to specific pathogen-free chickens by the subcutaneous route.

To study the pathogenicity of new duck reovirus (NDRV) to chickens, eighty 3-day-old SPF chickens were equally divided into two groups. The experimental group was inoculated with a NDRV challenge strain of 100 µL (10-5.00 ELD50/0.1 mL) by the subcutaneous (s.c.) route, and the control group was inoculated with 100 µL of sterile phosphate-buffered saline (PBS) by the same route. In the experimental group, chickens exhibited introflexion of claws, performing of splits, stunting syndrome, weight loss and death. Gross lesions such as enlargement and yellowish-white focal necroses were observed in the liver and spleen. Microscopic changes were typical including varying degrees of hepatocyte steatosis and necrosis, splenic lymphocyte necrosis, interstitial pneumonia. Viral loads were detected in lung, liver, heart, spleen, duodenum, burse and kidney. The liver and spleen viral loads remained a much higher level and maintained for a longer time, suggesting that these tissues might be the target organs. In summary, NDRV can cause systemic infections and death in chickens, which indicated that chickens may be infected by NDRV in poultry production.

Experimental co-infection of variant infectious bursal disease virus and fowl adenovirus serotype 4 increases mortality and reduces immune response in chickens.

Infectious bursal disease virus (IBDV) and fowl adenovirus serotype 4 (FAdV-4) cause infectious bursal disease (IBD) and hydropericardium-hepatitis syndrome, respectively. Recently, studies have reported co-infections of poultry with IBDV and FAdV-4, which is an important problem in the poultry industry. Here, the variant IBDV strain ZD-2018-1 and FAdV-4 isolate HB1501 were used to assess the pathogenicity of co-infection in 1-day-old specific pathogen-free (SPF) chickens. Compared with chickens infected with only FAdV-4, those coinfected with IBDV and FAdV-4 showed enhanced clinical symptoms, higher mortality, more severe tissue lesions, and higher biochemical index levels. Furthermore, the expression of interleukin (IL)-6, IL-1ß, and interferon-γ mRNAs in the IBDV-FAdV-4 coinfected chickens was delayed, and the antibody response levels were significantly lower in those birds compared with the FAdV-4-infected chickens. These results indicate that co-infection with variant IBDV ZD-2018-1 and FAdV-4 HB1501 could significantly promote the pathogenicity of FAdV-4 and reduce the immune response in chickens. This study provides the foundation for further investigation of the interaction mechanism in IBDV and FAdV-4 co-infection.

Dexamethasone reduces infectious bursal disease mortality in chickens.

Very virulent infectious bursal disease virus (vvIBDV) causes high mortality in chickens but measures to reduce the mortality have not been explored. Chickens (8-9 weeks) were treated with 3 agents before and during vvIBDV inoculation. Dexamethasone treatment reduced the mortality of infected chickens (40.7% vs. 3.7%; p < 0.001), but treatment with aspirin or vitamin E plus selenium did not affect the mortality. The bursa of Fabricius appeared to have shrunk in both dead and surviving chickens (p < 0.01). The results indicate that dexamethasone can reduce mortality in vvIBDV-infected chickens and may provide therapeutic clues for saving individual birds infected by the virus.

Pathogenesis and shedding of Usutu virus in juvenile chickens.

Usutu virus (USUV; family: Flaviviridae, genus: Flavivirus), is an emerging zoonotic arbovirus that causes severe neuroinvasive disease in humans and has been implicated in the loss of breeding bird populations in Europe. USUV is maintained in an enzootic cycle between ornithophilic mosquitos and wild birds. As a member of the Japanese encephalitis serocomplex, USUV is closely related to West Nile virus (WNV) and St. Louis encephalitis virus (SLEV), both neuroinvasive arboviruses endemic in wild bird populations in the United States. An avian model for USUV is essential to understanding zoonotic transmission. Here we describe the first avian models of USUV infection with the development of viremia. Juvenile commercial ISA Brown chickens were susceptible to infection by multiple USUV strains with evidence of cardiac lesions. Juvenile chickens from two chicken lines selected for high (HAS) or low (LAS) antibody production against sheep red blood cells showed markedly different responses to USUV infection. Morbidity and mortality were observed in the LAS chickens, but not HAS chickens. LAS chickens had significantly higher viral titers in blood and other tissues, as well as oral secretions, and significantly lower development of neutralizing antibody responses compared to HAS chickens. Mathematical modelling of virus-host interactions showed that the viral clearance rate is a stronger mitigating factor for USUV viremia than neutralizing antibody response in this avian model. These chicken models provide a tool for further understanding USUV pathogenesis in birds and evaluating transmission dynamics between avian hosts and mosquito vectors.

Poultry disease occurrences and their impacts in Ethiopia.

Poultry production contributes significantly to the livelihoods of Ethiopian farmers and to the national economy although it is hampered by different factors, including poultry diseases. There is scarcity of published evidences on the occurrence and impacts of poultry diseases although such evidences are important for policy makers in designing appropriate interventions. A total of 595 households were interviewed and 11 FGDs were conducted to collect data on the occurrence of diseases and the number of dead chickens in the last 12 months. Hence, respiratory diseases, sudden death, and eye-face-head diseases were mentioned in all of the FGDs as the most frequently occurring disease in the districts. Of households interviewed, 86.1% reported poultry disease occurrence in the last 12 months, and gastrointestinal, eye-face-head, and neurological diseases were identified to be the top three ranked diseases of chickens in the districts. Flocks with access to diagnostic services (Adj. OR = 4.16; P = 0.004) and/or access to animal health providers (Adj. OR = 10.50; P = 0.001) were more likely to report disease occurrence. In the studied population, the diseases resulted in deaths of 2219 chickens valued at 352,219.5 Birr (11,740.65 USD) and a mean crude mortality of 31.87%. Female-lead households (mean difference = 5.95%; P = 0.018) and multiple age units present on the farm (mean difference = 20.92%; P = < 0.000) had higher chicken mortality. Similarly, higher mortality was reported in flocks without access to diagnosis (mean difference = 9.97%; P = < 0.000) and vaccination (mean difference = 12.34%; P = < 0.000) services. The high occurrence of disease and mortalities might be explained by a lack of an organized poultry health service delivery system in the country. Therefore, a carefully designed health service delivery system addressing needs of poultry producers, supported by relevant policy and corresponding strategies, is recommended to address the indicated challenges. Moreover, private health providers with well-defined roles need to be engaged to successfully and sustainably solve the poultry disease problems.

Isolation of Campylobacter hepaticus from free-range poultry with spotty liver disease in New Zealand.

Case history: In October 2019, a free-range egg laying flock suffering an outbreak of spotty liver disease was investigated. Eight 32-week-old hens were examined post-mortem. Clinical and pathological findings: Five of the eight hens had sparse, focal, gross hepatic lesions typical of spotty liver disease. Histopathology of the liver showed random, focal hepatic necrosis, lymphoplasmacytic cholangitis/pericholangitis and, in one hen, severe lymphoplasmacytic cholecystitis. Campylobacter-like organisms were grown from all eight bile samples which were confirmed by PCR as Campylobacter hepaticus. The genome of C. hepaticus isolates from the outbreak were sequenced and compared to those of isolates from Australia and the United Kingdom. Phylogenetic analysis based on single nucleotide polymorphisms showed that the C. hepaticus isolates from this outbreak were most closely related to isolates from Australia. Diagnosis: Campylobacter hepaticus focal hepatic necrosis. Clinical relevance: This is the first report of an outbreak of spotty liver disease confirmed to be caused by C. hepaticus in poultry in New Zealand. Therefore infection with C. hepaticus should be considered as a differential diagnosis for mortality in laying hens around peak lay in New Zealand.

Bursa atrophy at 28 days old caused by variant infectious bursal disease virus has a negative economic impact on broiler farms in Japan.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is highly contagious, immunosuppressive and causes a negative economic impact on poultry industry. IBDV-vaccinated broiler farms at south Kyushu, Japan had a bursa-to-bodyweight ratio (BB ratio) reduction at 28 days (d) old, followed by high mortality 30 d later. We analysed the influence of the IBDV on atrophy of the bursa of fabricius (BF) and the subsequent mortality after 30 d. Ten broilers were sampled at each timepoint from the farm with high mortality at 21, 25, 28 and 35 d. A second flock from the same farm was sampled at 14, 21, 25, 28, 35 and 42 d. IBDV was detected in BF samples at 25, 28 and 35 d and at 21, 25, 28 and 35 d in the first and second flocks, respectively, using immunohistochemical staining and RT-PCR. IBDV isolates from both flocks were closely related to the China KM523643 strain. Histopathology and TUNEL assay indicated apoptosis, severe lymphoid depletion, vacuoles within follicles, lymphoid follicle atrophy and fibrosis in the BF. We observed 75% of the polyserositis and 10% of the airsacculitis at 30 D in dead broilers. The antigenic variant IBDV infection was appeared to be the main influencing factor on BF atrophy and BB ratio reduction in the broilers. High mortality in the broilers after 30 d could be due to secondary infection. The disease caused by IBDV had a negative economic impact in the farm. RESEARCH HIGHLIGHTS New variant IBDV caused bursa atrophy and reduced BB ratio in 28-day-old broilers. After vIBDV had infected broilers, at 21 days old they became immunosuppressed. High mortality at 30 days old in broilers was due to secondary infection. New vIBDV has a negative economic impact on broiler farms in Japan.

A highly pathogenic recombinant infectious bronchitis virus with adaptability in cultured cells.

Infectious bronchitis virus (IBV) of GI-19 (QX), GI-7 (TW), GI-13 (4/91) and GI-1 (Mass) lineages have been frequently detected in China in recent years. Here, An IBV strain, referred as GD17/04, was isolated from the dead yellow feather chicken vaccinated with H52 and 4/91 vaccines, whose genome sequence was obtained through high-throughput sequencing. Then it has been confirmed by the RDP and SimPlot analysis that GD17/04 is a recombinant strain deriving from YX10, 4/91, TW 2575/98 and H52 strains. Therein S1 gene of GD17/04 consists of sequences of TW2575/98 and 4/91, the former for the region of 20,371 to 21,072 nt and 21,847 to 21,975 nt, the latter for the sandwiched region of 21,073 to 21,846 nt. Moreover, as a nephropathogenic variant which caused high morbidity of 100 % and mortality of 60 %, unlike most other IBV strains, GD17/04 can cause obvious cell lesion in primary CEK cell, and even in DF-1 cells, without the process of continuous passage. As the few IBV strain can infect avian passage cell line, GD17/04 provides a material basis for further study of the interaction mechanism between IBV and avian host. Collectively, the findings highlight the significance that biological characteristics of novel strain should be studied, in addition to constant epidemiologic and molecular surveillance for IBV.

Correlation Between Clostridium perfringens Alpha- and NetB-Toxin and Chick Mortality in Commercial Broiler Farms During Different Anticoccidial Control Programs.

The purpose of the present study was to determine whether a correlation existed between chick mortality and the presence of Clostridium perfringens alpha-toxin and NetB-toxin genes (cpa and netB) in C. perfringens recovered from litter in commercial broiler houses. Because coccidiosis predisposes chickens to necrotic enteritis, the concentration of Eimeria oocysts in these samples was measured, and the numbers were used in similar correlation analyses. Litter samples were collected at 0, 2, and 4 wk growout from six broiler farms (18 houses total) during an anticoccidial drug (ACD) control program and from nine broiler farms (23 houses total) during an Eimeria vaccine (VAC) control program. Of these, litter samples were collected from five farms during both ACD and VAC programs. The litter samples were processed for Eimeria oocyst and C. perfringens spore enumerations by standard parasitologic and microbiologic techniques. DNA was also extracted for C. perfringens DNA for PCR detection of genes coding for alpha- and NetB-toxin. A general trend during the ACD programs was a transient decrease in both Eimeria maxima and non-E. maxima (Eamipt) numbers at 2 wk growout. The pattern was slightly different during VAC with E. maxima and Eamipt levels increasing over time. Average concentrations of C. perfringens in litter were highest at 2 wk (∼105-106 spores/g) during ACD and at placement during VAC (∼105-106 spores/g). During the ACD program, a strong correlation was observed between 0 and 3-wk chick mortality and the presence at placement (0 wk) of netB (r = 0.42-0.48) or cpa (r = 0.55-0.67). A very strong correlation was observed in 0-5-wk chick mortality and the presence of netB at 4 wk growout (0.73-0.95). During a VAC program, a strong correlation was only observed between the presence of netB at placement and 0-1-wk chick mortality (r = 0.67).

Concurrent infection of Avibacterium paragallinarum and fowl adenovirus in layer chickens.

The diagnosis of a concurrent infection of Avibacterium paragallinarum and fowl adenovirus (FAdV) in an infectious coryza-like outbreak in the outskirt of Beijing is reported. The primary signs of the infection were acute respiratory signs, a drop in egg production, and the presence of hydropericardium-hepatitis syndrome-like gross lesions. Laboratory examination confirmed the presence of A. paragallinarum by bacterial isolation and a species-specific PCR test. In addition, conventional serotyping identified the isolates as Page serovar A. Fowl adenovirus was isolated from chicken liver specimen and identified by hexon gene amplification. In addition, histopathologic analysis and transmission electron microscopy examination further confirmed the presence of the virus. Both hexon gene sequencing and phylogenetic analysis defined the viral isolate as FAdV-4. The pathogenic role of A. paragallinarum and FAdV was evaluated by experimental infection of specific-pathogen-free chickens. The challenge trial showed that combined A. paragallinarum and FAdV infection resulted in more severe clinical signs than that by FAdV infection alone. The concurrent infection caused 50% mortality compared with 40% mortality by FAdV infection alone and zero mortality by A. paragallinarum infection alone. To our knowledge, this is the first report of A. paragallinarum coinfection with FAdV. The case implies that concurrent infections with these 2 agents do occur and more attention should be given to the potential of multiple agents during disease diagnosis and treatment.

Risk factors for poor health and performance in European broiler production systems.

BACKGROUND: Conventional broilers are currently one of the most efficient protein converters. Although decades of progress in genetic selection and feed formulation have lead to high standards of efficient broiler production, still a lot of variability is found between farms and between successive flocks. The aim of this study was to investigate risk- and/or protective factors for poor health and performance in conventional broiler-farms in Europe by developing eight multivariable linear mixed models. Three different models were used to investigate mortality (overall, first week, after first week), three models for performance variables (growth, feed conversion, European production index) and two models were related to slaughterhouse data (i.e. dead on arrival and condemnation rate). RESULTS: Several factors related to management and housing were significantly associated with health and performance of broilers. The following factors were associated with increased mortality: floor quality, neonatal septicemia, ventilation type and other professional activities of the farmer. The factors associated with performance were chick sex, coccidiosis infections, necrotic enteritis, dysbacteriosis, light intensity adaptations, ventilation type, comparing daily flock results with previous flock results by farmer, daily check of feed and water system and type of feed. For dead on arrival three risk factors were identified i.e. daily growth, type of light adaptation and type of drinkers system. For condemnation rate seven risk factors were found, i.e. type of drinking system, daily growth, feed withdrawal time, type of ventilation, house size, septicemia after seven days and type of feed. CONCLUSIONS: These results imply that a multifactorial approach is required with adaptations involving both improvements in management, housing, health programs and an increasing level of professionalism of the farmer in order to improve broiler performance and health.

Co-infection of Salmonella enteritidis with H9N2 avian influenza virus in chickens.

Salmonella and avian influenza virus are important pathogens affecting the poultry industry and human health worldwide. In this experimental study, we evaluated the consequences of co-infection of Salmonella enteritidis (SE) with H9N2 avian influenza virus (H9N2-AIV) in chickens. Four groups were included: control group, H9N2-AIV group, H9N2-AIV + SE group, and SE group. Infected chickens were intranasally inoculated with H9N2-AIV at 21 days of age and then orally administered SE on the same day. The birds were monitored for clinical signs, mortality rates, and alterations in body weight. Sera, intestinal fluids, oropharyngeal, and cloacal swabs, and tissue samples were collected at 2, 6, 10, and 14 days post-infection (dpi). Significant increases in clinical signs and mortality rates were observed in the H9N2-AIV + SE group. Moreover, chickens with co-infection showed a significant change in body weight. SE faecal shedding and organ colonization were significantly higher in the H9N2-AIV + SE group than in the SE group. H9N2-AIV infection compromised the systemic and mucosal immunity against SE, as evidenced by a significant decrease in lymphoid organ indices as well as systemic antibody and intestinal immunoglobulin A (IgA) responses to SE and a significant increase in splenic and bursal lesion scores. Moreover, SE infection significantly increased shedding titres and duration of H9N2-AIV. In conclusion, this is the first report of co-infection of SE with H9N2-AIV in chickens, which leads to increased pathogenicity, SE faecal shedding and organ colonization, and H9N2-AIV shedding titre and duration, resulting in substantial economic losses and environmental contamination, ultimately leading to increased zoonoses.

Age-Dependent Lethality in Ducks Caused by Highly Pathogenic H5N6 Avian Influenza Virus.

Ducks show notably higher resistance to highly pathogenic avian influenza viruses as compared to chickens. Here, we studied the age-dependent susceptibility in ducks to the infections caused by highly pathogenic avian influenza viruses. We intranasally infected ducks aged 1, 2, 4, and 8 weeks with highly pathogenic H5N6 avian influenza viruses isolated in South Korea in 2016. All the 1-and 2-week-old ducks died after infection, 20% of 3-week-old ducks died, and from the ducks aged 4 and 8 weeks, all of them survived. We performed microarray analysis and quantitative real-time PCR using total RNA isolated from the lungs of infected 2- and 4-week-old ducks to determine the mechanism underlying the age-dependent susceptibility to highly pathogenic avian influenza virus. Limited genes were found to be differentially expressed between the lungs of 2- and 4-week-old ducks. Cell damage-related genes, such as CIDEA and ND2, and the immune response-related gene NR4A3 were notably induced in the lungs of infected 2-week-old ducks compared to those in the lungs of infected 4-week-old ducks.

Usutu Virus Infection of Embryonated Chicken Eggs and a Chicken Embryo-Derived Primary Cell Line.

Usutu virus (USUV) is a mosquito-borne flavivirus, closely related to the West Nile virus (WNV). Similar to WNV, USUV may cause infections in humans, with occasional, but sometimes severe, neurological complications. Further, USUV can be highly pathogenic in wild and captive birds and its circulation in Europe has given rise to substantial avian death. Adequate study models of this virus are still lacking but are critically needed to understand its pathogenesis and virulence spectrum. The chicken embryo is a low-cost, easy-to-manipulate and ethically acceptable model that closely reflects mammalian fetal development and allows immune response investigations, drug screening, and high-throughput virus production for vaccine development. While former studies suggested that this model was refractory to USUV infection, we unexpectedly found that high doses of four phylogenetically distinct USUV strains caused embryonic lethality. By employing immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction, we demonstrated that USUV was widely distributed in embryonic tissues, including the brain, retina, and feather follicles. We then successfully developed a primary cell line from the chorioallantoic membrane that was permissive to the virus without the need for viral adaptation. We believe the future use of these models would foster a significant understanding of USUV-induced neuropathogenesis and immune response and allow the future development of drugs and vaccines against USUV.

Genotyping and reassortment analysis of highly pathogenic avian influenza viruses H5N8 and H5N2 from Egypt reveals successive annual replacement of genotypes.

Highly pathogenic (HP) H5N1, clade 2.2.1, and low pathogenic avian influenza (LPAI) H9N2 viruses, G1-B lineage, are endemic in poultry in Egypt and have co-circulated for almost a decade. Surprisingly, no inter-subtypic reassortment events have been reported from the field during that time. After the introduction of HPAIV H5N8, clade 2.3.4.4b, in Egyptian poultry in 2016, suddenly HP H5N2 reassortants with H9N2 viruses emerged. The current analyses focussed on studying 32 duck flocks, 4 broiler chicken flocks, and 1 turkey flock, suffering from respiratory manifestations with moderate to high mortality reared in two Egyptian governorates during 2019. Real-time RT-PCR substantiated the presence of HP H5N8 in 21 of the 37 investigated flocks with mixed infection of H9N2 in two of them. HP H5N1 was not detected. Full hemagglutinin (HA) sequencing of 10 samples with full-genome sequencing of three of them revealed presence of a single genotype. Very few substituting mutations in the HA protein were detected versus previous Egyptian HA sequences of that clade. Interestingly, amino acid substitutions in the Matrix (M2) and the Neuraminidase (NA) proteins associated with conferring both Amantadine and Oseltamivir resistance were present. Systematic reassortment analysis of all publicly available Egyptian whole genome sequences of HP H5N8 (n = 23), reassortant HP H5N2 (n = 2) and LP H9N2 (n = 53) viruses revealed presence of at least seven different genotypes of HPAI H5Nx viruses of clade 2.3.4.4b in Egypt since 2016. For H9N2 viruses, at least three genotypes were distinguishable. Heat mapping and tanglegram analyses suggested that several internal gene segments in both HP H5Nx and H9N2 viruses originated from avian influenza viruses circulating in wild bird species in Egypt. Based on the limited set of whole genome sequences available, annual replacement patterns of HP H5Nx genotypes emerged and suggested selective advantages of certain genotypes since 2016.

Pathological and virological analysis of concurrent disease of chicken anemia virus infection and infectious bronchitis in Japanese native chicks.

A concurrent infection of chicken anemia virus (CAV) and infectious bronchitis virus (IBV) was detected in Japanese native chicks in 2017, in which a high mortality rate (97.7%) was recorded in a small flock of 130 chicks exhibiting poor growth. Histological examination revealed that the affected chicks exhibited two different pathological entities: one was severe hematopoietic and lymphocytic depletion with abnormally large cells containing intranuclear inclusion bodies of CAV, whereas the other was renal tubular necrosis due to IBV infection. Immunohistochemistry detected CAV antigens in the bone marrow, liver, and spleen as well as IBV antigens in the kidneys, trachea, and air sacs. CAV was isolated from the liver sample of the chicks, and the isolated strain was designated as CAV/Japan/HS1/17. A phylogenetic analysis of the CAV VP1 gene revealed that CAV/Japan/HS1/17 is genetically similar to Chinese strains collected from 2014 to 2016. An experimental infection was performed using CAV/Japan/HS1/17 and specific-pathogen-free chicks to determine the pathogenicity of CAV/Japan/HS1/17. The isolate caused 100% anemia and 70% mortality to chicks inoculated at one day old, 80% of chicks inoculated at seven days old also developed anemia, and 10% died from CAV infection. These results suggest that the unusually high mortality in Japanese native chicks can be attributed to dual infection with both CAV and IBV. The results of the experimental infection suggest that CAV/Japan/HS1/17 has a pathogenic potential to specific-pathogen-free chicks and a relatively higher pathogenicity than previous Japanese CAV strains.

Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis.

Tembusu virus (TMUV) is a flavivirus responsible for panzootic outbreaks of severe egg-drop and fatal encephalitis of domestic waterfowl in China. Although TMUV can be attenuated by in vitro passaging, experimental evidence supporting the role of specific genetic changes in virulence attenuation is currently lacking. Here, we performed site-directed mutagenesis on five envelope (E) protein amino acid residues in accordance with the attenuated TMUV generated in our recent study. Our results showed that the Thr-to-Lys mutation of residue 367 in E protein (E367) plays a predominant role in viral cell adaptation and virulence attenuation in ducks compared with mutations in other residues. We further demonstrated that the positively charged basic amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the efficiency of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K mutation increased viral neutralization sensitivity to the early immune sera. Together, our findings provide the first evidence that a basic amino acid substitution at E367 strongly impacts the in vitro and in vivo infection of TMUV.IMPORTANCE Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses in the production of domestic waterfowl since the virus was first recognized in China in 2010. To control TMUV infection, a live-attenuated vaccine candidate of TMUV was developed in our previous study, but the mechanisms of virulence attenuation are not fully understood. Here, we found that the Thr-to-Lys substitution at E367 is a crucial determinant of TMUV virulence attenuation in ducks. We demonstrated that the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide new insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines.

Recombinant infectious bronchitis coronavirus H120 with the spike protein S1 gene of the nephropathogenic IBYZ strain remains attenuated but induces protective immunity.

Infectious bronchitis (IB) is a highly infectious viral disease responsible for major economic losses in the poultry industry. A reverse genetic vaccine is a safe, rapid, and effective method of achieving IB prevention and control. In this study, we constructed the recombinant strain, rH120-S1/YZ, using a reverse genetic system, based on the backbone of the H120 vaccine strain, with the S1 gene replaced with that of the QX-like nephropathogenic strain, ck/CH/IBYZ/2011, isolated in China. The results of dwarf chicken embryos, growth kinetics, and viral titration in the embryos demonstrated that the biological characteristics of the recombinant virus remained unchanged. Like the rH120-infected group and in contrast to the rIBYZ-infected group, no mortality, clinical signs, or lesions were observed in the lungs or kidneys of young chickens inoculated with rH120-S1/YZ. The viral loads in various tissues, cloacal, and oral swabs was lower in most types of samples, indicating that the rH120-S1/YZ strain was highly safe in chicks. Compared to rH120 vaccination group, when the efficacy of this strain was evaluated against the QX-like IBV strain, better protection, with 100% survival rate and no disease symptom or gross lesion was observed in the chickens vaccinated with rH120-S1/YZ. Increased levels of IBV-specific antibodies were detected in the serum of the rH120-S1/YZ-vaccinated animals 14 days post-vaccination. Collectively, our results suggest that the recombinant strain, rH120-S1/YZ, may represent a promising vaccine candidate against QX-like IBVs.