Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1.

BACKGROUND: Infectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain's gC, TK, gG, gD, and gE genes. RESULTS: The study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/µL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28. CONCLUSIONS: The established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

First report of large-berry coffee (Coffea liberica) anthracnose caused by Colletotrichum kahawae in China.

Large-berry coffee (Coffea liberica) is one of the three cultivated coffee species and a precious breeding germplasm in China (Yan et al, 2019). Anthracnose is a damaging epidemic disease on coffee worldwide (Mohammed et al. 2015). Between June and September 2022, anthracnose was observed on coffee plants in Puer area, Yunnan, China and disease incidence (% plants diseased) of 8.5%-28.2% was recorded in the field. The disease symptoms were observed at all growth stages. Lesions on leaves were circular or oval, with a white to gray central zone outlined by a brown margin and surrounded by a chlorotic halo, Φ5.1-18.5 mm; some lesions extended and coalesced later to form large, blighted areas, leading to complete leaf senescence, defoliation and bare blighted branches on heavily infected trees. The spots on coffee berries were oval or fusiform, sunken and brown-black; diseased berries became gray-black and dried-out but remained on the tree. Leaves with typical anthracnose lesions were collected from fields in Simao ( 22.07°E,100.98°N) to isolate the pathogen. Leaf pieces (5×5mm) from the lesion margin were cut, surface-sterilized with 75% ethanol and 2% NaClO, and cultured on PDA at 25°C. Three isolates with the same colony morphology were obtained by hyphal tip purification. Detached and intact leaves of 6-month coffee seedlings were inoculated with Φ5mm mycelial discs of the isolates. Anthracnose lesions developed on the inoculated leaves, with all 3 isolates, 7d after incubation in a growth chamber (25°C, > 90% RH and lighting 8 h/d at 11000 lux). Pathogens with the same colony morphology as those of the original isolates were re-isolated from the infected tissues of inoculated leaves, thus fulfilling Koch's Postulates. The ITS sequence (PP550861) for the isolate was PCR-amplified and Blast-n analyses showed 100 % (554/554bp) identity to Colletotrichum kahawae LWTJ01; so they were the same population and coded as KFTJ02. The actin (ACT), calmodulin(CAL), glyceraldehydes-3-phosphate dehydrogenase (GAPHD) and histone 3 (HIS3) genes (Qiu et al. 2020) were amplified from one of KFTJ02 isolates, sequenced and deposited in NCBI GenBank (OR842543, OR842544, OR842545 & OR842546). A phylogenetic tree was generated based on the concatenated sequences of the four genes and those of related Colletotrichum spp. using MEGA 6.0 and KFTJ02 clustered in the same clade with C. kahawae IMI319418 on the tree (Bootstrap sup.=88%). When cultured at 25°C on PDA for 7 days, its colonies were near round or ovoid, gray-white, contoured, Φ73.2-80.1 (76.2±2.3)mm or growth rate 10.2-11.1(8.1) mm/d (n=10). The hyphae were hyaline, septated, branching at near right angles. Conidial masses formed 14 days after incubation. The conidia were elliptical, hyaline, monocellular, 10.2-15.5 (12.7±1.06)×3.8-5.2 (4.3±0.52) µm (n=50). The appressoria were black-brown, oval or irregular, 7.8-9.3 (8.5±0.81)µm (n= 50). These morphological characteristics were consistent with those of C. kahawae (Bridge et al, 2008). Therefore, KFTJ02 was identified as C. kahawae, which has been found to infect Camellia oleifera, Areca catechu and Ficus microcarpa (Wei et al, 2023; Zhang et al, 2020; Lin 2023). The coffee berry disease pathogen (C. kahawae) is a quarantine species which has not been recorded and so it is first reported on coffee crops in China. Results of the present study provide important references for further studies on this disease.

Phylogenetic analysis of swine influenza A (H1N2) viruses isolated in Jinju City, Republic of Korea.

Genomic sequences of the swine influenza A (H1N2) viruses "A/Swine/South Korea/GN-1/2018" and "A/Swine/South Korea/GNJJ/2020" sampled from Jinju City, Republic of Korea, are reported here. The sequences of these viruses were 99% similar. These included eight genes from each of the H3N2pM, A(H1N1)2009pdm, and North American swine lineages.

Genomic and Pathologic Characterization of the First FAdV-C Serotype 4 Isolate from Black-Necked Crane.

Fowl adenoviruses (FAdVs) are distributed worldwide in poultry and incriminated as the etiological agents for several health problems in fowls, and are capable of crossing species barriers between domestic and wild fowls. An FAdV strain was, for the first time, isolated from black-necked crane in this study, and was designated as serotype 4 Fowl aviadenovirus C (abbreviated as BNC2021) according to the phylogenetic analysis of its DNA polymerase and hexon gene. The viral genomic sequence analysis demonstrated that the isolate possessed the ORF deletions that are present in FAdV4 strains circulating in poultry fowls in China and the amino acid mutations associated with viral pathogenicity in the hexon and fiber 2 proteins. A viral challenge experiment with mallard ducks demonstrated systemic viral infection and horizontal transmission. BNC2021 induced the typical clinical signs of hepatitis-hydropericardium syndrome (HHS) with swelling and inflammation in multiple organs and showed significant viral replication in all eight organs tested in the virus-inoculated ducks and their contactees at 6 dpi. The findings highlight the importance of surveillance of FAdVs in wild birds.

[Identification and expression of uridine diphosphate glycosyltransferase(UGT) gene family from Dendrobium officinale].

Uridine diphosphate glycosyltransferase(UGT) is a highly conserved protein in plants, which usually functions in secondary metabolic pathways. This study used the Hidden Markov Model(HMM) to screen out members of UGT gene family in the whole genome of Dendrobium officinale, and 44 UGT genes were identified. Bioinformatics was used to analyze the structure, phylogeny, and promoter region components of D. officinale genes. The results showed that UGT gene family could be divided into four subfamilies, and UGT gene structure was relatively conserved in each subfamily, with nine conserved domains. The upstream promoter region of UGT gene contained a variety of cis-acting elements related to plant hormones and environmental factors, indicating that UGT gene expression may be induced by plant hormones and external environmental factors. UGT gene expression in different tissues of D. officinale was compared, and UGT gene expression was found in all parts of D. officinale. It was speculated that UGT gene played an important role in many tissues of D. officinale. Through transcriptome analysis of D. officinale mycorrhizal symbiosis environment, low temperature stress, and phosphorus deficiency stress, this study found that only one gene was up-regulated in all three conditions. The results of this study can help understand the functions of UGT gene family in Orchidaceae plants and provide a basis for further study on the molecular regulation mechanism of polysaccharide metabolism pathway in D. officinale.

Insights into the Genes Involved in ABA Biosynthesis and Perception during Development and Ripening of the Chilean Strawberry Fruit.

Hormones act as master ripening regulators. In non-climacteric fruit, ABA plays a key role in ripening. Recently, we confirmed in Fragaria chiloensis fruit that in response to ABA treatment the fruit induces ripening-associated changes such as softening and color development. In consequence of these phenotypic changes, transcriptional variations associated with cell wall disassembly and anthocyanins biosynthesis were reported. As ABA stimulates the ripening of F. chiloensis fruit, the molecular network involved in ABA metabolism was analyzed. Therefore, the expression level of genes involved in ABA biosynthesis and ABA perception was quantified during the development of the fruit. Four NCED/CCDs and six PYR/PYLs family members were identified in F. chiloensis. Bioinformatics analyses confirmed the existence of key domains related to functional properties. Through RT-qPCR analyses, the level of transcripts was quantified. FcNCED1 codifies a protein that displays crucial functional domains, and the level of transcripts increases as the fruit develops and ripens, in parallel with the increment in ABA. In addition, FcPYL4 codifies for a functional ABA receptor, and its expression follows an incremental pattern during ripening. The study concludes that FcNCED1 is involved in ABA biosynthesis; meanwhile, FcPYL4 participates in ABA perception during the ripening of F. chiloensis fruit.

Diversity and phylogeography of begomoviruses and DNA satellites associated with the leaf curl and mosaic disease complex of eggplant.

Eggplant is one of the important vegetable crops grown across the world, and its production is threatened by both biotic and abiotic stresses. Diseases caused by viruses are becoming major limiting factors for its successful cultivation. A survey for begomovirus-like symptoms in 72 eggplant fields located in six different Indian states revealed a prevalence of disease ranging from 5.2 to 40.2%, and the symptoms recorded were mosaic, mottling, petiole bending, yellowing, and upward curling, vein thickening, and enation of the leaves, and stunting of plants. The causal agent associated with these plants was transmitted from infected leaf samples to healthy eggplant seedlings via grafting and whiteflies (Bemisia tabaci). The presence of begomovirus was confirmed in 72 infected eggplant samples collected from the surveyed fields exhibiting leaf curl and mosaic disease by PCR using begomovirus specifc primers (DNA-A componet), which resulted in an expected amplicon of 1.2 kb. The partial genome sequence obtained from amplified 1.2 kb from all samples indicated that they are closely related begomovirus species, tomato leaf Karnataka virus (ToLCKV, two samples), tomato leaf curl Palampur virus (ToLCPalV, fifty eggplant samples), and chilli leaf curl virus (ChLCuV, twenty samples). Based on the partial genome sequence analysis, fourteen representative samples were selected for full viral genome amplification by the rolling circle DNA amplification (RCA) technique. Analyses of fourteen eggplant isolates genome sequences using the Sequence Demarcation Tool (SDT) indicated that one isolate had the maximum nucleotide (nt) identity with ToLCKV and eight isolates with ToLCPalV. Whereas, four isolates four isolates (BLC1-CH, BLC2-CH, BLC3-CH, BLC4-CH) are showing nucleotide identity of less than 91% with chilli infecting viruses begomoviruses with chilli infecting begomoviruses and as per the guidelines given by the ICTV study group for the classification of begomoviruses these isolates are considered as one novel begomovirus species, for which name, Eggplant leaf curl Chhattisgarh virus (EgLCuChV) is proposed. For DNA-B component, seven eggplant isolates had the highest nt identity with ToLCPalV infecting other crops. Further, DNA satellites sequence analysis indicated that four betasatellites identified shared maximum nucleotide identity with the tomato leaf curl betasatellite and five alphasatellites shared maximum nucleotide identity with the ageratum enation alphasatellite. Recombination and GC plot analyses indicated that the bulk of begomovirus genome and associated satellites presumably originated from of previously known mono and bipartite begomoviruses and DNA satellites. To the best of our knowledge, this is India's first report of ToLCKV and a noval virus, eggplant leaf curl Chhattisgarh virus associated with eggplant leaf curl disease.

Isolation, molecular characterization, and disinfectants susceptibility of swine-carried mammalian orthoreoviruses in Japan in 2020-2022.

Biosecurity enhancement contributes to the reduction of various microbial pathogens. Mammalian orthoreoviruses (MRVs) which are increasingly recognized as potentially serious problems on swine industry were used as indicators of biosecurity enhancement on two pig farms. Twelve MRVs were detected and isolated from fecal specimens of healthy pigs collected from one of the two farms in Japan. By sequencing based on the partial S1 gene, MRV isolates were classified as MRV1 and MRV2. Additionally, the virucidal activities of disinfectants toward the isolated MRV1 were evaluated using quaternary ammonium compound (QAC) diluted 500 times with water (QAC-500), 0.17% food additive glade calcium hydroxide (FdCa(OH)2) solution, QAC diluted with 0.17% FdCa(OH)2 solution (Mix-500), sodium hypochlorite at 100 or 1,000 parts per million (ppm) of total chlorine (NaClO-100 or NaClO-1000, respectively). To efficiently inactivate MRV1 (≥3 log10 reductions), 0.17% FdCa(OH)2, Mix-500 and NaClO-1000 required 5 min, whereas it took 30 min for QAC-500. The number of MRV detections has decreased over time, after using Mix-500 for disinfection on the positive farm. These results suggest that different serotypes of MRVs are circulating among pigs, and that the occurrence of MRVs in the farms decreased consequent to more effective disinfection.

Phylogenetic and antigenic analysis of HA gene of influenza virus B (Victoria) in Beijing during 2021-2022 surveillance season / 中华微生物学和免疫学杂志

Objective:To investigate the phylogenetic and antigenic characteristics of hemagglutinin (HA) gene of influenza B/Victoria lineage (BV) viruses in Beijing during the 2021-2022 influenza surveillance season, and to analyze whether the circulating BV viruses match the vaccine strain.Methods:Pharyngeal swab specimens from influenza like-illness (ILI) cases in the 2021-2022 influenza surveillance season were collected from surveillance network labs in Beijing and cultured in MDCK cells and chicken embryo to isolate BV viruses. Nucleic acids of the viruses were extracted, and the HA gene was amplified and sequenced. The nucleotide and amino acid sequence identity of the HA gene was analyzed using MEGA5.0 software. A phylogenetic tree of HA gene was constructed using the maximum likelihood method. The N-glycosylation sites in HA were predicted online. Three-dimensional structure of HA was constructed using SWISS-MODEL homologous modeling. Hemagglutination inhibition (HI) test was performed to analyze the antigenicity of BV viruses.Results:A total of 402 BV viruses were collected and 58 strains with full-length HA gene sequences were chosen for further analysis. Compared with the HA gene of this year′s vaccine strain (B/Washington/02/2019), there were 27 amino acid mutations, 11 of which were located in four different antigenic determinants. The phylogenetic analysis revealed that three subgroups of 1A.3, 1A.3a1, and 1A.3a2 co-circulated in Beijing with 54 strains (54/58, 93.10%) clustered to the Clade 1A.3a2, two strains (2/58, 3.45%) clustered to the Clade 1A.3a1, and two strains (2/58, 3.45%) in the same subgroup (Clade 1A.3) as the vaccine component BV strain in 2021-2022. Compared with the vaccine strain (B/Washington/02/2019), two BV strains had an additional N-glycosylation site at residue 197, while the other 56 strains showed no change in N-glycosylation sites. Antigenic analysis showed that 35 BV strains (35/58, 60.34%) were antigenically similar to the vaccine strain and 23 strains (23/58, 39.66%) were low-response strains.Conclusions:Three subgroups of BV viruses co-circulated in Beijing during the 2021-2022 influenza surveillance season. The predominant subgroup was Clade 1A.3a2 (93.10%), showing a certain genetic distance with the vaccine strain (B/Washington/02/2019). Nearly 40% (39.66%) of the viruses were low-response strains. This study indicated that continuous monitoring of the variations of influenza epidemic strains and timely providing laboratory basis for screening vaccine component strains were the basic technical guarantee for coping with influenza pandemic.

Identification and expression of uridine diphosphate glycosyltransferase(UGT) gene family from Dendrobium officinale / 中国中药杂志

Uridine diphosphate glycosyltransferase(UGT) is a highly conserved protein in plants, which usually functions in secondary metabolic pathways. This study used the Hidden Markov Model(HMM) to screen out members of UGT gene family in the whole genome of Dendrobium officinale, and 44 UGT genes were identified. Bioinformatics was used to analyze the structure, phylogeny, and promoter region components of D. officinale genes. The results showed that UGT gene family could be divided into four subfamilies, and UGT gene structure was relatively conserved in each subfamily, with nine conserved domains. The upstream promoter region of UGT gene contained a variety of cis-acting elements related to plant hormones and environmental factors, indicating that UGT gene expression may be induced by plant hormones and external environmental factors. UGT gene expression in different tissues of D. officinale was compared, and UGT gene expression was found in all parts of D. officinale. It was speculated that UGT gene played an important role in many tissues of D. officinale. Through transcriptome analysis of D. officinale mycorrhizal symbiosis environment, low temperature stress, and phosphorus deficiency stress, this study found that only one gene was up-regulated in all three conditions. The results of this study can help understand the functions of UGT gene family in Orchidaceae plants and provide a basis for further study on the molecular regulation mechanism of polysaccharide metabolism pathway in D. officinale.

Molecular characterization of canine and feline kobuvirus infections in Iran.

Kobuviruses are viral pathogens with broad host range presented in human gastroenteritis cases; but, the pathogenesis of these viruses in companion animals is not well described. In the present study, the presence of canine (CaKVs) and feline kobuviruses (FeKVs) was detected in the 100 fecal samples of diarrhoeic and healthy companion dogs and cats by polymerase chain reaction in Tehran, Iran. The prevalence of infection was estimated as 8.00% and 4.00% in dogs and cats, respectively. All positive samples were belonged to non-diarrhoeic animals except for a feline sample being co-infected with panleukopenia. Sequence analysis showed multiple point mutations in canine and feline Iranian strains and new feline strain was detected in the present study. This is the first detection of CaKVs and FeKVs in Iran; but, the exact role of these enteric viral pathogens and their zoonotic risks are better to be clarified in all endemic regions.

Molecular epidemiology and genetic characterization of Clostridium perfringens infections in lambs.

Clostridium perfringens is gram positive bacterium, wide spread in environment causing various diseases in animals and human. The current study was conducted to indentify the genetic identity of C. perfringens isolates from lambs from Egypt. Using specific primers amplifying genes associated to the toxins produced by C. perfringens, multiplex PCR was used to confirm C. perfringens in 87 out of 140 samples were collected from diseased and suspected lambs. The isolates were classified as type A in 49.4%, type B in 31.1% and type D in 19.5% of isolates. The phylogenetic analysis for the partial sequences of C. perfringens strains based on plc gene, cpb gene and etx gene obtained in the present study showed high degree of similarity with other sequences of C. perfringens strains in GenBank, isolating from sheep from Egypt and other countries. According to the findings, lambs with enterotoxaemia more frequently have C. perfringens type A and an efficient hygienic control program is necessary to reduce the infection spreading among susceptible animals.

Characterization of the complete chloroplast genome of Pellionia scabra (Urticaceae).

Pellionia scabra Benth. 1861 (Urticaceae) is distributed in east and southeast Asian countries, including China, Vietnam, and Japan, and has important applications in construction, medicine, and the food industry. We sequenced the genome using Illumina DNA sequencing technology. The genome was 153,220 bp long. Annotation of the genome showed that it encoded 130 genes, including 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. In addition, 15 of the genes contained a single intron and two contained two introns. Furthermore, rps12 consists of 3 exons that are expected to be trans-spliced together. Subsequent phylogenetic analysis revealed that P. scabra is closely related to the Elatostema species (i.e. E. stewardii [MZ292972], E. dissectum [MK227819], and E. laevissimum var. laevissimum [MN189961]).

Comprehensive Comparative Analysis and Development of Molecular Markers for Dianthus Species Based on Complete Chloroplast Genome Sequences.

Dianthus spp. is a genus with high economic and ornamental value in the Caryophyllaceae, which include the famous fresh-cut carnation and the traditional Chinese herbal medicine, D. superbus. Despite the Dianthus species being seen everywhere in our daily lives, its genome information and phylogenetic relationships remain elusive. Thus, we performed the assembly and annotation of chloroplast genomes for 12 individuals from seven Dianthus species. On this basis, we carried out the first comprehensive and systematic analysis of the chloroplast genome sequence characteristics and the phylogenetic evolution of Dianthus. The chloroplast genome of 12 Dianthus individuals ranged from 149,192 bp to 149,800 bp, containing 124 to 126 functional genes. Sequence repetition analysis showed the number of simple sequence repeats (SSRs) ranged from 75 to 80, tandem repeats ranged from 23 to 41, and pair-dispersed repeats ranged from 28 to 43. Next, we calculated the synonymous nucleotide substitution rates (Ks) of all 76 protein coding genes to obtain the evolution rate of these coding genes in Dianthus species; rpl22 showed the highest Ks (0.0471), which suggested that it evolved the swiftest. By reconstructing the phylogenetic relationships within Dianthus and other species of Caryophyllales, 16 Dianthus individuals (12 individuals reported in this study and four individuals downloaded from NCBI) were divided into two strongly supported sister clades (Clade A and Clade B). The Clade A contained five species, namely D. caryophyllus, D. barbatus, D. gratianopolitanus, and two cultivars ('HY' and 'WC'). The Clade B included four species, in which D. superbus was a sister branch with D. chinensis, D. longicalyx, and F1 '87M' (the hybrid offspring F1 from D. chinensis and 'HY'). Further, based on sequence divergence analysis and hypervariable region analysis, we selected several regions that had more divergent sequences, to develop DNA markers. Additionally, we found that one DNA marker can be used to differentiate Clade A and Clade B in Dianthus. Taken together, our results provide useful information for our understanding of Dianthus classification and chloroplast genome evolution.

The complete chloroplast genome of Nageia fleuryi (Hickel) de Laub. (Podocarpaceae).

Nageia fleuryi (Hickel) de Laub. 1987 belongs to the genus Nageia in the family Podocarpaceae and is distributed throughout southeast Asia, including China, Vietnam, and Cambodia. It is a plant with high economic beneficial for food and construction industries. Here, we report on the complete chloroplast (cp) genome of N. fleuryi for the first time. The complete cp genome is similar to many gymnosperm plants, however, it lacks inverted repeat regions and does not possess a typical quadripartite structure. The complete cp genome is 133,870 bp in size and the overall guanine-cytosine (GC) content was found to be 37.27%. The total number of genes is 119, including 82 protein-coding genes, 33 tRNA genes, and 4 rRNA genes. Of these, 14 genes contain one intron, two genes contain two introns, and rps12 possessed a trans-splicing mechanism. Finally, the phylogenic tree demonstrated that N. fleuryi is closely related to Nageia nagi (AB830885.1 and LC572156.1).

Molecular and Antigenic Characterization of Avian H9N2 Viruses in Southern China.

The H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in BJ94 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 × 10-3 substitutions/site/year, which was 1.5-fold faster than that of the average H9N2 HA rate (3.95 × 10-3 substitutions/site/year). Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses. IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry (especially in chicken in South Korea and China). Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 AIVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.

Mechanochemical Evolution of Disulfide Bonds in Proteins.

Disulfide bonds play a pivotal role in the mechanical stability of proteins. Numerous proteins that are known to be exposed to mechanical forces in vivo contain disulfide bonds. The presence of cryptic disulfide bonds in a protein structure may be related to its resistance to an applied mechanical force. Disulfide bonds in proteins tend to be highly conserved but their evolution might be directly related to the evolution of the protein mechanical stability. Hence, tracking the evolution of disulfide bonds in a protein can help to derive crucial stability/function correlations in proteins that are exposed to mechanical forces. Phylogenic analysis and ancestral sequence reconstruction (ASR) allow tracking the evolution of proteins from the past ancestors to our modern days and also establish correlations between proteins from different species. In addition, ASR can be combined with single-molecule force spectroscopy (smFS) to investigate the mechanical properties of proteins including the occurrence and function of disulfide bonds. Here we present a detailed protocol to study the mechanochemical evolution of proteins using a fragment of the giant muscle protein titin as example. The protocol can be easily adapted to AFS studies of any resurrected mechanical force bearing protein of interest.

A Comprehensive Study of the Genus Sanguisorba (Rosaceae) Based on the Floral Micromorphology, Palynology, and Plastome Analysis.

Sanguisorba, commonly known as burnet, is a genus in the family Rosaceae native to the temperate regions of the Northern hemisphere. Five of its thirty species are distributed in Korea: Sanguisorba officinalis, S. stipulata, S. hakusanensis, S. longifolia, and S. tenuifolia. S. officinalis has been designated as a medicinal remedy in the Chinese and Korean Herbal Pharmacopeias. Despite being a valuable medicinal resource, the morphological and genomic information, as well as the genetic characteristics of Sanguisorba, are still elusive. Therefore, we carried out the first comprehensive study on the floral micromorphology, palynology, and complete chloroplast (cp) genome of the Sanguisorba species. The outer sepal waxes and hypanthium characters showed diagnostic value, despite a similar floral micromorphology across different species. All the studied Sanguisorba pollen were small to medium, oblate to prolate-spheroidal, and their exine ornamentation was microechinate. The orbicules, which are possibly synapomorphic, were consistently absent in this genus. Additionally, the cp genomes of S. officinalis, S. stipulata, and S. hakusanensis have been completely sequenced. The comparative analysis of the reported Sanguisorba cp genomes revealed local divergence regions. The nucleotide diversity of trnH-psbA and rps2-rpoC2, referred to as hotspot regions, revealed the highest pi values in six Sanguisorba. The ndhG indicated positive selection pressures as a species-specific variation in S. filiformis. The S. stipulata and S. tenuifolia species had psbK genes at the selected pressures. We developed new DNA barcodes that distinguish the typical S. officinalis and S. officinalis var. longifolia, important herbal medicinal plants, from other similar Sanguisorba species with species-specific distinctive markers. The phylogenetic trees showed the positions of the reported Sanguisorba species; S. officinalis, S. tenuifolia, and S. stipulata showed the nearest genetic distance. The results of our comprehensive study on micromorphology, pollen chemistry, cp genome analysis, and the development of species identification markers can provide valuable information for future studies on S. officinalis, including those highlighting it as an important medicinal resource.

Molecular Characterization, Virulence and Antimicrobial Susceptibility Testing of Riemerella anatipestifer Isolated from Ducklings.

This pilot study aimed to characterize Riemerella anatipestifer from ducklings, testing their susceptibility to antimicrobial agents and to detect their virulence markers. Seven R. anatipestifer isolates with 11.67% infection rate were identified out of sixty freshly dead ducklings and confirmed by PCR assay targeting gyrB gene. The gyrB gene sequences of R. anatipestifer isolates were 100% identical to each other and also showed 100% sequence similarity to the published gyrB genes. Four virulence genes namely ompA, prtC, hagA, and sspA were identified in all isolates except sspA was detected in 5 isolates. The antibiogram revealed higher sensitive to imipenem, amikacin, and rifampin, while, a remarkably high resistance was displayed against ampicillin, penicillin, cefipime, trimethoprim/sulfamethoxazole, gentamicin, ceftazidime, streptomycin and cefoperazone. Proper and rapid identification of R. anatipestifer with detection of their antimicrobial susceptibility and its virulence potential is essential for understanding the epidemiology of R. anatipestifer and to apply the effective control strategies.

Etiology and clinical characteristics of SARS-CoV-2 and other human coronaviruses among children in Zhejiang Province, China 2017-2019.

BACKGROUND: A novel coronavirus (SARS-CoV-2) emerging has put global public health institutes on high alert. Little is known about the epidemiology and clinical characteristics of human coronaviruses infections in relation to infections with other respiratory viruses. METHODS: From February 2017 to December 2019, 3660 respiratory samples submitted to Zhejiang Children Hospital with acute respiratory symptoms were tested for four human coronaviruses RNA by a novel two-tube multiplex reverse transcription polymerase chain reaction assays. Samples were also screened for the occurrence of SARS-CoV-2 by reverse transcription-PCR analysis. RESULTS: Coronavirus RNAs were detected in 144 (3.93%) specimens: HCoV-HKU1 in 38 specimens, HCoV-NL63 in 62 specimens, HCoV-OC43 in 38 specimens and HCoV-229E in 8 specimens. Genomes for SARS-CoV-2 were absent in all specimens by RT-PCR analysis during the study period. The majority of HCoV infections occurred during fall months. No significant differences in gender, sample type, year were seen across species. 37.5 to 52.6% of coronaviruses detected were in specimens testing positive for other respiratory viruses. Phylogenic analysis identified that Zhejiang coronaviruses belong to multiple lineages of the coronaviruses circulating in other countries and areas. CONCLUSION: Common HCoVs may have annual peaks of circulation in fall months in the Zhejiang province, China. Genetic relatedness to the coronaviruses in other regions suggests further surveillance on human coronaviruses in clinical samples are clearly needed to understand their patterns of activity and role in the emergence of novel coronaviruses.