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.

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.

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.

Analysis of Correlation Between Vertebral Endplate Change and Lumbar Disc Degeneration.

BACKGROUND To evaluate the correlation between vertebral endplate change and the level of lumbar disc degeneration via magnetic resonance imaging (MRI). MATERIAL AND METHODS A total of 345 patients who were recruited from our hospital from May 2012 to May 2016 were evaluated for the presence of intervertebral disc degeneration or herniation. The degree of degeneration was assessed according to Pfirrmann grade. Vertebral endplate change was evaluated based on the endplate concave angle (ECA), and Modic change on sagittal MRI. The correlation between ECA and lumbar disc degeneration or Modic change and lumbar disc degeneration was analyzed. RESULTS The results showed that there was no statistically significant difference in comparison of the ECAs in adjacent L3-5 vertebra between males and females. With the aggravation in degenerative changes of L3-5 discs, the ECAs of adjacent L3 superior endplate, L4 inferior and superior endplates and L5 inferior endplate were gradually enlarged, indicating the positive correlation between the lumbar disc degeneration and ECAs. The rate of Modic change in females was higher than that in males without a statistically significant difference. Area of Modic change was positively correlated with the degree of lumbar disc degeneration. Additionally, we also identified the positive correlation between the rate of Modic change and the degree of lumbar disc degeneration. CONCLUSIONS Endplate angle and lumbar disc degeneration are positively correlated. The endplates and endplate signal changes can reflect the degree of disc degeneration and Modic changes can reflect the rate of clinical lumbar disc degeneration degree.

[Effects of different gene segments of influenza virus on the growth of recombinants].

OBJECTIVE: We explored which internal genes of influenza virus that affect the titer of recombinant viruses and contribute to the high yield of Influenza A seed virus in ovo. METHODS: Six internal genes or mutant or polymerase complex of A/Puerto Rico/8/1934 (H1N1) (PR8) virus genes were replaced individually by corresponding gene of A/ chicken/ZJ/China/2013 ( H5N1) virus, and the hemagglutination titers of recombinant viruses were compared by HA assay. RESULTS: PB2 gene had the greatest influence, its replace failed to generate recombinant virus. When PB1, PA, or M gene was replaced, the titers of recombinant viruses dropped by 3.7, 3.4, 3.0 (log2), respectively. NS gene had little influence upon HA titer. When polymerase complex genes were replaced, virus titer dropped slightly to 7.6 log2, and it did not confer the same growth characteristics (8.4 log2) found when a complete polymerase complex was of PR8 origin. When amino acids of position 627 of PR8 PB2 gene were mutated to glutamic acid, virus titer rose from 8.4 log2 to 8.7 log2. CONCLUSION: The optimal gene combinations may facilitate replication through viral RNA and protein interaction with cellular components as well as interaction of viral RNA and protein or protein-protein interactions within the virus. These multi-factorial contributions resulted in selection of a high replication competent reassortant in embryonated chicken eggs in comparison to the respective low yield wild type viruses, and laid the foundation for high

Subclinical highly pathogenic avian influenza virus infection among vaccinated chickens, China.

Subclinical infection of vaccinated chickens with a highly pathogenic avian influenza A(H5N2) virus was identified through routine surveillance in China. Investigation suggested that the virus has evolved into multiple genotypes. To better control transmission of the virus, we recommend a strengthened program of education, biosecurity, rapid diagnostics, surveillance, and elimination of infected poultry.

Identification of a potential novel type of influenza virus in Bovine in China.

Bovine influenza virus was first identified in the USA in 2013, and the virus represents a potential novel type of influenza viruses. However, the distribution and evolution of the virus remain unknown. We conducted a pilot survey of bovine influenza virus in China, and identified three bovine influenza viruses which are highly homogenous to the ones identified in the USA, suggesting that the bovine influenza virus likely circulates widely and evolves slowly in the world.

A clinical survey of common avian infectious diseases in China.

Multiple common avian infectious diseases (CAIDs), namely, avian infectious diseases excluding highly pathogenic avian influenza and Newcastle disease, such as avian salmonellosis and coccidiosis, cause huge economic loss in poultry production and are of great significance in public health. However, they are usually not covered in the systems for reporting of animal diseases. Consequently, the distribution of CAIDs is not clear in many countries. Here, we report a clinical survey of CAIDs in China based on clinical diagnosis of eight veterinary clinics in 2011 and 2012. This survey provided the distribution data of viral, bacterial, and parasitic CAIDs in different types of avian flocks, seasons, and regions, data that are of great value in the research, prevention, and control of poultry diseases. This survey suggested that avian colibacillosis, infectious serositis in ducks caused by Riemerella anatipestifer, avian salmonellosis, fowl cholera, avian mycoplasmosis, avian aspergillosis, coccidiosis, low pathogenic avian influenza, infectious bronchitis, infectious bursal disease, and infectious laryngotracheitis are likely to be prevalent in the poultry in China.

Epidemiological and risk analysis of the H7N9 subtype influenza outbreak in China at its early stage.

Dozens of human cases infected with H7N9 subtype avian influenza virus (AIV) have been confirmed in China since March, 2013. Distribution data of sexes, ages, professions and regions of the cases were analyzed in this report. The results showed that the elderly cases, especially the male elderly, were significantly more than expected, which is different from human cases of H5N1 avian influenza and human cases of the pandemic H1N1 influenza. The outbreak was rated as a Grade III (severe) outbreak, and it would evolve into a Grade IV (very severe) outbreak soon, using a method reported previously. The H7N9 AIV will probably circulate in humans, birds and pigs for years. Moreover, with the driving force of natural selection, the virus will probably evolve into highly pathogenic AIV in birds, and into a deadly pandemic influenza virus in humans. Therefore, the H7N9 outbreak has been assumed severe, and it is likely to become very or extremely severe in the future, highlighting the emergent need of forceful scientific measures to eliminate any infected animal flocks. We also described two possible mild scenarios of the future evolution of the outbreak.

Physiological and metabolic toxicity of polystyrene microplastics to Dunaliella salina.

The toxicity of microplastics (MPs) to marine microalgae has raised much concern. However, research at metabolic level is quite limited. In this study, the physiological and metabolic effects of polystyrene (PS) and aged polystyrene (A-PS) MPs on Dunaliella salina were investigated. Both PS and A-PS inhibited the growth of microalgae, but promoted the pigment synthesis in algal cells. The oxidative stress analysis indicated that PS and A-PS induced high production of reactive oxygen species (ROS), and caused oxidative damage to algal cells. Particularly, the highest ROS level in PS and A-PS groups were 1.70- and 2.24-fold of that in the control group, respectively. Untargeted metabolomics analysis indicated that PS and A-PS significantly increased the differential metabolites. Compared with the control group, the significant upregulation of glycerophospholipids metabolites illustrated that severe membrane lipid peroxidation occurred in the MPs groups. Metabolic pathways analysis showed that PS and A-PS perturbed the amino acid-related metabolic pathways. In particular, the amino acid biosynthesis and ATP-binding cassette (ABC) transporter pathways were significantly upregulated, thus promoting nitrogen storage and transmembrane transport in Dunaliella salina. Transmembrane transport requires a large amount of ATP; as a result, algal cell division is inhibited. In addition, A-PS stimulated more active glutathione metabolism than PS. These results enrich the understanding of the toxicity of PS MPs to microalgae at the metabolic level, and are helpful for further assessing the ecological impacts of MPs on microalgae.

A ROS/GAS5/SIRT1 reinforcing feedback promotes oxidative stress-induced adipogenesis in bone marrow-derived mesenchymal stem cells during osteoporosis.

BACKGROUND: LincGAS5 have been reported to regulate the progression of osteoporosis (OP). However, the relationship between LincGAS5 and reactive oxygen species (ROS) in osteoporosis were still unclear. METHODS: Bilateral ovariectomy (OVX) rat were established as OP model and verified by the Micro-computed tomography. The ROS level of BMSCs derived from OVX and control rat were detected by Immunofluorescence (IF) and flow cytometry. The role of GAS5, miR-23b-3p and SIRT1 on the osteogenic differentiation were dectected by ARS saining and ALP staining, while the The Oil Red O staining and flow cytometry (FCM) were hired to determine adipogenic differentiation of BMSCs under different treatment. The expression of GAS5,miR-23b-3p and SIRT1 in BMSCs was detected by RT-qPCR and the correlation among them was analyzed. In addition, Luciferase activity was used to detect whether miR-23b-3p combined with GAS5 and SIRT1 in OP mice BMSCs. RESULTS: We established the OVX rat model and found higher ROS level in BMSCs isolated from OVX rats. Meanwhile, GAS5 was down-regulated by ROS and remarkably lowly expressed in OVX rat comparing with the negative control. We confirmed GAS5 inhibited adipogenesis and promoted osteoporosis progression. Mechanically, GAS5 bound with miR-23b-3p and suppressed its biological function. We also identified that miR-23b-3p bound with Sirtuin 1 (SIRT1) and decreased its stability. Furthermore, SIRT1 suppressed ROS production in BMSCs, which in turn un-regulated GAS5 expression through ROS-GAS5 axis. CONCLUSION: We identified a negative feedback loop, ROS-GAS5-SIRT1, in osteoporosis progression. Our findings provided potential targets and biomarkers for osteoporosis prevention and treatment.

Correction: Establishment and application of a quadruple real-time RT-PCR for detecting avian metapneumovirus.

[This corrects the article DOI: 10.1371/journal.pone.0270708.].

Single and combined toxicity of polystyrene nanoplastics and copper on Platymonas helgolandica var. tsingtaoensis: Perspectives from growth inhibition, chlorophyll content and oxidative stress.

The combined toxic effects of nanoplastics and heavy metals on aquatic organisms have attracted widespread attention; however, the results are inconsistent and the mechanisms remain unclear. In this study, the single and combined toxicity effects of Cu and two types of polystyrene nanoplastics (PS-NPs; 50 nm PS and 55 nm PS-COOH) on Platymonas helgolandica var. tsingtaoensis were investigated, including growth inhibition, chlorophyll content, and oxidative stress. An adverse dose-response relationship on growth inhibition was found in the Cu treatment groups, which was related to the decrease in chlorophyll content and damage to cell membranes. The growth inhibitory effect of PS-NPs on microalgae increased with exposure time and concentration, and no significant difference was found in the two types of PS-NPs because of the negligible contribution of functional groups. A more significant increase in chlorophyll content was found in PS treatments than in PS-COOH treatments at 96 h because of the microscale aggregates formed by PS. Higher concentrations (≥ 50 mg/L) of PS-NPs caused membrane lipid peroxidation, which might be responsible for growth inhibition. In the combined exposure experiments, a synergistic effect on the growth inhibition rate was obtained using the independent action model and Abbott model. Combined exposure triggered more severe oxidative damage to the microalgae. Adsorption experiment results showed that there was no adsorption between PS-NPs and Cu, while the interaction of Cu and algal cells could be promoted due to the presence of the PS-NPs, which explained the increasing combined toxicity. This study could improve our understanding of the combined toxicity of nanoplastics and heavy metals and could provide a new explanation for the mechanism of combined toxicity.