Progress on CRISPR/Cas9 system in the genetic improvement of livestock and poultry.

CRISPR/Cas9 gene editing technology, as a highly efficient genome editing method, has been extensively employed in the realm of animal husbandry for genetic improvement. With its remarkable efficiency and precision, this technology has revolutionized the field of animal husbandry. Currently, CRISPR/Cas9-based gene knockout, gene knock-in and gene modification techniques are widely employed to achieve precise enhancements in crucial production traits of livestock and poultry species. In this review, we summarize the operational principle and development history of CRISPR/Cas9 technology. Additionally, we highlight the research advancements utilizing this technology in muscle growth and development, fiber growth, milk quality composition, disease resistance breeding, and animal welfare within the livestock and poultry sectors. Our aim is to provide a more comprehensive understanding of the application of CRISPR/Cas9 technology in gene editing for livestock and poultry.

Massively Parallel CRISPR-Cas9 Knockout Screening in Sheep Granulosa Cells for FSH Response Genes.

Follicle-stimulating hormone (FSH) regulates ovarian follicle development through specific gene expression programs. Granulosa cells (GCs) are somatic cells surrounding the oocytes, secreting gonadotropins to regulate ovulation and promote follicular development. By analyzing the effects of different doses of FSH on the proliferation of GCs, we found that adding 10 ng/mL of FSH, as the optimal concentration, could promote the growth of GCs. Furthermore, we have successfully constructed the first CRISPR-Cas9 knockout library targeting the genes on chromosomes 2 and 3 and the X chromosomes of the sheep massively parallel coding gene, as well as an ovarian GCs knockout cell library. For the first time, we have exposed the knockout cell library to a concentration of 10 ng/mL FSH to explore the underlying mechanisms. Through this screening, we have identified 836 positive-negative screening genes that are responsive to FSH, thereby revealing the regulatory mechanisms and screening the functionality of candidate genes. Next, RNA-Seq of control (0 ng/mL), low (10 ng/mL), and high (100 ng/mL) doses of FSH revealed 1708 differentially expressed genes, and combined with 836 genes, we obtained 129 FSH dose-dependent genes with extremely significant differences. This enables us to delve deeper into investigating and identifying the mechanisms by which FSH regulates GCs. More generally, we have discovered new regulatory factors and identified reproductivity-associated major effectors. These findings provide novel research directions for further studies on sheep reproduction.

Integrated Multi-Tissue Transcriptome Profiling Characterizes the Genetic Basis and Biomarkers Affecting Reproduction in Sheep (Ovis aries).

The heritability of litter size in sheep is low and controlled by multiple genes, but the research on its related genes is not sufficient. Here, to explore the expression pattern of multi-tissue genes in Chinese native sheep, we selected 10 tissues of the three adult ewes with the highest estimated breeding value in the early study of the prolific Xinggao sheep population. The global gene expression analysis showed that the ovary, uterus, and hypothalamus expressed the most genes. Using the Uniform Manifold Approximation and Projection (UMAP) cluster analysis, these samples were clustered into eight clusters. The functional enrichment analysis showed that the genes expressed in the spleen, uterus, and ovary were significantly enriched in the Ataxia Telangiectasia Mutated Protein (ATM) signaling pathway, and most genes in the liver, spleen, and ovary were enriched in the immune response pathway. Moreover, we focus on the expression genes of the hypothalamic-pituitary-ovarian axis (HPO) and found that 11,016 genes were co-expressed in the three tissues, and different tissues have different functions, but the oxytocin signaling pathway was widely enriched. To further explore the differences in the expression genes (DEGs) of HPO in different sheep breeds, we downloaded the transcriptome data in the public data, and the analysis of DEGs (Xinggao sheep vs. Sunite sheep in Hypothalamus, Xinggao sheep vs. Sunite sheep in Pituitary, and Xinggao sheep vs. Suffolk sheep in Ovary) revealed the neuroactive ligand-receptor interactions. In addition, the gene subsets of the transcription factors (TFs) of DEGs were identified. The results suggest that 51 TF genes and the homeobox TF may play an important role in transcriptional variation across the HPO. Altogether, our study provided the first fundamental resource to investigate the physiological functions and regulation mechanisms in sheep. This important data contributes to improving our understanding of the reproductive biology of sheep and isolating effecting molecular markers that can be used for genetic selection in sheep.

Effect of statin treatment on mortality in elderly patients with type 2 diabetes mellitus patients: a retrospective cohort study.

BACKGROUND: The effects of statins on the reduction of mortality in individuals aged 75 years or older remain controversial. We conducted this study to investigate whether there is an association between statin therapy and mortality in patients with type 2 diabetes mellitus (T2DM) who are over the age of 75 years. METHODS: The present study used data from the Staged Diabetes Targeting Management Study, which began in 2005. A total of 518 T2DM patients older than 75 years were included. Cox regression analyses were used to evaluate the association between statins and specific causes of death in patients with T2DM. RESULTS: After a follow-up period of 6.09 years (interquartile range 3.94-8.81 years), 111 out of 518 patients died. The results of Cox regression analyses showed that there was no significant association between statin use and all-cause mortality (HR 0.75; 95% CI 0.47, 1.19) after adjustment for all potential confounders. Subgroup analysis indicated that statins had no association with the risk of all-cause mortality or deaths caused by ischemic cardiovascular diseases in T2DM patients with or without coronary heart disease. CONCLUSIONS: Our study found no significant association between all-cause mortality and statin use in T2DM patients over the age of 75 years. More evidence is needed to support the use of statins in the elderly T2DM patients.

Identification of milk-related genes and regulatory networks in Bactrian camel either supplemented or under grazing.

Using gene co-expression networks to understand dynamic characterizations in lactating animals becomes a common method. However, there are rarely reporters focusing on milk traits in Bactrian camel by high-throughput sequencing. We used RNA-seq to generate the camel transcriptome from the blood of 16 lactating Alxa Bactrian camel in different feeding groups. In total, we obtained 1185 milk-related genes correlated with milk yield, milk protein, milk fat, and milk lactose across the WGCNA analysis. Moreover, 364 milk-related genes were differentially expressed between supplementation and grazing feeding groups. The differential expression-camel milk-related genes CMRGs (DE-CMRGs) in supplement direct an intensive gene co-expression network to improve milk performance in lactating camels. This study provides a non-invasive method to identify the camel milk-related genes in camel blood for four primary milk traits and valuable theoretical basis and research ideas for the study of the milk performance regulation mechanism of camelid animals.

RBP7 Regulated by EBF1 Affects Th2 Cells and the Oocyte Meiosis Pathway in Bone Metastases of Bladder Urothelial Carcinoma.

BACKGROUND: Bladder urothelial carcinoma (BLCA) is a malignancy with a high incidence worldwide. One-third of patients may experience aggressive progression later on, and 70% of patients who have undergone surgical intervention will still suffer from metastasis. MATERIALS AND METHODS: RNA sequencing profiles of BLCA samples were obtained from The Cancer Genome Atlas (TCGA) database. Differential expression and univariate Cox regression analyses were performed to identify prognosis-related differentially expressed immune genes (DEIGs). Subsequently, a proportional hazards model of DEIGs was then constructed by univariate regression analysis. Differential expression and correlation analyses, CIBERSORT, Single Sample Gene Set Enrichment Analysis (ssGSEA), GSVA were conducted on transcription factors (TFs), immune cells/pathways and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The regulation network was then constructed. Eventually, ATAC-seq, ChIP-seq, scRNA-seq, and multiple online databases were employed for further validation. RESULTS: A proportional hazards model of 31 DEIGs was constructed and risk score was calculated and proven to be a independent prognostic factor. Then 5 immune genes were characterized to be significantly correlated with bone metastasis, stage and TF expression simultaneously. 4 TFs were identified to be significantly correlated with prognosis and RBP7 expression. 5 immune cells/pathways were revealed to be significantly correlated with RBP7 expression. Only 1 KEGG pathway was identified to be significant in Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) analyses. The regulatory relationship was then constructed, in which the correlation between EBF1 and RBP7 (R = 0.677, p < 0.001), Th2 cells and RBP7 (R = 0.23, p < 0.001), the oocyte meiosis pathway and RBP7 (R = 0.14, p = 0.042) were the most statistically significant. The results were further confirmed by Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), single-cell RNA sequencing (scRNA-seq), and multiple online databases validation. CONCLUSIONS: This study revealed that the EBF1-RBP7 regulatory relationship had potential importance in the bone metastasis in BLCA through Th2 cells and the oocyte meiosis pathway.

Association between TyG index and long-term mortality of critically ill patients: a retrospective study based on the MIMIC Database.

OBJECTIVE: To evaluate the association of triglyceride-glucose (TyG) index on admission with outcomes of critically ill patients. DESIGN: A retrospective study. SETTING: A population-based cohort study of Medical Information Mart for Intensive Care III Database (MIMIC III). PARTICIPANTS: All intensive care unit admissions were extracted from MIMIC III. MAIN OUTCOME MEASURES: The TyG index was calculated as ln [triglycerides (mg/dL)×glucose (mg/dL)/2]. The primary endpoint was 360-day mortality. RESULTS: A total of 3902 patients with an average age of 63.1±15.9 years old were enrolled, including 1623 (41.6%) women. The 360-day mortality was lower in a higher TyG group. Compared with the lowest TyG group, the HR of 360-day mortality was 0.79 (95% CI (0.66, 0.95); p=0.011) in the fully adjusted Cox model and 0.71 (95% CI (0.59, 0.85); p<0.001) in the stepwise Cox model. In the subgroup analysis, an interaction effect was detected between TyG index and gender. CONCLUSIONS: A lower TyG index was associated with the risk of 360-day mortality in critically ill patients, which could be a predictor of long-term survival of critically ill patients.

Estimates of Genomic Heritability and the Marker-Derived Gene for Re(Production) Traits in Xinggao Sheep.

Xinggao sheep are a breed of Chinese domestic sheep that are adapted to the extremely cold climatic features of the Hinggan League in China. The economically vital reproductive trait of ewes (litter size, LS) and productive traits of lambs (birth weight, BWT; weaning weight, WWT; and average daily gain, ADG) are expressed in females and later in life after most of the selection decisions have been made. This study estimated the genetic parameters for four traits to explore the genetic mechanisms underlying the variation, and we performed genome-wide association study (GWAS) tests on a small sample size to identify novel marker trait associations (MTAs) associated with prolificacy and growth. We detected two suggestive significant single-nucleotide polymorphisms (SNPs) associated with LS and eight significant SNPs for BWT, WWT, and ADG. These candidate loci and genes also provide valuable information for further fine-mapping of QTLs and improvement of reproductive and productive traits in sheep.

Multi-Tissue Transcriptome Study of Innate Immune Gene Expression Profiling Reveals Negative Energy Balance Altered the Defense and Promoted System Inflammation of Dairy Cows.

Negative energy balance (NEB) during the perinatal period leads to metabolic and immunological disorders in dairy cows, resulting in systemic responses and inflammation. The innate immune system is crucial for the host's protection and inflammatory response. However, systematic research is still lacking on how NEB affects the innate immune system to alter the 'host defense capability and inflammatory response. In this investigation, raw transcriptome data of adipose, blood, endometrial, hypothalamus, and liver tissues were downloaded from a public database, cleaned, aligned, quantified, and batch-corrected. The innate immune gene list was retrieved from innateDB, followed by the expression matrix of innate immune genes in various tissues for differential expression analysis, principle component analysis (PCA), and gene set enrichment analysis (GSEA). Under the effect of NEB, adipose tissue had the most differentially expressed genes, which were predominantly up-regulated, whereas blood GSEA had the most enriched biological processes, which were predominantly down-regulated. The gene sets shared by different tissues, which are predominantly involved in biological processes associated with defense responses and inflammation, were dramatically down-regulated in endometrial tissues and highly up-regulated in other tissues. Under the impact of NEB, LBP, PTX3, S100A12, and LCN2 play essential roles in metabolism and immunological control. In conclusion, NEB can downregulate the defensive response of innate immune genes in endometrial, upregulate the immune and inflammatory response of other tissues, activate the host defense response, and increase the systemic inflammatory response. The analysis of the effects of NEB on innate immune genes from the multiple tissues analysis provides new insights into the crosstalk between metabolism and immunity and also provides potential molecular targets for disease diagnosis and disease resistance breeding in dairy cows.

Urinary Nickel Was Associated with the Prevalence of Diabetes: Results from NHANES.

The study aimed to investigate the association between nickel exposure and the presence of diabetes (DM). The participants were analyzed from the National Health and Nutrition Examination Surveys (NHANES) 2017-2018. Urinary nickel exposure was measured using inductively coupled-plasma mass spectrometry. DM was diagnosed based on the WHO standards. The association between nickel and DM or fasting glucose was examined using multivariable linear regression models and logistic regression models. A total of 1899 participants (933 men and 966 women) were included in our study, of whom 342 (18.0%) were diagnosed with DM. There was a significant positive association between nickel level and DM (OR: 1.27, 95%CI: 1.04-1.56) after adjusting for potential confounding factors. Comparing with the lowest quartile, the highest quartile independently increased a 0.66-fold higher risk of DM (OR: 1.66, 95%CI: 1.13-2.44]). In addition, nickel exposure was independently related to the level of fasting glucose. The exposure to nickel was associated with a higher risk of DM in general population.

Effects of exogenous melatonin on expressional differences of immune-related genes in cashmere goats.

The interplay between melatonin and immune system is well recognized in humans. The true integration of research on cashmere goat is still far from clear, especially for cashmere goat maintained in wool and cashmere growth. In this study, we applied various approaches to identify the complex regulated network between the immune-related genes and transcription factors (TFs) and to explore the relationship between melatonin and gene expression in cashmere goats. In total, 1,599 and 1756 immune-related genes were found in the blood and skin of cashmere goats, respectively, and 24 differentially expressed immune-related GO terms were highly expressed in blood after melatonin implantation. We studied the melatonin-dependent networks between the TFs and immune-related genes in cashmere goat. The 3 major regulatory networks were interconnected through TFs. The TFs, such as PHF5A, REXO4, STRAP, JUNB, GATAD2A, ZNF710, and VDR, were also expressed in the blood and skin tissue of cashmere goat. In addition, most genes in these networks, such as VDR, JUNB, and Trib3, were involved in WNT pathway, which is related to cashmere wool growth regulation. On the network basis, we developed a knockout mouse model to identify the network interaction. We observed that 8 high-sulfur protein genes, 12 keratin (KRT) genes, and 19 keratin associated protein (KRTAP) genes related to the growth of cashmere wool were almost not expressed in Trib3 -/- rat skin. Our results suggested that the expression of genes related to wool and cashmere growth may be regulated by the interaction network between genes affected by melatonin and immune-related genes. In summary, we outlined some particularly promising ways for future research on immune-related genes of cashmere goats and the role of melatonin in wool and cashmere growth.

The clinical characteristics of Chinese elderly patients with different durations of type 2 diabetes mellitus.

Aims: To explore the clinical characteristics among elderly (aged ≥60 years) patients with type 2 diabetes (T2DM) of different durations. Methods: Clinical characteristics were investigated in 3840 elderly T2DM patients according to their different durations of diabetes (< 1 year, 1~5 years, 5~10 years, and ≥ 10 years). Kruskal-Wallis and Dunn tests were used to assess the differences among groups for continuous variables. The chi-square and post hoc tests were carried out for dichotomous variables. The logistic regression was adopted to investigate the relationships between various durations of diabetes and the control rates of achieving the control targets for T2DM as well as diabetic vascular complications. Results: There were 972, 896, 875 and 1097 patients with a duration of diabetes of <1, 1~5, 5~10 and ≥10 years, respectively. In logistic regression models adjusted for age, sex, education, BMI, smoking and family history of diabetes, elderly T2DM patients with a duration of diabetes of ≥10 years were more likely to reach the comprehensive control targets for TC (ORTC = 1.36, 95% CI =1.14-1.63), LDL-C (ORLDL-C = 1.39, 95% CI =1.17-1.66), TG (ORTG = 1.76, 95% CI =1.46-2.12) and BMI (ORBMI = 1.82, 95% CI =1.52-2.18). Elderly T2DM patients with a duration of diabetes of 1~5 years were more likely to achieve the HbA1c control target (ORHbA1c = 1.92, 95% CI = 1.59-2.31) than elderly T2DM patients with a duration of diabetes of <1 year. Furthermore, in elderly T2DM patients with a duration of diabetes of 5~10 years or ≥ 10 years, the duration of diabetes was positively associated with diabetic macrovascular complications (coronary heart disease and peripheral artery disease). In elderly T2DM patients with a duration of diabetes of over 10 years, the duration of diabetes was associated with diabetes kidney disease (all P < 0.05). Conclusions: It is worth noting that the clinical characteristics of elderly patients with type 2 diabetes in different durations of diabetes are different.

A review on the effect of gut microbiota on metabolic diseases.

Human gut microbiota are a huge and complex microbial community, which is recognized to play a significant role in regulating host metabolism. However, the destruction of gut microbiota leads to the pathological response of host, and thus results in a variety of metabolic diseases. This article gives a brief review of research progress on gut microbiota and some main metabolic diseases, including osteoporosis, obesity, type 2 diabetes, non-alcoholic fatty liver, and hypertension, with a specific focus on the effect of gut microbiota on diseases' occurrence and development. In addition, this review article also shows some case studies on the regulation of gut microbiota by new means, such as fecal microbiota transplantation and oral probiotics. Although gut microbiota are considered as a promising novel target for the treatment of metabolic diseases, it is also necessary to encourage further studies to provide more valuable data for guiding the application of gut microbiota on disease therapy in future.

Role of Sulfur Metabolism Gene and High-Sulfur Gene Expression in Wool Growth Regulation in the Cashmere Goat.

Sulfur, an essential mineral element for animals, mainly exists in the form of organic sulfur-containing amino acids (SAAs), such as cystine, methionine, and cysteine, within the body. The content, form, and structure of sulfur play an important role in determining the wool fiber quality. In addition, keratin-associated proteins, one of the most crucial wool fiber components, are rich in SAAs. However, sulfur metabolism from the blood to the skin and hair follicles remains unclear. In this study, we analyzed high-sulfur protein gene and sulfur metabolism genes in the cashmere goat and explored the effects of melatonin on their expression. In total, 53 high-sulfur protein genes and 321 sulfur metabolism genes were identified. We found that high-sulfur protein genes were distributed in the 3-4 and 144M regions of chromosome 1 and the 40-41M region of chromosome 19 in goats. Moreover, all year round, allele-specific expression (ASE) is higher in the 40-41M region of chromosome 19 than in the other regions. Total of 47 high-sulfur protein genes showed interaction with transcription factors and cofactors with ASE. These transcription factors and cofactors were inhibited after melatonin implantation. The network analysis revealed that melatonin may activate the sulfur metabolism process via the regulation of the genes related to cell energy metabolism and cell cycle in the skin, which provided sufficient SAAs for wool and cashmere growth. In conclusion, our findings provide a new insight into wool growth regulation by sulfur metabolism genes and high-sulfur protein genes in cashmere goats.

Effect of Tai Chi and Resistance Training on Cancer-Related Fatigue and Quality of Life in Middle-Aged and Elderly Cancer Patients.

OBJECTIVE: To study the effect of Tai Chi (TC) and resistance training (RT) with different intensity on the cancer-related fatigue (CRF) and quality of life (QoL) of middle-aged and elderly cancer patients. METHODS: Totally 120 cancer patients were enrolled and randomly assigned to 4 groups by a random number table, including TC group, high-intensity 60% one repetition maximum (1-RM) RT group (HIRT), low-intensity (30% 1-RM) RT group (LIRT) and control group, 30 patients in each group. Participants in the TC group received 24-form simplified Yang-style TC training at a frequency of 40 min per day, 3 days per week for 12 weeks. Patients in the two RT groups received 10 sessions, 6 designated movements per day, 3 days per week for 12 weeks. The 1-RM of 6 muscle groups, fat mass (FM), lean body mass (LBM), along with the scores of Brief Fatigue Inventory (BFI), QoL questionnaire for Chinese cancer patients receiving chemobiotherapy (QLQ-CCC), Generalized Anxiety Disorder-7 (GAD-7), Patient Health Questionnaire-9 (PHQ-9) and Pittsburgh Sleep Quality Index (PSQI) were measured before and after training. The adverse effect was also observed. RESULTS: After 12-week intervention, patients in both TC and RT groups showed significant improvements in CRF and QLQ-CCC compared to pre-treatment (P<0.05). Compared with the LIRT and TC groups, patients in the HIRT group improved more significantly in increasing muscle strength and LBM, and reducing in FM (P<0.05). Patients in the TC group significantly increased in lower limb muscle strength compared with the LIRT group (P<0.05). In addition, patients in the TC group showed more significant improvements in scores of GAD-7, PHQ-9 and PSQI than 2 RT groups (P<0.05). CONCLUSIONS: TC and RT, both low- and high-intensity training, can significantly increase muscle strength, reduce CRF and improve QoL in the middle-aged and elderly cancer patients. TC has a better effect than RT in terms of sleep quality and mental health. The long-term application is needed to substantiate the effect of TC as an alternative exercise in cancer patients.

The achievement of comprehensive control targets among type 2 diabetes mellitus patients of different ages.

OBJECTIVE: To evaluate achievement of comprehensive controls among patients with type 2 diabetes mellitus (T2DM) in different age groups. RESULTS: The elderly patients had higher control rates for BMI (44.36%), TC (50.83%) and LDL-C (48.27%) than those aged 60-80 years and younger patients (all P <0.05). Multiple logistic regression revealed that elderly patients were more likely to achieve control targets for HbA1c (odd ratio (OR) = 2.19), TC (OR = 1.32), HDL-C (OR = 1.35), and TG (OR = 1.74) than younger patients. This effect was stronger in males (ORHbA1c = 2.27; ORTC = 1.41; ORHDL-C = 1.51; ORTG = 1.80). By contrast, elderly females were only more likely to achieve HbA1c < 7.0% (OR=1.88). CONCLUSIONS: Our findings suggest that comprehensive control strategies still should be strengthened. METHODS: A total of 3126 T2DM patients were included, and detected blood pressure (BP), body mass index (BMI), glycosylated hemoglobin (HbA1c), fasting plasma glucose (FPG), postprandial plasma glucose (PPG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C). We divided patients into three age groups (<60, 60-80 and ≥ 80 years), to assess the differences in achieving the control targets.

Long non-coding RNA DANCR alleviates hypoxia-caused H9c2 cells damage through up regulation of HIF-1α.

Hypoxia is an important cause of myocardial cell loss, further inducing various heart illnesses, including acute myocardial infraction (AMI). Long non-coding RNA (LncRNA) discrimination antagonising non-protein coding RNA (DANCR) was firstly identified as epidermal cell differentiation suppressor. Here, we aimed to explore the effects and mechanism of DNACR in hypoxia-induced H9c2 cells. Hypoxic cells were made through 94% N2, 5% CO2, and 1% O2 environment for 24 h. Cell viability and apoptosis were detected via methyl thiazolyl tetrazolium (MTT) method and flow cytometry analysis, respectively. The expression of DANCR and HIF-1α was examined via qRT-PCR. The expression of proteins related to cell apoptosis and PI3K/AKT/mTOR and ERK1/2 signal pathways was examined through western blot analysis. We found that hypoxia induced obvious cell activity inhibition and apoptosis increasing in H9c2 cells. DANCR was negatively regulated under hypoxia condition. Overexpression of DANCR rescued activity and attenuated apoptosis. Moreover, the overexpression of DANCR elevated the activation of PI3K/AKT/mTOR and ERK1/2 pathways. Further study indicated that DANCR could up-regulate the expression of HIF-1α. Si-HIF-1α transfection could remove the beneficial effects of DANCR overexpression in hypoxia-caused H9c2 cells damage. In conclusion, DANCR alleviated hypoxia-caused H9c2 cells damage through positive regulation of HIF-1α.

Genetic evolution of equine influenza viruses isolated in China.

China experienced an outbreak of equine influenza during 2007-2008. Meanwhile, its neighbor countries, such as Mongolia, India and Japan, have also been affected by various influenza virus strains in each country. Phylogenetic analysis showed that the newly emerging Chinese strains belong to Florida sublineage clade 2, as well as the Indian strain Jammu-Katra/6/08 and the Mongolian strain Mongolia/1/08. All of these strains were derived from European strains of this clade, such as the Newmarket/1/07 and Cheshire/1/07 strains, but these were not related to Japanese strains isolated around the same time (Florida sublineage clade 1) or to Chinese strains isolated in the 1990s (European lineage). Some unique amino acid changes were found in the antigenic sites in Asian strains of Florida sublineage clade 2. Moreover, the loss of a glycosylation site was found in the Liaoning/9/08 strain. From these studies, we have determined that equine influenza viruses in China have evolved with some new characteristics during recent years, and this emphasizes the importance of continued equine influenza virus surveillance in China.

Isolation and genetic characterization of H3N8 equine influenza virus from donkeys in China.

During the 2007 outbreak of equine influenza (EIV) in China, an influenza virus designated A/donkey/Xinjiang/5/2007 (donkey/Xinjiang/2007) was isolated from a symptomatic donkey in Xinjiang Uygur Autonomous Region, China. To analyze the genetic evolution of the new isolate, the hemagglutinin (HA) gene of donkey/Xinjiang/2007 was amplified and sequenced. Sequence alignment, prediction of glycosylation sites and phylogenetic analysis of the HA1 protein of donkey/Xinjiang/2007 showed most similarity to the Florida sublineage clade 2 of the American lineage of equine influenza viruses. The HA1 sequence of donkey/Xinjiang/2007 had high identity with the Chinese EIV strains isolated in recent years and had low identity with A/swine/Anhui/1/2006, a horse-derived influenza virus isolated from pigs in China. Compared with vaccine strains, the HA1 amino acid sequence of donkey/Xinjiang/2007 varied in the five putative antigenic sites to different degrees and distributed in a different branch of the HA1 phylogenetic tree. Our findings provide further evidence of an American H3N8 equine influenza virus in China in addition to the European H3N8 equine influenza virus. Accordingly, a vaccine against equine influenza in China should contain components of both strains of viruses to prevent widespread infection.