Research Note: A recombinant duck-derived H6N2 subtype avian influenza virus can replicate and shed in young chickens and cause disease.

The H6N2 subtype avian influenza virus (AIV) is commonly detected in the migratory waterfowl reservoirs. Previously, H6N2 AIV was believed to be nonpathogenic to young chickens and could not infect or shed in their respiratory tract under experimental conditions. However, in present study, a highly recombinant strain of duck-derived H6N2 AIV was discovered and isolated for pathogenicity tests. The results revealed that H6N2 could induce seroconversion in chickens and high morbidity of over 86.7%, along with evident upper respiratory tract hemorrhage. Moreover, 5 substitutions were detected in the upper respiratory tract shedding reisolated virus, with a high viral load in the target organs of infected chickens. In contrast, ducks failed to exhibit any symptoms, pathological lesions, or viral shedding, while demonstrated seroconversion and high viral load in the livers. These findings indicate that H6N2 AIV could also show pathogenicity to chickens under experimental conditions, thereby effectively replicating and shedding in chickens. Therefore, the study provides further elucidations on the pathogenicity of H6N2 AIV.

Evolution of prevalent H9N2 subtype of avian influenza virus during 2019 to 2022 for the development of a control strategy in China.

The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused significant losses in chicken flocks throughout China. At present, consensus has been reached that field isolates of H9N2 underwent antigenic drift to evolve into distinct groups with significant antigenic divergence from the commercially available vaccines in China. This project continues to monitor the evolution characteristics of H9N2 hemagglutinin (HA) genes in China over the past 3 yr. The results showed that the current circling H9N2 viruses were diversified into h9.4.2.5 subclade, which was genetically distant from commonly used commercial vaccine strains. Compared with vaccine strains or 2014 strains, more than 42.1% of the variable antigenic sites in recent 3 yr' strains have shown significant changes and these stacked changes have caused significant differences in antigenicity. We constructed a recombinant vaccine strain rCQY-GHHA, which uses A/Chicken/China/SichuanCQY/2014 as the framework and A/Chicken/China/SichuanGH/2020 strain, which meets the recent viral antigenic characteristics, as the HA gene donor. The recombinant strain was prepared as an oil-adjuvant inactivated vaccine following an industrial process. The results of the immune protection experiment showed that the rCQY-GHHA vaccine was better than the commercial vaccine strain SS in reducing the morbidity, pathological lesion, virus shedding, and viral load. These results provide a reference for the control of H9N2 AIV in China.

The conserved L1089 in the S2 subunit of avian infectious bronchitis virus determines viral kidney tropism by disrupting virus-cell fusion.

The virulence of avian gamma-coronavirus infectious bronchitis viruses (IBV) for the kidney has led to high mortality in dominant-genotype isolations, but the key sites of viral protein that determine kidney tropism are still not fully clear. In this study, the amino acid sequences of the S2 subunit of IBVs with opposing adaptivity to chicken embryonic kidney cells (CEKs) were aligned to identify putative sites associated with differences in viral adaptability. The S2 gene and the putative sites of the non-adapted CN strain were introduced into the CEKs-adapted SczyC30 strain to rescue seven mutants. Analysis of growth characteristics showed that the replacement of the entire S2 subunit and the L1089I substitution in the S2 subunit entirely abolished the proliferation of recombinant IBV in CEKs as well as in primary chicken oviduct epithelial cells. Pathogenicity assays also support the decisive role of this L1089 for viral nephrotropism, and this non-nephrotropic L1089I substitution significantly attenuates pathogenicity. Analysis of the putative cause of proliferation inhibition in CEKs suggests that the L1089I substitution affects neither virus attachment nor endocytosis, but instead fails to form double-membrane vesicles to initiate the viral replication and translation. Position 1089 of the IBV S2 subunit is conservative and predicted to lie in heptad repeat 2 domains. It is therefore reasonable to conclude that the L1089I substitution alters the nephrotropism of parent strain by affecting virus-cell fusion. These findings provide crucial insights into the adaptive mechanisms of IBV and have applications in the development of vaccines and drugs against IB.

Mutation of D201G near the receptor binding site significantly drives antigenic drift of circulating H9N2 subtype avian influenza virus.

The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused significant losses in chicken flocks throughout China. Our previous research has shown that field isolates of H9N2 underwent antigenic drift to evolve into distinct groups with significant antigenic divergence from the commercially available vaccines. The present study sought to identify which single mutations that have naturally appeared in isolates from the past 5 years have driven antigenic drift. Six high-frequency mutation sites in/near the receptor binding site region were screened by comparing amino acid alignments of the H9N2 AIVs isolated from China between 2014 and 2019. Two substitutions (A168N and D201G) were demonstrated to have a significant impact on the antigenicity but did not change the growth kinetics of the virus. It is worth noting that the D201G substitution not only significantly changed the antigenicity but also caused immune escape against the parental virus. In conclusion, A168N and D201G substitution are newly discovered determinants that can significantly change the antigenicity of H9N2 AIV, which should be tracked during outbreaks.

Research Note: Effects of Escherichia coli co-infection on the protective efficacy assessment of two common infectious bronchitis vaccines.

Avian infectious bronchitis (IB), a highly contagious disease hazardous to the poultry industry, is caused by an etiological agent called the infectious bronchitis virus (IBV). Some IBV strains (IBVs) alone usually do not cause high mortality in field conditions if not with secondary pathogens including Escherichia coli (E. coli). Herein, we established an IBV and E. coli co-infection model to evaluate the protective efficacy of two IBV vaccine strains against a new emerging genotype GVI-1 with mild virulence in experimental conditions. Chickens were inoculated with IBV field isolate ZQX (genotype GVI-1) and challenged 4 dlater with the E. coli strain MS160427 (serotype O8). Subsequently, these chickens were euthanized at seven days postchallenge (d.p.c.) with E. coli. An autopsy revealed that lesions in the IBV plus E. coli co-infection group were more severe than those in the IBV-infected group. This pathological model was used to assess the protective effect of two commonly used vaccine strains (H120 and 4/91) against the IBV ZQX strain, and a significantly better protective efficacy was observed for 4/91 compared with H120. Thus, IBV and E. coli co-infection could be employed in assessing the protective efficacy of IBV vaccines.

A class â lentogenic newcastle disease virus strain confers effective protection against the prevalent strains.

The traditional vaccine strains, such as LaSota, do not completely prevent the shedding of NDV. An ideal vaccine which could not only prevent the clinical signs, but significantly reduce the shedding of NDV is urgently needed for the eradication of ND. In this study, an NDV isolate APMV-1/Chicken/China (SC)/PT3/2016 (hereafter referred as PT3) was identified as a class Ⅰ NDV and a lentogenic strain. The antigenic relationship between PT3 and 3 other NDV strains, including vaccine strain LaSota and 2 prevalent genotype Ⅶd and Ⅵb strains were analyzed. The protective efficacy of PT3 and LaSota against challenge with genotype Ⅶd and Ⅵb strains were assessed. The antigenic analysis result showed that 4 strains belong to the single serotype and the PT3 antiserum exhibited the highest HI titer against 3 other NDV strains. The results of protective efficacy showed that both of LaSota and PT3 could provide 100% survivability for infected chickens. However, PT3 performed better in inducing higher humoral responses and reducing virus shedding than the LaSota strain. Lentogenic strains from Class I NDV appear to be promising vaccine candidates for the control of ND, and allows for the easy discrimination of field NDV and vaccine strains.

Hsp40 Protein DNAJB6 Interacts with Viral NS3 and Inhibits the Replication of the Japanese Encephalitis Virus.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus prevalent in east and southeast Asia, the Western Pacific, and northern Australia. Since viruses are obligatory intracellular pathogens, the dynamic processes of viral entry, replication, and assembly are dependent on numerous host-pathogen interactions. Efforts to identify JEV-interacting host factors are ongoing because their identification and characterization remain incomplete. Three enzymatic activities of flavivirus non-structural protein 3 (NS3), including serine protease, RNA helicase, and triphosphatase, play major roles in the flaviviruses lifecycle. To identify cellular factors that interact with NS3, we screened a human brain cDNA library using a yeast two-hybrid assay, and identified eight proteins that putatively interact with NS3: COPS5, FBLN5, PPP2CB, CRBN, DNAJB6, UBE2N, ZNF350, and GPR137B. We demonstrated that the DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6) colocalizes and interacts with NS3, and has a negative regulatory function in JEV replication. We also show that loss of DNAJB6 function results in significantly increased viral replication, but does not affect viral binding or internalization. Moreover, the time-course of DNAJB6 disruption during JEV infection varies in a viral load-dependent manner, suggesting that JEV targets this host chaperone protein for viral benefit. Deciphering the modes of NS3-interacting host proteins functions in virion production will shed light on JEV pathogenic mechanisms and may also reveal new avenues for antiviral therapeutics.

Acute oral toxicity test and assessment of combined toxicity of cadmium and aflatoxin B1 in kunming mice.

Cadmium and aflatoxin B1 (AFB1) are both common and widespread pollutants in food and feed. There are several reports on toxicity induced by Cadmium or AFB1 alone, but few address the toxicity caused by co-exposure to the two substances. In this study, 42 female and 42 male Kunming (KM) mice were divided into seven groups to test the acute oral toxicity of CdCl2 and AFB1, using Karber's method. The combined toxicity was assessed using the Keplinger evaluation system. Acute toxicity symptoms, deaths, and body and organ weights were evaluated, and hematological, blood biochemical, and histopathological analyses were conducted. The results revealed the following median lethal doses (LD50): LD50(Female KM mice) = 62.56 mg/kg; LD50(Male KM mice) = 48.79 mg/kg; LD50(KM mice)=55.27 mg/kg. The combined toxicity of AFB1 and CdCl2 showed an additive effect in mice, and an increase in the mixed dose of AFB1 and CdCl2 resulted in greater toxicity. These results demonstrated that the combined toxicity of AFB1 and CdCl2 was greater than the toxicities of the individual components in mice; thus, this may cause particular challenges when addressing these hazards in food and feed and the associated risk to human and animal health.

Preparation and protective efficacy of a chicken embryo kidney cell-attenuation GI-19/QX-like avian infectious bronchitis virus vaccine.

Avian infectious bronchitis (IB) is a highly contagious disease, and hazardous to the poultry industry. Immune failure often occurs due to the emergence of new serotypes or field strains antigenically different from the vaccine strains. To prepare a candidate vaccine against the prevalent avian infectious bronchitis virus (IBV) in China, the GI-19/QX-like field isolate Sczy3 was selected as the progenitor strain and attenuated via passaging in chicken embryo kidney (CEK) cells for 100 times. The 100th generation of CEK-adapted strain, designated SczyC100, was safe to use on one-day old specific pathogen-free (SPF) chicken as determined by pathogenicity and virulence reversion test. The efficacies of SczyC100 and two commonly used commercial vaccines (H120 and 4/91) against prevalent GI-19/QX and GI-7/TWI type virulent strains were evaluated. Sczy3C100 effectively reduced the morbidity, mortality, mean lesion scores (MLSs), and viral load of trachea of chickens challenged by GI-19/QX and GI-7/TWI strains. CEK-adapted SczyC100 is therefore a potential vaccine candidate for the control of IB in China.

Isolation and molecular characterization of prevalent Fowl adenovirus strains in southwestern China during 2015-2016 for the development of a control strategy.

Fowl adenovirus (FAdV) has caused significant losses in chicken flocks throughout China in recent years. However, the current understanding of the genetic and pathogenic characteristics of the FAdV epidemic in southwestern China remains poorly understood. In this study, a total of 22 strains were isolated from liver samples of diseased chickens from farms in southwestern China. Phylogenetic analysis based on the hexon loop-1 gene showed that the 22 isolates were clustered into four distinct serotypes: FAdV serotype 4 (FAdV-4) (86.4%, 19/22), FAdV-2 (4.5%, 1/22), FAdV-8a (4.5%, 1/22), and FAdV-8b (4.5%, 1/22). FAdV-4 was the predominant serotype in southwestern China. Pathogenicity testing showed that the FAdV-4 serotype strain CH/GZXF/1602 and FAdV-8a strain CH/CQBS/1504 were pathogenic to chickens, with mortality rates reaching as high as 80% and 20%, respectively. The primary clinical feature observed following infection with strain CH/GZXF/1602 (FAdV-4) was hepatitis-hydropericardium syndrome, and that of strain CH/CQBS/1504 (FAdV-8a) was inclusion body hepatitis. Conversely, the FAdV-2 serotype strain CH/GZXF/1511 and FAdV-8b serotype strain CH/CQBS/1512 was not observed to be pathogenic in chickens. Then, CH/GZXF/1602 (FAdV-4) was selected for the preparation of an inactivated oil-emulsion vaccine. Immune studies on Partridge Shank broilers showed that a single dose immunization at 17 days of age could not only protect against homologous challenge with virulent FAdV-4 but also provided protection against clinical disease following challenge with the heterologous FAdV-8b virulent strain until 70 days of age. The characterization of newly prevalent FAdV strains provides a valuable reference for the development of an efficacious control strategy.

Genetic and antigenic evolution of H9N2 subtype avian influenza virus in domestic chickens in southwestern China, 2013-2016.

H9N2 avian influenza virus (AIV) has caused significant losses in chicken flocks throughout china in recent years. There is a limited understanding of the genetic and antigenic characteristics of the H9N2 virus isolated in chickens in southwestern China. In this study a total of 12 field strains were isolated from tissue samples from diseased chickens between 2013 and 2016. Phylogenetic analysis of the Hemagglutinin (HA) and Neuraminidase (NA) nucleotide sequences from the 12 field isolates and other reference strains showed that most of the isolates in the past four years could be clustered into a major branch (HA-branch A and NA-branch I) in the Clade h9.4.2 lineages. These sequences are accompanied by nine and seven new amino acids mutations in the HA and NA proteins, respectively, when compared with those previous to 2013. In addition, four new isolates were grouped into a minor branch (HA-branch B) in the Clade h9.4.2 lineages and two potential N-glycosylation sites were observed due to amino acid mutations in the HA protein. Three antigenic groups (1-3), which had low antigenic relatedness with two commonly used vaccines in China, were identified among the 12 isolates by antigenMap analysis. Immunoprotection testing showed that those two vaccines could efficiently prevent the shedding of branch A viruses but not branch B viruses. In conclusion, these results indicate the genotype of branch B may become epidemic in the next few years and that a new vaccine should be developed for the prevention of H9N2 AIV.

Phylogenetic and antigenic analysis of avian infectious bronchitis virus in southwestern China, 2012-2016.

The aim of this study was to decipher the molecular epidemiological and antigenic characteristics of infectious bronchitis virus strains (IBVs) isolated in recent years in southwestern China. A total of 24 field strains were isolated from diseased chickens between 2012 and 2016. Phylogenetic analysis based on S1 nucleotide sequences showed that 16 of the 24 isolates were clustered into four distinct genotypes: QX (37.5%), TW (16.7%, TWI and TWII), Mass (8.3%), and J2 (4.2%). The QX genotype was still the prevalent genotype in southwestern China. Recombination analysis of the S1 subunit gene showed that eight of the 24 field strains were recombinant variants that originated from field strains and vaccine strains. A new potential recombination hotspot [ATTTT(T/A)] was identified, implying that recombination events may become more and more common. The antigenicity of ten IBVs, including seven field strains and commonly used vaccine strains, were assayed with a viral cross-neutralization assay in chicken embryonated kidney cells (CEK). The results showed that the ten IBVs could be divided into four serotypes (Massachusetts, 793B, Sczy3, and SCYB). Sczy3 and 793B were the predominant serotypes. Six of the seven field isolates (all except for cK/CH/SCYB/140913) cross-reacted well with anti-sera against other field strains. In conclusion, the genetic and antigenic features of IBVs from southwestern China in recent years have changed when compared to the previous reports. The results could provide a reference for vaccine development and the prevention of infectious bronchitis in southwestern China.

Modelling Growth and Bacteriocin Production by Lactobacillus plantarum BC-25 in Response to Temperature and pH in Batch Fermentation.

The use of bacteriocin-producing probiotics to improve food fermentation processes seems promising. However, lack of fundamental information about their functionality and specific characteristics may hinder their industrial use. Predictive microbiology may help to solve this problem by simulating the kinetics of bacteriocin-producing strains and optimising the cell growth and production of beneficial metabolites. In this study, a combined model was developed which could estimate, from a given initial condition of temperature and pH, the growth and bacteriocin production of Lactobacillus plantarum BC-25 in MRS broth. A logistic model was used to model the growth of cells, and the Luedeking-Piret model was used to simulate the biomass and bacteriocin production. The parameters generated from these primary models were used in a response surface model to describe the combined influence on cell growth, biomass and bacteriocin production. Both the temperature and pH influenced cell and bacteriocin production significantly. The optimal temperature and pH for cell growth is 35 °C and 6.8, and the optimal bacteriocin production condition is a range dependent on two growth-associated constants (YA/X and K), where temperature is from 27 to 34 °C, and pH is 6.35 to 6.65. The developed model is consistent with similar studies and could be a useful tool to control and increase the production of lactic acid bacteria in bioreactors.