The genetic and biological characterization of the first avian paramyxovirus serotype 14 isolated from chicken in China.

In October 2020, an avian paramyxovirus serotype 14 (APMV-14)-designated chicken/Fujian/2160/2020 (FJ2160) was isolated from tracheal and cloacal swab sample of chicken collected from live bird market in Fujian province in China during the active surveillance program. The complete genome of FJ2160 comprised 15,444 nucleotides (nt) complying with the paramyxovirus "rule of six" and encoded six non-overlapping structural proteins in the order of 3'-NP-P-M-F-HN-L-'5. The complete genome sequence analysis showed that FJ2160 had the highest identity (90.0%) with the APMV-14 isolated from Japan, while the nucleotide sequence identities of FJ2160 and other APMVs ranged from 42.4 to 51.1%. The F protein cleavage site was TREGR↓L, which resembled a lentogenic strain of APMV-1. Phylogenetic analysis revealed that the FJ2160 closest relative was APMV-14. The intracerebral pathogenicity index (ICPI) tests indicated that the virus was lentogenic. This is the first report of APMV-14 in China. These results provide evidence that APMV-14 could infect chickens and reveal the genetic characteristics and biological properties of the virus, which can help to better understand this new emerging APMV-14.

Reverse transcription recombinase-aided amplification assay for avian influenza virus.

Avian influenza virus (AIV) infection can lead to severe economic losses in the poultry industry and causes a serious risk for humans. A rapid and simple test for suspected viral infection cases is crucial. In this study, a reverse transcription recombinase-aided amplification assay (RT-RAA) for the rapid detection of all AIV subtypes was developed. The reaction temperature of the assays is at 39 °C and the detection process can be completed in less than 20 min. The specificity results of the assay showed that this method had no cross-reaction with other main respiratory viruses that affect birds, including Newcastle disease virus (NDV) and infectious bronchitis virus (IBV). The analytical sensitivity at a 95% confidence interval was 102 RNA copies per reaction. In comparison with a published assay for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), the κ value of the RT-RAA assay in 384 avian clinical samples was 0.942 (p < 0.001). The sensitivity and specificity of the RT-RAA assay for avian clinical sample detection was determined as 97.59% (95% CI 93.55-99.23%) and 96.79% (95% CI 93.22-98.59%), respectively. The RT-RAA assay for AIV in this study provided an effective and practicable tool for AIV molecular detection.

Molecular epidemiology analysis of fowl adenovirus detected from apparently healthy birds in eastern China.

BACKGROUND: Fowl adenovirus is of major concern to the poultry industry worldwidely. In order to monitor the prevalent status of Fowl adenovirus in China, a total of 1920 clinical samples from apparently healthy birds in the 25 sites of poultry flocks, Slaughterhouse and living bird markets from 8 provinces in eastern China were collected and detected by PCR, sequencing, and phylogenetic analysis. RESULTS: The epidemiological survey showed that Fowl adenoviruses were detected in living bird markets, and circulating in a variety of fowl species, including chickens, ducks, goose and pigeons. Among the 1920 clinical samples, 166 samples (8.65%) were positive in the fowl adenovirus PCR detection. In this study, totally all the 12 serotypes (serotypes of 1, 2, 3, 4, 5, 6, 7, 8A, 8B, 9, 10 and 11) fowl adenoviruses were detected, the most prevalent serotype was serotype 1. Phylogenetic analysis indicated that 166 FAdVs of 12 serotypes were divided into 5 fowl adenovirus species (Fowl aviadenovirus A, B, C, D, E). CONCLUSIONS: In the epidemiological survey, 8.65% of the clinical samples from apparently healthy birds were positive in the fowl adenovirus PCR detection. Totally all the 12 serotypes fowl adenoviruses were detected in a variety of fowl species, which provided abundant resources for the research of fowl adenoviruses in China. The newly prevalent FAdV serotypes provides valuable information for the development of an effective control strategy for FAdV infections in fowls.

KCNN4 may weaken anti-tumor immune response via raising Tregs and diminishing resting mast cells in clear cell renal cell carcinoma.

BACKGROUND: Studies over the past decade have shown that competitive endogenous RNA (ceRNA) plays an essential role in the tumorigenesis and progression of clear cell renal cell carcinoma (ccRCC). Meanwhile, immune checkpoint blocker is gradually moving towards the first-line treatment of ccRCC. Hence, it's urgent to develop a new prediction model for the efficiency of immunotherapy. At present, there is no study to reveal the effect of ceRNA network on the efficiency of immunotherapy for ccRCC. METHODS: To systematically analyze the effect of ceRNA hub genes in ccRCCon immune response, we constructed prognosis models based on ceRNAs and immune cells, respectively. We constructed ceRNA network using hypergeometric distribution test and correlation analysis with R script based on The Cancer Genome Atlas (TCGA) database. We then applied the Cibersort algorithm to simulate the infiltration overview of immune cells in kidney renal clear carcinoma (KIRC) samples. Prognosis-related immune cells were screened and a predictive model of these cells was constructed. Prognosis-related immune cells and ceRNA hub genes were performed with co-expression analysis. Finally, qRT-PCR and immunofluorescence assays were performed to validate the results. RESULTS: The construction of ceRNA related prognosis model contained 8 hub genes, including RELT, MYO9B, KCNN4, SIX1, OTOGL, MALAT1, hsa-miR-130b-3p, and hsa-miR-21-5p. The area under the receiver operating characteristic curve (AUC) was 0.77 at 5 years. For the construction of immune cells prognosis model, 3 immune cells (T cells regulatory, Macrophages, Mast cells resting) were adopted, and the AUC was 0.65 at 5 years. We then merged the two models by correlation analysis and co-expression analysis. Finally, we found that KCNN4 positively correlates with T cells regulatory (Tregs) and negatively correlates with mast cells resting significantly. Furthermore, higher expression of KCNN4 may lead to a higher potential for immune evasion and lower efficiency for immune checkpoint inhibitors (ICIs). CONCLUSIONS: Generally, this is the first study to assess the prognostic value of immune related ceRNA hub genes in ccRCC, and KCNN4 was finally demonstrated to be a key regulatory factor with strong correlation with Tregs and mast cells resting.

Reverse transcription recombinase-aided amplification assay for H5 subtype avian influenza virus.

BACKGROUND: The H5 subtype avian influenza virus (AIV) has caused huge economic losses to the poultry industry and is a threat to human health. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks. METHODS: In this study, we developed a reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of H5 subtype AIV. Assays were performed at a single temperature (39 °C), and the results were obtained within 20 min. RESULTS: The assay showed no cross-detection with Newcastle disease virus or infectious bronchitis virus. The analytical sensitivity was 103 RNA copies/µL at a 95% confidence interval according to probit regression analysis, with 100% specificity. Compared with published reverse transcription quantitative real-time polymerase chain reaction assays, the κ value of the RT-RAA assay in 420 avian clinical samples was 0.983 (p < 0.001). The sensitivity for avian clinical sample detection was 97.26% (95% CI, 89.56-99.52%), and the specificity was 100% (95% CI, 98.64-100%). CONCLUSIONS: These results indicated that our RT-RAA assay may be a valuable tool for detecting H5 subtype AIV.

Antigenic Variant of Highly Pathogenic Avian Influenza A(H7N9) Virus, China, 2019.

In China, influenza A(H7N9) virus appeared in 2013, then mutated into a highly pathogenic virus, causing outbreaks among poultry and cases in humans. Since September 2017, extensive use of the corresponding vaccine, H7-Re1, successfully reduced virus prevalence. However, in 2019, a novel antigenic variant emerged, posing considerable economic and public health threats.A.

Diversity and distribution of type A influenza viruses: an updated panorama analysis based on protein sequences.

BACKGROUND: Type A influenza viruses (IAVs) cause significant infections in humans and multiple species of animals including pigs, horses, birds, dogs and some marine animals. They are of complicated phylogenetic diversity and distribution, and analysis of their phylogenetic diversity and distribution from a panorama view has not been updated for multiple years. METHODS: 139,872 protein sequences of IAVs from GenBank were selected, and they were aligned and phylogenetically analyzed using the software tool MEGA 7.0. Lineages and subordinate lineages were classified according to the topology of the phylogenetic trees and the host, temporal and spatial distribution of the viruses, and designated using a novel universal nomenclature system. RESULTS: Large phylogenetic trees of the two external viral genes (HA and NA) and six internal genes (PB2, PB1, PA, NP, MP and NS) were constructed, and the diversity and the host, temporal and spatial distribution of these genes were calculated and statistically analyzed. Various features regarding the diversity and distribution of IAVs were confirmed, revised or added through this study, as compared with previous reports. Lineages and subordinate lineages were classified and designated for each of the genes based on the updated panorama views. CONCLUSIONS: The panorama views of phylogenetic diversity and distribution of IAVs and their nomenclature system were updated and assumed to be of significance for studies and communication of IAVs.

Isolation and characterization of a novel H7N8 avian influenza virus from domestic ducks in Central China in 2017.

In 2017, an H7N8 avian influenza virus (AIV) was isolated from a domestic duck from a farm in Central China. Sequences analysis showed that this strain received its genes from H7, H1, H2, H3, H5, and H6 AIVs of domestic poultry and wild birds in Asia. It exhibited low pathogenicity in chickens and mild pathogenicity in mice. These results suggest the importance of continued surveillance of the H7N8 virus to better understand the ecology and evolution of the AIVs in poultry and wild birds and the potential threat to human health.

Protective efficacy of an inactivated chimeric H5 avian influenza vaccine against H5 highly pathogenic avian influenza virus clades 2.3.4.4 and 2.3.2.1.

The H5 subtype of highly pathogenic avian influenza (HPAI) viruses pose a serious challenge to public health and the poultry industry in China. In this study, we generated a chimeric QH/KJ recombinant virus expressing the entire haemagglutinin (HA)-1 region of the HPAI virus A/chicken/China/QH/2017(H5N6) (clade 2.3.4.4) and the HA2 region of the HPAI virus A/chicken/China/KJ/2017(H5N1) (clade 2.3.2.1). The resulting chimeric PR8-QH/KJ virus exhibited similar in vitro growth kinetics as the parental PR8-QH and PR8-KJ viruses. The chimeric PR8-QH/KJ virus induced specific, cross-reactive haemagglutination-inhibition and serum-neutralizing antibodies against both QH and KJ viruses, although PR8-QH and PR8-KJ exhibited no cross-reactivity with each other. Furthermore, the chimeric PR8-QH/KJ vaccine significantly reduced virus shedding and completely protected chickens from challenge with HPAI H5N6 and H5N1 viruses. However, the Re-8 vaccine against clade 2.3.4.4 viruses provided specific-pathogen-free chickens only partial protection when challenged with QH virus. Our results suggest that the antigenic variation of these epidemic viruses occurred and they can escape the current vaccine immunization. The Re-8 vaccine needs an update. The chimeric PR8-QH/KJ vaccine is effective against H5 HPAI virus clades 2.3.4.4 and 2.3.2.1 in chickens.

Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.