Long stem revision versus short stem revision with plate osteosynthesis for Vancouver type B2 periprosthetic femoral fracture: a comparative study of eighty five cases.

PURPOSE: Periprosthetic femoral fractures (PPFs) around the hip are challenging complications in orthopaedic surgery, particularly Vancouver type B2 (VTB2) fractures. The surgical management of these fractures is crucial and depends on various factors. Cementless short taper stem with plate osteosynthesis is an alternative surgical technique. This study aims to compare the outcomes of this surgical technique with revision arthroplasty (RA) with long stem in the treatment of VTB2 PPFs. METHODS: This retrospective study was conducted in a single medical institute from February 2010 to May 2019. Patients who had received either total hip arthroplasty or bipolar hemiarthroplasty and subsequently developed a VTB2 PPF were included; patients who sustained intra-operative fractures or received a cemented stem previously were excluded from the analysis. The patients were divided into two groups: group I received RA with cementless long stem, while group II underwent RA with cementless short taper stem with plate osteosynthesis. Demographic data, radiographic and functional outcomes, and complications were analyzed between the two groups. RESULTS: A total of 85 patients diagnosed with VTB2 PPFs were included in the study. There were no significant differences between the two groups in terms of demographic data, including age, gender, mean follow-up times, estimated blood loss, and operative times. The radiographic results showed that there was no significant difference in the incidence of subsidence and implant stability between the two groups. However, group II tended to have less subsidence and periprosthetic osteolysis. Patients in group II had significantly better functional scores (mean Harris hip score: post-operative: 60.2 in group I and 66.7 in group ii; last follow-up: 77.4 in group 1 and 83.2 in group II (both p < 0.05)). There were no significant differences in the overall complication rate, including infection, dislocation, re-fracture, and revision surgery, between the two groups. CONCLUSIONS: Both surgical techniques, cementless long stem and cementless short taper stem with plate osteosynthesis, are effective in the treatment of Vancouver B2 PPFs, with no significant differences in outcomes or complications. However, patients in cementless short taper stem with plate osteosynthesis had better functional scores at both post-operative and the last follow-up.

Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control.

Host-derived cellular pathways can provide an unfavorable environment for virus replication. These pathways have been a subject of interest for herpesviruses, including the betaherpesvirus human cytomegalovirus (HCMV). Here, we demonstrate that a compound, ARP101, induces the noncanonical sequestosome 1 (SQSTM1)/p62-Keap1-Nrf2 pathway for HCMV suppression. ARP101 increased the levels of both LC3 II and SQSTM1/p62 and induced phosphorylation of p62 at the C-terminal domain, resulting in its increased affinity for Keap1. ARP101 treatment resulted in Nrf2 stabilization and translocation into the nucleus, binding to specific promoter sites and transcription of antioxidant enzymes under the antioxidant response element (ARE), and HCMV suppression. Knockdown of Nrf2 recovered HCMV replication following ARP101 treatment, indicating the role of the Keap1-Nrf2 axis in HCMV inhibition by ARP101. SQSTM1/p62 phosphorylation was not modulated by the mTOR kinase or casein kinase 1 or 2, indicating ARP101 engages other kinases. Together, the data uncover a novel antiviral strategy for SQSTM1/p62 through the noncanonical Keap1-Nrf2 axis. This pathway could be further exploited, including the identification of the responsible kinases, to define the biological events during HCMV replication. IMPORTANCE Antiviral treatment for human cytomegalovirus (HCMV) is limited and suffers from the selection of drug-resistant viruses. Several cellular pathways have been shown to modulate HCMV replication. The autophagy receptor sequestosome 1 (SQSTM1)/p62 has been reported to interact with several HCMV proteins, particularly with components of HCMV capsid, suggesting it plays a role in viral replication. Here, we report on a new and unexpected role for SQSTM1/p62, in HCMV suppression. Using a small-molecule probe, ARP101, we show SQSTM1/p62 phosphorylation at its C terminus domain initiates the noncanonical Keap1-Nrf2 axis, leading to transcription of genes under the antioxidant response element, resulting in HCMV inhibition in vitro. Our study highlights the dynamic nature of SQSTM1/p62 during HCMV infection and how its phosphorylation activates a new pathway that can be exploited for antiviral intervention.

Protein detection in blood with single-molecule imaging.

The ability to characterize individual biomarker protein molecules in patient blood samples could enable diagnosis of diseases at an earlier stage, when treatment is typically more effective. Single-molecule imaging offers a promising approach to accomplish this goal. However, thus far, single-molecule imaging methods have not been translated into the clinical setting. The detection limit of these methods has been confined to the picomolar (10-12 M) range, several orders of magnitude higher than the circulating concentrations of biomarker proteins present in many diseases. Here, we describe single-molecule augmented capture (SMAC), a single-molecule imaging technique to quantify and characterize individual protein molecules of interest down to the subfemtomolar (<10-15 M) range. We demonstrate SMAC in a variety of applications with human blood samples, including the analysis of disease-associated secreted proteins, membrane proteins, and rare intracellular proteins. SMAC opens the door to the application of single-molecule imaging in noninvasive disease profiling.

Platelet-endothelial associations may promote cytomegalovirus replication in the salivary gland in mice.

Platelet decline is a feature of many acute viral infections, including cytomegalovirus (CMV) infection in humans and mice. Platelet sequestration in association with other cells, including endothelium and circulating leukocytes, can contribute to this decline and influence the immune response to and pathogenesis of viral infection. We sought to determine if platelet-endothelial associations (PEAs) contribute to platelet decline during acute murine CMV (mCMV) infection, and if these associations affect viral load and production. Male BALB/c mice were infected with mCMV (Smith strain), euthanized at timepoints throughout acute infection and compared to uninfected controls. An increase in PEA formation was confirmed in the salivary gland at all post-inoculation timepoints using immunohistochemistry for CD41+ platelets co-localizing with CD34+ vessels. Platelet depletion did not change amount of viral DNA or timecourse of infection, as measured by qPCR. However, platelet depletion reduced viral titer of mCMV in the salivary glands while undepleted controls demonstrated robust replication in the tissue by plaque assay. Thus, platelet associations with endothelium may enhance the ability of mCMV to replicate within the salivary gland. Further work is needed to determine the mechanisms behind this effect and if pharmacologic inhibition of PEAs may reduce CMV production in acutely infected patients.

Displaced isolated greater tuberosity fractures of elder adults treated with plate osteosynthesis.

BACKGROUND: We elucidated the effect of open reduction and internal fixation with locking plate for acute isolated displaced greater tuberosity fractures of humerus in elder adults (aged >60 years). METHODS: From 2009 to 2015, data from 32 patients, aged between 60 and 88 years, who had acute unilaterally displaced greater tuberosity fractures of humerus were collected and evaluated retrospectively. All the fractures were managed with open reduction and internal fixation with locking plate. The follow-up period was 50.8 months on an average (range 22-80 months). Finally, 25 patients were available for final evaluation of radiographic and functional results. RESULTS: All the 32 fractures had union with the average union time of 14 weeks (range 10-18 weeks) and with no complications. The average of preoperative Visual Analogue Scale (VAS) was 6.2 (range 4-8), ASES was 30.4 (range 13-45), and Constant score was 30.4 (range 20-45). At the last follow-up, the mean VAS was 1.3 (range 0-2.5), the mean ASES score was 90.1 (range 72-100), and the mean Constant score was 90.3 (range 80-100). There were statistically significant differences between preoperative and final follow-up in VAS, ASES, and Constant score. Hundred percent of patients had good or excellent results by Constant score, with excellent results (86-100) in 17 (68%) patients and good result (71-85) in 8 (32%). CONCLUSION: In conclusion, open reduction and internal fixation with locking plate is an effective treatment for acute displaced greater tuberosity fractures of humerus in elder adults.

Inhibition of Cytomegalovirus Replication with Extended-Half-Life Synthetic Ozonides.

Artesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replication in vitro, but therapeutic success in humans has been variable. We hypothesized that the short in vivo half-life of AS may contribute to the different treatment outcomes. We tested novel synthetic ozonides with longer half-lives against HCMV in vitro and mouse cytomegalovirus (MCMV) in vivo Screening of the activities of four ozonides against a pp28-luciferase-expressing HCMV Towne recombinant identified OZ418 to have the best selectivity; its effective concentration inhibiting viral growth by 50% (EC50) was 9.8 ± 0.2 µM, and cytotoxicity in noninfected human fibroblasts (the concentration inhibiting cell growth by 50% [CC50]) was 128.1 ± 8.0 µM. In plaque reduction assays, OZ418 inhibited HCMV TB40 in a concentration-dependent manner as well as a ganciclovir (GCV)-resistant HCMV isolate. The combination of OZ418 and GCV was synergistic in HCMV inhibition in vitro Virus inhibition by OZ418 occurred at an early stage and was dependent on the cell density at the time of infection. OZ418 treatment reversed HCMV-mediated cell cycle progression and correlated with the reduction of HCMV-induced expression of pRb, E2F1, and cyclin-dependent kinases 1, 2, 4, and 6. In an MCMV model, once-daily oral administration of OZ418 had significantly improved efficacy against MCMV compared to that of twice-daily oral AS. A parallel pharmacokinetic study with a single oral dose of OZ418 or AS showed a prolonged plasma half-life and higher unbound concentrations of OZ418 than unbound concentrations of AS. In summary, ozonides are proposed to be potential therapeutics, alone or in combination with GCV, for HCMV infection in humans.

Staged protocol for the treatment of chronic femoral shaft osteomyelitis with Ilizarov's technique followed by the use of intramedullary locked nail.

BACKGROUND: Infected nonunion of the femoral shaft is uncommon, and usually presents with challenging therapeutic and reconstructive problems. There are still controversies over treating infected nonunion of the femoral shaft. The purposes of this retrospective study were to review the treatment outcomes and describe a staged protocol for spontaneous wound healing. METHODS: Six patients with chronic femoral shaft infected-nonunion from October 2002 to September 2010 were included in this retrospective study. Serial plain films and triple films of lower legs were performed to evaluate the alignment of the treated femoral shaft and bony union following our staged protocol of Ilizarov distraction osteogenesis and intramedullary nailing. RESULTS: An average bone defect of 7 cm was noted after staged osteotomy. Mean follow-up was 87.5 (range, 38-133) months. Union was achieved in all six patients, with an average external fixation time of 6.8 (range, 5-11) months. There was no reinfection. One complication of a 4-cm leg discrepancy was noted, with an initial shortening of 15 cm. The mean knee ranges of motion (ROM) before staged protocols and at final follow-up were 64.2±8.6 (range, 60-75)° and 53.3±9.3 (range, 40-65)°, respectively. The ROM at the knee joint statistically decreased following staged protocols. CONCLUSION: In the treatment of chronic femur osteomyelitis, the staged protocol of Ilizarov distraction osteogenesis followed by intramedullary nailing was safe and successful, and allowed for union, realignment, reorientation, and leg-length restoration. With regard to the soft tissue, this technique provides a unique type of reconstructive closure for infected wounds. It is suggested that the staged protocol is reliable in providing successful simultaneous reconstruction for bone and soft tissue defects without flap coverage.

Evaluating a Cobalt-Tetraphenylporphyrin Complex, Functionalized with a Reduced Graphene Oxide Nanocomposite, for Improved Tooth Whitening.

OBJECTIVES: To study using cobalt-tetraphenylporphyrin/reduced graphene oxide (CoTPP/RGO), a nanocomposite highly catalytic both for oxidizing and for reducing hydrogen peroxide (H2 O2 ), to whiten teeth. METHODS: Dyes (D&C Red 34, Orange No. 4), tea, and betel nuts (Areca catechu) were used to stain the tooth specimens for three days and subsequently bleached using H2 O2 alone, H2 O2 plus CoTPP, or H2 O2 plus CoTPP/RGO for 0.5-70 hours. The process was photographed using an electronic chromometer and a camera. Color change was assessed after each session. The results of bleaching teeth with and without photoirradiation were compared. RESULTS: CoTPP/RGO increased the tooth-whitening efficacy of H2 O2 for specimens stained with dyes, tea, and betel nuts. Using lamps (photoactivation) (wavelengths: 254 nm and 310 nm) to catalyze the H2 O2 bleaching agent reduced the bleaching treatment time. CLINICAL SIGNIFICANCE: First, the Co/TPP/RGO complex allows improved tooth bleaching and shorter treatment times. Second, because the Co/TPP/RGO complex bleached D&C Red 34 at a similar rate and to a similar degree as tea and other natural agents stain teeth, it is a convenient staining agent for studying many vital aspects of tooth bleaching. (J Esthet Restor Dent 28:321-329, 2016).

Efficacy and Mechanism of Action of Low Dose Emetine against Human Cytomegalovirus.

Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50-40±1.72 nM, CC50-8±0.56 µM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism.

Dependence of coronavirus RNA replication on an NH2-terminal partial nonstructural protein 1 in cis.

UNLABELLED: Genomes of positive (+)-strand RNA viruses use cis-acting signals to direct both translation and replication. Here we examine two 5'-proximal cis-replication signals of different character in a defective interfering (DI) RNA of the bovine coronavirus (BCoV) that map within a 322-nucleotide (nt) sequence (136 nt from the genomic 5' untranslated region and 186 nt from the nonstructural protein 1 [nsp1]-coding region) not found in the otherwise-identical nonreplicating subgenomic mRNA7 (sgmRNA7). The natural DI RNA is structurally a fusion of the two ends of the BCoV genome that results in a single open reading frame between a partial nsp1-coding region and the entire N gene. (i) In the first examination, mutation analyses of a recently discovered long-range RNA-RNA base-paired structure between the 5' untranslated region and the partial nsp1-coding region showed that it, possibly in concert with adjacent stem-loops, is a cis-acting replication signal in the (+) strand. We postulate that the higher-order structure promotes (+)-strand synthesis. (ii) In the second examination, analyses of multiple frame shifts, truncations, and point mutations within the partial nsp1-coding region showed that synthesis of a PEFP core amino acid sequence within a group A lineage betacoronavirus-conserved NH2-proximal WAPEFPWM domain is required in cis for DI RNA replication. We postulate that the nascent protein, as part of an RNA-associated translating complex, acts to direct the DI RNA to a critical site, enabling RNA replication. We suggest that these results have implications for viral genome replication and explain, in part, why coronavirus sgmRNAs fail to replicate. IMPORTANCE: cis-Acting RNA and protein structures that regulate (+)-strand RNA virus genome synthesis are potential sites for blocking virus replication. Here we describe two: a previously suspected 5'-proximal long-range higher-order RNA structure and a novel nascent NH2-terminal protein component of nsp1 that are common among betacoronaviruses of group A lineage.

Reselection of a genomic upstream open reading frame in mouse hepatitis coronavirus 5'-untranslated-region mutants.

An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5' untranslated region (UTR) of >75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5'-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5'-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro, the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture.

Staged protocol for the treatment of chronic tibial shaft osteomyelitis with Ilizarov's technique followed by the application of intramedullary locked nail.

Open tibial shaft fractures are the most common open fractures, and many complications can occur. During the treatment period, infection leading to osteomyelitis was the most common complication. However, no consensus exists regarding the ideal management for such cases in the literature.The purposes of this retrospective study were to review the treatment of patients with chronic tibial shaft osteomyelitis over the past 14 years who were referred to the authors' institution and to provide a staged protocol for spontaneous wound healing. The staged protocol included: (1) radical debridement for infected bone and soft tissue; (2) immediate application of Ilizarov's apparatus for all patients except those needing delayed application; (3) osteotomy in healthy bone; (4) simultaneous distraction-compression osteogenesis and histogenesis; (5) additional docking-site bone grafting; and (6) shifting the external fixator to a locked nail when callus formation was visible at the distraction site. Union was achieved in 15 of 16 patients, with an average external fixation time of 4.5 months (range, 3-6 months). No deformity or leg-length discrepancy greater than 1 cm occurred.In the treatment of chronic osteomyelitis, this staged protocol was safe and successful and allowed for union, realignment, reorientation, and leg-length restoration. Regarding the soft tissues, this technique provides a unique type of reconstructive closure for infected wounds. It is suggested that the staged protocol is reliable in providing successful simultaneous reconstruction for bone and soft tissue defects without flap coverage.

Genetic evidence of a long-range RNA-RNA interaction between the genomic 5' untranslated region and the nonstructural protein 1 coding region in murine and bovine coronaviruses.

Higher-order RNA structures in the 5' untranslated regions (UTRs) of the mouse hepatitis coronavirus (MHV) and bovine coronavirus (BCoV), separate species in the betacoronavirus genus, appear to be largely conserved despite an ∼36% nucleotide sequence divergence. In a previous study, each of three 5'-end-proximal cis-acting stem-loop domains in the BCoV genome, I/II, III, and IV, yielded near-wild-type (wt) MHV phenotypes when used by reverse genetics to replace its counterpart in the MHV genome. Replacement with the BCoV 32-nucleotide (nt) inter-stem-loop fourth domain between stem-loops III and IV, however, required blind cell passaging for virus recovery. Here, we describe suppressor mutations within the transplanted BCoV 32-nt domain that along with appearance of potential base pairings identify an RNA-RNA interaction between this domain and a 32-nt region ∼200 nt downstream within the nonstructural protein 1 (Nsp1)-coding region. Mfold and phylogenetic covariation patterns among similarly grouped betacoronaviruses support this interaction, as does cotransplantation of the BCoV 5' UTR and its downstream base-pairing domain. Interestingly, cotransplantation of the BCoV 5' UTR and BCoV Nsp1 coding region directly yielded an MHV wt-like phenotype, which demonstrates a cognate interaction between these two BCoV regions, which in the MHV genome act in a fully interspecies-compliant manner. Surprisingly, the 30-nt inter-stem-loop domain in the MHV genome can be deleted and viral progeny, although debilitated, are still produced. These results together identify a previously undescribed long-range RNA-RNA interaction between the 5' UTR and Nsp1 coding region in MHV-like and BCoV-like betacoronaviruses that is cis acting for viral fitness but is not absolutely required for viral replication in cell culture.

Dynamic compression plate and cancellous bone graft for aseptic nonunion after intramedullary nailing of femoral fracture.

We evaluated the effect of revision with dynamic compression plate and cancellous bone graft for aseptic nonunion after intramedullary nailing of femoral shaft fracture. Fifty patients with aseptic nonunion of femoral shaft fracture after intramedullary nailing were reviewed and analyzed retrospectively between 1996 and 2007. There were 40 men and 10 women with an average age of 44 years (range, 19-76 years). Thirty-five were diaphyseal fractures, 8 were distal fractures, and 7 were proximal fractures. Twenty-eight fractures were defined as atrophic nonunion, 13 fractures were hypertrophic nonunion, and 9 fractures could not be defined clearly. All fractures were managed by retaining previous implants, open reduction and internal fixation with dynamic compression plate, and supplementation by cancellous bone graft. The average follow-up period was 76 months (range, 24-128 months). Functional evaluations were done by Harris Hip score and Hospital for Special Surgery knee score. All nonunions united on average at 24 weeks (range, 18-32 weeks). One superficial wound infection occurred. At follow-up, each patient was evaluated to have satisfactory function results, with near normal hip/knee functions without noticeable pain, and full return to preinjury activities/work without pain.Augmentative dynamic compression plate with cancellous bone graft is a reliable and effective treatment for revision of aseptic nonunion of femoral shaft fracture after intramedullary nailing.

Avian reovirus core protein muA expressed in Escherichia coli possesses both NTPase and RTPase activities.

Analysis of the amino acid sequence of core protein muA of avian reovirus has indicated that it may share similar functions to protein mu2 of mammalian reovirus. Since mu2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant muA ( muA) was designed and used to test these activities. muA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that muA possessed NTPase activity that enabled the protein to hydrolyse the beta-gamma phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k(cat) values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of muA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein muA. The activity of muA is dependent on the divalent cations Mg(2+) or Mn(2+), but not Ca(2+) or Zn(2+). Optimal NTPase activity of muA was achieved between pH 5.5 and 6.0. In addition, muA enzymic activity increased with temperature up to 40 degrees C and was almost totally inhibited at temperatures higher than 55 degrees C. Tests of phosphate release from RNA substrates with muA or K408A/K412A muA indicated that muA, but not K408A/K412A muA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of muA might be carried out at the same active site, and that protein muA could play important roles during viral RNA synthesis.

Genetic variation of the lambdaA and lambdaC protein encoding genes of avian reoviruses.

Sequence and phylogenetic analysis of lambdaA and lambdaC protein encoding genes of 12 avian reoviruses is described. The sequence of lambdaA possesses a variable region (residues 19-51) located within a conserved hydrophilic region (residues 1-110) and a C(2)H(2) zinc-binding motif (residues 182-202). lambdaC shows the two conserved K residues at positions 169 and 188 indicative of guanylyltransferase activity, an ATP/GTP-binding site motif A (residues 379-386), and a conserved S-adenosyl-l-methionine-binding motif (residues 822-830). Pairwise sequence comparisons show that the mean sequence identities of lambdaA encoding genes and lambdaA proteins are 92% and 98%, respectively, and those of lambdaC encoding genes and lambdaC proteins are 91% and 95%, respectively. Phylogenetic analysis of lambdaA and lambdaC encoding genes reveals that both encoding genes have diverged into three distinct lineages, respectively, and that there is no correlation between lineages and viral serotypes or pathotypes. Also, reassortment of gene segments L1 and L3 has been observed between viruses.

The sequence and phylogenetic analysis of avian reovirus genome segments M1, M2, and M3 encoding the minor core protein muA, the major outer capsid protein muB, and the nonstructural protein muNS.

The sequences and phylogenetic analyses of the M-class genome segments of 12 avian reovirus strains are described. The S1133 M1 genome segment is 2283 base pairs long, encoding a protein muA consisted of 732 amino acids. Each M2 or M3 genome segment of 12 avian reovirus strains is 2158 or 1996 base pairs long, respectively, encoding a protein muB or muNS consisted of 676 and 635 amino acids, respectively. The S1133 genome segment has the 5' GCUUUU terminal motif, but each M2 and M3 genome segment displays the 5' GCUUUUU terminal motif which is common to other known avian reovirus genome segments. The UCAUC 3'-terminal sequences of the M-class genome segments are shared by both avian and mammalian reoviruses. Noncoding regions of both 5'- and 3'-termini of the S1133 M1 genome segment consist of 12 and 72 nucleotides, respectively, those of each M2 genome segment consist of 29 and 98 nucleotides, respectively, and those of each M3 genome segment are 24 and 64 nucleotides, respectively. Analysis of the average degree of the M-class gene and the deduced mu-class protein sequence identities indicated that the M2 genes and the muB proteins have the greatest level of sequence divergence. Computer searches revealed that the muA possesses a sequence motif (NH(2)-Leu-Ala-Leu-Asp-Pro-Pro-Phe-COOH) (residues 458-464) indicative of N-6 adenine-specific DNA methylase. Examination of the muB amino acid sequences indicated that the cleavage site of muB into muBN and muBC is between positions 42 and 43 near the N-terminus of the protein, and this site is conserved for each protein. During in vitro treatment of virions with trypsin to yield infectious subviral particles, both the N-terminal fragment delta and the C-terminal fragment phi were shown to be generated. The site of trypsin cleavage was identified in the deduced amino acid sequence of muB by determining the amino-terminal sequences of phi proteins: between arginine 582 and glycine 583. The predicted length of delta generated from muBC is very similar to that of delta generated from mammalian reovirus mu1C. Taken together, protein muB is structurally, and probably functionally, similar to its mammalian homolog, mu1. In addition, two regions near the C-terminal and with a propensity to form alpha-helical coiled-coil structures as previously indicated are observed for each protein muB. Phylogenetic analysis of the M-class genes revealed that the predicted phylograms delineated 3 M1, 5 M2, and 2 M3 lineages, no correlation with serotype or pathotype of the viruses. The results also showed that M2 lineages I-V consist of a mixture of viruses from the M1 and M3 genes of lineages I-III, reflecting frequent reassortment of these genes among virus strains.

Evidence of nucleotidyl phosphatase activity associated with core protein sigma A of avian reovirus S1133.

Both avian reovirus core protein sigma A purified from virus-infected cell extracts and the purified bacterially expressed protein sigma A (e sigma A) were characterized for their nucleoside triphosphate (NTP) hydrolysis activity by thin-layer chromotography. Protein sigma A from both preparations has a nonspecific nucleotidyl phosphatase activity that hydrolyzes four types of NTP to their corresponding nucleoside di- and monophosphates and free phosphate. The divalent cation requirement for this activity of e sigma A was further examined by the addition of Mn(2+), Mg(2+), Ca(2+), and Zn(2+) ions. NTP hydrolysis by e sigma A was maximal when Mn(2+), Mg(2+), or Ca(2+) concentrations were 5, 4, or 1 mM, respectively. Addition of Mn(2+) or Mg(2+) stimulated the reactions up to 4- or 3-fold, respectively, higher than Ca(2+) (2.2-fold). However, Zn(2+) ion inhibited this activity of e sigma A. The results suggest that nucleotidyl phosphatase activity of e sigma A is absolutely dependent on the divalent cations Mn(2+), Mg(2+), or Ca(2+), but not Zn(2+). Similar results were obtained from the analysis of divalent cation requirements for the protein sigma A nucleotidyl phosphatase activity. Optimal pH for nucleotidyl phosphatase activity of protein sigma A from both preparations was determined using reaction mixtures buffered at different pH. The results show that the optimal activities of both proteins were similar and were achieved between pH 7.5 and 8.5.