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To exploit a bioactive material as components of functional products, juices from purple perilla; purple potato and blueberry were abstracted and concentrated. These concentrated juices were diluted to optimize the composition by evalustion of lavor. A bioactive material with 30% concentrated juice from purple perilla,50% from blueberry and 20% from purple potato, which contains 3.72% anthocyanin and shows antioxidant activity. This biomaterial can be use in processing functional products which have the functions of plant anthocyanin.

Little is known about the virulence and clinical impact on humans from infection with Anaeroglobus geminates, an anaerobic gram-negative coccus belonging to the family Veillonellaceae. We report the first case of an Anaeroglobus geminates invasive infection in humans characterized by pneumonia complicated with empyema. The pathogen was initially identified as Veillonella spp. by an automatic identification system (Becton-Dickinson and Company, Franklin Lakes, NJ, USA) and definitively identified following 16S ribosomal RNA gene sequence analysis. The patient was cured by surgical decortication and antimicrobial therapy. In this case, the combination of effective antibiotics, surgical intervention, and adequate drainage successfully cured the patient.

Detection of cytosolic nucleic acids by pattern recognition receptors leads to the induction of type I interferons (IFNs) and elicits the innate immune response. We report here the identification of RIOK3 as a novel adaptor protein that is essential for the cytosolic nucleic acid-induced type I IFN production and for the antiviral response to gammaherpesvirus through two independent kinome-wide RNA interference screens. RIOK3 knockdown blocks both cytosolic double-stranded B-form DNA and double-stranded RNA-induced IRF3 activation and IFN-β production. In contrast, the overexpression of RIOK3 activates IRF3 and induces IFN-β. RIOK3 functions downstream of TBK1 and upstream of IRF3 activation. Furthermore, RIOK3 physically interacts with both IRF3 and TBK1 and is necessary for the interaction between TBK1 and IRF3. In addition, global transcriptome analysis shows that the expression of many gene involved antiviral responses is dependent on RIOK3. Thus, knockdown of RIOK3 inhibits cellular antiviral responses against both DNA and RNA viruses (herpesvirus and influenza A virus). Our data suggest that RIOK3 plays a critical role in the antiviral type I IFN pathway by bridging TBK1 and IRF3. IMPORTANCE The innate immune response, such as the production of type I interferons, acts as the first line of defense, limiting infectious pathogens directly and shaping the adaptive immune response. In this study, we identified RIOK3 as a novel regulator of the antiviral type I interferon pathway. Specifically, we found that RIOK3 physically interacts with TBK1 and IRF3 and bridges the functions between TBK1 and IRF3 in the activation of type I interferon pathway. The identification of a cellular kinase that plays a role the type I interferon pathway adds another level of complexity in the regulation of innate immunity and will have implications for developing novel strategies to combat viral infection.

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2′,3′-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs.

Tigecycline (TGC)-resistant extensively drug-resistant Acinetobacter baumannii (XDRAB) is an increasing threat in regard to nosocomial infections. The resistance-nodulation-cell division (RND) efflux pump has played an important role in TGC resistance. In this study, total 81 TGC-resistant XDRAB isolates were analyzed for their responses to the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). We found that NMP could reduce by 4-fold or greater than 4-fold the minimum inhibitory concentration (MIC) of TGC in 45 isolates (55.6 %). After typing with pulsed-field gel electrophoresis (PFGE), group A appeared to be the major cluster with good synergistic response to NMP. Transcripts of the AdeABC efflux pump gene were consistently more correlated with TGC resistance than transcripts of the AdeFGJ or AdeIJK efflux pump genes in these isolates. Of the 81 isolates, the amino acid sequences of AdeR and AdeS were further classified and combined into 31 different codes. Although the dissemination of TGC-resistant XDRAB isolates was genetically diverse in our hospital, their responses to NMP conversion were still strain-dependent. We found that AdeRS combination codes were better than PFGE typing in separating groups of isolates with different sensitivity to NMP conversion.

Ledum palustre is an important perfume plant in northeast China, and widely distributed at Changbai mountain of Jilin province. The components and contents of essential oils in Ledumpalustre were highly correlated with their growth environment. By using GC-MS, about 32 compounds were identified from Ledumpalustre essential oils of Changbai mountain. The chemical components were L-bornyl acetate (20.2%), 1,2,3,4-tetramethyl-benzene (16.5%), (−)-myrtenal (4.4%), terpinen-4-ol (4.3%), 2,6-dimethyl-2,6-octadiene (3.8%), trans-pinocarveol (3.7%), L(-)-borneol (3.0%), geranyl acetate (2.8%), (+)-aromandendrene (2.3%), ledol (1.7%), sabenene (1.6%), β-pinene (1.4%), camphene (1.1%), caryophyllene oxide (1.1%), and citronellyl formate (0.5%), which indicate the application of Ledumpalustre essential oils from Changbai mountain should be different from other areas.

Using ginseng, wild wine and acetic acid to the orthogonal experiment method to determine the fermentation process, low temperature and cryogenic grinding fermentation brewing vinegar. The results showed that: cryogenic grinding with a significant impact on the quality of the wine, the best ginseng vinegar fermentation parameters for initial dosage of 6% alcohol, fermentation temperature was 31 °C, fermentation time was 96h.By mixed fermentation Changbai Mountain forest berries and ginseng significantly alter the flavor of vinegar.

In this paper, the production technology and application of carbohydrate-based fat mimics (CBFM) from starch of mung beans was studied for further exploitation. The starch of mung beans was hydrolyzed by enzyme and used in processing of low-fat sausage as fat mimics. The condition of enzyme hydrolysis is that enzyme recruitment was 6 U/g, the substrate concentration was 15%, the hydrolysis time was 15min, the reaction temperature of 85°C. The best formula is adding amount of CBFM 14%, starch 15%, water 16%, carrageenan 0.9%. Compared to the non-simulated fat sausage, the cooking loss of the low-fat sausage was reduction, adhesion increases, water retention rate increases, fat content reduces, freeze-thaw stability has increased, which avoids sausage retrogradation and increases the storage stability of the products.

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of pyrimidine and purine deoxyribonucleosides, and are essential for maintaining mitochondrial dNTP pools for mitochondrial DNA replication. Here the expression of mitochondrial TK2 and dGK in relation to cell growth phases in cultured cells was investigated. TK2 and dGK protein levels in isolated mitochondria and TK2 activity in total cell extracts from U2OS and TK1 deficient L929 cells were determined. We found that TK2 levels were negatively correlated with cell growth rates and there was an exponential increase in TK2 levels in cells entering stationary phase. The expression of dGK did not change and appeared to be constitutive.

Summary Previous research revealed a 6-fold higher incidence of tuberculosis (TB) amongst aborigines compared to Han Chinese in Taiwan. To investigate the reasons for this disparity, we genotyped Mycobacterium tuberculosis (MTB) strains obtained from members of different aboriginal tribes in different geographical regions of Taiwan by using molecular methods. In total, 177 isolates of MTB collected from patients at four hospitals in Taiwan from January 2006 to December 2011 were analysed by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem-repeat (MIRU-VNTR) typing. The most prevalent strains in the eastern and central regions of Taiwan were Beijing (45.7% in eastern) and Haarlem (39.1% in eastern, 37.1% in central) lineages, whereas in southern regions the most prevalent strains were EAI (47.7%) and Haarlem (20.5%) lineages. The high prevalence of EAI in southern Taiwan aborigines may be closely associated with Austronesian culture. This study provides a first overview of the M. tuberculosis strains circulating in aboriginal populations in Taiwan. The high prevalences of certain MTB lineages within aboriginal sub-populations suggest that transmission of MTB may have been restricted to close contacts.

Let a, b, k, r be nonnegative integers with 1 ≤ a ≤ b and r ≥ 2. Let G be a graph of order n with $n > \tfrac{{(a + b)(r(a + b) - 2) + ak}} {a} $ . In this paper, we first show a characterization for all fractional (a, b, k)-critical graphs. Then using the result, we prove that G is all fractional (a, b, k)-critical if $\delta (G) \geqslant \tfrac{{(r - 1)b^2 }} {a} + k $ and $|N_G (x_1 ) \cup N_G (x_2 ) \cup \cdots \cup N_G (x_r )| \geqslant \tfrac{{bn + ak}} {{a + b}} $ for any independent subset {x 1, x 2, …, x r } in G. Furthermore, it is shown that the lower bound on the condition $|N_G (x_1 ) \cup N_G (x_2 ) \cup \cdots \cup N_G (x_r )| \geqslant \tfrac{{bn + ak}} {{a + b}} $ is best possible in some sense, and it is an extension of Lu’s previous result.

Acinetobacter nosocomialis is increasingly identified as a causative agent of nosocomial infections in recent year. Among the 111 bloodstream infection isolates, we observed 36.9% (41/ 111) of Acinetobacter nosocomialis isolates were imipenem non-susceptible. MLST type Sequence type 410 (ST410) harboring the plasmid pAB-NCGM253 which encodes the blaOXA-72 gene was the major group in the 41 imipenem non-susceptible Acinetobacter nosocomialis. To our knowledge, this is the first time that pAB-NCGM253 was found in Acinetobacter nosocomialis. Finally, we demonstrated that the plasmid pAB-NCGM253 has the potential to spread carbapenem resistance between Acinetobacter species.

The herpes virus entry mediator (HVEM) a member of the TNFR superfamily, is a key immunoregulator with both proinflammatory and inhibitory signaling functions. Both α and β-herpes viruses exploit HVEM to establish persistent infections, however little is known about whether the oncogenic γ-herpesviruses modulate HVEM. Here, we investigate the role of HVEM in a mouse model of γ-herpesvirus (MHV68) infection. Control C57BL/6 and HVEM-deficient (Tnfrsf14−/−) mice were intranasally infected with MHV68. We found that virus replication in the lungs of Tnfrsf14−/− mice increased significantly indicating HVEM expression is required to control MHV68 infection. We found that alveolar macrophages were drastically reduced in Tnfrsf14−/− mice compared to C57/BL6 controls suggesting their survival requires HVEM signaling. Importantly, we showed that IL-1β levels in the lungs of MHV68 infected Tnfrsf14−/−mice were significantly higher than C57BL/6 controls. Bone marrow-derived macrophage from Tnfrsf14−/− showed that HVEM is necessary to prevent IL-1β production in response to inflammasome activator. Antibodies that block IL-1R or IL-1β drastically increased the viral load in the lungs suggested that IL-1β is necessary to control viral replication. Taken together, our results highlight the role for HVEM in innate immune response evasion of MHV68 that could involve the inflammasome.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

Objective To explore the mechanism of transcription factor Twist on promoting peritoneal fibrosis of human peritoneal mesothelial cells (HPMCs) in patients undergoing peritoneal dialysis (PD). Methods The HPMCs in peritoneal fluid after dialysis from patients underwent peritoneal dialysis was collected and cultured. The expression and subcellular localization of E-cadherin, α-SMA, Twist and Bmi-1 were assayed by immunofluorescence and Western blotting. The expression of Twist, Bmi-1, E-cadherin and α-SMA were determined by immunofluorescence and Western blotting after the plasmids pcDNA3.1-twist and empty vector pcDNA3.1 were transfected into HPMCs with Lipofectamine 2000 in vitro. Twist, Bmi-1, E-cadherin and α-SMA were determined by Western blotting and immunofluorescence after silencing the expression of Twist by small interfering RNA (siRNA) and empty vector pSlience. Bmi-1, E-cadherin and α-SMA were assessed by Western blotting and immunofluorescence after silencing the expression of Bmi-1 by siRNA. Results The expression of E-cadherin decreased gradually and the expression of Twist, α-SMA and Bmi-1 increased along with the increase in PD time as determined with immunofluorescence and Western blotting. Compared with that expressed in the HPMCs trasfected with empty vector pcDNA3.1, the expression of E-cadherin significantly reduced and the expressions of α-SMA, Twist and Bmi-1 increased significantly after transfection of the plasmids pcDNA3.1-twist (P

Using the Annual Survey of Chinese Industrial Firm over the period 1998-2007, we have investigated the impact of the public subsidy on the firm survival rate of new exporting firms by the survival analysis techniques. We find that the median survival time of Chinese exporting is short, just 2 years and the mean duration is 2.5-2.6 years , which is consistent with the present survival literatures. Conditioning on the firm-specific variables , the public subsidy has negative effect on the survival rate and the ownership of enterprise matters. It also documents that the longer firms stay in the exporting market , the lower the hazard rate and the firms productivity, export intensity, the amount of the foreign capital have significantly positive effect on the survival probability.

Essential oils were extracted from biomaterials of root bark, stem bark, twigs, leaves and flowers fromMagnolia sieboldiiby the means of steam distillation. Their chemical compositions of these essential oils were analyzed by GC-MS method and their flavor properties were evaluated based on the basic media of cold creams. The results showed that the highest yield of essential oils belonged to the essential oil from root bark and the lowest belonged to that from leaves; As to flavor, the parts of leaves and flower are more suitable for exploiting cosmetic products; Twigs, flowers and leaves belonged to the same cluster by Hierarchical cluster analysis on chemical components from these essential oils. The essential oil from leaves should be further exploited due to its good flavor for cosmetic products and a kind of newable natural resources, although this essential oil had lower extraction yeilds.

Dandelion (Taraxacum spp) is an medical active materials used in many traditional and modern herbal medical systems, its polysaccharides have many bioactivities. Taraxacum mongolicum is one of the most widely consumed as Chinese herbal medicine and root tea in Changbai Mountains area, Northeast China. In this study, a procedure for the extraction of polysaccharides from the root of T mongolicum(TMRPS) was established. Based on single-variable tests experiment to determine the ranges of the extraction parameters (extraction temperature (X1), extraction time (X2), and ratio of solvent to raw material (X3)), response surface methodology (RSM) was used to optimize for these extraction parameters (X1, X2, X3) enhancing the forward extraction efficiency of TMRPS by a three-level three-variable Box-Behnken experimental design. The results showed that the ranges of the extraction parameters were determined as 60°C-80°C(X1),1h-3h (X2),20-40(v/m)(X3) by single-variable tests and the maximal yield (9.01%) of TMRPS was reached at extraction temperature 70.2°C, extraction time 1.79 h and ratio of solvent to raw material 28.0 (v):1 (m). Convenient for experiment, appropriate readjustment of these extraction parameters was made. Under these readjusted conditions, the mean extraction yield of TMRPS was 8.85%, which was near to theoretical calculation and indicates that these results were agree closely with model predictions.

In this study, Strontium (Sr) was added as modifier during the casting of Al-11.6Si-0.5Mg alloy, and the effect of T6 heat treatment on microstructure and performance of alloy was also investigated. The results showed that the 0.3% Al-8%Sr master alloy can refine effectively the α-Al dendrite and eutectic structure; the best economic process of T6 heat treatment is solution at 535°C for 6 hrs., and water cooling at 50~60°C,aging at 160°C for 6 hrs , then air cooling. After Sr modification and T6 heat treatment, the mechanical properties of alloy are improved remarkably, i.e., the tensile strength increased to 348MPa from 183MPa before modification and the elongation raises from 3.0% to 6.5%. So this alloy is applied to the strain clamp products in electric power fitting industry.

One kind of deformed magnesium alloy with high strength and high plasticity was made to be bicycle parts. Meantime, the microstructure and mechanical properties of the magnesium alloy were analyzed. In addition, the deformation and welding technology of taper pipe and shaped tube of bicycle were studied. The results showed that the bicycle parts manufactured through extruding and welding of the new type magnesium alloy have high strength, good plasticity. The tensile strength can reach 321.3 MPa and the elongation is more than 20%. By adopting the new welding process of internal mending and cuttage grafting, the after-welding-strength is 90.2%. of the base metal Meanwhile, the industrialization level can meet the requirements of European Union (EU) vehicle standards.

We collected and analyzed the time to detection (TTD) of blood cultures in the BacT/Alert automated system from 2002 to 2007. Among the 10,893 monomicrobial isolates from a total of 133,735 blood culture sets, the recoveries of aerobic bottles were compared with those of anaerobic bottles in this study. Significantly more Gram-positive cocci (except Staphylococcus aureus and enterococci), glucose nonfermentative Gram-negative bacteria, and yeast were recovered from aerobic bottles than from anaerobic bottles. The average TTD was 19.0 hr and 20.1 hr for the aerobic and anaerobic bottles, respectively, and 96.8% of the microorganisms were detected within the first 72 hr. Of the 5,489 microorganisms recovered from both of the blood culture bottle pair, microbial growth was significantly more often detected first in the anaerobic bottles than the aerobic bottles for Enterobacteriaceae except Serratia marcescens, while S. aureus, coagulase-negative staphylococci and Pseudomonas aeruginosa were more often detected first in the aerobic bottles. According to these data, we conclude that the earlier positivity of anaerobic bottles is a useful marker for rapid presumptive identification of Enterobacteriaceae infection. J. Clin. Lab. Anal. 27:113–120, 2013. © 2013 Wiley Periodicals, Inc.

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.

The regulation of the late viral gene expression in betaherpesviruses is largely undefined. We have previously shown that the murine cytomegalovirus proteins pM79 and pM92 are required for late gene transcription. Here, we provide insight into the mechanism of pM79 and pM92 activity by determining their interaction partners during infection. Co-immunoprecipitation-coupled MS studies demonstrate that pM79 and pM92 interact with an array of cellular and viral proteins involved in transcription. Specifically, we identify RNA polymerase II as a cellular target for both pM79 and pM92. We use inter-protein coevolution analysis to show how pM79 and pM92 likely assemble into a late transcription complex composed of late transcription regulators pM49, pM87 and pM95. Combining proteomic methods with coevolution computational analysis provides novel insights into the relationship between pM79, pM92 and RNA polymerase II and allows the generation of a model of the multi-component viral complex that regulates late gene transcription.

Purpose: Bloodstream infections (BSIs) caused by Acinetobacter species have been extensively reported, however, which majorly focused on respiratory tract infections. The risk of mortality and the effect of early catheter removal on survival in catheter-related BSIs (CRBSIs) caused by Acinetobacter spp. remain unclear. This study aims to investigate that. Methods: This is a retrospective multicentric study conducted in Taiwan from 2012 to 2014. Patients with at least 1 positive blood culture and catheter culture for the same Acinetobacter spp., showing symptoms and signs of CRBSIs, were included (n = 119). Risk factors for 30-day mortality were analyzed using a logistic regression model. The characteristics of patients with early catheter removal (within 48 hours after CRBSIs) were compared to those without removal matching for age, sex, and disease severity. Results: There were no differences in 30-day mortality with regard to causative Acinetobacter spp., catheter type, site, and appropriateness of antimicrobial therapy. Patients with higher Acute Physiologic and Chronic Health Evaluation (APACHE) II scores (odds ratio [OR]: 1.12; 95% confidence interval [CI]: 1.02-1.23; P = .014), shock (OR: 6.43; 95% CI: 1.28-32.33; P = .024), and longer hospitalization before CRBSIs (OR: 1.04; 95% CI: 1.00-1.08; P = .027) had a significantly higher 30-day mortality rate. Early removal of catheters after CRBSIs was not associated with better survival benefits. Conclusion: Higher disease severity (APACHE II score), shock, and longer hospitalization before bacteremia were independently associated with a higher 30-day mortality in CRBSIs caused by Acinetobacter spp. In previous published guidelines, infected catheters were suggested to be removed in CRBSIs caused by gram-negative bacilli. Even though early removal of catheters did not associate with a better survival outcome in current results, it should be judiciously evaluated according to the clinical conditions and risks individually. For better elucidation of these issues, further well-controlled prospective study may be warranted.

Zika virus (ZIKV) causes microcephaly in congenital infection, neurological disorders, and poor pregnancy outcome and no vaccine is available for use in humans or approved. Although ZIKV was first discovered in 1947, the exact mechanism of virus replication and pathogenesis remains unknown. Recent outbreaks of Zika virus in the Americas clearly suggest a human-mosquito cycle or urban cycle of transmission. Understanding the conserved and adaptive features in the evolution of ZIKV genome will provide a hint on the mechanism of ZIKV adaptation to a new cycle of transmission. Here, we show comprehensive analysis of protein evolution of ZIKV strains including the current 2015–16 outbreak. To identify the constraints on ZIKV evolution, selection pressure at individual codons, immune epitopes and co-evolving sites were analyzed. Phylogenetic trees show that the ZIKV strains of the Asian genotype form distinct cluster and share a common ancestor with African genotype. The TMRCA (Time to the Most Recent Common Ancestor) for the Asian lineage and the subsequently evolved Asian human strains was calculated at 88 and 34 years ago, respectively. The proteome of current 2015/16 epidemic ZIKV strains of Asian genotype was found to be genetically conserved due to genome-wide negative selection, with limited positive selection. We identified a total of 16 amino acid substitutions in the epidemic and pre-epidemic strains from human, mosquito, and monkey hosts. Negatively selected amino acid sites of Envelope protein (E-protein) (positions 69, 166, and 174) and NS5 (292, 345, and 587) were located in central dimerization domains and C-terminal RNA-directed RNA polymerase regions, respectively. The predicted 137 (92 CD4 TCEs; 45 CD8 TCEs) immunogenic peptide chains comprising negatively selected amino acid sites can be considered as suitable target for sub-unit vaccine development, as these sites are less likely to generate immune-escape variants due to strong functional constrains operating on them. The targeted changes at the amino acid level may contribute to better adaptation of ZIKV strains to human-mosquito cycle or urban cycle of transmission.

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available., IMPORTANCE To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available.

We theoretically investigate the sensing performance of the dielectric-metal core-shell resonators (DMCSRs) that support multipolar sharp magnetic and electric-based cavity plasmon resonances. We show that at the cavity resonances the ability of the DMCSRs to strongly confine the optical fields inside the cavity is robust against the existence of nano-openings in the metal shell layer. As a result, both the perfect DMCSRs having a complete metal shell layer and the non-perfect DMCSRs with nano-openings in the metal shell layers exhibit high refractive index sensitivities of 700 ~1200 nm/RIU. Furthermore, we demonstrate that such high refractive index sensitivities could be well maintained in an array of interconnected non-perfect DMCSRs. The narrow linewidths of the cavity plasmon resonances coupled with their high index sensitivities make the array of non-perfect DMCSRs possess high figure of merit (FOM) values up to ~88, approaching the theoretically estimated upper limit (FOM ≈108) for gold standard prism coupled surface-plasmon sensors.

The structure of fitness landscapes is critical for understanding adaptive protein evolution. Previous empirical studies on fitness landscapes were confined to either the neighborhood around the wild type sequence, involving mostly single and double mutants, or a combinatorially complete subgraph involving only two amino acids at each site. In reality, the dimensionality of protein sequence space is higher (20L) and there may be higher-order interactions among more than two sites. Here we experimentally characterized the fitness landscape of four sites in protein GB1, containing 204 = 160,000 variants. We found that while reciprocal sign epistasis blocked many direct paths of adaptation, such evolutionary traps could be circumvented by indirect paths through genotype space involving gain and subsequent loss of mutations. These indirect paths alleviate the constraint on adaptive protein evolution, suggesting that the heretofore neglected dimensions of sequence space may change our views on how proteins evolve. DOI: http://dx.doi.org/10.7554/eLife.16965.001, eLife digest Proteins can evolve over time by changing their component parts, which are called amino acids. These changes usually happen one at a time and natural selection tends to preserve those changes that make the protein more efficient at its specific tasks, while discarding those that impair the protein’s activity. However the effect of each change depends on the protein as a whole, and so two changes that separately make the protein worse can make it much better if they occur together. This phenomenon is called epistasis and in some cases it can trap proteins in a sub-optimal form and prevent them from improving further. Proteins are made from twenty different kinds of amino acid, and there are millions of different combinations of amino acids that could, in theory, make a protein of a given length. Studying protein evolution involves making variants of the same protein, each with just a few changes, and comparing how efficient, or “fit”, they are. Previous studies only measured the fitness of a few variants and showed that epistasis could block protein evolution by requiring the protein to lose some fitness before it could improve further. However, new techniques have now made it easier to study protein evolution by testing many more protein variants. Wu, Dai et al. focused on four amino acids in part of a protein called GB1 and tested the efficiency of every possible combination of these four amino acids, a total of 160,000 (204) variants. Contrary to expectations, the results suggested that the protein could evolve quickly to maximise fitness despite there being epistasis between the four amino acids. Overcoming epistasis typically involved making a change to one amino acid that paved the way for further changes while avoiding the need to lose fitness. The original change could then be reversed once the epistasis was overcome. The complexity of this solution means it can only be seen by studying a large number of protein variants that represent many alternative sequences of protein changes. Wu, Dai et al. conclude that proteins are able to achieve a higher level of fitness through evolution by exploring a large number of changes. There are many possible changes for each protein and it is this variety that, despite epistasis, allows proteins to become naturally optimised for the tasks that they perform. While the full complexity of protein evolution cannot be explored at the moment, as technology advances it will become possible to study more protein variants. Such advances would therefore hopefully allow researchers to discover even more about the natural mechanisms of protein evolution. DOI: http://dx.doi.org/10.7554/eLife.16965.002

Based on data from the air quality monitoring stations in the Pearl River Delta during the 2015 Spring Festival, the regional air quality was investigated and the impact of burning fireworks on urban air quality was assessed. The results showed that: Zhaoqing was the worst polluted city in PM₁₀, PM₂.₅, SO₂ and CO in terms of concentrations in the region during the period, Huizhou was the worst polluted city in O₃ and Guangzhou was the most polluted city in NO₂ at the same time. Compared to the data of last year, the SO₂, NO₂, CO, PM₁₀ and PM₂.₅ concentrations had decreased significantly, but the O₃ concentration had increased during the Spring Festival. Burning fireworks during the Spring Festival were mainly concentrated in the suburbs. The concentrated discharge of fireworks made the SO₂, PM₁₀ and PM₂.₅ concentrations increased significantly in the New Year's Eve night, even multiplied, but had no significant effect on CO, O₃ and NO₂. The rapid decline in PM₂.₅/PM₁₀ proportion was caused by the discharge of fireworks, and the ratio of PM₂.₅/PM₁₀ reached the minimum when concentration of particles reached the peak. By assessing, the maximum contribution of hourly concentration from burning fireworks in each city was between 16 µg · m⁻³ and 65 µg · m⁻³ for PM₂.₅, between 28 µg · m⁻³ and 138 µg · m⁻³ for PM₁₀ and between 9 µg · m⁻³ and 43 µg · m⁻³ for SO₂.

The purpose of this review is to objectively evaluate the biochemical and pathophysiological properties of 0.9% saline (henceforth: saline) and to discuss the impact of saline infusion, specifically on systemic acid-base balance and renal hemodynamics. Studies have shown that electrolyte balance, including effects of saline infusion on serum electrolytes, is often poorly understood among practicing physicians and inappropriate saline prescribing can cause increased morbidity and mortality. Large-volume (>2 L) saline infusion in healthy adults induces hyperchloremia which is associated with metabolic acidosis, hyperkalemia, and negative protein balance. Saline overload (80 ml/kg) in rodents can cause intestinal edema and contractile dysfunction associated with activation of sodium-proton exchanger (NHE) and decrease in myosin light chain phosphorylation. Saline infusion can also adversely affect renal hemodynamics. Microperfusion experiments and real-time imaging studies have demonstrated a reduction in renal perfusion and an expansion in kidney volume, compromising O2 delivery to the renal parenchyma following saline infusion. Clinically, saline infusion for patients post abdominal and cardiovascular surgery is associated with a greater number of adverse effects including more frequent blood product transfusion and bicarbonate therapy, reduced gastric blood flow, delayed recovery of gut function, impaired cardiac contractility in response to inotropes, prolonged hospital stay, and possibly increased mortality. In critically ill patients, saline infusion, compared to balanced fluid infusions, increases the occurrence of acute kidney injury. In summary, saline is a highly acidic fluid. With the exception of saline infusion for patients with hypochloremic metabolic alkalosis and volume depletion due to vomiting or upper gastrointestinal suction, indiscriminate use, especially for acutely ill patients, may cause unnecessary complications and should be avoided. More education regarding saline-related effects and adequate electrolyte management is needed.

To observe the effect of electroacupuncture (EA) therapy on levels of oxygen free radicals (OFR) and hippocampal apoptosis-related protein expression in ischemic learning-memory disorder rats so as to investigate its mechanisms underlying improvement of ischemic learning-memory impairment.A total of 60 SD rats were randomly divided into sham operation (sham), model, medication, and EA groups, with 15 rats in each group. The learning-memory disorder model was made by occlusion of bilateral carotid arteries. EA (2- 3 Hz, 2 mA) was applied to "Zhi San Zhen" ["Shenting" (GV 24) and bilateral "Benshen" (GB 13)] for 30 min, once a day for 3 weeks. The rats of the medication group were treated by lavage of Aricept (0.03 mg . kg(-1) . d(-1)), once daily for 3 weeks. The rats' learning-memory ability was detected by Morris water maze tests and the state of hippocampal apoptosis cells was observed by light microscope after TUNEL staining and the expression of hippocampal Bcl-2, Bax and Caspase-3 proteins was detected by immunohistochemistry. Serum and hippocampal superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) contents were detected by chemical colorimetric analysis.Compared with the sham group, the escape latencies (place-navigation) after modeling were evidently prolonged, and the times of target-platform crossing in 90 sec (spatial probe test) considerably reduced in the model group (P0.01), suggesting an impairment of learning-memory ability. After the treatment for 21 d, the increased escape latency and the reduced target-platform crossing time in both EA and medication groups were reversed in comparison with the model group (P0.01), suggesting an improvement of memory ability, and the effect of the EA group was significantly superior to that of the medication group (P0.05). Compared with the sham group, the number of apoptotic cells in hippocampal CA 1- CA 3 regions, and the expression levels of hippocampal Bcl-2, Bax and Caspase-3 proteins, and serum and hippocampal MDA contents were significantly increased in the model group (P0.01), while serum and hippocampal SOD and GSH-Px levels obviously decreased in the model group (P0.01). After the treatment for 21 days, compared to the model group, the number of the apoptotic cells, the expression levels of hippocampal Bax and Caspase--3 proteins, and the contents of serum and hippocampal MDA were notably decreased in the EA and medication groups (P0.01), whereas, Bcl-2 protein expression levels, and serum and hippocampal SOD and GSH-Px activity were notably up-regulated in the EA and medication groups (P0.01). The effects of EA group were obviously superior to those of medication group in increasing hippocampal Bcl-2 immunoactivity, serum SOD and GSH-Px and hippocampal GSH-Px activity and in down-regulating serum MDA level (P0.01, P0.05).Electroacupuncture intervention can improve learning-memory ability in ischemic learning-memory disorder rats which may be associated with its effects in reducing blood and hippocampal OFR contents and hippocampal cellular apoptosis.

Background The high error rate of next generation sequencing (NGS) restricts some of its applications, such as monitoring virus mutations and detecting rare mutations in tumors. There are two commonly employed sequencing library preparation strategies to improve sequencing accuracy by correcting sequencing errors: read-pairing method and tag-clustering method (i.e. primer ID or UID). Here, we constructed a homogeneous library from a single clone, and compared the variant calling accuracy of these error-correction methods. Result We comprehensively described the strengths and pitfalls of these methods. We found that both read-pairing and tag-clustering methods significantly decreased sequencing error rate. While the read-pairing method was more effective than the tag-clustering method at correcting insertion and deletion errors, it was not as effective as the tag-clustering method at correcting substitution errors. In addition, we observed that when the read quality was poor, the tag-clustering method led to huge coverage loss. We also tested the effect of applying quality score filtering to the error-correction methods and demonstrated that quality score filtering was able to impose a minor, yet statistically significant improvement to the error-correction methods tested in this study. Conclusion Our study provides a benchmark for researchers to select suitable error-correction methods based on the goal of the experiment by balancing the trade-off between sequencing cost (i.e. sequencing coverage requirement) and detection sensitivity. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2388-9) contains supplementary material, which is available to authorized users.

Background Epistasis is one of the central themes in viral evolution due to its importance in drug resistance, immune escape, and interspecies transmission. However, there is a lack of experimental approach to systematically probe for epistatic residues. Results By utilizing the information from natural occurring sequences and high-throughput genetics, this study established a novel strategy to identify epistatic residues. The rationale is that a substitution that is deleterious in one strain may be prevalent in nature due to the presence of a naturally occurring compensatory substitution. Here, high-throughput genetics was applied to influenza A virus M segment to systematically identify deleterious substitutions. Comparison with natural sequence variation showed that a deleterious substitution M1 Q214H was prevalent in circulating strains. A coevolution analysis was then performed and indicated that M1 residues 121, 207, 209, and 214 naturally coevolved as a group. Subsequently, we experimentally validated that M1 A209T was a compensatory substitution for M1 Q214H. Conclusions This work provided a proof-of-concept to identify epistatic residues by coupling high-throughput genetics with phylogenetic information. In particular, we were able to identify an epistatic interaction between M1 substitutions A209T and Q214H. This analytic strategy can potentially be adapted to study any protein of interest, provided that the information on natural sequence variants is available. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2358-7) contains supplementary material, which is available to authorized users.

Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agentLeifsonia xylisubsp.xyli(Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification ofLxxdetection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting thePart1gene ofLxxwere used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg ofLxxgenomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods forLxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

This paper proposes a new position distribution method——Information Flow Configuration Method Based on Fuzzy Clustering Analysis, aiming at improving the precision and reliability of the airport operation command simulation system. It analyzed samples through the fuzzy clustering method, with the operation command information used as the clustering samples, and 7 kinds of information attribute used as clustering index. Then by using Matlab, it realized the cluster of the command information, and the cluster pedigree chart was drawn as well. At last, the correlation coefficient confirms that this approach is feasible in distributing the positions of the simulation system.

Non-tuberculous mycobacteria (NTM) are increasingly important opportunistic pathogens responsible for a variety of clinical diseases. The aim of this study was to evaluate a novel technique, real-time PCR coupled with high-resolution melting analysis (real-time PCR-HRMA), for NTM identification. Two pairs of unique primers targeted to the 16S rRNA gene and the 16S-23S internal transcribed spacer region were selected for further evaluation. A total of 149 mycobacterial clinical isolates were subjected to analysis using the real-time PCR-HRMA system. Overall, 134 NTM identified by the 16S rRNA full-gene sequencing method were categorized into four major groups: Mycobacterium avium complex, Mycobacterium chelonae group, Mycobacterium gordonae and Mycobacterium fortuitum group. Of the 134 prevalent mycobacterial isolates, 101 mycobacteria (75.4 %) could be identified correctly by the real-time PCR-HRMA system. The individual sensitivities for the M. avium complex, M. chelonae group, M. gordonae and M. fortuitum groups were 90.9, 89.1, 100 and 36.8 %, respectively. The specificity of identifying these groups varied from 96.4 to 100 %. When identification failed, mostly it was attributable to various species in the M. fortuitum group. The real-time PCR-HRMA system is therefore a rapid and sensitive method for identifying prevalent NTM in a clinical laboratory.

Herpesviruses maintain a dynamic balance between latency and productive infection. This is a complex process regulated by viral and cellular factors. We have developed a Murine gammaherpesvirus 68 (MHV-68) model system in which to study mechanisms underlying balance between latency and lytic infection. We have generated an epithelial cell line that carries MHV-68 in a tightly latent form by using a bacterial artificial chromosome clone of the virus genome with a mutation in the MHV-68 major lytic R transactivator gene. Complementation of this defect in trans by transfection with a plasmid encoding R transactivator initiated and restored the productive cycle. This cell line model was used to investigate transcription factor occupancy (CCCTC binding factor [CTCF] and Sp1) of the two internal repeat elements in the viral genome during latency and reactivation using chromatin immunoprecipitation. Our results show that CTCF can bind to the 40-bp and the 100-bp repeat sequences during latency, whereas binding is reduced upon reactivation. In contrast, Sp1 only bound to the 100-bp repeat after reactivation. Our results indicate that the large internal repeat sequences in MHV-68 have different functions. We hypothesise that the 40-bp repeat may be involved in regulation of gene expression during the maintenance of latency, while the 100-bp repeat domain may be involved in regulation of the lytic cycle.

The human γ-herpesviruses EBV and Kaposi’s sarcoma-associated herpesvirus establish lifelong latent infections, can reactivate in immunocompromised individuals, and are associated with the development of malignancies. Murine γ-herpesvirus-68 (γHV68), a rodent pathogen related to EBV and Kaposi’s sarcoma-associated herpesvirus, provides an important model to dissect mechanisms of immune control and investigate vaccine strategies. Infection of mice with γHV68 elicits robust antiviral immunity, and long-term protection from γHV68 reactivation requires both cellular and humoral immune responses. Vaccination of mice with AC-replication and transcription activator (RTA), a highly lytic latency-null recombinant γHV68, results in complete protection from wild-type γHV68 infection that lasts for at least 10 mo. In this report, we examine the immune correlates of AC-RTA–mediated protection and show that sterilizing immunity requires both T cells and Ab. Importantly, Ab was also critical for mitigating viral infection in the brain, and in the absence of Ab-mediated control, amplification of the AC-RTA virus in the brain resulted in fatality. Our results highlight important considerations in the development of vaccination strategies based on live-attenuated viruses.

Human gammaherpesvirus infections of the central nervous system (CNS) have been linked to various neurological diseases. Murine gammaherpesvirus 68 (MHV-68), genetically related and biologically similar to human gammaherpesviruses, infects the CNS in laboratory mice. However, viral persistency of MHV-68 has not been studied following CNS infection. In this study, we undertook the noninvasive bioluminescence imaging of a recombinant MHV-68 expressing the firefly luciferase (M3FL) to monitor virus progression after CNS infection. The M3FL virus inoculated in the brain systemically spread to the abdominal area in bioluminescence imaging, which was further confirmed by detection of viral genome and transcripts. The disseminated wild-type virus established latency in the spleen. Moreover, the treatment of the infected mice with CsA induced reactivation of latent MHV-68 from the brain and the spleen. Our results suggest that MHV-68 may persist both inside and outside the CNS once it gains access to the CNS.

The new process electroless Sn deposition on cast AZ61 was revealed, which employed to AZ61 magnesium alloy automobile parts and could provide high wear-resistant and high hardness. The pretreatment processes before electroless Sn deposition could ensure better adhesion between Sn coating and magnesium alloy. The speed of the Sn deposition increases quickly with increasing the content of composite catalysts in the plating bath. The technology is environment friendly and cannot cause hazard to environment because of absence of nickel and chromate in the whole process. Wastewater treatment technology of electroless Sn bath was investigated as well.

Replication and transcription activator (RTA), an immediate-early gene, is a key molecular switch to evoke lytic replication of gammaherpesviruses. Open reading frame 49 (ORF49) is conserved among gammaherpesviruses and shown to cooperate with RTA in regulating virus lytic replication. Here we show a molecular mechanism and in vivo functions of murine gammaherpesvirus 68 (MHV-68 or γHV-68) ORF49. MHV-68 ORF49 was transcribed and translated as a late gene. The ORF49 protein was associated with a virion, interacting with the ORF64 large tegument protein and the ORF25 capsid protein. Moreover, ORF49 directly bound to RTA and its negative cellular regulator, poly(ADP-ribose) polymerase-1 (PARP-1), and disrupted the interactions of RTA and PARP-1. Productive replication of an ORF49-deficient mutant virus (49S) was attenuated in vivo as well as in vitro . Likewise, latent infection was also impaired in the spleen of 49S-infected mice. Taken together, our results suggest that the virion-associated ORF49 protein may promote virus replication both in vitro and in vivo by providing an optimal environment in the early phase of virus infection as a derepressor of RTA.

Control of the motion process of acceleration/deceleration (Acc/Dec) is one of key complicated problems in CNC system development. The excellent Acc/Dec control can improve the processing precision. The traditional Acc/Dec algorithms such as the linear and the exponential are not suitable for high speed motion because there would be flexible impacts existing in feed motion, especially that the jerk of the S-shape curve Acc/Dec is discontinuous. A new flexible Acc/Dec algorithm for high speed motion system based on the squared sine function is introduced. The modeling of the Acc/Dec algorithm with Trigonometric Function Squared Sine(TFSS) shape curve is constructed.In this algorithm, the sine curve in jerk, acceleration, velocity and displacement are all continuous. The simulation experiments result demonstrate that the Acc/Dec algorithm with TFSS shape curve is a new method of continuous Jerk for the design flexible Acc/Dec.

Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in the salvage of pyrimidine deoxynucleosides needed for mitochondrial DNA synthesis. TK2 phosphorylates thymidine (dThd), deoxycytidine (dCyd), and many other antiviral pyrimidine nucleoside analogs. Zidovudine (AZT) is the first nucleoside analog approved for anti-HIV therapy, and it is still used in combination with other drugs. One of the side effects of long-term treatment with nucleoside analogs is mitochondrial DNA depletion, which has been ascribed to competition by AZT for the endogenous dThd phosphorylation carried out by TK2. Here we studied the kinetics of AZT and 3′-fluorothymidine phosphorylation by recombinant human TK2 and the effects of these and other pyrimidine nucleoside analogs on the phosphorylation of dThd and dCyd. Thymidine analogs strongly inhibited dThd phosphorylation but not dCyd phosphorylation, which instead was stimulated ∼30%. We found that recombinant human TK2 contained the feedback inhibitor dTTP in a 1:1 molar ratio and that incubation with dThd and AZT could completely remove the enzyme-bound dTTP, but dCyd was less efficient in this regard. The release of feedback inhibitor by dThd and dThd analogs most likely accounts for the observed kinetics. Similar effects were also observed with native rat liver mitochondrial TK2, strongly indicating a physiologic role for this process, which most likely is an important factor in the mitochondrial toxicity observed with antiviral nucleoside analogs.

The ability to obtain sequence-specific genetic information about rare target organisms directly from complex biological samples at the point-of-care would transform many areas of biotechnology. Microfluidics technology offers compelling tools for integrating multiple biochemical processes in a single device, but despite significant progress, only limited examples have shown specific, genetic analysis of clinical samples within the context of a fully integrated, portable platform. Herein we present the Magnetic Integrated Microfluidic Electrochemical Detector (MIMED) that integrates sample preparation and electrochemical sensors in a monolithic disposable device to detect RNA-based virus directly from throat swab samples. By combining immunomagnetic target capture, concentration, and purification, reverse-transcriptase polymerase chain reaction (RT-PCR) and single-stranded DNA (ssDNA) generation in the sample preparation chamber, as well as sequence-specific electrochemical DNA detection in the electrochemical cell, we demonstrate the detection of influenza H1N1 in throat swab samples at loads as low as 10 TCID(50), 4 orders of magnitude below the clinical titer for this virus. Given the availability of affinity reagents for a broad range of pathogens, our system offers a general approach for multitarget diagnostics at the point-of-care.

A series of a-C:H films have been prepared by plasma-based ion implantation (PBII) with acetylene on AISI 321 substrates. The effect of negative bias pulse on the characteristics of these films was investigated. The structures of the films were analyzed by X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. The surface hardness was measured by microindentation tests. The results indicated that the characteristics of these films are strongly depended on the negative bias pulse. When the bias pulse ranges from -10kV to -40kV, the films are typical diamond like carbon (DLC) films, while the films deposited at -5kV are polymer films. The peak intensity ratio of the D-band to that of the G-band (ID/IG) of the DLC films changes with the negative bias pulse. The minimum value of ID/IG (1.02) was gotten at -10kV.

Here we describe the cloning of a sequenced WUMS isolate of murine gammaherpesvirus-68 (MHV-68, γHV-68, also known as MuHV-4) as a bacterial artificial chromosome (BAC). We engineered the insertion of the BAC sequence flanked by loxP sites into the left end of the viral genome before the M1 open reading frame. The infectious viruses were reconstituted following transfection of the MHV-68 BAC DNA into cells. The MHV-68 BAC-derived virus replicated indistinguishably from the wild-type virus in cultured cells. Excision of the BAC insert was efficiently achieved by coexpressing the Cre recombinase. Although the BAC insertion did not significantly affect acute productive infection in the lung, it severely compromised the ability of MHV-68 to establish splenic latency. Removal of the BAC sequence restored the wild-type level of latency. Site-specific mutagenesis was carried out by RecA-mediated recombination to demonstrate that this infectious BAC clone can be used for genetic studies of MHV-68.