Dynamic changes in intestinal microbiota and metabolite composition of pre-weaned beef calves.

Gut microbes and their metabolites are essential for maintaining host health and production. The intestinal microflora of pre-weaned calves gradually tends to mature with growth and development and has high plasticity, but few studies have explored the dynamic changes of intestinal microbiota and metabolites in pre-weaned beef calves. In this study, we tracked the dynamics of faecal microbiota in 13 new-born calves by 16S rRNA gene sequencing and analysed changes in faecal amino acid levels using metabolomics. Calves were divided into the relatively high average daily gain group (HA) and the relatively low average daily gain group (LA) for comparison. The results demonstrated that the alpha diversity of the faecal microbiota increased with calf growth and development. The abundance of Porphyromonadaceae bacterium DJF B175 increased in the HA group, while that of Lactobacillus reuteri decreased. The results of the LEfSe analysis showed that the microbiota of faeces of HA calves at eight weeks of age was enriched with P. bacterium DJF B175, while Escherichia coli and L. reuteri were enriched in the microbiota of faeces of LA calves. Besides, the total amino acid concentration decreased significantly in the eighth week compared with that in the first week (P < 0.05). Overall, even under the same management conditions, microorganisms and their metabolites interact to play different dynamic regulatory roles. Our results provide new insights into changes in the gut microbiota and metabolites of pre-weaned calves.

Effects of myostatin gene knockout on porcine extraocular muscles.

Myostatin (MSTN), a negative regulator of skeletal muscle mass, is not well known in extraocular muscles (EOMs). EOMs are specialized skeletal muscles. Hence, in this study, the effect of MSTN on the superior rectus (SR) and superior oblique (SO) of 2-month-old MSTN knockout (MSTN-/-) and wild-type (WT) pigs of the same genotype was investigated. SR (P < 0.01) and SO (P < 0.001) fiber cross-sectional areas of MSTN-/- pigs were significantly larger than those of WT pigs. Compared with WT pigs, MSTN-/- SO displayed a decrease in type I fibers (WT: 27.24%, MSTN-/-: 10.32%, P < 0.001). Type IIb fibers were higher in MSTN-/- pigs than in WT pigs (WT: 30.38%, MSTN-/-: 62.24%, P < 0.001). The trend in SR was the same as that in SO, although the trend in SO was greater than that in SR. The expression of myogenic differentiation factor (MyoD) and myogenic (MyoG) showed a significant increase in MSTN-/- SO (about 2.5-fold and 2-fold, respectively at the gene expression level, about 1.5-fold at the protein level) compared with WT pigs. MSTN plays an important role in the development of EOMs and regulates the muscle fiber type by modulating the gene expression of MyoD and MyoG in pigs.

Letter to the Editor: An ultra-sensitive assay using cell-free DNA fragmentomics for multi-cancer early detection.

Early detection can benefit cancer patients with more effective treatments and better prognosis, but existing early screening tests are limited, especially for multi-cancer detection. This study investigated the most prevalent and lethal cancer types, including primary liver cancer (PLC), colorectal adenocarcinoma (CRC), and lung adenocarcinoma (LUAD). Leveraging the emerging cell-free DNA (cfDNA) fragmentomics, we developed a robust machine learning model for multi-cancer early detection. 1,214 participants, including 381 PLC, 298 CRC, 292 LUAD patients, and 243 healthy volunteers, were enrolled. The majority of patients (N = 971) were at early stages (stage 0, N = 34; stage I, N = 799). The participants were randomly divided into a training cohort and a test cohort in a 1:1 ratio while maintaining the ratio for the major histology subtypes. An ensemble stacked machine learning approach was developed using multiple plasma cfDNA fragmentomic features. The model was trained solely in the training cohort and then evaluated in the test cohort. Our model showed an Area Under the Curve (AUC) of 0.983 for differentiating cancer patients from healthy individuals. At 95.0% specificity, the sensitivity of detecting all cancer reached 95.5%, while 100%, 94.6%, and 90.4% for PLC, CRC, and LUAD, individually. The cancer origin model demonstrated an overall 93.1% accuracy for predicting cancer origin in the test cohort (97.4%, 94.3%, and 85.6% for PLC, CRC, and LUAD, respectively). Our model sensitivity is consistently high for early-stage and small-size tumors. Furthermore, its detection and origin classification power remained superior when reducing sequencing depth to 1× (cancer detection: ≥ 91.5% sensitivity at 95.0% specificity; cancer origin: ≥ 91.6% accuracy). In conclusion, we have incorporated plasma cfDNA fragmentomics into the ensemble stacked model and established an ultrasensitive assay for multi-cancer early detection, shedding light on developing cancer early screening in clinical practice.

Role of env gene and LTR sequence in the pathogenesis of subgroup K avian leukosis virus.

Avian leukosis virus (ALV) is a retrovirus that induces tumours in infected birds; ALV is divided into different subgroups according to the env gene and cellular tropism. In general, ALV subgroup J (ALV-J) is considered to be the most pathogenic and prevalent subgroup while subgroup K (ALV-K), a newly identified subgroup, only causes mild symptoms. To illuminate the roles of the env viral gene and LTR sequence in pathogenic differences between ALV-J and ALV-K, rescued ALV-J strain rSDAU1005, rescued ALV-K strain rJS11C1, and recombinant strains rENV(J)-LTR(K) and rENV(K)-LTR(J) were characterized and investigated in this study. Among rescued viruses, rSDAU1005 had the highest replication efficiency while rJS11C1 replicated the slowest (replication efficiency rankings were rSDAU1005 >rENV(K)-LTR(J)>rENV(J)-LTR(K)>rJS11 C1). The luciferase reporter gene assay results showed that the promoter activity of ALV-K LTR was lower than that of the ALV-J LTR promoter, which may have accounted for the slower replication efficiency of ALV-K. Pathogenicity of the four rescued viruses was determined via inoculating the yolk sacs of specific-pathogen-free chickens. The results demonstrated that all four viruses were pathogenic; rSDAU1005 caused the most severe growth retardation and immunosuppression. rENV(J)-LTR(K) was more pathogenic when compared to rENV(K)-LTR(J), indicating that env and the LTR sequence play important roles in pathogenicity between ALV-K and ALV-J. Additionally, env seemed to especially play a role in ALV-K pathogenesis. This study provided scientific data and insight to improve detection methods and judgement criteria in ALV clearance and surveillance.

m6 A transferase KIAA1429-stabilized LINC00958 accelerates gastric cancer aerobic glycolysis through targeting GLUT1.

Emerging evidence has demonstrated that N6 -methyladenosine (m6 A) and long noncoding RNAs (lncRNAs) are both crucial regulators in gastric cancer (GC) tumorigenesis. However, the interaction of m6 A and lncRNAs in GC progression are still unclear. Here, our team discovered that lncRNA LINC00958 expression up-regulated in GC tissue and cells. Clinically, high-expression of LINC00958 was clinically correlated to lower survival of GC patients. Functionally, in vitro assays demonstrated that LINC00958 promoted the GC cells' aerobic glycolysis. Mechanistically, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) found that there were m6 A-modificated sites in LINC00958, and moreover m6 A methyltransferase KIAA1429 catalyzed the m6 A modification on LINC00958 loci. Moreover, LINC00958 interacted with GLUT1 mRNA via the m6 A-dependent manner to enhance GLUT1 mRNA transcript stability, thereby positively regulating the aerobic glycolysis of GC. In conclusion, our findings reveal the function and mechanism of KIAA1429-induced LINC00958 in GC, delineating novel understanding of m6 A-lncRNA in cancer biology.

Endogenous Avian Leukosis Virus in Combination with Serotype 2 Marek's Disease Virus Significantly Boosted the Incidence of Lymphoid Leukosis-Like Bursal Lymphomas in Susceptible Chickens.

In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek's disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes.IMPORTANCE Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.

Marek's disease vaccines-induced differential expression of known and novel microRNAs in primary lymphoid organ bursae of White Leghorn.

Marek's disease (MD) is a contagious disease of domestic chickens caused by MD viruses. MD has been controlled primarily by vaccinations, yet sporadic outbreaks of MD take place worldwide. Commonly used MD vaccines include HVT, SB-1 and CVI988/Rispens and their efficacies are reportedly dependent of multiple factors including host genetics. Our previous studies showed protective efficacy of a MD vaccine can differ drastically from one chicken line to the next. Advanced understanding on the underlying genetic and epigenetic factors that modulate vaccine efficacy would greatly improve the strategy in design and development of more potent vaccines. Two highly inbred lines of White Leghorn were inoculated with HVT and CVI988/Rispens. Bursa samples were taken 26 days post-vaccination and subjected to small RNA sequencing analysis to profile microRNAs (miRNA). A total of 589 and 519 miRNAs was identified in one line, known as line 63, 490 and 630 miRNAs were identified in the other, known as line 72, in response to HVT or CVI988/Rispens inoculation, respectively. HVT and CVI988/Rispens induced mutually exclusive 4 and 13 differentially expressed (DE) miRNAs in line 63 birds in contrast to a non-vaccinated group of the same line. HVT failed to induce any DE miRNA and CVI988/Rispens induced a single DE miRNA in line 72 birds. Thousands of target genes for the DE miRNAs were predicted, which were enriched in a variety of gene ontology terms and pathways. This finding suggests the epigenetic factor, microRNA, is highly likely involved in modulating vaccine protective efficacy in chicken.

Designing of CuS growing on Bi2WO6 nanosheet heterostructures based on a photoelectrochemical aptasensor for detecting ofloxacin.

Bi2WO6 (BW) was compounded with different contents of copper sulfide (CuS) by a two-step procedure. The chemical composition and morphology of the materials were characterized by X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The results of photoelectrochemical (PEC) tests showed that CuS can improve the PEC performance of semiconductor materials and it has the best performance when the CuS mass fraction is 5%. Therefore, CuS/BW-5% nanocomposite has been constructed as ofloxacin (OFL) drug PEC aptasensors by binding of aptamer receptors. The PEC aptasensor based on CuS/BW-5% has a linear relationship for OFL of 1-12,000 nM and a determination limit of 0.35 nM. Since the photoelectron potential generated by CuS/BW-5% heterojunction reduces the combination of photogenerated electrons and holes CuS/BW-5% has a better photoelectrocatalytic performance. Graphical abstract Schematic presentation of a photoelectrochemical aptasensor based on CuS/Bi2WO6 for the determination of OFL.

cxcl12 gene engineered endothelial progenitor cells further improve the functions of oligodendrocyte precursor cells.

Oligodendrocyte precursor cells (OPCs) are needed for white matter repair after various brain injury. Means that promote OPC functions could benefit white matter recovery after injury. Chemokine CXCL12 and endothelial progenitor cells (EPCs) both have been shown to promote remyelination. We hypothesize that the beneficial effects of EPCs and CXCL12 can be harnessed by genetically modifying EPCs with cxcl12 to synergistically improve the functions of OPCs. In this work, CXCL12-EPC was generated using virus-mediated gene transfer. OPCs were cultured with CXCL12-EPC conditioned media (CM) to analyze its impact on the proliferation, migration, differentiation and survival properties of OPCs. We blocked or knocked-down the receptors of CXCL12, namely CXCR4 and CXCR7, respectively to investigate their functions in regulating OPCs properties. Results revealed that CXCL12-EPC CM further promoted OPCs behavioral properties and upregulated the expression of PDGFR-α, bFGF, CXCR4 and CXCR7 in OPCs, albeit following different time course. Blocking CXCR4 diminished the beneficial effects of CXCL12 on OPCs proliferation and migration, while knocking down CXCR7 inhibited OPCs differentiation. Our results supported that cxcl12 gene modification of EPCs further promoted EPCs' ability in augmenting the remyelination properties of OPCs, suggesting that CXCL12-EPC hold great potential in white matter repair.

The experimental transmission of reticuloendotheliosis virus by cock semen.

Following artificial insemination, the egg-laying rate of a large-scale breeder chicken flock declined by10-15 %. Real-time quantitative polymerase chain reaction (qPCR) analysis detected the presence of reticuloendotheliosis virus (REV) in semen from the breeder cocks used. Six REV strains were successfully isolated from semen randomly extracted from those cocks. Additionally, the whole sequence of SDAUR-S1 was sequenced and analysed. Cock models with continuous production of REV-positive semen were established by intravenous injection with SDAUR-S1. Eggs were then collected from hens after artificial insemination with REV-positive semen, for virus detection. The positive REV antibody rate for egg albumen was 58.3 % and the REV-positive rate for hatched embryos was 8.3 %, which suggested not only that REV can infect cock semen, but can also infect the offspring. In conclusion, the present study is the first to report on the isolation, genome analysis and transmission of REV in cock semen.

Protection induced by a gp90 protein-based vaccine derived from a Reticuloendotheliosis virus strain isolated from a contaminated IBD vaccine.

BACKGROUND: Reticuloendotheliosis is an immunosuppressive disease caused by avian reticuloendotheliosis virus (REV). It is commonly found in poultry farms and has caused a notable economic loss worldwide. Despite this, there is currently no effective vaccine available to protect against REV infection. METHOD: In this study, gp90 protein derived from an REV isolated from a contaminated vaccine was co-administered with cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) adjuvant to hens to determine if it protects their chicks against REV infection. To synthesize the gp90 protein, the gp90 gene was amplified using polymerase chain reaction, expressed in Escherichia coli, and purified. The resulting recombinant protein was injected intramuscularly into breeder hens along with CpG-ODN adjuvant and then serum antibody levels were regularly evaluated. After the fertilized eggs from these vaccinated hens had hatched, the resulting chicks were challenged with a 102.7 50% tissue culture infectious dose (TCID50) of REV at 1 day old and the REV antibody levels in these hatched chickens were evaluated before and after the challenge. Viremia and growth rate were measured weekly and statistically analyzed. RESULTS: The results suggest that the gp90 recombinant protein was successfully prepared and, when used with CpG-ODN adjuvant to immunize breeder hens, induced serological antibody production against REV in both hens and their hatched chicks. In addition, the maternal antibodies induced by the gp90 protein vaccine effectively protected majority of the chicks from REV infection. CONCLUSIONS: Overall, we found the gp90 protein obtained in this study may be a potential vaccine candidate that had good immunogenicity and could be an auxiliary measure to accelerate the eradication of REV.

Characterization of subgroup J avian Leukosis virus isolated from Chinese indigenous chickens.

BACKGROUND: In spite of the purification of the laying hens and broilers of avian leukosis virus (ALV) has made remarkable achievements, the infection of ALV was still serious in Chinese indigenous chickens. METHODS: In order to assess the epidemic state of avian leukosis virus in indigenous chickens in China, 10 novel strains of ALV subgroup J (ALV-J), named JS16JH01 to JS16JH10, were isolated and identified by virus isolation and immunofluorescence antibody assays from a Chinese local breed farm with a sporadic incidence of tumors. To understand their virological characteristics further, the proviral genome of ENV-LTR was sequenced and compared with the reference strains. RESULTS: The homology of the gp85 gene between the ten ALV-J strains and NX0101 was in the range from 89.7-94.8% at the nuclear acid level. In addition, their gp85 genes were quite varied, with identities of 92-98% with themselves at the nuclear acid level. There were several snp and indel sites in the amino acid sequence of gp85 genes after comparison with other reference strains of ALV. Interestingly, a novel insertion in the gp85 region was found in two strains, JS16JH01 and JS16JH07, compared with NX0101 and HPRS-103. DISCUSSION: At present, owing to the large-scale purification of ALV in China, laying hens and broiler chickens with ALV infection are rarely detected, but ALVs are still frequently detected in the local chickens, which suggests that more efforts should be applied to the purification of ALV from indigenous chickens.

Newcastle disease virus-attenuated vaccine LaSota played a key role in the pathogenicity of contaminated exogenous virus.

Newcastle disease virus (NDV)-attenuated vaccine has been widely used since the 1950s and made great progress in preventing and controlling Newcastle disease. However, many reports mention exogenous virus contamination in attenuated vaccines, while co-contamination with fowl adenovirus (FAdV) and chicken infectious anaemia virus (CIAV) in the NDV-attenuated vaccine also emerged in China recently, which proved to be an important reason for the outbreaks of inclusion body hepatitis-hydropericardium syndrome in some flocks. It is amazing that exogenous virus contamination at extremely low doses still infected chickens and induced severe disease; thus, we speculated that there must be some interaction between the NDV-attenuated vaccine and the contaminated exogenous viruses within. Accordingly, simulation experiments were launched using FAdV and CIAV isolated from the abovementioned vaccine. The results showed that the pathogenicity of FAdV and CIAV co-infection through the contaminated vaccine was significantly higher than that of direct oral infection, while the synergistic reaction of these viruses and LaSota prompted their multiplication in vivo and disturbed the production of antibodies against each other. This study showed the interactions of FAdV, CIAV and LaSota after using contaminated NDV-attenuated vaccine, helping us to understand how the contaminated exogenous viruses cause infection and induce severe disease at a relatively low dose through the oral route.

Monomeric CXCL12 outperforms its dimeric and wild type variants in the promotion of human endothelial progenitor cells' function.

CXCL12 overexpression improves neurobehavioral recovery during post-ischemic stroke through multiple mechanisms including promoting endothelial progenitor cells function in animal models. It has been proposed that the monomer and dimer forms possess differential chemotactic and regulatory function. The aim of present study is to explore whether a monomeric or dimeric CXCL12 plays a different role in the endothelial progenitor cells proliferation, migration, and tube-formation in vitro. In this study, we transferred monomeric, dimeric and wild type CXCL12 gene into endothelial progenitor cells via lentiviral vectors. We investigated endothelial progenitor cells function following the interaction of CXCL12/CXCR4 or CXCL12/CXCR7 and downstream signaling pathways. Our results showed that the monomeric CXCL12 transfected endothelial progenitor cells had enhanced ability in cell proliferation, migration, and tube-formation compared to that in dimeric or wild type controls (p < 0.05). Both CXCR4 and CXCR7 were significantly overexpressed in the monomeric CXCL12 transfected endothelial progenitor cells compared to that in the dimeric or wide type controls (p < 0.05). The function of migration, but not proliferation or tube-formation, was significantly reduced in the monomeric CXCL12 transfected endothelial progenitor cells when the cells were pre-treated with either CXCR4 inhibitor AMD3100 or siCXCR7 (p < 0.05), suggesting this cell function was partially regulated by CXCL12/CXCR4 and CXCL12/CXCR7 signal pathways. Our study demonstrated that monomeric CXCL12 was the fundamental form, which played important roles in endothelial progenitor cells' proliferation, migration, and tube-formation.

Vertical transmission of avian leukosis virus subgroup J (ALV-J) from hens infected through artificial insemination with ALV-J infected semen.

BACKGROUND: Avian leukosis virus (ALV) is one of the main causes of tumour development within the poultry industry in China. The subgroup J avian leukosis viruses (ALV-J), which induce erythroblastosis and myelocytomatosis, have the greatest pathogenicity and transmission ability within this class of viruses. ALV can be transmitted both horizontally and vertically; however, the effects of ALV infection in chickens-especially roosters-during the propagation, on future generations is not clear. Knowing the role of the cock in the transmission of ALV from generation to generation might contribute to the eradication programs for ALV. RESULTS: The results showed that two hens inseminated with ALV-J-positive semen developed temporary antibody responses to ALV-J at 4-5 weeks post insemination. The p27 antigen was detected in cloacal swabs of six hens, and in 3 of 26 egg albumens at 1-6 weeks after insemination. Moreover, no viremia was detected at 6 weeks after insemination even when virus isolation had been conducted six times at weekly intervals for each of the 12 females. However, ALV-J was isolated from 1 of their 34 progeny chicks at 1 week of age, and its gp85 had 98.4%-99.2% sequence identity with the gp85 of ALV-J isolated from semen samples of the six cocks. CONCLUSIONS: Our findings indicated that females that were late horizontally infected with ALV-J by artificial insemination might transmit the virus to progeny through eggs, which amounts to vertical transmission.

Genome analysis and pathogenicity of reticuloendotheliosis virus isolated from a contaminated vaccine seed against infectious bursal disease virus: first report in China.

Specific-pathogen-free (SPF) chickens were inoculated with the virus seed of an infectious bursal disease virus (IBDV)-attenuated vaccine, and positive reticuloendotheliosis virus (REV) antibody levels were subsequently detected in the chicken sera, indicating potential REV contamination of the vaccine. After neutralization with IBDV-positive blood serum, the vaccine was inoculated into DF-1 cells for REV isolation and identification. An REV strain, designated IBD-C1605, was identified using an immunofluorescence assay test. Three pairs of primers were employed for the amplification, cloning and sequencing of three overlapping fragments of the IBD-C1605 genome, and the whole-genome sequence of this isolate was obtained after gene assembly. The genome was 8362 base pairs (nt) in length and its homology with the nucleotide sequences of different reference strains varied between 94.2 and 99.2 %. Isolate IBD-C1605 was inoculated into 1-day-old SPF chickens to observe its pathogenicity. Infection with this organism slowed down the weight gain of SPF chickens and caused atrophy of their immune organs, such as the bursa of Fabricius and thymus gland. Furthermore, the chicken antibody levels decreased significantly after Newcastle disease virus and avian influenza virus subtype H9 vaccine immunization. This is the first report on the isolation and identification of REV from attenuated vaccine virus seeds in China, and is also the first study on the pathogenicity of REV from a contaminated vaccine in China. Our findings contribute towards a better understanding of the detrimental effects of vaccine contamination with exogenous viruses such as REV.

Identification of avian leukosis virus subgroup J-associated acutely transforming viruses carrying the v-src oncogene in layer chickens.

To elucidate the molecular basis for the rapid oncogenicity of an acutely transforming avian leukosis virus (ALV), isolated from fibrosarcomas in Hy-Line Brown commercial layer chickens infected with ALV subgroup J (ALV-J), the complete genomic structure of the provirus was determined. In addition to ALV-J replication-complete virus SDAU1102, five proviral DNA genomes, named SJ-1, SJ-2, SJ-3, SJ-4 and SJ-5, carrying different lengths of the v-src oncogene were amplified from original tumours and chicken embryo fibroblasts (CEFs) infected with viral stocks. The genomic sequences of the SJ-1-SJ-5 provirus were closely related to that of SDAU1102 but were defective. The results of Western blot analysis and immunohistochemical staining also showed overexpression of the p60v-src protein in infected CEFs and tumour tissue. To the best of our knowledge, this is the first report of the isolation and identification of acutely transforming viruses carrying the v-src oncogene with ALV-J as the helper virus. It also offers insight into the generation of acutely transforming ALVs carrying the v-src oncogene.

Analysis of Quasispecies of Avain Leukosis Virus Subgroup J Using Sanger and High-throughput Sequencing.

BACKGROUND: Avian leukosis viruses subgroup J (ALV-J) exists as a complex mixture of different, but closely related genomes named quasispecies subjected to continuous change according to the Principles of Darwinian evolution. METHOD: The present study seeks to compare conventional Sanger sequencing with deep sequencing using MiSeq platform to study quasispecies dynamics of ALV-J. RESULTS: The accuracy and reproducibility of MiSeq sequencing was determined better than Sanger sequencing by running each experiment in duplicate. According to the mutational rate of single position and the ability to distinguish dominant quasispecies with two sequencing methods, conventional Sanger sequencing technique displayed high randomness due to few sequencing samples, while deep sequencing could reflect the composition of the quasispecies more accurately. In the mean time, the research of quasispecies via Sanger sequencing was simulated and analyzed with the aid of re-sampling strategy with replacement for 1000 times repeat from high-throughput sequencing data, which indicated that the higher antibody titer, the higher sequence entropy, the harder analyzing with the conventional Sanger sequencing, resulted in lower ratios of dominant variants. CONCLUSIONS: In sum, deep sequencing is better suited for detecting rare variants comprehensively. The simulation of Sanger sequencing that we propose here will also help to standardize quasispecies researching under different selection pressure based on next-generation sequencing data.

Rescue of avian leukosis subgroup-J-associated acutely transforming viruses carrying different lengths of the v-fps oncogene and analysis of their tumorigenicity.

In our previous study, six subgroup J strains of avian leukosis virus (ALV-J)-associated acutely transforming viruses carrying different lengths of the v-fps oncogene, designated as Fu-J and Fu-J1-5, were isolated and characterized from fibrosarcomas in ALV-J-infected chickens. In the present study, the oncogenic potential of Fu-J and Fu-J1-5 was investigated using a reverse genetics technique. Six replication-defective viruses, named rFu-J and rFu-J1-5, were rescued with the replication-competent rescued ALV-J strain rSDAU1005 as a helper virus by co-transfection of chicken embryo fibroblast monolayers with infectious clone plasmids. Experimental bird studies were performed, demonstrating that only the rescued rFu-J virus carrying the complete v-fps oncogene with rSDAU1005 as the helper virus could induce acute fibrosarcoma after inoculation in specific-pathogen-free (SPF) chickens. These results provide direct evidence that the replication-defective acutely transforming Fu-J virus, with the complete v-fps oncogene, was associated with acute fibrosarcoma in chickens infected with ALV-J in the field, as reported previously.