Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation

BACKGROUND: The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome. RESULTS: The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads. CONCLUSIONS: The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories

Instrumentation in Maxillofacial Surgery: Few Practical Tips

When a newly inducted plastic surgery resident embarks on maxillofacial surgery, with drills, screws, plates and burrs, it seems like a new domain altogether. As a new resident, it is truly fascinating as to how such wide variety of bony work is done without scarring over the face. Here we discuss a few practical tips which the author has learned during his surgical sojourn in residency. It is hoped that the readers who are new to maxillofacial surgery, shall find these useful

Education in plastic surgery: Are we headed in the right direction?

Introduction: Plastic surgery in India is in an era of transition. The speciality faces many challenges as it grows. The present study attempts to identify these challenges and the prevalent mood among the teachers and the trainees. Materials and Methods: The study was conducted from September 2011 to June 2012. In an E-mail based survey a questionnaire was mailed to professionals actively involved in teaching and training of residents in plastic surgery in many institutes running MCh courses in plastic surgery (Group I) [Appendix 1]. Another questionnaire was mailed to residents undergoing training in plastic surgery and those who had completed their training within past 2 years (Group II) [Appendix 2]. Chi-square test was applied to test for statistical significance. Observations: 29 Group I and 33 Group II subjects responded to the questionnaire. While 72.4% teachers believed that the current system is producing plastic surgeons with enough skill level, only 9.1% of the respondents in Group II thought the same (Chi-square = 28.1; df = 2; P < 0.001). Whereas 58.6% Group I respondents thought that their student is sufficiently equipped to compete in today’s scenario [Figure 1], only 18.2% Group II respondents thought that their training is enough [Figure 2]. (Chi-square = 16.4; df = 2; P < 0.001). Nearly 28% respondents in Group I and only 3% in Group II thought that scientific research and publications should be made mandatory for successful completion of plastic surgery training (Chi-square = 9.4; df = 2; P = 0.009). Adequate exposure was thought to be available in general plastic surgery (Group I: 92% Group II: 81%), maxillofacial surgery (Group I: 72% Group II: 68%) and hand surgery (Group I: 84% Group II: 69%). Both groups agreed that exposure is lacking in craniofacial surgery, aesthetic surgery and microvascular surgery. Aesthetic surgery (38.7%) and microvascular surgery (32.6%) were the most frequent response when the Group II respondents were enquired about the subspeciality they would like to focus on in their practice. Inter-departmental exchange of students for limited period of time was favoured by 86.2% of Group I respondents and 93.9% Group II respondents (Chi-square = 1.3; df = 2; P = 0.49). Conclusion: The current training programme is differently perceived by teachers and the trainees. We recommend that constant deliberations at national and regional forums should take place regarding our education and training programmes