Analysis of the Brucella melitensis epidemic in Xinjiang: genotyping, polymorphism, antibiotic resistance and tracing.

Brucella spp. are facultative intracellular pathogens that cause zoonosis- brucellosis worldwide. There has been a trend of the re-emergence of brucellosis worldwide in recent years. The epidemic situation of brucellosis is serious in Xinjiang. To analyze the epidemic situation of Brucella spp. in Xinjiang among humans and animals, this study identified 144 Brucella isolates from Xinjiang using classical identification and 16 S rRNA sequencing. MLVA, drug resistance testing, and wgSNP detection were also performed. At the same time, analysis was conducted based on the published data of Brucella isolates worldwide. The results showed that the dominant species was B. melitensis biovar 3, which belonged to GT42 (MLVA-8 typing) and the East Mediterranean lineage. The correlation among isolates was high both in humans or animals. The isolates in Xinjiang exhibited higher polymorphism compared to other locations in China, with polymorphism increasing each year since 2010. No amikacin/kanamycin-resistant strains were detected, but six rifampicin-intermediate isolates were identified without rpoB gene variation. The NJ tree of the wgSNP results indicated that there were three main complexes of the B. melitensis epidemic in Xinjiang. Based on the results of this study, the prevention and control of brucellosis in Xinjiang should focus on B. melitensis, particularly strains belonging to B. melitensis bv.3 GT42 (MLVA-8 typing) and East Mediterranean lineage. Additionally, the rifampicin- and trimethoprim-sulfamethoxazole- resistance of isolates in Xinjiang should be closely monitored to avoid compromising the therapeutic efficacy and causing greater losses. These results provide essential data for the prevention and control of brucellosis in Xinjiang and China. Although the isolates from Xinjiang have significant characteristics among Chinese isolates and can reflect the epidemiological situation of brucellosis in China to some extent, this study cannot represent the characteristics of isolates from other regions.

Transcriptome and the gut microbiome analysis of the impacts of Brucella abortus oral infection in BALB/c mice.

Brucellosis is a zoonotic infectious disease caused by Brucella spp, which could cause serious economic losses to animal husbandry and threaten human public health. Ingestion of contaminated animal products is a common way to acquire Brucella infection in humans, while research on effect of oral Brucella infection on host gut microbiota and the gene expression in intestinal tissues is limited. In the present study, 16S rRNA sequencing and RNA sequencing were conducted to explore gut microbiota and expression profiles of mRNAs in the colon of BALB/c mice, which were infected by Brucella abortus 2308. The fecal samples were collected at 7 and 28 days post infection to observe changes in the gut microbiota during Brucella infection. In the alpha diversity analysis, significantly increased Chao 1 index was observed at 28 days after Brucella infection. The Bray-Curtis distancebased principal coordinate analysis indicated that the WT group showed a separation from the Brucella infection groups. In addition, analysis of composition of microbes revealed that Prevotellaceae_NK3B31_group were more abundant in 1 week and 4 week infection groups, while Turicibacter was only more abundant in 4 week infection group. Based on the RNA-seq assay, a total of 45 differentially expressed genes were detected between Brucella abortus infection group and control group. Furthermore, KEGG pathway enrichment analysis showed that protein processing in endoplasmic reticulum, Legionellosis, Spliceosome, Hippo signaling pathway and Influenza A were significantly enriched in response to Brucella abortus infection. Our finding will help to improve the knowledge of the mechanisms underlying Brucella infection and may provide novel targets for future treatment of this pathogen infection.

ClpP protease modulates bacterial growth, stress response, and bacterial virulence in Brucella abortus.

The process of intracellular proteolysis through ATP-dependent proteases is a biologically conserved phenomenon. The stress responses and bacterial virulence of various pathogenic bacteria are associated with the ATP-dependent Clp protease. In this study, a Brucella abortus 2308 strain, ΔclpP, was constructed to characterize the function of ClpP peptidase. The growth of the ΔclpP mutant strain was significantly impaired in the TSB medium. The results showed that the ΔclpP mutant was sensitive to acidic pH stress, oxidative stress, high temperature, detergents, high osmotic environment, and iron deficient environment. Additionally, the deletion of clpP significantly affected Brucella virulence in macrophage and mouse infection models. Integrated transcriptomic and proteomic analyses of the ΔclpP strain showed that 1965 genes were significantly affected at the mRNA and/or protein levels. The RNA-seq analysis indicated that the ΔclpP strain exhibited distinct gene expression patterns related to energy production and conversion, cell wall/membrane/envelope biogenesis, carbohydrate transport, and metabolism. The iTRAQ analysis revealed that the differentially expressed proteins primarily participated in amino acid transport and metabolism, energy production and conversion, and secondary metabolites biosynthesis, transport and catabolism. This study provided insights into the preliminary molecular mechanism between Clp protease to bacterial growth, stress response, and bacterial virulence in Brucella strains.

The Pseudogene BMEA_B0173 Deficiency in Brucella melitensis Contributes to M-epitope Formation and Potentiates Virulence in a Mice Infection Model.

It is widely acknowledged that pseudogenes play important roles in bacterial diversification and evolution and participate in gene regulation and RNA interference (RNAi). However, the function of most pseudogenes in Brucella spp remains poorly understood, warranting further studies.To comprehensively analyze the function of the pseudogenes BMEA_B0173 in Brucella melitensis strain 63/9, a BMEA_B0173 in-frame deleted mutant strain was constructed. Then, the phenotypes of the mutant strain, such as growth characteristics and bacterial virulence, were assessed in mice infection models. Finally, iTRAQ analysis was performed to investigate the gene expression profile affected by the pseudogenes BMEA_B0173. In this study, we found that BMEA_B0173 deletion exhibited increased agglutination with M monospecific sera. In a mouse model of chronic infection, the BMEA_B0173 deletion strain displayed increased colonization in the spleen compared to the wild-type pathogen. The iTRAQ assay revealed that 252 proteins were differentially expressed between the BMEA_B0173 deletion and the wild-type strains. In addition, deletion of BMEA_B0173 significantly increased the expression of proteins involved in the denitrification pathway, iron metabolism, and several transcriptional regulators, which might cause increased virulence of the mutant strain. In conclusion, this study preliminary uncovered the function of the pseudogene BMEA_B0173 in Brucella melitensis 63/9 and provided novel insights for studying the pathogenesis of Brucella strains.

Mutations in PB2 and HA enhanced pathogenicity of H4N6 avian influenza virus in mice.

The H4 subtype avian influenza virus (AIV) continues to circulate in both wild birds and poultry, and occasionally infects mammals (e.g. pigs). H4-specific antibodies have also been detected in poultry farm workers, which suggests that H4 AIV poses a potential threat to public health. However, the molecular mechanism by which H4 AIVs could gain adaptation to mammals and whether this has occurred remain largely unknown. To better understand this mechanism, an avirulent H4N6 strain (A/mallard/Beijing/21/2011, BJ21) was serially passaged in mice and mutations were characterized after passaging. A virulent mouse-adapted strain was generated after 12 passages, which was tentatively designated BJ21-MA. The BJ21-MA strain replicated more efficiently than the parental BJ21, both in vivo and in vitro. Molecular analysis of BJ21-MA identified four mutations, located in proteins PB2 (E158K and E627K) and HA (L331I and G453R, H3 numbering). Further studies showed that the introduction of E158K and/or E627K substitutions into PB2 significantly increased polymerase activity, which led to the enhanced replication and virulence of BJ21-MA. Although individual L331I or G453R substitutions in HA did not change the pathogenicity of BJ21 in mice, both mutations significantly enhanced virulence. In conclusion, our data presented in this study demonstrate that avian H4 virus can adapt to mammals by point mutations in PB2 or HA, which consequently poses a potential threat to public health.

Deletion of the LuxR-type regulator VjbR in Brucella canis affects expression of type IV secretion system and bacterial virulence, and the mutant strain confers protection against Brucella canis challenge in mice.

Brucella spp. are facultative intracellular pathogens and zoonotic agents which pose a huge threat to human health and animal husbandry. The B. melitensis, B. abortus, and B. suis cause undulant fever and influenza-like symptoms in humans. However, the effects of B. canis have not been extensively studied. The quorum sensing-dependent transcriptional regulator VjbR influences the Brucella virulence in smooth type Brucella strains, such as B. melitensis, B. abortus and rough type Brucella ovis. However, the function of VjbR in the rough-type B. canis is unknown. In the present study, we discovered that deletion of this regulator significantly affected Brucella virulence in macrophage and mice infection models. The expression levels of virB operon and the ftcR gene were significantly altered in the vjbR mutant strain. We further investigated the protective effect of different doses of the vjbR mutant in mice and the results indicated that VjbR conferred protection against the virulent B. canis strain. This study presents the first evidence that the transcriptional regulator VjbR has important function in B. canis. In addition, according to its reduced virulence and the protective immunity it induces in mice, it can be a potential live attenuated vaccine against B. canis.

Interleukin 8 and Pentaxin (C-Reactive Protein) as Potential New Biomarkers of Bovine Tuberculosis.

Bovine tuberculosis (bTB) is caused by Mycobacterium bovis During the early stage of infection, greater than 15% of M. bovis-infected cattle shed mycobacteria through nasal secretions, which can be detected by nested PCR. To compare the differences in the protein profiles of M. bovis-infected cattle that were nested PCR positive (bTBPCR-P) and M. bovis-infected cattle that were nested PCR negative (bTBPCR-N) and to screen for biomarkers that will facilitate the early and accurate detection of bTB, we investigated the protein expression profiles of serum and bovine purified protein derivative (PPD-B)-stimulated plasma among bTBPCR-P (n = 20), bTBPCR-N (n = 20), and uninfected cattle (NC; n = 20) by iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2D LC-MS/MS). After comprehensive analysis, we selected 15 putative differentially expressed serum proteins and 15 plasma proteins for validation by parallel reaction monitoring (PRM) with the same cohort used in the iTRAQ analysis. Four serum and five PPD-B-stimulated proteins were confirmed in follow-up enzyme-linked immunosorbent assays. PPD-B-stimulated interleukin 8 (IL-8) displayed the potential to differentiate M. bovis-infected cattle from NC, with an area under the curve (AUC) value of 0.9662, while PPD-B-stimulated C-reactive protein (CRP) displayed the potential to differentiate bTBPCR-P from bTBPCR-N, with an AUC value of 1.00. Finally, double-blind testing with 244 cattle indicated that the PPD-B-stimulated IL-8 test exhibited good agreement with traditional tests (κ > 0.877) with a >90% relative sensitivity and a >98% relative specificity; the PPD-B-stimulated CRP test displayed good agreement with nested PCR (κ = 0.9117), with an observed 94% relative sensitivity and 97% relative specificity. Therefore, the PPD-B-stimulated IL-8 and CRP tests could be used to detect bTB and to differentiate bTBPCR-P from bTBPCR-N.

RNA-seq reveals the critical role of Lon protease in stress response and Brucella virulence.

The Brucella spp encounter stressful environment inside their host cells. The Lon protein is an important protease related to cellular protein degradation and resistance to stress in Brucella. However, the molecular mechanism between Lon protein and stress response was still unknown. In this study, it was found that the lon mutant exhibited obvious growth defect in TSB medium, compared with its parent strain. In addition, our results indicated that Lon protein was involved in resistance to various stress conditions and all the ß-lactam antibiotics tested. Although deletion of this protease did not affect Brucella virulence in macrophage, the mutant strain was significantly attenuated in mice infection model at 1 week post infection, and the expression level of several cytokine genes was significantly changed in vivo. To gain insight into the genetic basis for the distinctive phenotypic properties exhibited by the lon mutant strain, RNA-seq was performed, and the result showed that various genes involved in stress response, quorum sensing and transcriptional regulation were significantly altered in Δlon strain. Overall, these studies have preliminary uncovered the molecular mechanism between Lon protease, stress response and bacterial virulence.

Transcriptome analysis of gene expression profiling of infected macrophages between Brucella suis 1330 and live attenuated vaccine strain S2 displays mechanistic implication for regulation of virulence.

Brucellosis is one of the most common zoonotic epidemics worldwide. Vaccination against brucellosis is an important control strategy to prevent the disease in many high-prevalence regions. At present, Brucella vaccine strain S2 is the most widely used vaccine in China. To uncover the mechanisms underlying virulence attenuation of S2, in this study we characterized the transcriptional profile of S2 and 1330 infected macrophages by transcriptome analysis. The results revealed that expressions of 440 genes were significantly different between macrophages infected by 1330 and S2. Data analysis showed that in the gene ontology term, the different expressed genes involved in innate immune response, phagoctyosis, recognition, and inflammatory response were significantly enriched. Pathway enrichment analysis indicated that the genes involved in transcriptional misregulation in cancer, staphylococcus aureus infection pathways and NF-kappa B signaling pathway were significantly affected. To reveal the molecular mechanisms related to different expression profiles of infected macrophages, the transcription levels of the different genes between the two bacterial genomes were detected. In total, the transcription of 29 different genes was significantly changed in either culture medium or infected microphages. The results of this study can be conducive to the promotion of better understanding of the related mechanisms underlying virulence attenuation of S2 and interactions between host cells and Brucella strains.

A Phage-Like IncY Plasmid Carrying the mcr-1 Gene in Escherichia coli from a Pig Farm in China.

We report here a new type of plasmid that carries the mcr-1 gene, the pMCR-1-P3 plasmid, harbored in an Escherichia coli strain isolated from a pig farm in China. pMCR-1-P3 belongs to the IncY incompatibility group and is a phage-like plasmid that contains a large portion of phage-related sequences. The backbone of this plasmid is different from that of other mcr-1-carrying plasmids reported previously.

Rough brucella strain RM57 is attenuated and confers protection against Brucella melitensis.

Brucella melitensis (B. melitensis) is a facultative intracellular pathogen, which is the main epidemic strain in China. To overcome disadvantages of traditional live attenuated vaccines, in this study a rough mutant RM57 was induced from a B. melitensis isolate M1981. In order to uncover the reason of changes in the LPS of RM57, the nucleotide sequences and transcription levels of all known genes related to LPS synthesis were detected. As LPS plays an important role in outer membrane integrity, the sensitivities of RM57 to SDS and polymyxin B were examined. The results showed that the expression level of LPS genes of RM57 was not significantly changed, and RM57 was sensitive to polymyxin B, compared to its parent strain. In further study, the virulence and protective efficacy of RM57 in mice and guinea pigs were determined, and our data indicated that RM57 was attenuated and had good protection effects, especially in guinea pigs model. Overall, these results demonstrated that the artificially induced rough mutant strain RM57 was an efficacious vaccine candidate against the challenge of virulent B. melitensis. Our data presented here provided additional insight into the mechanism of LPS synthesis of Brucella spp.

[Immune response of body after Mycobacterium avium subsp. paratuberculosis infection and advances in detection methods - A review].

The progressive form of clinical Mycobacterium avium subsp. paratuberculosis (MAP) is characterized by production losses, weight loss, chronic intractable diarrhea, and severe emaciation leading to death in cattle. Substantial economic losses to the animal husbandry are a result of infection. Cattles are usually infected in their youth through the oral route and will experience a long subclinical stage. At the early stage of infection, cellular immunity is the main immune response with bacterium excretion increased significantly after a subclinical period of 2 to 5 years. The majority of methods currently used to detect MAP are based on etiological detection, cellular and humoral immune response. Owing to the different mechanism of diagnostic methods varies a lot at a particular infection period. In this review, we illustrate the transmission route and the characteristic of immune responses of MAP, and also summarize the diagnostic methods of MAP.

Influence of avian leukosis virus long terminal repeat on biological activities of Marek's disease virus.

GX0101 was the first reported field strain of recombinant Marek's disease virus (MDV) that contained a long terminal repeat (LTR) from the reticuloendotheliosis virus (REV). It is a very virulent MDV strain, with relatively high horizontal transmission ability. The REV LTR in GX0101 genome was proved to decrease the pathogenicity but increase the potential for horizontal transmission of the virus. Here we constructed a recombinant MDV GX0101-ALV-LTR to study stability of avian leukosis virus (ALV) LTR at the REV LTR insertion site in GX0101 genome and its influence on biological activities of the recombinant virus. The results showed that GX0101-ALV-LTR was able to replicate stably both in vitro and in vivo. ALV LTR remained stable in chickens infected either by inoculation with the recombinant virus GX0101-ALV-LTR or by horizontal transmission, as well as in cell culture. The pathogenic properties of GX0101-ALV-LTR virus were evaluated in infected specific-pathogen-free chickens. The present study demonstrated that the GX0101-ALV-LTR virus had a weaker inhibitory effect on the growth rates of the infected chickens and induced weaker immunosuppressive effects. Horizontal transmission ability of the GX0101-ALV-LTR virus appeared to be similar with its parental virus GX0101. In short, ALV LTR was stable in GX0101 after replacing REV LTR, and the recombinant virus showed similar horizontal transmission ability but decreased pathogenicity.

Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein.

BACKGROUND: Mycoplasma synoviae is an avian pathogen that can lead to respiratory tract infections and arthritis in chickens and turkeys, resulting in serious economic losses to the poultry industry. Enolase reportedly plays important roles in several bacterial pathogens, but its role in M. synoviae has not been established. Therefore, in this study, the enolase encoding gene (eno) of M. synoviae was amplified from strain WVU1853 and expressed in E. coli BL21 cells. Then the enzymatic activity, immunogenicity and binding activity with chicken plasminogen (Plg) and human fibronectin (Fn) was evaluated. RESULTS: We demonstrated that the recombinant M. synoviae enolase protein (rMsEno) can catalyze the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP), the Km and Vmax values of rMsEno were 1.1 × 10(-3) M and 0.739 µmol/L/min, respectively. Western blot and immuno-electron microscopy analyses confirmed that enolase was distributed on the surface and within the cytoplasm of M. synoviae cells. The binding assays demonstrated that rMsEno was able to bind to chicken Plg and human Fn proteins. A complement-dependent mycoplasmacidal assay demonstrated that rabbit anti-rMsEno serum had distinct mycoplasmacidal efficacy in the presence of complement, which also confirmed that enolase was distributed on the surface of M. synoviae. An inhibition assay showed that the adherence of M. synoviae to DF-1 cells pre-treated with Plg could be effectively inhibited by treatment with rabbit anti-rMsEno serum. CONCLUSION: These results reveal that M. synoviae enolase has good catalytic activity for conversion of 2-PGA to PEP, and binding activity with chicken Plg and human Fn. Rabbit anti-rMsEno serum displayed an obvious complement-dependent mycoplasmacidal effect and adherent inhibition effect. These results suggested that the M. synoviae enolase plays an important role in M. synoviae metabolism, and could potentially impact M. synoviae infection and immunity.

Recombinant production of SAG1 fused with xylanase in Pichia pastoris induced higher protective immunity against Eimeria tenella infection in chicken.

Chicken coccidiosis is an intestinal disease caused by the parasite Eimeria, which severely damages the growth of chickens and causes significant economic losses in the poultry industry. Improvement of the immune protective effect of antigens to develop high efficiency subunit vaccines is one of the hotspots in coccidiosis research. Sporozoite-specific surface antigen 1 (SAG1) of Eimeria tenella (E. tenella) is a well-known protective antigen and is one of the main target antigens for the development of subunit, DNA and vector vaccines. However, the production and immunoprotective effects of SAG1 need to be further improved. Here, we report that both SAG1 from E. tenella and its fusion protein with the xylanase XynCDBFV-SAG1 are recombinant expressed and produced in Pichia pastoris (P. pastoris). The substantial expression quantity of fusion protein XynCDBFV-SAG1 is achieved through fermentation in a 15-L bioreactor, reaching up to about 2 g/L. Moreover, chickens immunized with the fusion protein induced higher protective immunity as evidenced by a significant reduction in the shedding of oocysts after E. tenella challenge infection compared with immunized with recombinant SAG1. Our results indicate that the xylanase enhances the immunogenicity of subunit antigens and has the potential for developing novel molecular adjuvants. The high expression level of fusion protein XynCDBFV-SAG1 in P. pastoris holds promise for the development of effective recombinant anti-coccidial subunit vaccine.

Complete genome sequence of a recombinant Marek's disease virus field strain with one reticuloendotheliosis virus long terminal repeat insert.

Marek's disease virus (MDV) Chinese strain GX0101, isolated in 2001 from a vaccinated flock of layer chickens with severe tumors, was the first reported recombinant MDV field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. GX0101 belongs to very virulent MDV (vvMDV) but has higher horizontal transmission ability than the vvMDV strain Md5. The complete genome sequence of GX0101 is 178,101 nucleotides (nt) and contains only one REV-LTR insert at a site 267 nt upstream of the sorf2 gene. Moreover, GX0101 has 5 repeats of a 217-nt fragment in its terminal repeat short (TRS) region and 3 repeats in internal repeat short (IRS) region, compared to the other 10 strains with only 1 or 2 repeats in both TRS and IRS.

Sequence analysis of the whole genome of a recombinant Marek's disease virus strain, GX0101, with a reticuloendotheliosis virus LTR insert.

Marek's disease virus Chinese strain GX0101, isolated in 2001, is the first reported recombinant gallid herpesvirus type 2 (GaHV-2) field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. We constructed an infectious bacterial artificial chromosome (BAC) clone of GX0101, which showed characteristics very similar to those of the parental virus in replication and pathogenicity. Using the GX0101 BAC clone, the complete genome of GX0101 was sequenced and analyzed. The length of the GX0101 genome is 178,101 bp, and it contains only one REV-LTR insert at a site 267 bp upstream of the sorf2 gene.

[Preparation and identification of polyclonal antibody to serotype I Mareks disease virus sorf2 protein].

OBJECTIVE: To obtain mice and rabbit polyclonal antibody of serotype I Marek's disease virus (MDV) sorf2 protein with higher titer and to identify the specificity. METHODS: Using serotype I MDV GX0101 as template, we amplified sorf2 gene and then cloned it into pET-28a (+) and pET-32a (+) respectively. The recombinant plasmid pET-28a-sorf2 and pET-32a-sorf2 was separately transformed into Escherichia coli BL21 (Rosetta) competent cell which was induced with isopropylthio-beta-D-galactoside (IPTG). After purification, immuned 6-8 Balb/c mice and adult New Zealand white rabbit with the purified fusion protein and the anti-sorf2 polyclonal antibody were prepared. The specificity of the serum was detected by Western blot and the indirect immunofluorescence assay (IFA) method. RESULTS: Serotype I MDV sorf2 protein was expressed successfully in the recombinant E coli. Mice and rabbit anti-sorf2 polyclonal antibody with higher titer could react with sorf2 protein specifically. CONCLUSION: The prepared anti-sorf2 polyclonal antibody could identify MDV sorf2 gene deletion strain specifically. In addition, it could be used for differential of MDV vaccine poison HVT and serotype I MDV, which was useful for the separation and identification of clinical MDV.

Impact of MgtC on the Fitness of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis is an extracellular foodborne pathogen and usually causes self-limiting diarrhea in healthy humans. MgtC is known as a key subversion factor that contributes to intramacrophage adaptation and intracellular survival in certain important pathogens. Whether MgtC influences the fitness of Y. pseudotuberculosis is unclear. According to in silico analysis, MgtC in Y. pseudotuberculosis might share similar functions with other bacterial pathogens, such as Salmonella. Studies indicated that MgtC was clearly required for Y. pseudotuberculosis growth in vitro and bacterial survival in macrophages under Mg2+ starvation. Transcriptome analysis by RNA-seq indicated that 127 differentially expressed genes (DEGs) (fold change > 2 and p < 0.001) were discovered between wild-type PB1+ and mgtC mutant inside macrophages. However, a lack of MgtC only moderately, albeit significantly, reduced the virulence of Y. pseudotuberculosis in mice. Overall, this study provides additional insights for the role of MgtC in Y. pseudotuberculosis.

Molecular Characteristics of Bovine Viral Diarrhea Virus Strains Isolated from Persistently Infected Cattle.

In this study, we reported the isolation, identification, and molecular characteristics of nine BVDV strains that were isolated from the serum of persistently infected cattle. The new strains were designated as BVDV TJ2101, TJ2102, TJ2103, TJ2104, TJ2105, TJ2106, TJ2107, TJ2108 and TJ2109. The TJ2102 and TJ2104 strains were found to be cytopathic BVDV, and the other strains were non-cytopathic BVDV. An alignment and phylogenetic analysis showed that the new isolates share 92.2-96.3% homology with the CP7 strain and, thus, were classified as the BVDV-1b subgenotype. A recombination analysis of the genome sequences showed that the new strains could be recombined by the major parent BVDV-1a NADL strain and the minor parent BVDV-1m SD-15 strain. Some genome variations or unique amino acid mutations were found in 5'-UTR, E0 and E2 of these new isolates. In addition, a potential linear B cell epitopes prediction showed that the potential linear B cell epitope at positions 56-61 is highly variable in BVDV-1b. In conclusion, the present study has identified nine strains of BVDV from persistently infected cattle in China. Further studies on the virulence and pathogenesis of these new strains are recommended.