Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase

AbstractThe Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial

A Functional SNP in BNC2 Is Associated with Adolescent Idiopathic Scoliosis

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity. We previously conducted a genome-wide association study (GWAS) and detected two loci associated with AIS. To identify additional loci, we extended our GWAS by increasing the number of cohorts (2,109 affected subjects and 11,140 control subjects in total) and conducting a whole-genome imputation. Through the extended GWAS and replication studies using independent Japanese and Chinese populations, we identified a susceptibility locus on chromosome 9p22.2 (p = 2.46 × 10−13; odds ratio = 1.21). The most significantly associated SNPs were in intron 3 of BNC2, which encodes a zinc finger transcription factor, basonuclin-2. Expression quantitative trait loci data suggested that the associated SNPs have the potential to regulate the BNC2 transcriptional activity and that the susceptibility alleles increase BNC2 expression. We identified a functional SNP, rs10738445 in BNC2, whose susceptibility allele showed both higher binding to a transcription factor, YY1 (yin and yang 1), and higher BNC2 enhancer activity than the non-susceptibility allele. BNC2 overexpression produced body curvature in developing zebrafish in a gene-dosage-dependent manner. Our results suggest that increased BNC2 expression is implicated in the etiology of AIS

A novel mouse model of soft-tissue infection using bioluminescence imaging allows noninvasive, real-time monitoring of bacterial growth.

Musculoskeletal infections, including surgical-site and implant-associated infections, often cause progressive inflammation and destroy areas of the soft tissue. Treating infections, especially those caused by multi-antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) remains a challenge. Although there are a few animal models that enable the quantitative evaluation of infection in soft tissues, these models are not always reproducible or sustainable. Here, we successfully established a real-time, in vivo, quantitative mouse model of soft-tissue infection in the superficial gluteus muscle (SGM) using bioluminescence imaging. A bioluminescent strain of MRSA was inoculated into the SGM of BALB/c adult male mice, followed by sequential measurement of bacterial photon intensity and serological and histological analyses of the mice. The mean photon intensity in the mice peaked immediately after inoculation and remained stable until day 28. The serum levels of interleukin-6, interleukin-1 and C-reactive protein at 12 hours after inoculation were significantly higher than those prior to inoculation, and the C-reactive protein remained significantly elevated until day 21. Histological analyses showed marked neutrophil infiltration and abscesses containing necrotic and fibrous tissues in the SGM. With this SGM mouse model, we successfully visualized and quantified stable bacterial growth over an extended period of time with bioluminescence imaging, which allowed us to monitor the process of infection without euthanizing the experimental animals. This model is applicable to in vivo evaluations of the long-term efficacy of novel antibiotics or antibacterial implants

Correlation between the number of bacteria and bacterial PI <i>in vitro</i> and <i>in vivo</i>.

<p>BLI sensitivity was assessed using a CCD-based macroscopic detector to quantify the bacterial photon intensity (PI  =  photons/sec/cm²/steradian) for various numbers of bacteria (1.25×10⁵ to 2.0×10⁶ CFU per well). (A) <i>In vitro</i> study: the bacterial PI from colonies of bioluminescent MRSA was significantly correlated with the number of bacterial CFUs (<i>R</i>² = 0.9912). (B) <i>In vivo</i> study: various numbers of bacteria (1.25×10⁵–2.0×10⁶ CFU per inoculation) were inoculated into the SGM, and bioluminescence in the region of interest (ROI) was monitored by BLI. There was a significant correlation between the number of inoculated bacteria and the bacterial PI <i>in vivo</i> (<i>R</i>² = 0.9882).</p

Histological findings.

<p>Changes in histology over time in the SGM from infected mice. Axial sections of the infected SGM were prepared at 7 (A and B), 28 (C and D), and 42 (E and F) days after bacterial inoculation and stained with hematoxylin and eosin. Lower images: magnified views of the boxed regions in the upper images. On day 7, marked neutrophil infiltration and muscle destruction were detected at the injection sites. On day 28, an abscess that contained necrotic materials (asterisk) and covered by fibrous tissue was observed in the SGM. On day 42, scaring tissue (asterisk) observed in the subcutaneous tissue. Bars  = 200 µm (A, C, E), 10 µm (B, D, F).</p

Bacterial quantification.

<p>To evaluate the correlation between the bacterial PI and the number of bacteria in the infectious process <i>in vivo</i>, the positive area of gram-stained MRSA on the each tissue section from the different time-point animals (3, 7, and 28 days after inoculation) were calculated. Six axial sections which cover whole abscess in SGM were obtained from each animal. The total numbers of pixels in gram-positive area (blue stains) were measured in five fields from six sections (A). Image obtained from software (B) shows that gram-positive blue area has been replaced to red pixel dots (C).</p

Dual optical imaging in the mouse SGM model.

<p>(A) Bioluminescence and (B) fluorescence images in the same animal, 10 days after bacterial inoculation, are shown. (A) MRSA inoculated into the left SGM was sufficient to produce an observable bioluminescence signal. (B) A Cy5.5-conjugated inflammation probe accumulated at the inoculation site only 5 minutes after it was injected into the tail vein, and was monitored for over 2 weeks in the same animal. (C) In the merged image, the accumulation of the inflammation probe (yellow) appeared as an area of fluorescence that completely covered the entire region of bacterial bioluminescence signal (multi-color).</p

Changes in bacterial PI in the mouse SGM model and correlation between the number of inoculated bacteria and bacterial PI in the infection process.

<p>We inoculated MRSA Xen31 (3×10⁷ CFU/µl) in 5µl of medium into the SGM, and measured the bacterial PI in the ROI immediately after inoculation, and on days 1, 3, 7, 14, 21, and 28 (N = 6). (A) The mean bacterial PI in the SGM peaked immediately after inoculation (2.486×10⁴ PI) and remained high for at least 28 days (2.016×10⁴ PI). Means and SEM are shown. (B) There was a significant correlation between the pixels number of gram-positive area and the bacterial PI during 28 days <i>in vivo</i> (<i>R²</i> = 0.9788).</p

Serological data.

<p>Mean serum levels of IL-6, IL-1β, and CRP in MRSA-inoculated animals at various time points are shown (N = 4 each). Compared to the pre-inoculation level, the IL-6 level was significantly higher 12 hours after inoculation (<i>P</i><0.05). The IL-6 level gradually decreased, becoming normal by day 21. IL-1β was significantly elevated after 12 hours, and remained high for 7 days (P<0.05). CRP was significantly higher than the pre-inoculation level 0.5, 1, 3, and 21 days after inoculation (<i>P</i><0.05). The means and SEM are shown.</p