Identification and Characterization of a Novel Hypovirus from the Phytopathogenic Fungus Botryosphaeria dothidea.

Many mycoviruses have been accurately and successfully identified in plant pathogenic fungus Botryosphaeria dothidea. This study discovered three mycoviruses from a B. dothidea strain SXD111 using high-throughput sequencing technology. A novel hypovirus was tentatively named Botryosphaeria dothidea hypovirus 1 (BdHV1/SXD111). The other two were known viruses, which we named Botryosphaeria dothidea polymycovirus 1 strain SXD111 (BdPmV1/SXD111) and Botryosphaeria dothidea partitivirus 1 strain SXD111 (BdPV1/SXD111). The genome of BdHV1/SXD111 is 11,128 nucleotides long, excluding the poly (A) tail. A papain-like cysteine protease (Pro), a UDP-glucose/sterol glucosyltransferase (UGT), an RNA-dependent RNA polyprotein (RdRp), and a helicase (Hel) were detected in the polyprotein of BdHV1/SXD111. Phylogenetic analysis showed that BdHV1/SXD111 was clustered with betahypovirus and separated from members of the other genera in the family Hypoviridae. The BdPmV1/SXD111 genome comprised five dsRNA segments with 2396, 2232, 1967, 1131, and 1060 bp lengths. Additionally, BdPV1/SXD111 harbored three dsRNA segments with 1823, 1623, and 557 bp lengths. Furthermore, the smallest dsRNA was a novel satellite component of BdPV1/SXD111. BdHV1/SXD111 could be transmitted through conidia and hyphae contact, whereas it likely has no apparent impact on the morphologies and virulence of the host fungus. Thus, this study is the first report of a betahypovirus isolated from the fungus B. dothidea. Importantly, our results significantly enhance the diversity of the B. dothidea viruses.

Novel Mycoviruses Discovered from a Metatranscriptomics Survey of the Phytopathogenic Alternaria Fungus.

Alternaria fungus can cause notable diseases in cereals, ornamental plants, vegetables, and fruits around the world. To date, an increasing number of mycoviruses have been accurately and successfully identified in this fungus. In this study, we discovered mycoviruses from 78 strains in 6 species of the genus Alternaria, which were collected from 10 pear production areas using high-throughput sequencing technology. Using the total RNA-seq, we detected the RNA-dependent RNA polymerase of 19 potential viruses and the coat protein of two potential viruses. We successfully confirmed these viruses using reverse transcription polymerase chain reaction with RNA as the template. We identified 12 mycoviruses that were positive-sense single-stranded RNA (+ssRNA) viruses, 5 double-strand RNA (dsRNA) viruses, and 4 negative single-stranded RNA (-ssRNA) viruses. In these viruses, five +ssRNA and four -ssRNA viruses were novel mycoviruses classified into diverse the families Botourmiaviridae, Deltaflexivirus, Mymonaviridea, and Discoviridae. We identified a novel -ssRNA mycovirus isolated from an A. tenuissima strain HB-15 as Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2). Additionally, we characterized a novel +ssRNA mycovirus isolated from an A. tenuissima strain SC-8 as Alternaria tenuissima deltaflexivirus 1 (AtDFV1). According to phylogenetic and sequence analyses, we determined that AtNSRV2 was related to the viruses of the genus Sclerotimonavirus in the family Mymonaviridae. We also found that AtDFV1 was related to the virus family Deltaflexivirus. This study is the first to use total RNA sequencing to characterize viruses in Alternaria spp. These results expand the number of Alternaria viruses and demonstrate the diversity of these mycoviruses.

Molecular characterization of a novel victorivirus isolated from Botryosphaeria dothidea, the causal agent of longan leaf spot disease.

Mycoviruses are widespread in all major taxonomic groups of filamentous fungi. Previous research has indicated that mycoviruses are associated with the phytopathogenic fungus Botryosphaeria dothidea. In this study, three distinct double-stranded RNA viruses were detected in B. dothidea strain YCLYY11 isolated from a leaf spot of longan (Dimocarpus longana). The results of BLAST analysis revealed that the predicted amino acid sequences of those viruses were similar to those of Botryosphaeria dothidea chrysovirus 1, Botryosphaeria dothidea partitivirus 1, and an apparent novel victorivirus. Sequencing and analysis of the complete genome of the novel victorivirus indicated it is 5218 bp in length and contains two open reading frames (ORFs) that overlap at the tetranucleotide AUGA. BLASTp analysis of the proteins encoded by ORF1 and ORF2 showed that they were most similar to the coat protein and RNA-dependent RNA polymerase of Sphaeropsis sapinea RNA virus 2 (81.37% and 74.09% identical, respectively). A phylogenetic tree showed that the novel virus clustered together with victoriviruses and was separate from members of the other four genera of the family Totiviridae. Based on its genome structure and the results of phylogenetic analysis, we propose that this novel victorivirus should be named "Botryosphaeria dothidea victorivirus 3". This is also the first report of these three mycoviruses coinfecting a strain of B. dothidea.

First report of Fusarium oxysporum and Fusarium solani causing root rot on trifoliate orange rootstock in China.

Trifoliate orange (Poncirus trifoliata L) is a thorny tree of the Rue family, which is extensively used as citrus rootstock in China. In January 2021, several leaf yellowing, declining, and wilting citrus seedlings grafted on trifoliate orange rootstock with rotted main roots were observed in orchards located in Wuhan city, Hubei, China. In old orchards, the incidence of diseased roots was approximately 90%. Diseased roots from seven plants were collected and cut into small pieces (0.2 to 0.5 cm). These pieces were then surface-sterilized using 0.1% mercury bichloride for 3 min, 75% ethanol for 3 min, rinsed with sterile distilled water for several times, and then placed on potato dextrose agar (PDA) supplemented with 0.05% lactic acid (v/v), and incubated at at 25±2°C in dark. Fifty-threesingle-conidium isolates with morphological characteristics similar to Fusarium spp. were obtained (Leslie and Summerell 2006), which displayed two kinds of colony morphology. Thirty isolates showed white to orange-white abundant aerial mycelium in rings and acquired a yellow to orange pigmentation, tweenty-three isolates showed white to pink, fluffy aerial mycelium in rings and acquired an orange to red pigmentation. Isolate WG-1 and HrmY-9 from each group were used for future identification. The average colony growth rate of WG-1 and HrmY-9 on PDA was 0.95±0.06 and 0.69±0.11 mm/day, n=4, respectively. WG-1 produced numerous oval, unicellular microconidia without septa, 4.03-9.87×1.01-5.13 µm, n=80 and very few macroconidia with two to four septa, narrowed at both ends, 11.08-22.64×1.67-4.91 µm, n=30. HrmY-9 produced numerous curved macroconidia with three to four septa, 18.03-37.33×2.16-7.8 µm, n=80, microconidia were unicellular, oval, and 5.33-16.19×1.74-6.51 µm, n=50. Sequences of internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF-1α), and DNA-directed RNA polymerase largest subunit (RPB1) genes were amplified with the primers ITS1/ITS4, EF1a-F/EF1a-R, and RPB1-F5/RPB1-R8, respectively (White et al. 1990, O'Donnell et al. 1998, O'Donnell et al. 2010), sequenced and deposited in GenBank. Sequences of isolate WG-1 (GenBank accession No. ON045437, ON063232 and ON089664) and HrmY-9 (GenBank accession No. ON045438, ON063233 and ON089665) were 100% identical with the corresponding sequences of Fusarium oxysporum (OM876904, JF430180, and MT568959) and F. solani (MT605584, MK617767, and MT305110), respectively. Based on above results, WG-1 and HrmY-9 was identified as F. oxysporum and F. solani, respectively. Pathogenicity test were performed on healthy one-year-old trifoliate orange seedlings by dipping their injured roots into conidial suspension (50 ml, 1×106 conidia/mL) for 1 h and the rest of conidial suspension was added to the pot after replanting to make sure the inoculum was in contact with the roots. Roots of control plants were inoculated with sterilized water. All experiments were repeated twice. All plants were cultured at 26°C under a 16-h light/dark cycle. Typical symptoms developed on most of inoculated seedlings two months post inoculation. No disease symptoms appeared on control plants. Same colonies were reisolated from the inoculated roots, confirming Koch's postulates. To our knowledge, this is the first report of F. oxysporum and F. solani causing root rot on trifoliate orange rootstock in China. The identification of F. oxysporum and F. solani as the causal agents of the observed root rot on trifoliate orange rootstock is critical to the prevention and control of this disease in the future.

A Novel Heptasegmented Positive-Sense Single-Stranded RNA Virus from the Phytopathogenic Fungus Colletotrichum fructicola.

In this study, a novel positive-sense single-stranded RNA (+ssRNA) mycovirus, tentatively named Colletotrichum fructicola RNA virus 1 (CfRV1), was identified in the phytopathogenic fungus Colletotrichum fructicola. CfRV1 has seven genomic components, encoding seven proteins from open reading frames (ORFs) flanked by highly conserved untranslated regions (UTRs). Proteins encoded by ORFs 1, 2, 3, 5, and 6 are more similar to the putative RNA-dependent RNA polymerase (RdRp), hypothetical protein (P2), methyltransferase, and two hypothetical proteins of Hadaka virus 1 (HadV1), a capsidless 10- or 11-segmented +ssRNA virus, while proteins encoded by ORFs 4 and 7 showed no detectable similarity to any known proteins. Notably, proteins encoded by ORFs 1 to 3 also share considerably high similarity with the corresponding proteins of polymycoviruses. Phylogenetic analysis conducted based on the amino acid sequence of CfRV1 RdRp and related viruses placed CfRV1 and HadV1 together in the same clade, close to polymycoviruses and astroviruses. CfRV1-infected C. fructicola strains demonstrate a moderately attenuated growth rate and virulence compared to uninfected isolates. CfRV1 is capsidless and potentially encapsulated in vesicles inside fungal cells, as revealed by transmission electron microscopy. CfRV1 and HadV1 are +ssRNA mycoviruses closely related to polymycoviruses and astroviruses, represent a new linkage between +ssRNA viruses and the intermediate double-stranded RNA (dsRNA) polymycoviruses, and expand our understanding of virus diversity, taxonomy, evolution, and biological traits. IMPORTANCE A scenario proposing that dsRNA viruses evolved from +ssRNA viruses is still considered controversial due to intergroup knowledge gaps in virus diversity. Recently, polymycoviruses and hadakaviruses were found as intermediate dsRNA and +ssRNA stages, respectively, between +ssRNA and dsRNA viruses. Here, we identified a novel +ssRNA mycovirus, Colletotrichum fructicola RNA virus 1 (CfRV1), isolated from Colletotrichum fructicola in China. CfRV1 is phylogenetically related to the 10- or 11-segmented Hadaka virus 1 (HadV1) but consists of only seven genomic segments encoding two novel proteins. CfRV1 is naked and may be encapsulated in vesicles inside fungal cells, representing a potential novel lifestyle for multisegmented RNA viruses. CfRV1 and HadV1 are intermediate +ssRNA mycoviruses in the linkage between +ssRNA viruses and the intermediate dsRNA polymycoviruses and expand our understanding of virus diversity, taxonomy, and evolution.

[Surgical treatment of traumatic osteomyelitis of extremities with MRSA infection].

OBJECTIVE: To summarize and discuss the clinical efficacy and application value of intravenous drip of linezolid combined with local targeted sustained-release of vancomycin in the treatment of traumatic osteomyelitis of extremities infected with MRSA. METHODS: Thirty patients with traumatic osteomyelitis of extremities infected by MRSA from March 2015 to March 2017 were analyzed retrospectively, including 21 males and 9 females; aged 25 to 64 years old, with an average age of(47.94± 6.23) years old;the course of disease ranged from 9 to 23 months, with an average of (15.68±6.23) months. The lesions were located in tibia in 18 cases and calcaneus in 12 cases. The causes of injury were fall injury in 12 cases, trafficaccident injury in 9 cases and fall injury in 9 cases. There were 22 patients with closed fractures and 8 patients with open fractures. There were 13 cases of internal fixation. Twenty-two patients had sinustract, 8 patients had soft tissue defect with bone and internal fixation exposure, soft tissue defect area ranged from 2.0 cm × 3.0 cm to 8.2 cm × 12.3 cm;10 patients had bone defect, defect area ranged from 0.5 to 3.4 cm;bacterial culture of sinus tract or wound secretion in all patients was MRSA. On the basis of thorough debridement, calcium sulfate artificial bone loaded with vancomycin was implanted in the lesion, and linezolid and glucose injection was given intravenously during the perioperative period. The patients were followed up regularly according to the time of antibiotic use, blood routine, erythrocyte sedimentation rate, high-sensitivity C-reactive protein, liver and kidney function and other related laboratory indexes, X-ray, CT and other imaging examinations, bone healing, flap survival, joint function and McKee's osteomyelitis cure criteria. RESULTS: All the patients were followed up, and the duration ranged from 3 to 6 years, with a mean of (4.23±0.76) years. No recurrence of osteomyelitis occurred. Fracture healing, infection control, wound healing and functional recovery were achieved. CONCLUSION: Intravenous drip of linezolid combined with local targeted sustained-release of vancomycin for the treatment of MRSA infected traumatic osteomyelitis in limbs have significant effects and low recurrence rates.

Molecular characterization of a novel mycovirus infecting the phytopathogenic fungus Botryosphaeria dothidea.

In this study, we report the molecular characterization of a novel positive-sense single-stranded RNA (+ssRNA) mycovirus from Botryosphaeria dothidea strain G91, which we name "Botryosphaeria dothidea botourmiavirus 1" (BdBOV-1). The complete genome of BdBOV-1 is 2547 nucleotides (nt) long, contains one open reading frame (ORF) potentially encoding an RNA-dependent RNA polymerase (RdRp), and shows a close relationship to mycoviruses of the family Botourmiaviridae. Phylogenetic analysis based on the RdRp sequences confirmed that BdBOV-1 clustered together with the members of the family Botourmiaviridae. To the best of our knowledge, this is the first report of a +ssRNA mycovirus infecting B. dothidea.

[A comparative study of the curative effect of proximal fibular osteotomy and single condylar replacement for the treatment of knee osteoarthritis of different severity].

OBJECTIVE: To compare the clinical effect of proximal fibular osteotomy (PFO) and single condyle replacement (UKA) in the treatment of knee osteoarthritis of different severity. METHODS: From June 2015 to September 2017, 53 patients with knee osteoarthritis were analyzed retrospectively. According to the operation mode, they were divided into PFO group (26 cases) and UKA group (27 cases) . According to Kellygren-Lawrence imaging classification standard：PFO group, 5 cases of gradeⅡ, 11 cases of grade Ⅲ, 10 cases of grade Ⅳ; UKA group, 7 cases of gradeⅡ, 9 cases of grade Ⅲ, 11 cases of grade Ⅳ. The amount of intraoperative bleeding, operation time and postoperative hospital stay were compared between the two groups. The patients were followed up regularly in the outpatient clinic before operation, 3 months after operation and 1 year after operation. The WOMAC score and the angle of tibiofemoral angle at each time point in the same group were compared, and the OMAC score and the angle of tibiofemoral angle at each time between the two groups were compared. RESULTS: Fifty-three patients were followed up for 12 to 24 (16.6±4.8) months. Compared with UKA group, PFO group had less intraoperative bleeding, shorter operative time and shorter postoperative hospital stay (P<0.05) . The scores of pain, stiffness and body function in UKA group were better than those in PFO group (P<0.05) . After 3 months and 1 year, the WOMAC index in PFO group was significantly improved (P<0.05) ; after 3 months and 1 year, the WOMAC index in UKA group was significantly better than that in PFO group (P<0.05) ; after 3 months, the WOMAC index in PFO group was significantly better than that in UKA group (P<0.05) . The tibiofemoral angle of gradeⅡand Ⅲ patients in both groups decreased gradually (P<0.05) ; the tibiofemoral angle of grade Ⅳ patients in UKA group was smaller than that of grade Ⅳ patients in PFO group (P<0.05) . CONCLUSION: Compared with UKA, PFO has the advantages of small trauma, fast recovery and low cost. The curative effect of PFO is equal to or more than UKA in the patients with gradeⅡand Ⅲ knee osteoarthritis. It is an alternative surgical method for the treatment of knee osteoarthritis.

Molecular characterization of a novel emaravrius infecting Actinidia spp. in China.

Viruses in the genus Emaravirus contain 5-8 negative genomic RNAs and cause severe diseases of plants. In this study, a novel emaravirus, provisionally named Actinidia emaravirus 2 (AcEV-2), was identified from a kiwifruit tree showing leaf mottle and chlorosis symptoms. The genome of AcEV-2 consisted of at least six RNAs (RNAs 1-6) with sizes of 7079, 2252, 1387, 1514, 1744 and 1233 nucleotides (nts), respectively. Proteins encoded by RNAs1-4 of AcEV-2 shared the highest amino acid (aa) sequence identities of 62.2%-77.3% with the corresponding proteins of fig mosaic emaravirues (FMV) and pigeonpea sterility mosaic emaravirus 2 (PPSMV-2). Whilst, the P5 and P6 encoded by AcEV-2 exhibited the highest identities of 44.2% and 39.2% with the corresponding proteins of PPSMV-2. It was the second emaravirus infecting Actinidia trees in China. Preliminary virus detection disclosed the presence of AcEV-2 in three Actinidia species grown in three provinces in the central and southern China.

Characterization of a novel victorivirus isolated from the phytopathogenic fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is an important pathogenic fungus that causes serious diseases in fruits and trunks of many wood plant species worldwide. In this study, 28 B. dothidea strains isolated from pear trunk samples showing stem wart or canker symptoms were used to detect double-stranded RNA (dsRNA) viruses. The dsRNA bands with the size of ~ 1.0 to ~ 6.0 kbp were examined from ten strains. Here, we reported a novel dsRNA mycovirus, tentatively named as Botryosphaeria dothidea victorivirus 2 (BdVV2), isolated from the B. dothidea strain MSD53. BdVV2 contained spherical virions that were ~ 38 nm in diameter consisting of a single linear dsRNA genome of 5,090 bp in length. The BdVV2 genome contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp), which shared significant amino acid identities of 68% and 60% with the corresponding proteins of Sphaeropsis sapinea RNA viruses 1 (SsRV1). Phylogenetic analyses based on the aa sequences of CP and RdRp both showed that BdVV2 was phylogenetically related to the members of the genus Victorivirus in the family Totiviridae. BdVV2 is thus a novel victorivirus isolated from the phytopathogenic fungus B. dothidea.

Characterization of a Botybirnavirus Conferring Hypovirulence in the Phytopathogenic Fungus Botryosphaeria dothidea.

A double-stranded RNA (dsRNA) virus was isolated and characterized from strain EW220 of the phytopathogenic fungus Botryosphaeria dothidea. The full-length cDNAs of the dsRNAs were 6434 bp and 5986 bp in size, respectively. The largest dsRNA encodes a cap-pol fusion protein that contains a coat protein gene and an RNA-dependent RNA polymerase (RdRp) domain, and the second dsRNA encodes a hypothetical protein. Genome sequence analysis revealed that the sequences of the dsRNA virus shared 99% identity with Bipolaris maydis botybirnavirus 1(BmBRV1) isolated from the causal agent of corn southern leaf blight, Bipolaris maydis. Hence, the dsRNA virus constitutes a new strain of BmBRV1 and was named Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmBRV1-BdEW220). BmBRV1-BdEW220 contains spherical virions that are 37 nm in diameter and consist of two dsRNA segments. The structural proteins of the BmBRV1-BdEW220 virus particles were 110 kDa, 90 kDa, and 80 kDa and were encoded by dsRNA1 and 2-ORFs. Phylogenetic reconstruction indicated that BmBRV1 and BmBRV1-BdEW220 are phylogenetically related to the genus Botybirnavirus. Importantly, BmBRV1-BdEW220 influences the growth of B. dothidea and confers hypovirulence to the fungal host. To our knowledge, this is the first report of a botybirnavirus in B. dothidea.

Association of PAR-2 Gene Polymorphisms with the Inflammatory Response and Susceptibility to Knee Osteoarthritis in the Chinese Han Population.

OBJECTIVE: To investigate the relationship between single nucleotide polymorphisms (SNPs) of protease-activated receptor 2 (PAR-2) and the susceptibility to knee osteoarthritis (KOA) and synovial expression of inflammatory factors in the Chinese Han population. METHODS: Three hundred fifty KOA patients (KOA group) and 345 healthy volunteers (control group) were recruited for the study. Five milliliters of venous blood was taken from each subject to detect the PAR-2 rs1529505, rs631465, and rs2242991 locus genotypes. The expression of PAR-2 mRNA in the synovial tissue and the levels of matrix metalloproteinase (MMP-1), MMP-9, interleukin (IL)-6, and IL-1ß in the joint effusion were detected in 205 KOA patients who had undergone joint replacement surgery. RESULTS: The PAR-2 rs1529505 T allele and the rs2242991 G allele carriers had a higher risk of KOA (p < 0.001). The severity of KOA in patients with the PAR-2 rs1529505 and rs2242991 mutations were higher than in the wild-type controls (p < 0.05). The expression levels of the PAR-2 mRNA in wild types, heterozygotes, and homozygotes of the rs1529505 and rs2242991 loci increased in turn (p < 0.001). The levels of MMP-1, MMP-9, IL-6, and IL-1ß in the synovial fluid of the PAR-2 rs1529505 and rs2242991 locus mutants were distinctly higher than those with the wild-type alleles (p < 0.01). There was no correlation between the rs631465 SNP and susceptibility to KOA, severity of KOA, nor levels of PAR-2 mRNA, MMP-1, MMP-9, IL-6, and IL-1ß. CONCLUSIONS: The PAR-2 SNPs rs1529505 and rs2242991 are associated with the susceptibility to KOA. KOA is more severe in patients harboring the T and G alleles of these two SNPs, respectively. The levels of inflammatory factors in synovial tissue and blood are higher than those in wild-type patients.

[Surgical treatment of deep infection around the spine].

OBJECTIVE: To study the surgical method and clinical effect of deep infection around the spine. METHODS: The clinical data of 7 patients with deep infections around the spine treated from January 2015 to January 2018 were retrospectively analyzed. All patients were acute infection within 3 weeks after spinal surgery. There were 5 males and 2 females, aged from 29 to 67 years old with an average of 42 years old. Four of them had implants and the other three didn't. After infection was diagnosed, they accepted aggressive debridement with assistance of vacuum sealing drainage(VSD). The antibiotic artificial bones were put in wounds, combined with intravenous antibiotics. Blood-rich adjacent tissue flaps were used to reconstruct defect of wounds. The tissue flaps included 4 paraspinal muscle flaps, 1 thoracolumbar fascial flap, 1 latissimus dorsi flap and 1 paraspinal muscle combined with thoracolumbar fascial flap. RESULTS: All 7 patients were followed up from 6 to 24 months with an average of 13.28 months. During the follow-up period, among the 4 patients with instrument, except one patient of lumbar fusion removed internal fixation due to postoperative infection, the other 3 patients successfully kept the implants. One case of cervical fracture and dislocation was repaired by latissimus dorsi transposition. Its wound healed but hydrops accumulated under the flap. This patient was cured by puncture drainage and local pressure bandaging. The other 5 wounds' healing were first intention and no postoperative complications such as infection recurrence, hematoma, effusion or wound dehiscence occurred. CONCLUSIONS: Deep infection around the spine is a serious complication and should be treated aggressively once diagnosed. Thorough debridement with the help of negative pressure closed drainage, local application of antibiotic artificial bone combined with systemic intravenous antibiotics and repairing wounds with adjacent tissue flaps are effective procedures for the treatment of deep infection around the spine.

[Modified one-stage revision procedure for treating proximal femoral infected nonunion after intramedullary nail fixation].

OBJECTIVE: To explore the methods and results of modified one-stage revision procedure for treating proximal femoral infected nonunion after intramedullary nail fixation. METHODS: From June 2010 to June 2015, 10 patients of proximal femoral infected nonunion after intramedullary nail fixation were treated with modified one-stage revision procedure, including 9 males and 1 female, ranging in age from 35 to 77 years old. There were 3 cases of intertrochanteric fractures, 2 cases of intertrochanteric fractures accompanied with proximal femoral fractures and 5 cases of subtrochaneric fracures. The fractures ware fixed by LISS plate after radically debridement. The bone defects were repaired by free vascularized fibular graft and autogenous cancellous bone graft mixed artificial bone containing antibiotics. Postoperatively, ambulation without weight bearing was encouraged as early as possible. RESULTS: Ten patients were followed up from 9 to 30 months and all nonunions healed smoothly without wound infection recurrence or internal fixation failure at the final follow-up. The time for full weight bearing was from 12 to 28 weeks. The hip joint function was evaluated by Sanders Traumatic Hip Rating Scale, the result was excellent in 7 cases, good in 2, and fair in 1 at the final follow-up. CONCLUSIONS: Modified one-stage revision procedure is an effective treatment with a good functional result for proximal femoral infected nonunion after intramedullary nail fixation. On the basis of radical debridement, the combination of infection control and bone healing therapeutic techniques is key for success.

Identification and Characterization of a Novel Hepta-Segmented dsRNA Virus From the Phytopathogenic Fungus Colletotrichum fructicola.

A novel hepta-segmented double-stranded RNA (dsRNA) virus was isolated and characterized from the strain FJ-4 of the phytopathogenic fungus Colletotrichum fructicola, and was named Colletotrichum fructicola chrysovirus 1 (CfCV1). The full-length cDNAs of dsRNA1-7 were 3620, 2801, 2687, 2437, 1750, 1536, and 1211 bp, respectively. The 5'- and 3'-untranslated regions of the seven dsRNAs share highly similar internal sequence and contain conserved sequence stretches, indicating that they have a common virus origin. The 5'-and 3'-UTRs of the seven dsRNAs were predicted to fold into stable stem-loop structures. CfCV1 contains spherical virions that are 35 nm in diameter consisting of seven segments. The largest dsRNA of CfCV1 encodes an RNA-dependent RNA polymerase (RdRp), and the second dsRNA encodes a viral capsid protein (CP). The dsRNA5 encodes a C2H2-type zinc finger protein containing an R-rich region and a G-rich region. The smallest dsRNA is a satellite-like RNA. The functions of the other proteins encoded by dsRNA3, dsRNA4, dsRNA6 are unknown. Phylogenetic analysis, based on RdRp and CP, indicated that CfCV1 is phylogenetically related to Botryosphaeria dothidea chrysovirus 1 (BdCV1), and Penicillium janczewskii chrysovirus 2 (PjCV2), a cluster of an independent cluster II group in the family Chrysoviridae. Importantly, all the seven segments of CfCV1 were transmitted successfully to other virus-free strains with an all-or-none fashion. CfCV1 exerts minor influence on the growth of C. fructicola but can confer hypovirulence to the fungal host. To our knowledge, this is the first report of a hepta-segmented tentative chrysovirus in C. fructicola.

The Soybean GmNARK Affects ABA and Salt Responses in Transgenic Arabidopsis thaliana.

GmNARK (Glycine max nodule autoregulation receptor kinase) is the homolog of Arabidopsis thaliana CLAVATA1 (CLV1) and one of the most important regulators in the process of AON (Autoregulation of Nodulation), a process that restricts excessive nodule numbers in soybean. However, except for the function in AON, little is known about this gene. Here, we report that GmNARK plays important roles in process of plant response to abiotic stresses. Bioinformatic analysis and subcellular localization experiment results showed that GmNARK was a putative receptor like kinase and located at membrane. The promoter of GmNARK contains manifold cis regulatory elements that are responsive to hormone and stresses. Gene transcript expression pattern analysis in soybean revealed GmNARK was induced by ABA and NaCl treatment in both shoot and root. Overexpression of GmNARK in Arabidopsis resulted in higher sensitivity to ABA and salt treatment during seed germination and greening stages. We also checked the expression levels of some ABA response genes in the transgenic lines; the results showed that the transcript level of all the ABA response genes were much higher than that of wild type under ABA treatment. Our results revealed a novel role of GmNARK in response to abiotic stresses during plant growth and development.

[Comparison of clinical effects between anterior cervical discectomy combined with corpectomy and cervical posterior single open-door laminoplasty in treating three-segment cervical spondylotic myelopathy].

OBJECTIVE: To compare the clinical effects between anterior cervical discectomy and fusion(ACDF) combined with anterior cervical corpectomy and fusion(ACCF) and cervical posterior single open-door laminoplasty with mini-titanium plate fixation in treating three-segment cervical spondylotic myelopathy. METHODS: The clinical data of 63 patients (39 males and 24 females) with three-segment cervical spondylotic myelopathy underwent surgical treatment from March 2014 to March 2016 were retrospectively analyzed. Among them, 43 cases were treated by ACDF combined with ACCF(anterior group), and 20 cases were treated by cervical posterior single open-door laminoplasty with mini-titanium plate fixation(posterior group). Operative time, intraoperative blood loss, postoperative complications were compared between two groups. And according to JOA score to evaluate the clinical effect. RESULTS: All the patients were follow-up from 16 to 40 months with an average of 25.8 months. Operative time of anterior group and posterior group were (123.70±6.21) min and(118.70±5.41) min, respectively, there was no significant difference between two groups(P>0.05). Intraoperative blood loss of anterior group and posterior group were (85.23±7.51) ml and (107.18±9.41) ml, respectively, there was significant difference between two groups(P<0.05). In anterior group, axial symptoms occurred in 6 cases, dysphagia in 1 case, and no C5 nerve root palsy, hoarseness and choking cough were found, the incidence rate of complication was 16.3%(7/43); and in posterior group, axial symptoms occurred in 5 cases, C5 nerve root palsy in 1 case, and no dysphagia, hoarseness and choking cough were found, the incidence rate of complication was 30.0%(6/20); there was significant defference in incidence rate of complication between two group(P<0.05). At 1 week after operation and final follow-up, the JOA scores of anterior group were obviously better than that of posterior group(P<0.05). CONCLUSIONS: Above-mintioned two surgical treatment for cervical spondylotic myelopathy can provide instantly stability, the method of ACDF combined with ACCF was obviously better that of the method of cervical posterior single open-door laminoplasty in intraoperative blood loss, the incidence rate of complications, clinical effect.Thus, for the treatment of three-segment cervical spondylotic myelopathy, the method of ACDF combined with ACCF would be firstly chosen.

Characterization of a novel botybirnavirus isolated from a phytopathogenic Alternaria fungus.

Alternaria fungi are important pathogens infecting a wide variety of organisms. Here, we report a novel double-stranded RNA (dsRNA) mycovirus named Alternaria botybirnavirus 1 (ABRV1) isolated from a phytopathogenic Alternaria sp. strain (SCFS-3) infecting a pear tree in China. ABRV1 has two dsRNA components (dsRNAs 1 and 2) with the sizes of 6,188 and 5,903 bp, containing two putative open reading frames encoding two polyproteins (202 and 192 kDa, respectively). The polyprotein encoded by ABRV1 dsRNA1 shares 41% amino acid (aa) sequence identity with the one encoded by dsRNA2 (instead of dsRNA1) of Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1). Conversely, the polyprotein encoded by ABRV1 dsRNA2 shares 46% aa sequence identity with the one (i.e., cap-pol fusion protein) encoded by SsBRV1 dsRNA1. ABRV1 has isometric spherical virus particles (~40 nm in diameter), putatively composed of the 60-, 70- and 80-kDa structural proteins. The genomic organization and phylogenetic analyses revealed that ABRV1 belongs to a newly proposed family "Botybirnaviridae", and to our knowledge, this is the first report of a botybirnavirus infecting an Alternaria sp. strain.

[Total thigh musculocutaneous flap in reconstruction of refractory pressure ulcers around hips in patients with spinal cord injury].

OBJECTIVE: To discuss the treatment of life-threatening refractory pressure ulcers around hips in patients with spinal cord injuries(SCI)and evaluate its clinical outcomes. METHODS: From March 2012 to June 2015, eight paraplegic patients with life-threatening refractory pressure ulcers around hips were treated with total thigh musculocutaneous flaps following amputation of proximal femurs or hips, including 7 males and 1 female with an average age of (52.0±2.6) years old ranging from 35 to 68. The coures of disease was from 10 months to 7 years with a mean of(2.9±0.2) years. All the 8 patients had compound ulcers of more than two parts, 7 cases had hip infection on the same side. The area of superficial wounds ranged from 3.0 cm×3.0 cm to 12.0 cm×15.0 cm. The clinical effects were evaluated according to infection controlling, wound healing, improving of nutrition and life quality of patients. RESULTS: All patient were followed up for 3 months to 2 years with an average of 1.3 years. All flaps survived, 5 cases obtained wound healing at one-stage, 2 cases had wound dehiscence and the wounds were closed after a second operation, 1 case had partial flap necrosis which was healed by dressing change, 1 case had urethral injury that was repaired in operation. All wounds were cured successfully without infection and ulcer recurrence during the follow-up period. The nutrition and quality of life of all cases improved observably after operation. CONCLUSIONS: The total thigh musculocutaneous flap is effective to reconstruct the refractory pressure ulcers around hip of patient with SCI. It can rescue life at the cost of losing one lower limb. It is an operation of last resort for the patients.

[Surgical treatment of multiple ligament injuries of knee joints].

OBJECTIVE: To study operative effects for the treatment of multiple ligament injuries of knee joints. METHODS: From 2008 to 2013, 26 patients (17 males and 9 females) with multiple ligament injuries of knee joints were treated surgically. The average age was 40.7 years old, ranging from 29 to 55 years old. All the patients were treated with arthroscopic reconstruction of cruiate ligament with autogenous or allogeneic hamstrings and tendon, and at the same time received repair of medial collateral ligament and lateral collateral ligament, as well as the treatment of exterior and interior complex injuries. Nine patients received second stage operation after the initial operation for mistake or missed diagnosis, and other patients were treated at the first stage. The Lysholm scoring system was used to evaluate function and stability of knee joints before and after operation. RESULTS: All the patients were followed up for an average duration of 1.6 years (ranged, 0.8 to 3.2 years). The mean awaiting time for operation was 1.2 months. The Lysholm score was improved from preoperative 42.5 +/- 4.5 (ranged, 33 to 48) to the latest follow-up 78.1 +/- 3.9 (ranged, 57 to 95). The function of knee joint was improved obviously in the arthroscopic reconstruction patients, with joint range of motion exceeding 900 and with Varus & Valgus tests near to normal. All the patients had negative findings in the Lachman test at 70 degrees of flexion. CONCLUSION: Arthroscopic reconstruction should be the first choice in treating multiple ligament injuries of knee joints. If the anterior and posterior cruciate ligament injuries can't be treated simultaneously, the posterior cruciate ligament injuries should be treated preferentially at the first stage and the anterior cruciate ligament injuries should be treated at the second stage. The diagnosis of posterior cruciate ligament is easy to be missed.