Re-emergence of severe acute diarrhea syndrome coronavirus (SADS-CoV) in Henan, central China, 2023.

Severe acute diarrhea syndrome coronavirus (SADS-CoV) was first detected in Guangdong province of China in 2017. And yet from May 2021 to Jun 2023, there were no SADS-CoV outbreaks. In this study, we reported the recent outbreak of SADS-CoV in China on Jun 2023. Phylogenetic analysis showed the novel strain was derived from the ongoing transmission and evolution of SADS-CoV in China, rather than a separate cross-species transmission from bats. Also, the novel strain was found to participate in a recombant event as a minor parent and a missing base in the genome was discovered indicating an novel evolutionary pathway. Through virulence assays in piglets, we further determined that novel strain (SADS-CoV/HNNY/2023) was a highly virulent SADS-CoV strain with typical clinical symptoms: acute diarrhea, vomiting, rapid weight loss. Therefore, the re-emergence of SADS-CoV strains should be brought to people's attention.

Phylogenetic Analysis and Codon Usage Bias Reveal the Base of Feline and Canine Chaphamaparvovirus for Cross-Species Transmission.

Chaphamaparvoviruses (ChPVs) are ancient viruses that have been detected in a variety of hosts. In this study, through a phylogenetic analysis and the adaptability of ChPV to multiple hosts, we evaluated the basis for the ability of feline (FeChPV) and canine ChPV (CaChPV) for cross-species transmission. Phylogenetic analysis showed that FeChPV and CaChPV were closely related. Notably, two strains of ChPVs isolated from domestic cats and two from dogs clustered together with CaChPVs and FeChPVs, respectively, suggesting that the stringent boundaries between canine and feline ChPV may be broken. Further analysis revealed that CaChPV and FeChPV were more adapted to dogs than to cats. Mutation analysis identified several shared mutations in cross-species-transmissible strains. Furthermore, the VP structures of FeChPV and CaChPV exhibited a high degree of similarity across both cross-species-transmissible and non-cross-species-transmissible strains. However, it is crucial to note that these results are largely computational, and limitations exist in terms of the number and diversity of samples analyzed; the capacity for cross-species transmission should be approached with caution and elucidated in further studies.

Non-adaptive evolution in codon usage of human-origin monkeypox virus.

Monkeypox virus (Mpox) is a zoonotic infectious disease that threatens human and animal health, with a global outbreak of the low-pathogenic Mpox beginning from 2022. In this study, we analyzed the codon usage of Mpox between two clades, Clade-I and Clade-IIb-B, to understand changes in host adaptation. Clade-IIb-B of the Mpox genome underwent non-adaptive evolution making it less adapted to its host than Clade-I. The analysis of individual genes revealed that 48 genes exhibited non-adaptive mutation, while 38 genes underwent adaptive mutations. Genes involved in replication, transcription, and host-modulation exhibited a mix of adaptive and non-adaptive evolutionary patterns. This study also found that the mutations of Mpox led to changes in non-adaptative genes in different organs. Additionally, we found that codon usage of Mpox was less similar to that of up-regulated host genes and more similar to that of down-regulated host genes post-infection, indicating that codon usage affects host gene expression. Overall, the study highlights the non-adaptive changes in codon usage as a potential cause of differences in Mpox virulence and provides insights into the evolutionary and adaptive mechanisms of Mpox and its potential impact on pathogenicity and host adaptation.

Salicylic acid remodeling of the rhizosphere microbiome induces watermelon root resistance against Fusarium oxysporum f. sp. niveum infection.

Fusarium wilt disease poses a severe threat to watermelon cultivation by affecting the yield and quality of the fruit. We had previously found that the rhizosphere microbiome has a significant impact on the ability of watermelon plants to resist Fusarium wilt development and that salicylic acid (SA) is closely related to this phenomenon. Therefore, in this study, the role of SA as a mediator between plants and microbes in activating resistance against Fusarium oxysporum f. sp. niveum (FON) infection was explored through physiological, biochemical, and metagenomic sequencing experiments. We demonstrated that exogenous SA treatment could specifically increase some beneficial rhizosphere species that can confer resistance against FON inoculation, such as Rhodanobacter, Sphingomonas, and Micromonospora. Functional annotation analysis indicated that SA application significantly increased the relative abundance of glycoside hydrolase and polysaccharide lyase genes in the microbiome, which may play an essential role in increasing plant lipids. Moreover, network interaction analysis suggested that the highly expressed AAC6_IIC gene may be manipulated through SA signal transduction pathways. In conclusion, these results provide a novel strategy for controlling Fusarium wilt in watermelons from the perspective of environmental ecology, that is, by manipulating the rhizosphere microbiome through SA to control Fusarium wilt.

Facile, ultrasensitive, and highly specific diagnosis of goose astrovirus via reverse transcription-enzymatic recombinase amplification coupled with a CRISPR-Cas12a system detection.

Fatal gout in geese caused by goose astrovirus (GAstV) has been spreading rapidly in China since 2018, causing serious economic losses in the goose breeding industry. To achieve simple, convenient and sensitive detection of GAstV, a novel diagnostic test was developed by combining reverse transcription-enzymatic recombinase amplification (RT-ERA) and CRISPR-Cas12a technologies. RT-ERA primers were designed to pre-amplify the conserved region of the ORF2 gene of GAstV and the predefined target sequence detected using the Cas12a/crRNA complex at 37℃ for 30 min. Specific detection of GAstV was achieved with no cross-reaction with non-GAstV templates and a sensitivity detection limit of 2 copies. The experimental procedure could be completed within 1 h, including RNA extraction (15 min), RT-ERA reaction (20 min), CRISPR-Cas12a/crRNA detection (5 min) and result readout (within 2 min) steps. In conclusion, the combination of RT-ETA and CRISPR-Cas12a provides a rapid and specific method that should be effective for the control and surveillance of GAstV infections in farms from remote locations.

Rapid detection of porcine circovirus type 4 via multienzyme isothermal rapid amplification.

Porcine circovirus type 4 (PCV4) is a newly emerging pathogen that was first detected in 2019 and is associated with diverse clinical signs, including respiratory and gastrointestinal distress, dermatitis and various systemic inflammations. It was necessary to develop a sensitive and specific diagnostic method to detect PCV4 in clinical samples, so in this study, a multienzyme isothermal rapid amplification (MIRA) assay was developed for the rapid detection of PCV4 and evaluated for sensitivity, specificity and applicability. It was used to detect the conserved Cap gene of PCV4, operated at 41°C and completed in 20 min. With the screening of MIRA primer-probe combination, it could detect as low as 101 copies of PCV4 DNA per reaction and was highly specific, with no cross-reaction with other pathogens. Further assessment with clinical samples showed that the developed MIRA assay had good correlation with real-time polymerase chain reaction assay for the detection of PCV4. The developed MIRA assay will be a valuable tool for the detection of the novel PCV4 in clinical samples due to its high sensitivity and specificity, simplicity of operation and short testing time.

Rapid and Visual Detection of Porcine Parvovirus Using an ERA-CRISPR/Cas12a System Combined With Lateral Flow Dipstick Assay.

Porcine parvovirus (PPV) is one of the important causes of pig reproductive diseases. The most prevalent methods for PPV authentication are the polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and quantitative real-time PCR. However, these procedures have downsides, such as the fact that they take a long time and require expensive equipment. As a result, a rapid, visible, and economical clinical diagnostic strategy to detect PPV is necessary. In this study, three pairs of crRNA primers were designed to recognize the VP2 gene, and an ERA-CRISPR/Cas12a system for PPV detection was successfully developed. The approach involved isothermal detection at 37°C, and the method can be used for visual inspection. The detection limit of the ERA-CRISPR/Cas12a system was 3.75 × 102 copies/µL, and no cross reactions with other porcine viruses were found. In view of the preceding, a rapid, visible, and low-cost nucleic acid testing approach for PPV has been developed using the ERA-CRISPR/Cas12a system.

The Formation of Hollow Trait in Cucumber (Cucumis sativus L.) Fruit Is Controlled by CsALMT2.

The hollow trait is crucial for commercial quality of cucumber (Cucumis sativus L.) fruit, and its molecular regulatory mechanism is poorly understood due to its environmental sensitivity. In the previous research, we obtained the hollow and the non-hollow materials of ecotype cucumbers of South China, which were not easily affected by the external environment through a systematic breeding method. In this study, first, we proposed to use the percentage of the hollow area as the criterion to compare the hollow characteristics between two materials, and to analyze the formation mechanism of early hollow trait from the perspective of cytology. The results showed that the hollow trait occurred in the early stage of fruit development, and formed with the opening of carpel ventral zipped bi-cell layer, which formed rapidly from 2 to 4 days, and then slowed to a constant rate from 14 to 16 days. Meanwhile, the different genetic populations were constructed using these materials, and fine mapping was performed by bulked segregant analysis (BSA) and kompetitive allele specific PCR (KASP) method. The Csa1G630860 (CsALMT2), encoding protein ALMT2, was determined as a candidate gene for regulating the hollow trait in fruit. Furthermore, the expression profile of CsALMT2 was analyzed by qRT-PCR and fluorescence in situ hybridization. The expression of CsALMT2 had obvious tissue specificity, and it was abundantly expressed in the ovule development zone inside the fruit. In the hollow material of cucumber fruit, the expression of CsALMT2 was significantly downregulated. The subcellular localization in tobacco leaves indicated that CsALMT2 was distributed on the plasma membrane. In conclusion, in this study, for the first time, we found the regulatory gene of hollow trait in cucumber fruit, which laid the foundation for subsequent research on the molecular mechanism of hollow trait formation in cucumber fruit, and made it possible to apply this gene in cucumber breeding.

LncRNA LINC01270 aggravates the progression of gastric cancer through modulation of miR-326/EFNA3 axis.

Gastric cancer (GC) is lethal malignancy, which is associated with high mortality. Long noncoding RNA LINC01270 has been identified to act as a potential oncogene in several cancers. However, its role and related regulatory mechanism in GC are yet to be illustrated. The levels of lncRNA LINC01270, miR-326, and EphrinA3 (EFNA3) were assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) and colony formation assays were applied for analyzing cell proliferation. Transwell assay was used for measuring cellular migration and invasion. Western blot analysis was employed for evaluating the protein levels. Luciferase reporter and RNA pull-down assays were utilized to verify the binding ability between LINC01270 (or EFNA3) and miR-326. Our findings indicated that LINC01270 expression was significantly up-regulated in GC tissues and cell lines. Additionally, LINC01270 knockdown attenuated GC progression through inhibiting cell proliferation, migration, and invasion. Functional experiments identified that lncRNA LINC01270 could positively regulate EFNA3 expression by serving as a competing endogenous RNA (ceRNA) for miR-326. Through rescue assays, inhibition of GC progression caused by LINC01270 suppression was found to be reversed by the application of miR-326 inhibitor or EFNA3 overexpression. Overall, our work demonstrated that lncRNA LINC01270 can accelerate cell proliferation, migration, and invasion via modulating miR-326/EFNA3 axis. These findings might implicate the potential role of lncRNA LINC01270 in GC treatment.

Study on the Role of Phytohormones in Resistance to Watermelon Fusarium Wilt.

Fusarium wilt disease is one of the major diseases causing a decline in watermelon yield and quality. Researches have informed that phytohormones play essential roles in regulating plants growth, development, and stress defendants. However, the molecular mechanism of salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) in resistance to watermelon Fusarium wilt remains unknown. In this experiment, we established the SA, JA, and ABA determination system in watermelon roots, and analyzed their roles in against watermelon Fusarium wilt compared to the resistant and susceptible varieties using transcriptome sequencing and RT-qPCR. Our results revealed that the up-regulated expression of Cla97C09G174770, Cla97C05G089520, Cla97C05G081210, Cla97C04G071000, and Cla97C10G198890 genes in resistant variety were key factors against (Fusarium oxysporum f. sp. Niveum) FON infection at 7 dpi. Additionally, there might be crosstalk between SA, JA, and ABA, caused by those differentially expressed (non-pathogen-related) NPRs, (Jasmonate-resistant) JAR, and (Pyrabactin resistance 1-like) PYLs genes, to trigger the plant immune system against FON infection. Overall, our results provide a theoretical basis for watermelon resistance breeding, in which phytohormones participate.

Fowl adenovirus serotype 4 uses gga-miR-181a-5p expression to facilitate viral replication via targeting of STING.

Fowl adenovirus serotype 4 (FAdV-4) has caused substantial economic losses to the poultry industry and it has become a serious pathogen of poultry in China since 2015. MicroRNAs (miRNAs) play vital roles in regulating viral infection. However, how miRNAs regulate FAdV-4 replication in Leghorn male hepatocellular (LMH) cells remains unclear. This study aimed to elucidate the role of gga-miR-181a-5p in regulating FAdV-4 replication. The findings indicated that the expression of gga-miR-181a-5p was significantly upregulated in LMH cells during FAdV-4 infection. Also, the transfection of gga-miR-181a-5p mimics promoted FAdV-4 replication, while the opposite result was observed when gga-miR-181a-5p inhibitor was transfected in LMH cells. Moreover, the stimulator of interferon genes (STING) was found to be the target gene of gga-miR-181a-5p using software analysis, further confirming that STING was the target of gga-miR-181a-5p and gga-miR-181a-5p could negatively regulate the expression of STING at the mRNA and protein levels. Finally, the results showed that the overexpression of STING inhibited FAdV-4 replication and the knockout of STING promoted FAdV-4 replication. The collective findings revealed a novel host evasion mechanism adopted by FAdV-4 via gga-miR-181a-5p, suggesting novel strategies for designing miRNA-based vaccines and therapies.

Epidemiology and Evolution of Emerging Porcine Circovirus-like Viruses in Pigs with Hemorrhagic Dysentery and Diarrhea Symptoms in Central China from 2018 to 2021.

Porcine circovirus-like virus (PCLV) is a type of circular Rep-encoding single-stranded DNA virus and may be associated with the development of diarrheal symptoms in pigs. In this study, we retrospectively analyzed three years of past cases in Anhui, China, and reported a case of hemorrhagic enteritis and death in a pregnant sow possibly caused by PCLV. In addition, we analyzed the evolutionary characteristics of PCLV and found that mutation, recombination and selective pressure all played an important role in the evolution of PCLV. We identified N15D and T17S as well as L56T, T58R, K59Q, M62R, L75I and R190K mutations in two different branches, and we noted recombination events in the Rep of a group of Chinese strains. Analysis of selection pressure revealed that PCLV gained more positive selection, indicating that the virus is in a continuous evolutionary state. The PR2 plot, ENC-plot and neutrality analysis showed a greater role of natural selection than that of mutational pressure in the formation of codon usage patterns. This study is the first to identify PCLV in sows with hemorrhagic dysentery and death, and it provides new epidemiological information on PCLV infection in pigs in China.

Reverse transcription-enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription-enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation.

Enzymatic recombinase amplification coupled with CRISPR-Cas12a for ultrasensitive, rapid, and specific Porcine circovirus 3 detection.

Porcine circovirus type 3 (PCV3) is a disease associated with porcine dermatitis and nephrotic syndrome (PDNS) that has caused significant economic losses to swine herds since its discovery in 2016. To develop a simple, on-site, rapid, and sensitive assay to combat the spread of PCV3, we optimized the CRISPR/Cas12a (also known as Cpf1) system combined with enzymatic recombinase amplification (ERA) nucleic acid amplification to diagnose PCV3. The results showed that the ERA-CRISPR/Cas12a reaction could detect PCV3 within 1 h in genomic DNA harboring a minimum of seven copies. Additionally, we confirmed no cross-reactivity with PCV2, PCV4, or other porcine viruses, revealing the good specificity of this technique. These results demonstrated the ability of ERA-CRISPR/Cas12a to detect DNA at the single-molecule level and provide a rapid, simple, ultrasensitive, one-pot point-of-care test for PCV3 and suggest its potential for a variety of nucleic acid detection applications.

Complete Genetic Analysis of Plasmids Carrying mcr-1 and Other Resistance Genes in Avian Pathogenic Escherichia coli Isolates from Diseased Chickens in Anhui Province in China.

Antimicrobial resistance associated with colistin has emerged as a significant concern worldwide, threatening the use of one of the most important antimicrobials for treating human disease. This study aimed to investigate the prevalence of colistin-resistant avian-pathogenic Escherichia coli (APEC) and shed light on the possibility of transmission of mcr-1 (mobilized colistin resistance)-positive APEC. A total of 72 APEC isolates from Anhui Province in China were collected between March 2017 and December 2018 and screened for the mcr-1 gene. Antimicrobial susceptibility testing was performed using the broth dilution method. Pulsed-field gel electrophoresis, Southern blot analysis, and conjugation assay were performed to determine the location and conjugative ability of the mcr-1 gene. Whole-genome sequencing and analysis were performed using Illumina MiSeq and Nanopore MinION platforms. Three APEC isolates (AH25, AH62, and AH65) were found to be positive for the mcr-1 gene and showed multidrug resistance. The mcr-1 genes were located on IncI2 plasmids, and conjugation assays revealed that these plasmids were transferrable. Notably, strains AH62 and AH65, both belonging to ST1788, were collected from different places but carried the same drug resistance genes and shared highly similar plasmids. This study highlights the potential for a possible epidemic of mcr-1-positive APEC and the urgent need for continuous active monitoring.IMPORTANCE In this study, three plasmids carrying mcr-1 were isolated and characterized from APEC isolates from Anhui Province in China. The mcr-1 genes were located on IncI2 plasmids, and these plasmids were transferrable. These three IncI2 plasmids had high homology with the plasmids harbored by pathogenic bacteria isolated from other species. This finding showed that IncI2 plasmids poses a risk for the exchange of genetic material between different niches. Although colistin has been banned for use in food-producing animals in China, the coexistence of the broad-spectrum ß-lactamase and mcr-1 genes on a plasmid can also lead to the stable existence of mcr-1 genes. The findings illustrated the need to improve the monitoring of drug resistance in poultry systems so as to curb the transmission or persistence of multidrug-resistant bacteria.

Genetic characterization and phylogenetic analysis of feline astrovirus from Anhui province in eastern China.

This study aimed to explore the phylogenetic and molecular characteristics of feline astrovirus. A total of 33 fecal samples of domestic cats with or without diarrhea were collected from the Anhui province, and two positive samples were detected. The complete genome and ORF2 of the two strains were sequenced and phylogenetically analyzed. AH-1-2020 and AH-2-2020 displayed 83.4% homology, and their homologies with other reference strains were 75.3%-83.4% and 83.4%-95.0%, respectively. Phylogenetic tree analysis revealed that all strains could be classified into three different clusters; therefore, the mean amino acid genetic distances (p-dist) among the three clusters were estimated. The results suggested that the two strains and other FeAstV strains were grouped into three genotypes, with AH-1-2020 belonging to a novel genotype. High similarity was observed (65.9%-66.5% nucleotide identity and 63.8%-64.8% amino acid identity) in ORF2 between porcine astrovirus type 1 and AH-1-2020. Furthermore, inter-specific recombination between porcine astrovirus type 1 and FeAstV was observed. We, therefore, inferred that inter-specific transmission may exist between pigs and cats; however, further studies are required to verify this. This is the first report on the genetic characterization and phylogenetic analysis of FeAstVs in the Anhui province and would further the current understanding of the genetic diversity and epidemiology of FeAstVs.

Identification and full-genome sequencing of canine kobuvirus in canine fecal samples collected from Anhui Province, eastern China.

Canine kobuvirus (CaKoV), a newly described virus, is the causative agent of gastroenteritis in dogs. In this study, 57 fecal samples from dogs with diarrhea in Anhui Province, eastern China, were collected. Among these, five samples were identified to be infected with CaKoV, by polymerase chain reaction targeting the CaKoV 3D gene. The five CaKoV strains were subjected to phylogenetic analysis. The sequences of VP1 from the five CaKoV strains were 93.6%-96.1% identical to each other and 91.75%-97.95% identical to other reported CaKoV VP1 sequences. In addition, the complete genome of one strain was successfully amplified and sequenced. The genome consisted of 8223 nucleotides and shared 94.6%-97.0% nucleotide and 93.1%-94.0% amino acid sequence identity with other CaKoV isolates. Phylogenetic analysis revealed that the CaKoV strain from Anhui Province was similar to other Chinese strains, and it was more closely related to feline and mouse kobuviruses than to sheep and bovine kobuviruses. Interestingly, all of the CaKoV-positive samples were coinfected with canine parvovirus. The finding of CaKoV infection in dogs with diarrhea and coinfection with canine parvovirus are a cause for concern and highlight the need for management and preventive measures.

Molecular characterization and pathogenicity of highly pathogenic fowl adenovirus serotype 4 isolated from laying flock with hydropericardium-hepatitis syndrome.

Hydropericardium-hepatitis syndrome (HHS) is an important emerging disease responsible for huge economic losses to the poultry industry in China. HHS primarily affects 20 to 60-day-old broilers and rarely occurs in laying flock. In this study, the highly pathogenic fowl adenovirus (FAdV) strain, AH-F19, was isolated from the liver samples of 120-day-old laying flock with HHS and its phylogenetic information, genetic mutations, and pathogenicity was evaluated. The phylogenetic analysis revealed that AH-F19 belonged to the FAdV serotype 4 (FAdV-4) cluster, however, 100K differs from the other FAdV-4 strains and is divided into different branches. Amino acid variations in fiber-2 for pathogenic isolates and non-pathogenic isolates indicated that D219, T300, and T380 may not be responsible for virulence. Animal experiments revealed AH-F19 to be a highly pathogenic isolate that can cause 100% mortality in three-week-old specific pathogen-free (SPF) chickens, which exhibited typical hydropericardium and hepatitis. Microscopically, the presence of basophilic intranuclear inclusion bodies in hepatocytes, fractured heart muscle fibers, as well as kidney degeneration and necrosis was observed. Collectively, these findings enriched our understanding of FAdV-4 pathogenicity and provided a reference for further exploration into its pathogenicity.