Lasso peptide MccY alleviates non-typhoidal salmonellae-induced mouse gut inflammation via regulation of intestinal barrier function and gut microbiota.

IMPORTANCE: Diseases caused by Enterobacteriaceae multidrug-resistant strains have become increasingly difficult to manage. It is necessary to verify the new antibacterial drug MccY effect on non-typhoid Salmonella infection in mice since it is regarded as a promising microcin. The results demonstrated that MccY has a potential therapeutic application value in the protection against Salmonella-induced intestinal damage and alleviating related intestinal dysbiosis and metabolic disorders. MccY could be a promising candidate as an antimicrobial or anti-inflammatory agent for treating infectious diseases.

Chicken telomerase reverse transcriptase promotes the tumorigenicity of avian leukosis virus subgroup J by regulating the Wnt/ß-catenin signaling pathway.

This research aimed to analyze the regulatory effect of chicken telomerase reverse transcriptase (chTERT) on the Wnt/ß-catenin signaling pathway and its effect on the tumorigenicity of avian leukosis virus subgroup J (ALV-J) through in vivo experiments. The chTERT eukaryotic expression plasmid and its recombinant lentivirus particles were constructed for in vivo transfection of chTERT to analyze the effect of chTERT continuously overexpressed in chickens on the tumorigenicity of ALV-J. During 156 days of the artificial ALV-J tumor-inducing process, 7 solid tumors developed in 3 chickens in the chTERT-overexpression group (n = 26*2) and no tumors developed in the control group (n = 26*2). Another 18 tumors induced by ALV-J were confirmed and collected from breeding poultry farms. And we confirmed that chTERT was significantly highly expressed in ALV-J tumors. The ELISA data suggested that the protein levels of ß-catenin and c-Myc in the chicken plasma of the chTERT-overexpressing group with ALV-J infected were consistently and significantly higher than those of the control group. Compared with that of the tumor-adjacent tissues, the activity of the Wnt/ß-catenin signaling pathway and expression of the c-Myc was significantly increased in ALV-J tumors. And the percentage of apoptosis in ALV-J tumors significantly lower than that in tumor-adjacent tissues. Immunohistochemistry, Western blot and RT-qPCR suggested that the replication level of ALV-J in tumors was significantly higher than that in tumor-adjacent tissues. This study suggests that chTERT plays a critical role in the tumorigenicity of ALV-J by enhancing the Wnt/ß-catenin signaling pathway, which will contribute to further elucidating the tumor-inducing mechanism of ALV-J.

The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region.

Avian leukosis virus subgroup J (ALV-J) can cause neoplastic diseases in poultry and is still widely prevalent in China. Chicken telomerase reverse transcriptase (chTERT) is the core component of telomerase, which is closely related to the occurrence and development of tumors. Our previous studies showed that chTERT is overexpressed in ALV-J tumors, but the mechanism is still not completely clear. Therefore, this study aims to analyze the possible molecular mechanism of chTERT overexpression in ALV-J tumors from the perspective of DNA methylation and promoter mutation. Methylation sequencing of the chTERT amplicon showed that ALV-J replication promoted the methylation level of the chTERT promoter. And the methylation level of the chTERT promoter in ALV-J tumors was significantly higher than that in tumor-adjacent and normal tissues. Compared with the tumor-adjacent and normal tissues, the chTERT promoter in each ALV-J tumors tested had a mutation of -183 bp C > T, and 36.0% (9/25) of the tumors also had mutations of -184 bp T > C, -73 bp::GGCCC and -56 bp A > T in the chTERT promoter, which formed the binding sites for the transcription factors NFAT5, TFAP2A and ZEB1, respectively. The results of RT-qPCR and Western blotting showed that the occurrence of these mutations significantly increased the expression level of chTERT. In conclusion, this study demonstrated that the high expression of chTERT in ALV-J tumors is positively correlated with the level of hypermethylation and mutation in its promoter, which provides a new perspective for further research on the molecular mechanism of chTERT in ALV-J tumorigenesis.

First report of a novel goose astrovirus outbreak in Muscovy ducklings in China.

A highly acute disease characterized as visceral gout broke out in Muscovy ducklings in Henan province (China) in June 2020, with a mortality rate of up to 61%. In this study, common pathogenic agents were screened using reverse-transcription polymerase chain reaction or polymerase chain reaction. The results found the novel goose astrovirus (GoAstV) to be the pathogenic agent. We isolated the GoAstV, which has been designated as HNNY0620, using the Leghorn male chicken hepatocellular carcinoma (LMH) cell line and sequenced the complete genome. The phylogenetic tree showed that the amino acid (aa) sequences of ORF1a and ORF2 and the completed nucleotide sequences of the HNNY0620 strain were clustered in the GoAstV-I clade. ORF1a aa and whole-genome sequences were genetically close to TAstV-2 and DHV-3, whereas the ORF2 aa sequences were clustered with TAstV-2 and DHV2. Both the duck-origin GoAstVs and HNNY0620 harbored some special mutations, but ORF1a in 700 (I/T), ORF1b in 288 (F/L), and ORF2 in 306 (A/T) were only found in HNNY0620. These results suggest that the host range of GoAstV is diffusing, which can potentially affect other waterfowl.

Simple and Visible Detection of Novel Astroviruses Causing Fatal Gout in Goslings Using One-Step Reverse Transcription Polymerase Spiral Reaction Method.

In this study, a one-step isothermal method combining polymerase spiral reaction (PSR) with reverse transcription (RT-PSR) was established for rapid and specific detection of novel astroviruses causing fatal gout in goslings (N-GoAstV). The one-step RT-PSR was accomplished at the optimal temperature of 62°C and time of 40 min and used primers simply designed as conventional PCR primers, and the results of detection were visible to the naked eye. The detection limit of PSR was above 34.7 copies/µL at a 95% probability level according to probit regression analysis. The assay specifically detected N-GoAstV, and no other reference viruses were detected. These results suggest that the newly established RT-PSR assay could, in one step, accomplish reverse-transcription, amplification, and result determination providing a visible, convenient, rapid, and cost-effective test that can be carried out onsite, in order to ensure timely quarantine of N-GoAstV-infected birds, leading to effective disease control.

Molecular characterization of Cachavirus firstly detected in dogs in China.

Canine Cachavirus was novel parvovirus species has been firstly identified in dogs in USA and was classified within the proposed Chaphamaparvovirus genus. To investigate Cachavirus infection in dogs in China, 408 rectal swabs from healthy and diarrheic dogs obtained during 2018-2019 were screened. The rate of Cachavirus positivity was 0% and 1.55% in healthy or diarrheic dogs, respectively. However, statistical analysis suggested no association between the presence of the virus and clinical signs (p > 0.05). Nucleotide identity was 98.2%-98.9% for NS1 and 98.6%-99.1% for VP1, and amino acid identity was 97.9%-98.7% for NS1 and 98.8%-99.6% for VP1 between the five Chinese strains and Cachavirus-1A and Cachavirus-1B detected in the United States. Phylogenetic analysis also indicated that these Cachavirus strains are genetically related to Cachavirus-1A and Cachavirus-1B. This study confirms the presence of Cachavirus in pet dogs in China and provides novel findings on its molecular characteristics.

Rapid and visual detection of novel astroviruses causing fatal gout in goslings using one-step reverse transcription loop-mediated isothermal amplification.

To visually and rapidly detect a novel goose astrovirus (N-GoAstV) causing fatal gout in goslings, an isothermal detection method based on one-step reverse transcription loop-mediated isothermal amplification (one-step RT-LAMP) was established. The one-step RT-LAMP assay for N-GoAstV detection, using Bst 3.0 DNA polymerase with strong reverse transcription activity and primer sets targeting the opening reading frame 1b (ORF1b) of N-GoAstV, could be completed in 30 min using a water bath at 61°C; the detection results could be visually observed by adding a pH-sensitive dye containing phenol red and cresol red. The detection limit of the one-step RT-LAMP assay was 57.8 copies, which was similar to that of reverse transcription-quantitative polymerase chain reaction. The assay specifically detected N-GoAstV without any cross-reaction with other reference viruses, and this was further confirmed using enzyme digestion. These results indicated that the newly established RT-LAMP assay could accomplish reverse transcription, amplification, and visual result determination in one step, and the results obtained via this rapid and cost-effective method could be used to support disease control on farms in terms of N-GoAstV infection.

Novel genotype definition and genome characteristics of duck circovirus in central and Eastern China.

To explore genetic variations in duck circovirus (DuCV) and the molecular epidemiology of its infection, tissue samples were collected from 219 dead ducks from 20 farms in the central and eastern regions of China. All farms tested positive for DuCV, with duck-origin goose parvovirus, reovirus and Tembusu virus having co-infection rates of 100%, 0% and 0%, respectively. A total of 20 strains from the DuCV-positive flock were sequenced. The total sequence length was 1987-1996 nt, and the sequences shared 82% (JX499186, DuCV2 from Sichuan province, China) to 99.7% (KY328304, DuCV1 from Shandong Province, China) sequence identity with DuCV sequences available in GenBank. Hyper-variable regions were mainly located in open reading frame (ORF)2, ORF3 and intergenic regions. The tertiary structure of ORF2 from four provinces (Henan, Anhui, Zhejiang and Fujian) in China showed a canonical viral jelly roll and the antigenic epitope of ORF2 located in the bulge of the protein surface. Overall, 15 of the 20 DuCV strains are possibly derived through inter-genotypic and intragenotypic recombination. Based on sequence and phylogenetic analyses, six strains from Fujian Province clustered into a novel genotype-DuCV-1d. These findings may enrich our understanding of DuCV evolution and circulation and lay the foundation for vaccine strain selection.

Epidemiological investigation of avian infectious bronchitis and locally determined genotype diversity in central China: a 2016-2018 study.

Infectious bronchitis (IB), caused by avian IB virus (IBV), is an acute and highly contagious disease of chickens. From 2016 to 2018, 56 IBV strains were isolated and identified from clinical samples obtained from various chicken farms located in central China. The S1 sequencing of these strains revealed nucleotide and amino acid identities of 70.2 to 100% and 62.6 to 100%, respectively, compared with those of reference strains. Phylogenetic analysis indicated that the genotypes of the isolates included GI-13 (4/91), GI-7 (TW-I), GI-24 (Mass), GI-19 (QX), and GI-18 (LDT3-A), with GI-19 (QX) being the predominant genotype. Meanwhile, GI-13 (4/91) was the second most dominant genotype in Henan Province, whereas it was GI-7 (TW-I) in Hunan and Hubei provinces. Recombination analysis of 3 variant strains showed that CK/CH/HeN/20160113 might be a recombination of LDT3-A- and QX-type strains and that CK/CH/HeN/20160316 might be a recombination of Italy-02-type strain and CK-CH-LJS08II. The predicted tertiary structure between CK/CH/HeN/20160113 and LDT3-A-type strain revealed that the novel 336 (L-P) and 455 (S-A) mutations changed the structure from an alpha helix to a random crimp. In addition, the 275 (Y-F) site reduced the length of the ß-sheet, whereas the site 353 (A-T) extended the ß-sheet. These findings suggested that GI-19 (QX) remains the predominant genotype in central China, and a locally determined complex genotype associated with variable clinical symptoms exists related to gene recombination and mutations.

Characterization and genomic analysis of emerging astroviruses causing fatal gout in goslings.

Since February 2017, severe outbreaks of fatal gout caused by novel gosling astroviruses (GoAstVs) have occurred in several Chinese provinces, causing a considerable economic impact on the poultry industry. To assess the infection status of GoAstVs causing gout, 165 clinical samples were collected from goslings from seven farms located in different Chinese provinces, and they were screened for viral infection. Seven GoAstV strains were completely sequenced. The positive infection rates of GoAstV, goose parvovirus, reovirus, goose haemorrhagic polyomavirus and Tembusu virus were 100%, 9.69%, 3.64%, 0% and 0%, respectively, indicating the role of GoAstV in gout. The genomes of all seven GoAstV strains were 7170-nt long and encoded three open reading frames (ORFs), namely, ORF1a, ORF1b and ORF2. Sequence and phylogenetic analyses of the seven GoAstV strains showed that these were avastroviruses and were closely related to viruses classified within Avastrovirus 3 and turkey astrovirus 2. Moreover, the mutation rates of ORF1a and ORF2 were high, and ORF1a was highly mutated at amino acid loci 545-580. The tertiary structure of the mutated ORF2 protein was smooth, and its antigenic epitope was highly mutated, which may be related to the pathogenicity of the virus and caused by antibody pressure from the host. These findings enrich our understanding of the evolution of novel GoAstVs causing gout and their circulation as well as lay the foundation for the selection of vaccine strains.

Genetic Analysis of Avian Gyrovirus 2 Variant-Related Gyrovirus Detected in Farmed King Ratsnake (Elaphe carinata): The First Report from China.

Avian gyrovirus 2 (AGV2), which is similar to chicken infectious anemia virus, is a new member of the genus Gyrovirus. AGV2 has been detected not only in chicken but also in human tissues and feces. This study analyzed 91 samples (8 from liver tissue and 83 from fecal samples) collected from king ratsnakes (Elaphe carinata) from 7 separate farms in Hubei and Henan, China, for AGV2 DNA using PCR. The results demonstrated a low positive rate of AGV2 (6.59%, 6/91) in the snakes, and all the positive samples were collected from the same farm. The AGV2 strain HB2018S1 was sequenced, and its 2376 nt genome comprised three partially overlapping open reading frames: VP1, VP2, and VP3. Phylogenetic analysis revealed that the HB2018S1 and NX1506-1 strains from chickens in China belong to the same clade and that they have a nucleotide identity as high as 99.5%. Additionally, recombination analysis showed that HB2018S1 might originate from the recombination of viruses similar to those detected in chickens and a ferret. A total of 10 amino acid mutation sites (44(R/K), 74(T/A), 256 (C/R), 279(L/Q), and 373(V/A) in AGV2 VP1; 60(I/T), 125(T/I), 213(D/N), and 215(L/S) in AGV2 VP2; and 83(H/Y) in AGV2 VP3) different from those observed in most reference strains were found in the genome of HB2018S1, indicating that the differences may be related to a transboundary movement among hosts, which needs further elucidation. To the best of our knowledge, this study is the first report on an AGV2-infected poikilotherm, suggesting that cross-host transmission of viruses with circular single-stranded DNA genomes would be a public health concern.

Rapid and visible detection of Mycoplasma synoviae using a novel polymerase spiral reaction assay.

In this study, a rapid, specific, and sensitive detection assay for Mycoplasma synoviae (MS) was established using a polymerase spiral reaction (PSR) method. A pair of primers were designed according to the conserved region of the vlhA gene of MS, and PSR results were assessed using agarose gel electrophoresis and color rendering with a dye indicator. The optimum reaction temperature and time for PSR using the specific primers were 62°C and 40 min in a water bath, respectively. The sensitivity of the PSR assay for MS detection was 100 times more than that of the polymerase chain reaction assay based on agarose gel electrophoresis results and color change detected by the naked eye. Further experiments demonstrated that the primers specifically detected MS and showed no cross-reaction with other prevalent avian pathogens. Clinical sample testing confirmed that the MS-PSR assay is simple, rapid, specific, and sensitive, and thereby very suitable for application and promotion in the field and laboratories of grassroots units.

A monovalent anion affected multi-functional cellulase EGX from the mollusca, Ampullaria crossean.

A cellulose hydrolytic enzyme was isolated from the stomach juice of Ampullaria crossean, a kind of herbivorous mollusca. The enzyme was purified 45.3-fold to homogenety by ammonium sulfate precipitation, DEAE-Sephadex A-50 column, Bio-gel P-100 gel filtration column, and phenyl-Sepharose CL-4B column chromatography. The enzyme was designated as cellulase EGX. The purified enzyme is a multi-functional enzyme with the activities of exo-beta-1,4-glucanase (14.84 U/mg for p-nitrophenyl beta-D-cellobioside), endo-beta-1,4-glucanase (40.3 U/mg for carboxymethyl cellulose), and endo-beta-1,4-xylanase (196 U/mg for soluble xylan from birchwood). The monovalent anions such as F(-), Cl(-), Br(-), I(-), and NO(3)(-) are essential for its exo-beta-1,4-glucanase activity but have no effect on the activity for xylan, while I(-) higher than 5mM would inhibit the exo-beta-1,4-glucanase activity. The monovalent anions Cl(-) and Br(-) activate its endo-beta-1,4-glucanase activity. Binding of Cl(-) enhances the thermostability of EGX, but does not affect its fluorescence emission spectrum. The molecular mass of EGX is 41.5 kDa, as determined by SDS-PAGE. The pI value is about pH 7.35. The xylan hydrolytic activity of EGX reaches to the maximum between pH 4.8 and 6.0 and the pNPC hydrolytic activity reaches the maximum between pH 4.8 and 5.6, while that for CMC hydrolytic activity is between pH 4.4 and 4.8. Preliminary results showed that the enzyme was secreted by the mollusca itself.