Jersey-yak is a hybrid offspring of Jersey cattle and yak (Bos grunniens). Changing the feeding system of Jersey-yak can significantly improve its growth performance. In this study, tandem mass tag (TMT) proteomics technology was used to determine the differentially expressed proteins (DEPs) of the longissimus lumborum (LL) muscle of Jersey-yak fed different protein levels of diet. The results showed that compared with the traditional grazing feeding, the growth performance of Jersey-yaks was significantly improved by crude protein supplementation after grazing. A total of 3368 proteins were detected in these muscle samples, of which 3365 were quantified. A total of 434 DEPs were identified. Through analyses, it was found that some pathways related to muscle growth and development were significantly enriched, such as Rap1 signaling pathway, mTOR signaling pathway, and TGF-beta signaling pathway. A number of DEPs enriched in these pathways are related to muscle cell development, differentiation, and muscle development, including integrin subunit alpha 7 (ITGA7), myosin heavy chain 8 (MYH8), and collagen type XII alpha 1 chain (COL12A1). In conclusion, the results of this study provide insights into the proteomics of different feeding patterns of Jersey-yak, providing a stronger basis for further understanding the biological mechanism of hybrid varieties.

Cattle-yak, which is the hybrid F1 generation of cattle and yak, demonstrates better production performance compared to yak. However, there is limited research on the molecular mechanisms responsible for the muscle development of cattle-yak. To address this knowledge gap, a comprehensive transcriptomic survey of the longissimus dorsi muscle in cattle-yak was conducted. Three transcript types, namely lncRNAs, miRNAs, and circRNAs, along with protein-coding genes were characterized at two developmental stages (6 m, 18 m) of cattle-yak. The results revealed significant enrichment of these transcripts into pathways related to myoblast differentiation and muscle development signaling. Additionally, the study identified the TCONS00024465/circHIPK3-bta-miR-499-ADAMTS6 regulatory network, which may play a crucial role in the muscle development of cattle-yak by combining the transcriptome data of yak and constructing the ceRNA co-expression network. HEK 293 T cells were used to validate that TCONS00024465 and circHIPK3 are located upstream of bta-miR-499, and can competitively bind to bta-miR-499 as ceRNA. The study also verified that ADAMTS6 regulates skeletal muscle development by inhibiting myoblast proliferation, promoting myoblast differentiation, and positively regulating the apoptosis of myoblasts. Taken together, this study provides new insights into the advantages of cattle-yak production performance and offers a molecular basis for further research on muscle development.

Subgroup K avian leukosis virus (ALV-K) is a novel subgroup of ALV isolated from Chinese native chickens. As for a retrovirus, the interaction between its envelope protein and cellular receptor is a crucial step in ALV-K infection. Tva, a protein previously determined to be associated with vitamin B12/cobalamin uptake, has been identified as the receptor of ALV-K. However, the molecular mechanism underlying the interaction between Tva and the envelope protein of ALV-K remains unclear. In this study, we identified the C-terminal loop of the LDL-A module of Tva as the minimal functional domain that directly interacts with gp85, the surface component of the ALV-K envelope protein. Further point-mutation analysis revealed that E53, L55, H59, and G70, which are exposed on the surface of Tva and are spatially adjacent, are key residues for the binding of Tva and gp85 and facilitate the entry of ALV-K. Homology modeling analysis indicated that the substitution of these four residues did not significantly impact the Tva structure but impaired the interaction between Tva and gp85 of ALV-K. Importantly, the gene-edited DF-1 cell line with precisely substituted E53, L55, H59, and G70 was completely resistant to ALV-K infection and did not affect vitamin B12/cobalamin uptake. Collectively, these findings not only contribute to a better understanding of the mechanism of ALV-K entry into host cells but also provide an ideal gene-editing target for antiviral study.

Introduction:The crude protein level in the diet will affect the fermentation parameters, microflora, and metabolites in the rumen of ruminants. It is of great significance to study the effect of crude protein levels in supplementary diet on microbial community and metabolites for improving animal growth performance. At present, the effects of crude protein level in supplementary diet on rumen fermentation parameters, microbial community, and metabolites of Jersey-Yak (JY) are still unclear. Methods:The purpose of this experiment was to study the appropriate crude protein level in the diet of JY. The rumen fermentation indexes (volatile fatty acids and pH) were determined by supplementary diets with crude protein levels of 15.16 and 17.90%, respectively, and the microbial community and metabolites of JYs were analyzed by non-target metabonomics and metagenome sequencing technology, and the changes of rumen fermentation parameters, microbial flora, and metabolites in the three groups and their interactions were studied. Results and Discussion:The crude protein level in the supplementary diet had significant effects on pH, valeric acid, and the ratio of acetic acid to propionic acid (p < 0.05). The protein level had no significant effect on the dominant microflora at the phylum level (p > 0.05), and all three groups were Bacteroides and Firmicutes. The results of metabolite analysis showed that the crude protein level of supplementary diet significantly affected the metabolic pathways such as Bile secretion and styrene degradation (p < 0.05), and there were different metabolites between the LP group and HP group, and these different metabolites were related to the dominant microbial to some extent. To sum up, in this experiment, the effects of crude protein level in supplementary diet on rumen microorganisms and metabolites of JY and their relationship were studied, which provided the theoretical basis for formulating a more scientific and reasonable supplementary diet in the future.

Copy number variation (CNV) is a fundamental type of structural variation of the genome affecting the economic traits of livestock. The SOX6 gene (sex-determining region Y-box 6), as a transcription factor, has multiple functions with regard to sex determination, embryonic growth, the nervous system development, as well as bone, and various organ formation. This study employed quantitative real-time fluorescence quota PCR (qPCR) for detecting the SOX6-CNV of the 311 Ashidan yaks and analyzed the correlation of the SOX6-CNV with four phenotypes (including body weight, withers height, body length, and chest girth) of the yaks aged 6, 12, 18, and 30 months using ANOVA and multiple comparisons. Furthermore, the SOX6 gene expression was identified in seven different tissues of the yaks. The experiment results demonstrated the expression of SOX6 in each tissue, and the kidney and muscle tissue were found to have higher relative expression levels. Based on the processing by IBM SPSS software, SOX6-CNV was significantly correlated with the chest girth of the 6-months old yaks (p < 0.05) and 30-months yaks (p < 0.05), and withers height of 6 months yaks (p < 0.05) and 18-months yaks (p < 0.05), as well as the normal type of CNV, was chosen for yak breeding. In conclusion, SOX6 might be prominently involved in promoting growth and development of yaks, suggesting that the SOX6 gene can be used in breeding yaks by molecular marker-assisted selection (MAS). The study also offered some important insights into the references and clues for the genetic breeding of yaks.

The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density lipoprotein (LDL-A) module and is involved in the uptake of transcobalamin bound vitamin B12/cobalamin (Cbl). To map the functional determinants of Tva responsible for ALV-A receptor activity, a series of chimeric receptors were created by swapping the LDL-A module fragments between huCD320 and Tva. These chimeric receptors were then used for virus entry and binding assays to map the minimal ALV-A functional domain of Tva. The results showed that Tva residues 49 to 71 constituted the minimal functional domain that directly interacted with the ALV-A gp85 protein to mediate ALV-A entry. Single-residue substitution analysis revealed that L55 and W69, which were spatially adjacent on the surface of the Tva structure, were key residues that mediate ALV-A entry. Structural alignment results indicated that L55 and W69 substitutions did not affect the Tva protein structure but abolished the interaction force between Tva and gp85. Furthermore, substituting the corresponding residues of huCD320 with L55 and W69 of Tva converted huCD320 into a functional receptor of ALV-A. Importantly, soluble huCD320 harboring Tva L55 and W69 blocked ALV-A entry. Finally, we constructed a Tva gene-edited cell line with L55R and W69L substitutions that could fully resist ALV-A entry, while Cbl uptake was not affected. Collectively, our findings suggested that amino acids L55 and W69 of Tva were key for mediating virus entry. IMPORTANCE Retroviruses bind to cellular receptors through their envelope proteins, which is a crucial step in infection. While most retroviruses require two receptors for entry, ALV-A requires only one. Various Tva alleles conferring resistance to ALV-A, including Tvar1 (C40W substitution), Tvar2 (frame-shifting four-nucleotide insertion), Tvar3, Tvar4, Tvar5, and Tvar6 (deletion in the first intron), are known. However, the detailed entry mechanism of ALV-A in chickens remains to be explored. We demonstrated that Tva residues L55 and W69 were key for ALV-A entry and were important for correct interaction with ALV-A gp85. Soluble Tva and huCD320 harboring the Tva residues L55 and W69 effectively blocked ALV-A infection. Additionally, we constructed gene-edited cell lines targeting these two amino acids, which completely restricted ALV-A entry without affecting Cbl uptake. These findings contribute to a better understanding of the infection mechanism of ALV-A and provided novel insights into the prevention and control of ALV-A.

Copy number variations (CNVs) are a result of genomic rearrangement affecting DNA regions over 1 kb in length, and can include inversions, translocations, deletions, and duplications. The molecule interacting with CasL-like protein 2 (MICALL2) gene is primarily associated with mitochondrial protein targeting and exhibits predicted stress fiber colocalization. The monoacylglycerol O-acyltransferase 2 (MOGAT2) gene encodes an enzyme responsible for catalyzing diacylglycerol synthesis from 2-monoacylglycerol and fatty acyl-CoA. For this study, blood samples were obtained from 315 yaks, and the body weight, body length, withers height, and chest girth of these animals were measured at 6, 12, 18, and 30 months of age. Genomic DNA was harvested from the collected blood samples, and CNVs in these samples were detected by qPCR. The resultant data were compared using ANOVAs, revealing significant associations between MICALL2 gene CNVs and body weight at 6 months of age (p < 0.05), body length and chest girth at 30 months of age (p < 0.05), and withers height at 18 months of age (p < 0.01) in Ashidan yaks. Similarly, MOGAT2 CNVs were significantly associated with body weight at 6 and 30 months of age (p < 0.05), and with withers height at 18 months of age (p < 0.01) in these Ashidan yaks. MICALL2 and MOGAT2 gene expression was further analyzed in yak tissue samples, revealing that MICALL2 was most highly expressed in the adipose tissue, whereas MOGAT2 was most highly expressed in the lung. These results thus confirmed the relationship between CNVs in the MICALL2 and MOGAT2 genes and Ashidan yak growth traits, providing a valuable gene locus that can be leveraged for future marker-assisted yak breeding efforts.

为研究B亚型禽偏肺病毒病灭活疫苗对商品蛋鸡的免疫保护效果,本研究选取21日龄的商品蛋鸡,将本团队前期研制的B亚型禽偏肺病毒病灭活疫苗通过肌肉注射方式免疫蛋鸡(0.5 mL/只),并于首免后3周以相同剂量加强免疫1次.免疫后1周～6周每周采血,分离血清,采用ELISA检测及中和试验分别检测相应抗体,结果显示,初次免疫后6周,免疫组血清平均ELISA抗体效价达到1∶73 522,平均中和抗体效价达到7.6log2.加强免疫后3周通过滴鼻的方式攻毒(aMPV/BLN16强毒滴鼻200μL,5 000 TCID50/只),观察攻毒后1 d～8 d蛋鸡的临床症状.结果显示,攻毒对照组蛋鸡发病率为92.3％,免疫组蛋鸡不发病;攻毒后1d～8 d采集各组蛋鸡的鼻腔拭子,通过RT-qPCR方法检测病毒拷贝数,结果显示,免疫组蛋鸡鼻腔拭子中的病毒拷贝数较攻毒对照组下降约73.7％～95.0％;攻毒后9d,采集各组蛋鸡的鼻甲、气管、肺脏制备病理切片后进行病理组织学观察,结果显示,攻毒对照组蛋鸡鼻甲、气管、肺脏出现不同程度的病理损伤,免疫组蛋鸡各组织器官未见明显病理变化.本研究结果首次表明,B亚型禽偏肺病毒病灭活疫苗对商品蛋鸡具有良好的免疫保护效果.本研究为商品蛋鸡的B亚型禽偏肺病毒病的防控和免疫程序的制定提供了参考.

为研究B亚型禽偏肺病毒(aMPV/B)对蛋鸡的致病性,将aMPV/B LN16株经Vero细胞传代培养,通过点眼滴鼻的方式感染9周龄蛋鸡.感染后第2天,感染组蛋鸡开始出现流鼻涕,并伴有鼻痂等症状,发病率为92.3％.荧光定量PCR检测结果表明,感染组蛋鸡在感染后1～5 d排毒.感染后第4天,感染组蛋鸡的哈氏腺、气管、喉头及鼻甲骨内均有病毒分布.感染后7～21d,感染组蛋鸡的aMPV抗体水平逐渐升高.组织病理观察结果表明,感染组蛋鸡的鼻甲骨、肺脏、气管均表现出不同程度的病理损伤,主要表现为炎性细胞浸润.研究表明,aMPV/B可以感染蛋鸡并引起发病.

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, causes immunosuppression and high mortality in 3–6-week-old chickens. Innate immune defense is a physical barrier to restrict viral replication. After viral infection, the host shows crucial defense responses, such as stimulation of antiviral effectors to restrict viral replication. Here, we conducted RNA-seq in avian cells infected by IBDV and identified TRIM25 as a host restriction factor. Specifically, TRIM25 deficiency dramatically increased viral yields, whereas overexpression of TRIM25 significantly inhibited IBDV replication. Immunoprecipitation assays indicated that TRIM25 only interacted with VP3 among all viral proteins, mediating its K27-linked polyubiquitination and subsequent proteasomal degradation. Moreover, the Lys854 residue of VP3 was identified as the key target site for the ubiquitination catalyzed by TRIM25. The ubiquitination site destroyed enhanced the replication ability of IBDV in vitro and in vivo. These findings demonstrated that TRIM25 inhibited IBDV replication by specifically ubiquitinating and degrading the structural protein VP3.

Avian leukosis virus (ALV) can induce various tumors and cause serious production problems. ALVs isolated from chickens were divided into six subgroups (A-J). In 2012, a strain of a putative novel subgroup of ALVs was isolated from Chinese native chickens in Jiangsu Province and named as ALV-K. In this study, three ALV-K strains (JS14LH01, JS13LH14, and JS15SG01) were isolated from chickens with suspected ALV infection in Jiangsu Province. Their complete genomes were amplified, sequenced, and analyzed systematically. The results showed that JS14LH01 and JS13LH14 were ALV-K and ALV-E recombinant strains. Whereas JS15SG01 is an ALV-K, ALV-E, and ALV-J multiple recombinant strain containing the U3 region of ALV-J. The pathogenicity test of JS15SG01 revealed that, compared with previous ALV-K strains, the viremia and viral shedding level of JS15SG01-infected chickens were significantly increased, reaching 100 % and 59 %, respectively. More important, JS15SG01 induced significant proliferation of gliocytes in the cerebral cortex of infected chickens, accompanied by the neurotropic phenomenon. This is the first report about a multiple recombinant ALV-K strain that could invade and injure the brain tissue of chickens in China. Our findings enriched the epidemiologic data of ALV and helped to reveal the evolution of ALV strains prevalent in chicken fields.

Avian leukosis virus subgroup J (ALV-J) was first isolated from broiler chickens in China in 1999; subsequently, it was rapidly introduced into layer chickens and Chinese local chickens. Recently, the incidence of ALV-J in broiler and layer chickens has significantly decreased. However, it has caused substantial damage to Chinese local chickens, resulting in immense challenges to their production performance and breeding safety. To systematically analyze the molecular characteristics and the epidemic trend of ALV-J in Chinese local chickens, 260 clinical samples were collected for the period of 2013–2018; 18 ALV-J local chicken isolates were identified by antigen-capture enzyme-linked immunosorbent assay and subgroup A-, B-, and J-specific multiplex PCR. The whole genomic sequences of 18 isolates were amplified with PCR and submitted to GenBank. Approximately, 55.5% (10/18) of the 18 isolates demonstrated a relatively high homology (92.3–95.4%) with 20 ALV-J early-isolated local strains (genome sequences obtained from GenBank) in gp85 genes clustering in a separated branch. The 3ʹ untranslated region (3ʹ UTR) of the 18 isolates showed a 195–210 and 16–28 base pair deletion in the redundant transmembrane region and in direct repeat 1, respectively; 55.5% (10/18) of the 18 isolates retained the 147 residue E element. The U3 gene of 61.1% (11/18) of the 18 isolates shared high identity (94.6–97.3%) with ALV-J early-isolated local strains. These results implied that the gp85 and U3 of ALV-J local chicken isolates have rapidly evolved and formed a unique local chicken branch. In addition, it was determined that the gene deletion in the 3′UTR region currently serves as a unique molecular characteristic of ALV-J in China. Hence, the obtained results built on the existing ALV-J molecular epidemiological data and further elucidated the genetic evolution trend of ALV-J in Chinese local chickens.

Since subgroup J avian leukosis virus (ALV-J) was first isolated in the United Kingdom in 1988, it has seriously hindered the development of the poultry industry worldwide. Although cases of ALV-J infection have been reported as early as 2001 in Pakistan, there was no further research on the isolation and molecular characteristics of ALVs. In the present study, we first isolated two ALVs from suspicious clinical samples that were collected from a desi chicken farm in Pakistan. The results of multiplex PCR and indirect immunofluorescent antibody assays confirmed that the two isolates (PK19FA01 and PK19SA01) belonged to ALV-J. The complete genomes of the two isolates were amplified, sequenced, and systematically analyzed. We found that gp85 of PK19FA01 was more similar to that of the prototype strain HPRS103, whereas gp85 of PK19SA01 was more similar to that of American strains. The two isolates contained an intact E element of 147 residues and had a unique 135 bp deletion in the redundant transmembrane of the 3′ untranslated region. The U3 region of the two isolates was highly homologous to that of American ALV-J strains. To our knowledge, this is the first report of the isolation, complete genome sequencing, and systematic molecular epidemiological investigation of ALV-J in Pakistan. Our findings could enrich epidemiological data and might contributed to more effective measures to prevent and control avian leukosis in Pakistan.

Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of envelope (Env) surface proteins to cell receptors is a key step in triggering infections and has been the target of antiviral drug screening. ALV-J is an economically important avian pathogen that belongs to the genus Alpharetrovirus and has a wider host range than other ALV subgroups. Our results showed that the amino acids 38 to 131 of the N terminus and 159 to 283 of the C terminus of ALV-J gp85 controlled the efficiency of gp85 binding to chNHE1 and were critical for viral infection. In addition, the glycosylation sites (N6 and N11) and cysteines (C3 and C9) of gp85 played a crucial role in the receptor binding and viral entry. These findings might help elucidate the mechanism of the entry of ALV-J into host cells and provide antiviral targets for the control of ALV-J.

Avian leukosis virus subgroup J (ALV-J) is an important pathogen for various neoplasms and causes significant economic losses in the poultry industry. Serological detection of specific antibodies against ALV-J infection is important for successful clinical diagnosis. Here, a 293F stable cell line was established to stably express gp85 protein. In this cell line, gp85 protein was expressed at approximately 30 mg/L. A subgroup-specific indirect enzyme-linked immunosorbent assay (iELISA) was developed using ALV-J gp85 protein as coated antigen to detect antibodies against ALV-J. The sensitivity of the iELISA (1:51200 diluted in serum) was 16 times more than that of indirect immunofluorescence assay (IFA; 1:3200 diluted in serum). Moreover, there was no crossreactivity with antibodies against other common avian viruses and other avian leukosis virus subgroups, such as subgroups A and B. The practicality of the iELISA was further evaluated by experimental infection and clinical samples. The results from experimental infection indicated that anti-ALV-J antibodies were readily detected by iELISA as early as 4 weeks after ALV-J infection, and positive antibodies were detected until 20 weeks, with an antibody-positive rate of 11.1% to 33.3%. Moreover, analysis of clinical samples showed that 9.49% of samples were positive for anti-ALV-J antibodies, and the concordance rate of iELISA and IFA was 99.24%. Overall, these results suggested that the subgroup-specific iELISA developed in this study had good sensitivity, specificity, and feasibility. This iELISA will be very useful for epidemiological surveillance, diagnosis, and eradication of ALV-J in poultry farms.