MiR-339-5p inhibits replication of porcine reproductive and respiratory syndrome virus by targeting viral gene regions.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a variable virus, whose spread cannot be totally stopped by vaccination. PRRSV infection results in abortion and respiratory symptoms in pregnant pigs. One crucial component of the anti-viral infection strategy is microRNA (miRNA), a class of multifunctional small molecules. It is unknown whether miR-339-5p can specifically target the PRRSV gene and prevent the virus from replicating, despite the fact that miR-339-5p is markedly up-regulated during the PRRSV infection. In this pursuit, the present study revealed that the two PRRSV areas targeted by miR-339-5p were PRRSV nsp2-3378 to 3403 and PRRSV nsp2-3112 to 3133 using the miRanda program. Dual luciferase reporter assays showed that the miR-339-5p target region of the PRRSV gene sequence exhibited 100% homology and was highly conserved. Furthermore, the ability of miR-339-5p to target PRRSV gene areas was verified. It was found that the overexpression of miR-339-5p markedly reduced the PRRSV replication through PRRSV infection trials. The precursor sequence of ssc-miR-339-5p was amplified using the DNA of pig lung tissue as a template in order to create a fragment of 402 bp of porcine-derived miR-339-5p precursor sequence, which was then used to produce the eukaryotic expression plasmid of miR-339-5p. In conclusion, miR-339-5p can target the specific PRRSV gene areas and prevent PRRSV replication, offering fresh perspectives for the creation of medications that combat the PRRSV infection.

A multi-epitope subunit vaccine based on CU/ZN-SOD, OMP31 and BP26 against Brucella melitensis infection in BALB/C mice.

Brucellosis, a zoonosis caused by Brucella, is highly detrimental to both humans and animals. Most existing vaccines are live attenuated vaccines with safety flaws for people and animals. Therefore, it is advantageous to design a multi-epitope subunit vaccine (MEV) to prevent Brucella infection. To this end, we applied a reverse vaccinology approach. Six cytotoxic T cell (CTL) epitopes, seven T helper cell (HTL) epitopes, and four linear B cell epitopes from CU/ZN-SOD, Omp31, and BP26 were obtained. We linked the CTL, HTL, B-cell epitopes, the appropriate CTB molecular adjuvant, and the universal T helper lymphocyte epitope, PADRE, with linkers AAY, GPPGG, and KK, respectively. This yielded a 412-amino acid MEV construct, which we named MEVcob. The immunogenicity, stability, safety, and feasibility of the construct were evaluated by bioinformatics tools (including the AlphaFold2 prediction tool, the AlphaFold2 tool, NetMHC-I pan 4.0 server, IEDB MHC-I server, ABCpred service, and C-ImmSim server); the physicochemical properties, secondary and tertiary structures, and binding ability of MEVocb to toll-like receptor 4 (TLR4) was analyzed. Then, codon adaptation and computer cloning studies were performed. MEVocb is highly immunogenic in immunostimulation experiments, The proteins translated by these sequences were relatively stable, exhibiting a high antigenic index. Furthermore, mouse experiments confirmed that the MEVocb construct could raise IFN-γ, IgG, IgG2a, IgG1, IL-2, TNF-α levels in mice, indicating that induced a specific humoral and cellular immune response in BALB/c mice. This vaccine induced a statistically significant level of protection in BALB/c mice when challenged with Brucella melitensis 043 in Xinjiang. Briefly, we utilized immunoinformatic tools to design a novel multi-epitope subunit candidate vaccine against Brucella. This vaccine aims to induce host immune responses and confer specific protective effects. The study results offer a theoretical foundation for the development of a novel Brucella subunit vaccine.

Design of a multi-epitope vaccine against brucellosis fused to IgG-fc by an immunoinformatics approach.

Introduction: Brucella, a type of intracellular Gram-negative bacterium, has unique features and acts as a zoonotic pathogen. It can lead to abortion and infertility in animals. Eliminating brucellosis becomes very challenging once it spreads among both humans and animals, putting a heavy burden on livestock and people worldwide. Given the increasing spread of brucellosis, it is crucial to develop improved vaccines for susceptible animals to reduce the disease's impact. Methods: In this study, we effectively used an immunoinformatics approach with advanced computer software to carefully identify and analyze important antigenic parts of Brucella abortus. Subsequently, we skillfully designed chimeric peptides to enhance the vaccine's strength and effectiveness. We used computer programs to find four important parts of the Brucella bacteria that our immune system recognizes. Then, we carefully looked for eight parts that are recognized by a type of white blood cell called cytotoxic T cells, six parts recognized by T helper cells, and four parts recognized by B cells. We connected these parts together using a special link, creating a strong new vaccine. To make the vaccine even better, we added some extra parts called molecular adjuvants. These included something called human ß-defensins 3 (hBD-3) that we found in a database, and another part that helps the immune system called PADRE. We attached these extra parts to the beginning of the vaccine. In a new and clever way, we made the vaccine even stronger by attaching a part from a mouse's immune system to the end of it. This created a new kind of vaccine called MEV-Fc. We used advanced computer methods to study how well the MEV-Fc vaccine interacts with certain receptors in the body (TLR-2 and TLR-4). Results: In the end, Immunosimulation predictions showed that the MEV-Fc vaccine can make the immune system respond strongly, both in terms of cells and antibodies. Discussion: In summary, our results provide novel insights for the development of Brucella vaccines. Although further laboratory experiments are required to assess its protective effect.

Detection of emerging HoBi-like Pestivirus (BVD-3) during an epidemiological investigation of bovine viral diarrhea virus in Xinjiang: a first-of-its-kind report.

Xinjiang pastoral area is the second largest pastoral area in China, accounting for 26.8% of the available grassland area in the country, and the geographical advantage of cattle breeding industry is very obvious. Bovine viral diarrhea virus (BVDV) has always been one of the important viral diseases that have plagued the development of cattle farming industry in the world. As one of the main pastoral areas of China's cattle farming industry, the Xinjiang pastoral area has also been deeply affected. In this study, 6,153 bovine serum samples were collected from 18 large-scale cattle farms in 13 cities in Xinjiang. The antibodies and antigens of 6,153 and 588 serum samples were detected by serological detection methods, respectively. Ten serum samples, which were antigen-positive by ELISA, were randomly selected for RT-PCR detection, sequencing, and phylogenetic analysis of suspected HoBi-like Pestivirus (HoBiPeV) strains. The results showed that the positive rates of BVDV antibodies and antigens were 53.68% (3,303/6,153) and 6.12% (36/588), respectively. One of the 10 randomly selected seropositive samples was infected with the HoBiPeV strain. HoBiPeV, also referred to as BVDV-3, is an emerging atypical Pestivirus that occurs in cattle and small ruminants, and its clinical signs are similar to those of BVDV infection. Based on the whole genome of the BVDV-3 reference strain (JS12/01) on the GenBank, the homology of the detected strain was 96.02%. The whole genome nucleotide sequence was submitted to the GenBank database, and the gene accession number was obtained: OP210314. The whole genome of isolate OP210314 was 12.239 nucleotides and contained a 5'-UTR of 340 nucleotides, a 3'-UTR of 199 nucleotides, and a large open reading frame (ORF) encoding a polyprotein consisting of 3,899 amino acids. In conclusion, the prevalence rate of BVDV infection in Xinjiang dairy cows is high, and the genetic diversity is increasing. This study successfully identified and isolated HoBiPeV in Xinjiang for the first time, posing a potential threat to the cattle industry in Xinjiang.

Detection and Characterization of a Reassortant Mammalian Orthoreovirus Isolated from Bats in Xinjiang, China.

Mammalian orthoreoviruses (MRVs) are increasingly reported to cause various diseases in humans and other animals, with many possibly originating from bats, highlighting the urgent need to investigate the diversity of bat-borne MRVs (BtMRVs). Here, we report the detection and characterization of a reassortant MRV that was isolated from a bat colony in Xinjiang, China. The BtMRV showed a wide host and organ tropism and can efficiently propagate the cell lines of different animals. It caused mild damage in the lungs of the experimentally inoculated suckling mice and was able to replicate in multiple organs for up to three weeks post-inoculation. Complete genome analyses showed that the virus was closely related to MRVs in a wide range of animals. An intricate reassortment network was revealed between the BtMRV and MRVs of human, deer, cattle, civet and other bat species. Specifically, we found a bat-specific clade of segment M1 that provides a gene source for the reassortment of human MRVs. These data provide important insights to understand the diversity of MRVs and their natural circulation between bats, humans, and other animals. Further investigation and surveillance of MRV in bats and other animals are needed to control and prevent potential MRV-related diseases.

Using a Relative Quantitative Proteomic Method to Identify Differentially Abundant Proteins in Brucella melitensis Biovar 3 and Brucella melitensis M5-90.

Brucellosis, caused by Brucella spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, B. melitensis biovar 3 and compare it with those expressed by B. melitensis M5-90. The higher level of virulence for B. melitensis biovar 3 compared to B. melitensis M5-90 was validated in vitro and in vivo. A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the B. melitensis proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to B. melitensis M5-90 and B. melitensis biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in B. melitensis biovar 3 was 8-fold and 2-fold higher than in B. melitensis M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of B. melitensis M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified Brucella virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.

Abortion and various associated risk factors in dairy cow and sheep in Ili, China.

We studied livestock abortion and various associated risk factors in the Ili region of northwest China. Livestock abortion prevalence was estimated and correlated with infections (Brucellosis, Salmonellosis, Mycoplasma and Chlamydia seropositivity) and management (farming type and contact with other herds/flocks) risk factors. A total of 2996 serum samples (1406 cow, 1590 sheep) were identified by RBPT (Rose Bengal Plate Test) and c-ELISA (competitive-enzyme linked immunosorbent assay), and they showed the overall seroprevalence of brucellosis in the study area was cow 6.76%, sheep 9.50%. The seroprevalence of brucellosis in X county was cow 7.06%, sheep 9.12%; in H county was cow 11.70%, sheep 10.80%; and in Q county was cow 4.22%, sheep 9.11%. The overall seroprevalence of Mycoplasma in the study area was cow 3.20%, sheep 6.42%. The seroprevalence of Mycoplasma in X county was cow 3.39%, sheep 7.98%; in H county was cow 5.26%, sheep 9.97%; and in Q county was cow 2.11%, sheep 4.33%. The Odds ratio of brucellosis for cow and sheep, respectively, were 45.909 [95% CI 26.912-78.317, P<0.001] and 70.507 [95% CI 43.783-113.544, P<0.001] times higher than other abortion-related factors including mixed farming, contact with other flocks and Mycoplasma infection. A total of 54 samples, including aborted cow (22), sheep (30) fetuses and milk samples (2), were identified as Brucella melitensis (B. melitensis) positive. A total of 38 Brucella were isolated from 16 aborted cow, 20 sheep fetuses and 2 milk samples. All of these isolates were identified, and confirmed, as B. melitensis. A phylogenetic tree showed that the Brucella isolates closely matched the B. melitensis biovar 3 isolated in Inner Mongolia, China, and B. melitensis isolated from Norway and India. These results suggest that B. melitensis biovar 3 is the main pathogen responsible for cow and sheep abortion and also pose a human health risk. Additionally, livestock reproduction can also be influenced by Mycoplasma infection and managerial factors (farming type and contact with other herds/flocks), especially in remote areas.

A Novel Nanobody Directed against Ovine Myostatin to Enhance Muscle Growth in Mouse.

Myostatin (MSTN) is a member of the transforming growth factor beta superfamily and is a negative regulator of myogenesis. It has been shown to function by controlling the proliferation of myoblasts. MSTN inhibition is considered as a promising treatment for promoting animal growth in livestock. Nanobodies, a special antibody discovered in camel, have arisen as an alternative to conventional antibodies and have shown great potential when used as tools in different biotechnology fields, such as diagnostics and therapy. In this study, we examined the effect of MSTN inhibition by RMN on the muscle growth of mice. The results showed that RMN could specifically detect and bind MSTN, as well as inhibit MSTN activity. A significant increase in skeletal muscle mass was observed after intramuscular injection of RMN into mice. Enhanced muscle growth occurred because of myofiber hypertrophy. These results offer a promising approach to enhance muscle growth that warrants further investigation in domestic animals.

Extensive Genetic Diversity of Polyomaviruses in Sympatric Bat Communities: Host Switching versus Coevolution.

Polyomaviruses (PyVs) are small DNA viruses carried by diverse vertebrates. The evolutionary relationships of viruses and hosts remain largely unclear due to very limited surveillance in sympatric communities. In order to investigate whether PyVs can transmit among different mammalian species and to identify host-switching events in the field, we conducted a systematic study of a large collection of bats (n = 1,083) from 29 sympatric communities across China which contained multiple species with frequent contact. PyVs were detected in 21 bat communities, with 192 PyVs identified in 186 bats from 15 species within 6 families representing at least 28 newly described PyVs. Surveillance results and phylogenetic analyses surprisingly revealed three interfamily PyV host-switching events in these sympatric bat communities: two distinct PyVs were identified in two bat species in restricted geographical locations, while another PyV clustered phylogenetically with PyVs carried by bats from a different host family. Virus-host relationships of all discovered PyVs were also evaluated, and no additional host-switching events were found. PyVs were identified in different horseshoe bat species in sympatric communities without observation of host-switching events, showed high genomic identities, and clustered with each other. This suggested that even for PyVs with high genomic identities in closely related host species, the potential for host switching is low. In summary, our findings revealed that PyV host switching in sympatric bat communities can occur but is limited and that host switching of bat-borne PyVs is relatively rare on the predominantly evolutionary background of codivergence with their hosts.IMPORTANCE Since the discovery of murine polyomavirus in the 1950s, polyomaviruses (PyVs) have been considered highly host restricted in mammals. Sympatric bat communities commonly contain several different bat species in an ecological niche facilitating viral transmission, and they therefore represent a model to identify host-switching events of PyVs. In this study, we screened PyVs in a large number of bats in sympatric communities from diverse habitats across China. We provide evidence that cross-species bat-borne PyV transmission exists, though is limited, and that host-switching events appear relatively rare during the evolutionary history of these viruses. PyVs with close genomic identities were also identified in different bat species without host-switching events. Based on these findings, we propose an evolutionary scheme for bat-borne PyVs in which limited host-switching events occur on the background of codivergence and lineage duplication, generating the viral genetic diversity in bats.

Flea surveillance on wild mammals in northern region of Xinjiang, northwestern China.

Flea distribution in northern region of Xinjiang Uygur Autonomous Region (XUAR) and fluctuations of the annual fleas index in Alataw Pass were investigated. During a 4-year (2015-2018) study, 5789 fleas were collected directly from 15 mammals at eight counties in northern XUAR of northwestern China. Nineteen flea species, belonging to sixteen genera and seven families, were further confirmed by four genetic markers (18S rDNA, 28S rDNA, COI and COII) after morphological observation. Pulex irritans and Paraceras crispum parasitizing Asian badgers (Meles leucurus) were recorded for the first time. In addition, the fluctuations of the annual fleas index in Alataw Pass were surveyed. Xenopsylla gerbilli minax, Xenopsylla conformis conformis and Nosopsyllus laeviceps laeviceps were highly detected in the warm season while Paradoxopsyllus repandus, Ctenophthalmus dolichus dolichus and Coptopsylla lamellifer ardua were only found in the cold season. These findings extend our knowledge of flea species, distribution and annual fluctuations especially in China-Kazakhstan border.

High-frequency mutation and recombination are responsible for the emergence of novel porcine reproductive and respiratory syndrome virus in northwest China.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most highly infectious diseases in the pig industry, resulting in enormous economic losses worldwide. In this study, a PRRS virus (PRRSV) strain was isolated from primary porcine alveolar macrophage cells in Xinjiang in northwest China. This new strain was sequenced and designated as XJzx1-2015, and its sequence was then compared to those of other representative PRRSV strains from around the world. Complete genomic characterisation showed that the full-length nucleotide sequence of XJzx1-2015 exhibited low-level similarity to NB/04 (91.6%), JXA1 (90.5%), CH-1a (90.2%), VR-2332 (86.9%), QYYZ (85.7%), and JL580 (82.2%), with the highest similarity to HK13 (91.7%) sequence identity. Nonstructural protein 2 (NSP2) and glycosylated protein (GP) 2 of XJzx1-2015 had deletions of five and two amino acids, respectively, corresponding to strain VR-2332 positions 475-479 and 173-174. Phylogenetic analysis based on complete genome sequences showed that XJzx1-2015 and four other strains from China formed a new subgenotype closely related to other sublineage 8.7 (JXA1-like) strains belonging to the North American genotype. However, phylogenetic analysis based on NSP2 and GP5 showed that XJzx1-2015 clustered with sublineage 8.7 (JXA1-like, CH-1a-like) and lineage 3 (QYYZ-like) strains, respectively. Recombination analysis indicated that XJzx1-2015 is an intersubgenotype recombinant of CH-1a-like and QYYZ-like strains. Overall, our findings demonstrate that XJzx1-2015 is a novel PRRSV strain with a significantly high frequency of mutation and a recombinant between lineage 3 and sublineage 8.7 identified in northwest China. These results provide important insights into PRRSV evolution.

Tick distribution in border regions of Northwestern China.

Ticks are important vectors of emerging and re-emerging pathogens. The aim of this study was to determine tick species occurring in Xinjiang Uygur Autonomous Region (XUAR), especially on border regions. A total of 22,994 ticks (including 22,629 adults, 365 larvae and nymphs), belonging to six tick genera (i.e. Dermacentor, Hyalomma, Rhipicephalus, Haemaphysalis, Ixodes and Argas) and fourteen tick species, were collected from ten animal hosts in thirty-five counties (cities) in XUAR during 2011 - 2017. Rhipicephalus turanicus, Dermacentor niveus, Hyalomma asiaticum and Dermacentor marginatus were dominantly sampled from domestic animals while Dermacentor nuttalli, Haemaphysalis punctata, Haemaphysalis concinna, Rhipicephalus sanguineus sensu lato, Dermacentor silvarum, Hyalomma scupense and Argas persicus were sporadically found. Based on 16S rDNA, phylogenetic analyses showed that: i) R. turanicus genotypes in XUAR showed geographical separation, and belonged to clade I (major distribution in the Central Asian) rather than clade II (major distribution in the Mediterranean Basin); ii) Ixodes kaiseri, firstly sampled from Asian badgers (Meles leucurus), was in ancestral position compared to European tick species when combining COI haplotypes; and iii) Haemaphysalis erinacei from marbled polecats in China was a separate genotype compared with that in Mediterranean and Europe. Our findings suggest that geographical range plays a more important role than host-association in tick phylogeny, especially for R. turanicus, I. kaiseri and H. erinacei.

Advances and Application of DNA-functionalized Nanoparticles.

DNA-functionalized nanoparticle (DfNP) technology, the integration of DNA with nanotechnology, has emerged over recent decades as a promising biofunctionalization tool in the light of biotechnological approaches. The development of DfNPs has exhibited significant potential for several biological and biomedical applications. In this review, we focus on the mechanism of a series of DNA-NP nanocomposites and highlight the superstructures of DNA-based NPs. We also summarize the applications of these nanocomposites in cell imaging, cancer therapy and bioanalytical detection.

Occurrence and Multilocus Genotyping of Giardia duodenalis in Yunnan Black Goats in China.

Giardia duodenalis is an important zoonotic parasite which can parasitize in the intestines of humans and various animals. However, the information about the prevalence and genetic diversity of G. duodenalis in goats in China is limited. It is yet to be known whether Yunnan black goats, a unique goat breed in subtropical Yunnan province, southwestern China, are infected with G. duodenalis. Thus, a total of 907 fecal samples were collected from Yunnan black goats in five regions in Yunnan province, to estimate the prevalence and genotypes of G. duodenalis using a PCR-based approach. The G. duodenalis prevalence is 4.2% (38/907) in Yunnan black goats by nested amplification of the ß-giardin (bg) gene, and the genotypes are identified as assemblage E, with 5 novel subtypes (E11-E15). Multilocus sequence typing revealed that 11, 18, and 38 samples were amplifiable on tpi (triose phosphate isomerase), gdh (glutamate dehydrogenase), and bg locus, respectively, and identified three novel multilocus genotypes (MLGs): MLGE9-MLGE11. To our knowledge, this is the first report of G. duodenalis prevalence and genotypes in Yunnan black goats in China, which extended the host range of G. duodenalis and provided basic data for controlling G. duodenalis infection in Yunnan black goats.

Phages bearing specific peptides with affinity for porcine reproductive and respiratory syndrome virus GP4 protein prevent cell penetration of the virus.

Porcine reproductive and respiratory syndrome (PRRS) has caused significant economic losses to the pig industry worldwide over the last 30 years. GP4 is a minor highly glycosylated structural protein composed of 187 and 183 amino acids in types I and II porcine reproductive and respiratory syndrome virus (PRRSV), respectively. The GP4 protein co-localizes with cluster of differentiation 163 (CD163), the major receptor on the target cell membrane, to mediate PRRSV internalization and disassembly. However, it remains to be established whether blocking interactions between GP4 and host cells can inhibit viral proliferation. In the present study, recombinant GP4 protein prepared and purified using the Escherichia coli system effectively recognized PRRSV-positive serum. Phage display biopanning on GP4 protein showed that the specific phages obtained could distinguish PRRSV from the other viruses. The exogenous peptide WHEYPLVWLSGY displayed on one of the candidate phages showed high affinity for GP4 protein and exerted a significant inhibitory effect on PRRSV penetration in vitro. Moreover, the N-terminus of GP4 was predicted as the critical receptor binding site and the beginning of the fifth scavenger receptor cysteine-rich domain of CD163 as the critical ligand recognition site based on sequence alignment and model prediction analyses. The current study expands our understanding of PRRSV GP4 and its receptor CD163 and provides a fresh perspective for the development of novel peptide-based viral inhibition reagents.

The detection of brucellosis antibody in whole serum based on the low-fouling electrochemical immunosensor fabricated with magnetic Fe3O4@Au@PEG@HA nanoparticles.

It is a novel competitive challenge for electrochemical biosensor to directly, rapidly and ultrasensitively detect the disease markers in the whole serum due to biofouling caused by the complexity of actual samples. In this paper, poly (ethylene glycol) (PEG) and hyaluronic acid (HA) were utilized to modify Fe3O4 @Au nanoparticles (NPs). Based on the successfully preparation and characterization of Fe3O4 @Au@PEG@HA NPs with TEM, SEM, XRD, FTIR and EDS, respectively, a novel immunosensor of brucellosis with high selectivity, sensitivity and almost perfect protein-resistant properties in various external environments, especially, in complex biological systems was fabricated. More importantly, this immunosensor is capable of assaying brucellosis antibody in 100% serum without suffering from any significant biological interference. In addition, a wide linear response range from 10-15 g mL-1 to 10-11 g mL-1 towards antibody in 100% serum and a low limit of detection (LOD) of 0.36 fg mL-1 (3σ, n = 13) are demonstrated, which indicates that this immunosensor has a promising potential in clinical diagnosis.

Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein.

Bovine viral diarrhea-mucosal disease (BVD-MD) is caused by bovine viral diarrhea virus (BVDV), and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3), and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.

Loop-mediated isothermal amplification assay targeting the mpb70 gene for rapid differential detection of Mycobacterium bovis.

Loop-mediated isothermal amplification (LAMP) is a highly sensitive, rapid, cost-effective nucleic acid amplification method. Tuberculosis (TB) is widely popular in the world and it is difficult to cure. The fundamental treatment is to clear the types of TB pathogens such as Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M. tuberculosis). In order to detect and diagnose TB early, we constructed the differential diagnostic method of TB. In this study, we used LAMP for detection of M. bovis, based on amplification of the mpb70 gene which is a unique gene in M. bovis strain. The LAMP assay was able to detect only seven copies of the gene per reaction, whereas for the conventional PCR, it was 70 copies. The LAMP was evaluated for its specificity using six strains of five Mycobacterium species and 18 related non-Mycobacterium microorganism strains as controls. The target three Mycobacterium strains were all amplified, and no cross-reaction was found with 18 non-Mycobacterium microorganism strains. TB was detected by two methods, LAMP and conventional PCR (based on mpb70 gene); the positive rates of the two methods were 9.55 and 7.01 %, respectively. Our results indicate that the LAMP method should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of TB caused by M. bovis. Most importance is that the use of LAMP as diagnostic method in association with diagnostic tests based on mpb70 gene would allow the differentiation between M. bovis and other Mycobacterium in humans or animals. The LAMP method is actually in order to detect human TB, and it can be used for differential diagnosis in this paper.

Seoul virus in the Brown Rat ( Rattus norvegicus ) from Ürümqi, Xinjiang, Northwest of China.

Hantavirus infections among human populations are linked to the geographic distribution of the host rodents that carry the viruses. To determine the presence and distribution of hantaviruses in the northern region of Xinjiang Uygur Autonomous Region (XUAR), northwestern China, 844 rodents were captured from five locations in four dissimilar habitats during 2010-14 and examined for Hantavirus infection. Hantavirus nucleic acids were firstly detected in the brown rat ( Rattus norvegicus ) from Ürümqi, China, indicating that the Hantavirus was transmitted into Ürümqi in XUAR and circulated by the brown rat. Our results suggest that the brown rat may act as a natural reservoir for the virus in XUAR.