Identification of novel mammalian viruses in tree shrews ( Tupaia belangeri chinensis).

The Chinese tree shrew ( Tupaia belangeri chinensis), a member of the mammalian order Scandentia, exhibits considerable similarities with primates, including humans, in aspects of its nervous, immune, and metabolic systems. These similarities have established the tree shrew as a promising experimental model for biomedical research on cancer, infectious diseases, metabolic disorders, and mental health conditions. Herein, we used meta-transcriptomic sequencing to analyze plasma, as well as oral and anal swab samples, from 105 healthy asymptomatic tree shrews to identify the presence of potential zoonotic viruses. In total, eight mammalian viruses with complete genomes were identified, belonging to six viral families, including Flaviviridae, Hepeviridae, Parvovirinae, Picornaviridae, Sedoreoviridae, and Spinareoviridae. Notably, the presence of rotavirus was recorded in tree shrews for the first time. Three viruses - hepacivirus 1, parvovirus, and picornavirus - exhibited low genetic similarity (<70%) with previously reported viruses at the whole-genome scale, indicating novelty. Conversely, three other viruses - hepacivirus 2, hepatovirus A and hepevirus - exhibited high similarity (>94%) to known viral strains. Phylogenetic analyses also revealed that the rotavirus and mammalian orthoreovirus identified in this study may be novel reassortants. These findings provide insights into the diverse viral spectrum present in captive Chinese tree shrews, highlighting the necessity for further research into their potential for cross-species transmission.

Pathogenicity and Structural Basis of Zika Variants with Glycan Loop Deletions in the Envelope Protein.

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

Minimally invasive pedicle screw fixation, including the fractured vertebra, combined with percutaneous vertebroplasty for treatment of acute thoracolumbar osteoporotic compression fracture in middle-age and elderly individuals: A prospective clinical study.

ABSTRACT: To evaluate the feasibility, efficacy, and safety of minimally invasive pedicle screw (MIPS) fixation, including the fractured vertebra, combined with percutaneous vertebroplasty (PVP) for the treatment of acute thoracolumbar osteoporotic compression fracture in middle-age and elderly individuals.Between January 2016 and August 2019, a total of 30 patients, with a mean age of 69.4 years (range, 58-75 years), who experienced thoracic or lumbar fracture without neurological deficits, underwent the MIPS procedure combined with PVP. Preoperative and postoperative pain were assessed using a visual analog scale and Oswestry Disability Index. Cobb angles and anterior column height were measured on lateral radiographs before surgery and at 3 days, 1, 3, and 6 months, and 1 and 2 years at final follow-up after surgery.All patients underwent surgery successfully, with a mean follow-up of 18.2 ±â€Š5.7 months (range, 12-45 months). Mean preoperative visual analog scale score decreased from 7.3 ±â€Š2.2 to 1.4 ±â€Š0.3 at the final follow-up (P < .05). Mean preoperative Oswestry Disability Index decreased from 84.2 ±â€Š10.3 to 18.8 ±â€Š7.5 (P < .05) at the final follow-up. The Kyphosis angle of operative segment was improved from preoperative (21.38 ±â€Š1.68)° to (4.01 ±â€Š1.38)° 3 days postoperatively and (5.02 ±â€Š1.09)° at final follow-up (P < .05). The anterior vertebral height was improved from preoperative (49.86 ±â€Š6.50)% to (94.01 ±â€Š1.79)% 3 days postoperatively and (91.80 ±â€Š1.88)% at final follow-up (P < .05). No significant changes in vertebral body height restoration were observed during 2 years of follow-up after surgery. In addition, there were no instrumentation failures or complications in any of the patients.MIPS, including the fractured vertebra, combined with PVP, was a reliable and safe procedure, with satisfactory clinical and radiological results for the treatment of thoracolumbar osteoporotic compression fracture in patients without neurological deficits.

Identification of a recombinant equine coronavirus in donkey, China.

Equine coronavirus (ECoV) was first identified in the USA and has been previously described in several countries. In order to test the presence of ECoV in China, we collected 51 small intestinal samples from donkey foals with diarrhoea from a donkey farm in Shandong Province, China between August 2020 and April 2021. Two samples tested positive for ECoV and full-length genome sequences were successfully obtained using next-generation sequencing, one of which was further confirmed by Sanger sequencing. The two strains shared 100% sequence identity at the scale of whole genome. Bioinformatics analyses further showed that the two Chinese strains represent a novel genetic variant of ECoV and shared the highest sequence identity of 97.05% with the first identified ECoV strain - NC99. In addition, it may be a recombinant, with the recombination region around the NS2 gene. To our knowledge, this is the first documented report of ECoV in China, highlighting its risk to horse/donkey breeding. In addition, its potential risk to public health also warrants further investigation.

A novel SARS-CoV-2 related coronavirus with complex recombination isolated from bats in Yunnan province, China.

At the end of 2019, A new type of beta-CoV, SARS-CoV-2 emerged and triggered the COVID-19 pandemic, which spread overwhelmingly around the world in less than a year. However, the origin and direct ancestral viruses of SARS-CoV-2 remain unknown. RaTG13, a novel coronavirus found in bats in China's Yunnan Province, is the closest relative virus of the SARS-CoV-2 identified so far. In this study, a new SARS-CoV-2 related virus, provisionally named PrC31, was discovered in Yunnan province by retrospectively analyse bat next generation sequencing (NGS) data of intestinal samples collected in 2018. PrC31 shared 90.7% and 92.0% nucleotide identities to the genomes of SARS-CoV-2 and the bat SARSr-CoV ZC45, respectively. Sequence alignment of PrC31 showed that several genomic regions, especially orf1a and orf8 had the highest homology with those corresponding genomic regions of SARS-CoV-2 than any other related viruses. Phylogenetic analysis indicated that PrC31 shared a common ancestor with SARS-CoV-2 in evolutionary history. The differences between the PrC31 and SARS-CoV-2 genomes were mainly manifested in the spike genes. The amino acid homology between the receptor binding domains of PrC31 and SARS-CoV-2 was only 64.2%. Importantly, recombination analysis revealed that PrC31 underwent multiple complex recombination events (including three recombination breakpoints) involving the SARS-CoV and SARS-CoV-2 sub-lineages, indicating that PrC31 evolved from yet-to-be-identified intermediate recombination strains. Combined with previous studies, it is revealed that the beta-CoVs may possess a more complex recombination mechanism than we thought.

Genome Analysis of Coxsackievirus A4 Isolates From Hand, Foot, and Mouth Disease Cases in Shandong, China.

Coxsackievirus A4 (CVA4) is one of the most prevalent pathogens associated with hand, foot and mouth disease (HFMD), an acute febrile illness in children, and is also associated with acute localized exanthema, myocarditis, hepatitis and pancreatitis. Despite this, limited CVA4 genome sequences are currently available. Herein, complete genome sequences from CVA4 strains (n = 21), isolated from patients with HFMD in Shandong province, China between 2014 and 2016, were determined and phylogenetically characterized. Phylogenetic analysis of the VP1 gene from a larger CVA4 collection (n = 175) showed that CVA4 has evolved into four separable genotypes: A, B, C, and D; and genotype D could be further classified in to two sub-genotypes: D1 and D2. Each of the 21 newly described genomes derived from isolates that segregated with sub-genotype D2. The CVA4 genomes displayed significant intra-genotypic genetic diversity with frequent synonymous substitutions occurring at the third codon positions, particularly within the P2 region. However, VP1 was relatively stable and therefore represents a potential target for molecular diagnostics assays and also for the rational design of vaccine epitopes. The substitution rate of VP1 was estimated to be 5.12 × 10-3 substitutions/site/year, indicative of ongoing CVA4 evolution. Mutations at amino acid residue 169 in VP1 gene may be responsible for differing virulence of CVA4 strains. Bayesian skyline plot analysis showed that the population size of CVA4 has experienced several dynamic fluctuations since 1948. In summary, we describe the phylogenetic and molecular characterization of 21 complete genomes from CVA4 isolates which greatly enriches the known genomic diversity of CVA4 and underscores the need for further surveillance of CVA4 in China.

The evolution of Zika virus from Asia to the Americas.

Zika virus (ZIKV) was once considered an obscure member of the large and diverse family of mosquito-borne flaviviruses, and human infections with ZIKV were thought to be sporadic, with mild and self-limiting symptoms. The large-scale ZIKV epidemics in the Americas and the unexpected uncovering of a link to congenital birth defects escalated ZIKV infections to the status of a global public health emergency. Recent studies that combined reverse genetics with modelling in multiple systems have provided evidence that ZIKV has acquired additional amino acid substitutions at the same time as congenital Zika syndrome and other birth defects were detected. In this Progress article, we summarize the evolution of ZIKV during its spread from Asia to the Americas and discuss potential links to pathogenesis.

USP4 positively regulates RLR-induced NF-κB activation by targeting TRAF6 for K48-linked deubiquitination and inhibits enterovirus 71 replication.

Retinoic acid-inducible gene I-like receptor (RLR) is one of the most important pattern recognition receptors of the innate immune system that detects positive and/or negative stranded RNA viruses. Subsequently, it stimulates downstream transcription of interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) inducing the production of interferons (IFNs) and inflammatory cytokines. Tumour necrosis factor receptor associated factor 6 (TRAF6) is a key protein involved in the RLR-mediated antiviral signalling pathway, recruiting additional proteins to form a multiprotein complex capable of activating the NF-κB inflammatory pathway. Despite TRAF6 playing an important role in regulating host immunity and viral infection, the deubiquitination of TRAF6 induced by viral infection remains elusive. In this study, we found that enterovirus 71 (EV71) infection attenuated the expression of Ubiquitin-specific protease 4 (USP4) in vitro and in vivo, while overexpression of USP4 significantly suppressed EV71 replication. Furthermore, it was found that EV71 infection reduced the RLR signalling pathway and enhanced the degradation of TRAF6. USP4 was also found to interact with TRAF6 and positively regulate the RLR-induced NF-κB signalling pathway, inhibiting the replication of EV71. Therefore, as a novel positive regulator of TRAF6, USP4 plays an essential role in EV71 infection by deubiquitinating K48-linked ubiquitin chains.

Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin.

Cross-species transmission of viruses from wildlife animal reservoirs poses a marked threat to human and animal health 1 . Bats have been recognized as one of the most important reservoirs for emerging viruses and the transmission of a coronavirus that originated in bats to humans via intermediate hosts was responsible for the high-impact emerging zoonosis, severe acute respiratory syndrome (SARS) 2-10 . Here we provide virological, epidemiological, evolutionary and experimental evidence that a novel HKU2-related bat coronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), is the aetiological agent that was responsible for a large-scale outbreak of fatal disease in pigs in China that has caused the death of 24,693 piglets across four farms. Notably, the outbreak began in Guangdong province in the vicinity of the origin of the SARS pandemic. Furthermore, we identified SADS-related CoVs with 96-98% sequence identity in 9.8% (58 out of 591) of anal swabs collected from bats in Guangdong province during 2013-2016, predominantly in horseshoe bats (Rhinolophus spp.) that are known reservoirs of SARS-related CoVs. We found that there were striking similarities between the SADS and SARS outbreaks in geographical, temporal, ecological and aetiological settings. This study highlights the importance of identifying coronavirus diversity and distribution in bats to mitigate future outbreaks that could threaten livestock, public health and economic growth.

Murine model of acute myocarditis and cerebral cortical neuron edema induced by coxsackievirus B4.

Globally, coxsackievirus B4 (CV-B4) has been continuously isolated and evidence suggests an association with the development of pancreatitis and type I diabetes. In addition, CV-B4 is also associated with myocarditis and severe central nervous system (CNS) complications, which remain poorly studied and understood. In the present study, we established an Institute for Cancer Research (ICR) mouse model of CV-B4 infection and examined whether CV-B4 infection resulted in a predisposition to myocarditis and CNS infection. We found high survival in both the treatment and control group, with no significant differences in clinical outcomes observed. However, pathological lesions were evident in both brain and heart tissue of the CV-B4-infected mice. In addition, high viral loads were found in the neural and cardiac tissues as early as 2 days post infection. Expressions of IFN-γ and IL-6 in sera were significantly higher in CV-B4-infected mice compared to uninfected negative controls, suggesting the involvement of these cytokines in the development of histopathological lesions. Our murine model successfully reproduced the acute myocarditis and cerebral cortical neuron edema induced by CV-B4, and may be useful for the evaluation of vaccine candidates and potential antivirals against CV-B4 infection.

Intra-host dynamics of Ebola virus during 2014.

Since 2013, West Africa has encountered the largest Ebola virus (EBOV) disease outbreak on record, and Sierra Leone is the worst-affected country, with nearly half of the infections. By means of next-generation sequencing and phylogeographic analysis, the epidemiology and transmission of EBOV have been well elucidated. However, the intra-host dynamics that mainly reflect viral-host interactions still need to be studied. Here, we show a total of 710 intra-host single nucleotide variations (iSNVs) from deep-sequenced samples from EBOV-infected patients, through a well-tailored bioinformatics pipeline. We present a comprehensive distribution of iSNVs during this outbreak and along the EBOV genome. Analyses of iSNV and its allele frequency reveal that VP40 is the most conserved gene during this outbreak, and thus it would be an ideal therapeutic target. In the co-occurring iSNV network, varied iSNV sites present different selection features. Intriguingly, the T-to-C substitutions at the 3'-UTR of the nucleoprotein (NP; positions 3008 and 3011), observed in many patients, result in the upregulation of the transcription of NP through an Ebola mini-genome reporting system. Additionally, no iSNV enrichment within B-cell epitopes of GP has been observed.

Genetic diversity and evolutionary dynamics of Ebola virus in Sierra Leone.

A novel Ebola virus (EBOV) first identified in March 2014 has infected more than 25,000 people in West Africa, resulting in more than 10,000 deaths. Preliminary analyses of genome sequences of 81 EBOV collected from March to June 2014 from Guinea and Sierra Leone suggest that the 2014 EBOV originated from an independent transmission event from its natural reservoir followed by sustained human-to-human infections. It has been reported that the EBOV genome variation might have an effect on the efficacy of sequence-based virus detection and candidate therapeutics. However, only limited viral information has been available since July 2014, when the outbreak entered a rapid growth phase. Here we describe 175 full-length EBOV genome sequences from five severely stricken districts in Sierra Leone from 28 September to 11 November 2014. We found that the 2014 EBOV has become more phylogenetically and genetically diverse from July to November 2014, characterized by the emergence of multiple novel lineages. The substitution rate for the 2014 EBOV was estimated to be 1.23 × 10(-3) substitutions per site per year (95% highest posterior density interval, 1.04 × 10(-3) to 1.41 × 10(-3) substitutions per site per year), approximating to that observed between previous EBOV outbreaks. The sharp increase in genetic diversity of the 2014 EBOV warrants extensive EBOV surveillance in Sierra Leone, Guinea and Liberia to better understand the viral evolution and transmission dynamics of the ongoing outbreak. These data will facilitate the international efforts to develop vaccines and therapeutics.

Interoperative radiotherapy of seventy-two cases of early breast cancer patients during breast-conserving surgery.

OBJECTIVE: To evaluate interoperative radiotherapy after breast conservative surgery in early breast cancer patients in terms of postoperative complications, cosmetic outcome and recurrence events. METHODS: From June 2007 to Dec 2011, 143 early breast cancer patients received breast conservative surgery. Seventy-two (study group) received interoperative radiotherapy, compared with 71 patients (control group) given routine radiotherapy. Postoperative complications were evaluated 1 month after surgery; cosmetic outcome was evaluated 1 year postoperatively; recurrence and death events were followed up. RESULTS: The average wound healing time was 13~22 d in the study group and 9~14 d in the control group. In the study group, 2 patients developed lyponecrosis, 16 patients showed wound edema while no such side effects were found in the control group. No infection or hematomas were found in either group. In the study group (59 cases), overall cosmetic outcome in 53 patients was graded as excellent or good, and in 6 as fair or poor. Meanwhile in the control group (56 cases), 42 patients were graded as excellent or good, and 14 as fair or poor (P=0.032). After a follow-up from 3 to 54 months (median: 32 months), two patients (2.78%) in study group developed local relapses, one of them (1.39%) died, 2 patients (2.78%) developed bone metastases. In control group, one patient (1.41%) developed local relapse, 2 patients (2.82%) developed bone metastases, and no one died. CONCLUSION: Intraoperative radiotherapy is safe and reliable with good cosmetic outcome.

[Experimental study on invigorating kidney and activating blood on preventing and curing SD rats with knee osteoarthritis].

OBJECTIVE: To explore the effects and mechanism of invigorating kidney and activating blood, invigorating kidney and expelling wind on hemorheology, IL-1ß and TNF-α of SD rats with knee osteoarthritis, then definite the evolution of muscle certified turning into heumatism and compare the effect of Chinese herbal. METHODS: One hundred and eighty SD rats with 3-month-old (each weight was 185 to 215 g) received intra-articular injection of papain solution for establishing knee OA models. All rats were randomly divided into activating blood group, preventing group, expelling wind group, invigorating kidney group, invigorating kidney and activating blood group and model group. Laboratory indexes were obtained at the 30th, 60th, 90th days after gastric perfusion, which including state of mind, activity, fur, weight, joint swelling, largely image, hemorheology, inflammation and HE pathological appearance. RESULTS: After operation, rats appeared blood stasis and swelling and difficulty crawling. There was significant difference of hemorheology in invigorating kidney and activating blood group the content of IL-1ß and TNF-α was obviously lower than model group (P < 0.05 ). While the content of IL-1ß and TNF-α on the early stage was obviously higher than late stage (P < 0.05). CONCLUSION: Knee osteoarthritis mainly show synovial inflammation at the early stage, inflammation at early stage is more severe than late; invigorating kidney and activating blood decoction can inhibit the knee cartilage injury, improve blood circulation and prevent local inflammatory reaction. Activating blood decoction and invigorating kidney and activating blood Decoction have certain curative effect in early time, but the effects of invigorating kidney and activating blood Decoction is more effective than other on the late stage.

Transconjugation and genotyping of the plasmid-mediated AmpC beta-lactamase and extended-spectrum beta-lactamase genes in Klebsiella pneumoniae.

BACKGROUND: AmpC beta-lactamases and extended-spectrum beta-lactamases (ESBLs) are becoming predominant causes of resistance to third and forth-generation cephalosporins in Klebsiella pneumoniae (K. pneumoniae). It is very difficult to treat infectious diseases caused by multidrug-resistant K. pneumoniae. The purpose of the present study was to investigate transconjugation and characteristics of beta-lactamase genes in K. pneumoniae producing AmpC beta-lactamases and ESBLs. METHODS: AmpC beta-lactamases were detected by three-dimension test and ESBLs by disc confirmatory test. Minimum inhibitory concentrations (MICs) were determined by agar dilution. Transfer of resistance to EC600 (Rif(r)) was attempted by conjugation in broth and screened on agar containing cefotaxime (2 microg/ml) plus rifampin (1024 microg/ml). The genes encoding AmpC or ESBLs and their transconjugants were detected by PCR and verified by DNA sequencing. RESULTS: The resistant rates to ampicillin and piperacillin were 100% in 18 isolates of K. pneumoniae. However, imipenem was still of great bactericidal activity on K. pneumoniae, and its MIC(50) was 0.5 microg/mL. Eleven beta-lactamase genes, including TEM-1, TEM-11, SHV-13, SHV-28, CTX-M-9, CTX-M-22, CTX-M-55, OXA-1, LEN, OKP-6 and DHA-1, were found from 18 isolates. And at least one beta-lactamase gene occurred in each isolate. To our surprise, there were six beta-lactamase genes in the CZ04 strain. Among 18 isolates of K. pneumoniae, the partial resistant genes in 8 isolates were conjugated successfully, which had 100% homological sequence with donors by sequence analysis. Compared with donors, 8 transconjugants had attained resistance to most beta-lactams, including ampicillin, piperacillin, cefoxitin, cefotaxime and aztreonam, or even amikacin and gentamicin. CONCLUSIONS: R plasmids can be easily transferred between the resistant and sensitive negative bacilli. It is very difficult to block and prevent the spread of antimicrobial resistance. So more attention should be paid to reducing the frequency, times and dosage of antimicrobials, especially third or fourth cephalosporins.