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

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

Median cleft of mandible and lower lip with ankyloglossia and ectopic minor salivary gland on tongue

Median cleft of lower lip and mandible is a rare anomaly. This Cleft has also been described as Cleft No. 30 of Tessier′s classification. In minor forms only lower lip is cleft. Frequently, the cleft extends into the mandibular symphysis and the tongue is attached to the cleft alveolar margin. At times the tongue may be bifid or absent, hyoid absent, thyroid cartilage underdeveloped, strap muscles atrophic, manubrium sterni absent, clavicles widely spaced etc. The earliest report of this anomaly was by Couronne′ in 1819. Since then very few cases have been reported in literature with variations. We describe a male child who presented at the age of 6 months with an ectopic salivary gland on the dorsum of the tongue in addition to median cleft of lower lip, ankyloglossia and notching of the mandible. Excision of mass on dorsum of tongue, release of ankyloglossia and lip from the alveolus followed by repair was done. No bony work was done since the mandible was only notched. On post-operative follow-up at 18 months, dentition was delayed in both maxillary as well as mandibular teeth and there was a gap between the lower central incisors. At the age of 2 years 4 months, the dentition is still not complete and the gap between the lower central incisors is very apparent. There is a supernumerary upper central incisor on right side. There is no mobility between the two segments of mandible. Speech is normal. A regular follow-up will be done to study the eruption of permanent central incisors at the age of 7 years and till eruption of all permanent teeth to assess the occlusion and to decide whether any bony work is needed or not

Median cleft of mandible and lower lip with ankyloglossia and ectopic minor salivary gland on tongue

Median cleft of lower lip and mandible is a rare anomaly. This Cleft has also been described as Cleft No. 30 of Tessier's classification. In minor forms only lower lip is cleft. Frequently, the cleft extends into the mandibular symphysis and the tongue is attached to the cleft alveolar margin. At times the tongue may be bifid or absent, hyoid absent, thyroid cartilage underdeveloped, strap muscles atrophic, manubrium sterni absent, clavicles widely spaced etc. The earliest report of this anomaly was by Couronne' in 1819. Since then very few cases have been reported in literature with variations. We describe a male child who presented at the age of 6 months with an ectopic salivary gland on the dorsum of the tongue in addition to median cleft of lower lip, ankyloglossia and notching of the mandible. Excision of mass on dorsum of tongue, release of ankyloglossia and lip from the alveolus followed by repair was done. No bony work was done since the mandible was only notched. On post-operative follow-up at 18 months, dentition was delayed in both maxillary as well as mandibular teeth and there was a gap between the lower central incisors. At the age of 2 years 4 months, the dentition is still not complete and the gap between the lower central incisors is very apparent. There is a supernumerary upper central incisor on right side. There is no mobility between the two segments of mandible. Speech is normal. A regular follow-up will be done to study the eruption of permanent central incisors at the age of 7 years and till eruption of all permanent teeth to assess the occlusion and to decide whether any bony work is needed or not

Preparation of Plaster Moulage (Cast) in Plastic Surgery patients

The purpose of this paper is to describe the technique of making casts using alginate compound for negative and dental stone plaster for positive impressions. With certain modifications a cast could be made of any part of the body and one can make a museum of interesting cases. Casts serve as useful teaching material especially in cleft lip and palate patients to study the effect of surgery on growth and development of the cleft lip-palate-nose complex in relation to the remaining face. It also helps in planning reconstruction in cases of facial defects, recording serial changes in multistage surgery, pre-operative and post-operative comparison as in rhinoplasty, ear reconstruction, hand etc; for comparing results before and after treatment in keloid and hypertrophic scars, fabrication of implants and preparation of prosthesis. In spite of newer modalities like 3-D imaging and stereolithography, the usefulness of this old technique in certain interesting cases can not be denied

Median cleft of mandible and lower lip with ankyloglossia and ectopic minor salivary gland on tongue

Median cleft of lower lip and mandible is a rare anomaly. This Cleft has also been described as Cleft No. 30 of Tessier's classification. In minor forms only lower lip is cleft. Frequently, the cleft extends into the mandibular symphysis and the tongue is attached to the cleft alveolar margin. At times the tongue may be bifid or absent, hyoid absent, thyroid cartilage underdeveloped, strap muscles atrophic, manubrium sterni absent, clavicles widely spaced etc. The earliest report of this anomaly was by Couronne' in 1819. Since then very few cases have been reported in literature with variations. We describe a male child who presented at the age of 6 months with an ectopic salivary gland on the dorsum of the tongue in addition to median cleft of lower lip, ankyloglossia and notching of the mandible. Excision of mass on dorsum of tongue, release of ankyloglossia and lip from the alveolus followed by repair was done. No bony work was done since the mandible was only notched. On post-operative follow-up at 18 months, dentition was delayed in both maxillary as well as mandibular teeth and there was a gap between the lower central incisors. At the age of 2 years 4 months, the dentition is still not complete and the gap between the lower central incisors is very apparent. There is a supernumerary upper central incisor on right side. There is no mobility between the two segments of mandible. Speech is normal. A regular follow-up will be done to study the eruption of permanent central incisors at the age of 7 years and till eruption of all permanent teeth to assess the occlusion and to decide whether any bony work is needed or not

Case Report- Sweat gland tumor (Eccrine Porocarcinoma) of scalp: A rare tumor

Eccrine Porocarcinoma is a rare neoplasm arising from sweat glands. It was first described by Pinkus and Mehregan as ′Epidermotropic eccrine carcinoma′. It may occur de novo or as a malignant transformation of an eccrine poroma. It is commonly found in older age group and in the lower extremities. Clinically, it may present as a verrucous plaque, polypoid growth or an ulcerative lesion of long duration. Local recurrence and metastasis to skin, lymphnodes, viscera, and bone may occur. Treatment is wide local excision. Metastatic lesions can be treated with chemotherapy. We report a case of eccrine porocarcinoma of the scalp in a 50 years old female who presented to us with a bosselated, firm, painless, non-tender, freely mobile swelling over left fronto-parietal region of 12 years duration. It was excised and histopathological diagnosis was Eccrine Porocarcinoma. In literature, scalp porocarcinoma is a very rare tumor

The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing

<div><p>Ebola virus and Marburg virus are members of the <i>Filovirdae</i> family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.</p></div