Abstract H9N2 is the most prevalent low pathogenic avian influenza virus (LPAIV) in domestic poultry in the world. Two distinct H9N2 poultry lineages, G1-like (A/quail/Hong Kong/G1/97) and Y280-like (A/Duck/Hong Kong/Y280/1997) viruses, are usually associated with binding affinity for both α 2,3 and α 2,6 sialic acid receptors (avian and human receptors), raising concern whether these viruses possess pandemic potential. To explore the impact of mouse adaptation on the transmissibility of a Y280-like virus A/Chicken/Hubei/214/2017(H9N2) (abbreviated as WT), we performed serial lung-to-lung passages of the WT virus in mice. The mouse-adapted variant (MA) exhibited enhanced pathogenicity and advantaged transmissibility after passaging in mice. Sequence analysis of the complete genomes of the MA virus revealed a total of 16 amino acid substitutions. These mutations distributed across 7 segments including PB2, PB1, PA, NP, HA, NA and NS1 genes. Furthermore, we generated a panel of recombinant or mutant H9N2 viruses using reverse genetics technology and confirmed that the PB2 gene governing the increased pathogenicity and transmissibility. The combinations of 340 K and 588 V in PB2 were important in determining the altered features. Our findings elucidate the specific mutations in PB2 contribute to the phenotype differences and emphasize the importance of monitoring the identified amino acid substitutions due to their potential threat to human health.
Fluoroquinolone (FQ)-resistant Campylobacter jejuni is a serious problem worldwide that limits effective treatment of infections. The traditional detection method depends on bacterial isolation and MIC testing, or traditional PCR, which is time-consuming and hard to identify the FQ-resistant C. jejuni in a high abundance wild-type background. This study aimed to develop a rapid and accurate ddPCR assay to detect FQ-resistant C. jejuni mutants based on the crucial resistance mutation C257T (Thr-86-Ile) in gyrA. Our ddPCR gyrA assay showed high specificity and accuracy. Sanger sequencing and the qPCR assay could only recognize gyrA mutant sequences when the ratios of wild-type/mutant were 1:1 or 10:1, respectively. Our ddPCR gyrA assay was able to detect gyrA mutant sequences in the mixtures with up to at least 1000:1 wild-type/mutant ratios, which suggested a significant advantage to distinguish the low mutant signal from the wild-type background. We further monitored the occurrence of gyrA mutations under ciprofloxacin pressure using our ddPCR gyrA assay, and clearly showed that the transition of a dominant C. jejuni subpopulation from wild-type to gyrA C257T mutant, resulting in FQ-resistance. We tested 52 samples from live chickens and retail chicken meat and showed that four samples contained wild-type/mutant mixtures comprising 1.7%, 28.6%, 53.3%, and 87.0% gyrA C257T mutants, respectively. These results demonstrated that the ddPCR gyrA assay was a highly sensitive alternative method to distinguish and quantify FQ-resistant C. jejuni infections that could help guide the appropriate use of FQs in clinical practice. IMPORTANCE Campylobacter jejuni is considered to be the leading cause of human bacterial gastroenteritis worldwide, and fluoroquinolones (FQs) are the main choices for the treatment of bacterial gastroenteritis in clinical practice. In theory, antimicrobial susceptibility testing should help us to choose the most appropriate drugs for the treatment. However, to test the susceptibility of C. jejuni to FQs, the standardized method is bacteria isolation and MIC measurement, which will take more than 4 days. In addition, a low abundance of FQ-resistant C. jejuni is also hardly distinguished from a high abundance of wild-type background in the mixed infection. Therefore, the development of rapid and accurate detection technology for FQ-resistant C. jejuni is very important. This study provided a ddPCR gyrA assay, which is a highly sensitive alternative method to distinguish and quantify FQ-resistant C. jejuni infections that may help guide the appropriate use of FQs both in veterinary and human clinical practice.
Sequence analysis of 79 ciprofloxacin-resistant Campylobacter jejuni isolates collected in China showed resistance-related sequence variations in gyrA and CmeR-Box. All the isolates contain an identical Thr-86-Ile substitution in GyrA. Several novel CmeR-Box variations, including point substitutions, deletion, and insertion, were identified. The point insertion or deletion led to dramatically reduced binding of CmeR to the cmeABC promoter, which significantly increases the expression of cmeABC and contributes to the high fluoroquinolone resistance.
Pullorum disease, caused by Salmonella enterica serovar Pullorum (S. Pullorum), is one of the most important bacterial infections in the poultry industry in developing countries, including China. To examine the prevalence and characteristics of S. Pullorum, the Multilocus Sequence Typing (MLST) genotypes, fluoroquinolones resistance, and biofilm-forming abilities of S. Pullorum isolates were investigated, collected from 2011 to 2016 in China. Thirty S. Pullorum isolates collected from 2011 to 2016 were analyzed. Quinolones susceptibility testing showed that 90% of the isolates were resistant to the first generation of quinolines nalidixic acid, but the resistance rates to different fluoroquinolones agents were lower than 13.3%; for some there was even no resistance. Multilocus sequence typing (MLST) showed that ST-92 was the dominating genotype, accounting for 90.0% of all S. pullorum strains. The remaining three isolates were of the new reported sequence type ST-2151. Interestingly, the Asp87Gly substitution in quinolone resistance-determining regions (QRDR) of GyrA was only observed in the three strains of ST-2151, suggesting a potential correlation between Asp87Gly substitution and sequence type (p < 0.05). However, Asp87Gly substitution could not confer the resistant to ofloxacin and ciprofloxacin of these isolates. The plasmid-mediated quinolone resistance (PMQR) gene was not found in any of the tested isolates. Furthermore, an assay measuring biofilm-forming abilities showed that 46.7% of the isolates were non-biofilm producers, while 53.3% could form very weak biofilms, which might explain the relatively lower resistance to fluoroquinolones. We reported a high resistance rate to the first generation of quinolines nalidixic acid and relatively low resistance rates to fluoroquinolones in S. Pullorum isolates. In addition, weak biofilm-forming abilities were found, which might be an important reason of the low fluoroquinolones resistance rates of S. Pullorum isolates. ST-92 was the dominating genotype demonstrated by MLST, and the new sequence type ST-2151 showed a potential correlation with Asp87Gly substitution in QRDR of GyrA. We believe the characterization of these S. Pullorum isolates will be helpful to develop prevention and control strategies.
Infectious bronchitis (IB) and Newcastle disease (ND) are two major infectious diseases that are a threat to the domestic poultry industry. In this study, we successfully generated a recombinant LaSota candidate vaccine strain, rNDV-IBV-T/B, which expresses a short, synthetic, previously identified IBV S1 multi-epitope cassette using the reverse genetic system. The recombinant virus was propagated in nine-day-old embryonated chicken eggs for 20 passages and genetic stability was confirmed by whole genome DNA sequencing. The recombinant virus had a hemagglutination (HA) titer of 210, mean death time (MDT) of 118 hours, and intracerebral pathogenicity index (ICPI) of 0.05. None of these were significantly different from the parental Newcastle disease virus (NDV) LaSota strain (p > 0.05). Vaccination of white leghorn chickens at one day of age with 106 EID50 rNDV-IBV-T/B provided 90% protection against virulent IBV M41 challenge at three weeks of age, which was significantly higher than the protection of the control group vaccinated with phosphate-buffered saline (PBS) (p < 0.05). The ciliostasis scores of rNDV-IBV-T/B-vaccinated and LaSota-vaccinated groups were 4.2 and 37.6, respectively, which indicated that rNDV-IBV-T/B vaccination reduced the pathogenicity of IBV toward the trachea. Furthermore, real-time RT-PCR assay showed that the rNDV-IBV-T/B vaccination resulted in low levels of viral load (647.80 ± 49.65 RNA copies) in the trachea four days post-challenge, which is significantly lower than groups vaccinated with PBS (8591.25 ± 311.10 RNA copies) or LaSota (7742.60 ± 298.50 RNA copies) (p < 0.05). Meanwhile, the same dose of rNDV-IBV-T/B vaccination provided complete protection against velogenic NDV F48E9 challenge. These results demonstrate that the rNDV-IBV-T/B strain is a promising vaccine candidate to control both IB and ND simultaneously. Furthermore, epitope-based live vector vaccines provide an alternative strategy for the development of cost-effective and, broadly, cross-protective vaccines.
Influenza virus nucleoprotein (NP) is a promising target for universal influenza vaccines due to its conservation and high immunogenicity. Here, we uncovered a previously unknown factor that E. coli-produced NP carries bacterial RNA, which is crucial for its high immunogenicity but may pose safety and consistency concerns due to batch variability. To address these concerns, we developed a NP mutant (NPmut) that lacks RNA binding activity but can be loaded with CpG1826, a synthetic oligodeoxynucleotide adjuvant that has been used in the FDA-approved Hepatitis B vaccine. The CpG1826-loaded NPmut induced immune responses comparable to RNA-bound NP while eliminating safety risks. Additionally, the mixture of CpG1826-loaded NPmut and 3M2e protein (three tandem copies of the ectodomain of influenza M2 protein) provided enhanced protection against influenza viruses challenge. Our findings highlight the adjuvant activity of bacterial RNA in E. coli-produced NP and propose a safer strategy for developing universal influenza vaccines.
Thermostable Newcastle disease virus (NDV) vaccines have been used widely to control Newcastle disease for village poultry flocks, due to their independence of cold chains for delivery and storage. To explore the potential use of thermostable NDV as a vaccine vector, an infectious clone of thermostable avirulent NDV strain TS09-C was developed using reverse genetics technology. The GFP gene, along with the self-cleaving 2A gene of foot-and-mouth disease virus and ubiquitin monomer (2AUbi), were inserted immediately upstream of the NP (nucleocapsid protein), M (matrix protein) or L (large polymerase protein) gene translation start codon in the TS09-C infectious clone. Detection of GFP expression in the recombinant virus-infected cells showed that the recombinant virus, rTS-GFP/M, with the GFP gene inserted into the M gene expressed the highest level of GFP. The rTS-GFP/M virus retained the same thermostability, growth dynamics and pathogenicity as its parental rTS09-C virus. Vaccination of specific-pathogen-free chickens with the rTS-GFP/M virus conferred complete protection against virulent NDV challenge. Taken together, the data suggested that the rTS09-C virus could be used as a vaccine vector to develop bivalent thermostable vaccines against Newcastle disease and the target avian diseases for village chickens, especially in the developing and least-developed countries.
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