Towards Starting a Hand Transplant Unit and Achieving Success in a Hand Transplant: The Standard Operating Procedure.

Vascularized Composite Tissue Allotransplantation (VCA) allows replacement of lost body parts from brain-dead donors. These surgeries are laborious, time-intensive, and require vast planning. With the advent of better immunosuppressants, VCA will increasingly play an important role in the reconstructive field. In this paper, the authors share their standard operating protocol created after much deliberation.

Influence of Sex on Respiratory Syncytial Virus Genotype Infection Frequency and Nasopharyngeal Microbiome.

Respiratory syncytial virus (RSV) has a significant health burden in children, older adults, and the immunocompromised. However, limited effort has been made to identify emergence of new RSV genotypes' frequency of infection and how the combination of nasopharyngeal microbiome and viral genotypes impact RSV disease outcomes. In an observational cohort designed to capture the first infant RSV infection, we employed multi-omics approaches to sequence 349 RSV complete genomes and matched nasopharyngeal microbiomes, during which the 2012/2013 season was dominated by RSV-A, whereas 2013 and 2014 was dominated by RSV-B. We found non-G-72nt-duplicated RSV-A strains were more frequent in male infants (P = 0.02), whereas G-72nt-duplicated genotypes (which is ON1 lineage) were seen equally in both males and females. DESeq2 testing of the nasal microbiome showed Haemophilus was significantly more abundant in infants with RSV-A infection compared to infants with RSV-B infection (adjusted P = 0.002). In addition, the broad microbial clustering of the abundant genera was significantly associated with infant sex (P = 0.03). Overall, we show sex differences in infection by RSV genotype and host nasopharyngeal microbiome, suggesting an interaction between host genetics, virus genotype, and associated nasopharyngeal microbiome. IMPORTANCE Respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract infections in young children and is responsible for high hospitalization rates and morbidity in infants and the elderly. To understand how the emergence of RSV viral genotypes and viral-respiratory microbiome interactions contribute to infection frequency and severity, we utilized an observational cohort designed to capture the first infant RSV infection we employed multi-omics approaches to sequence 349 RSV complete genomes and matched nasopharyngeal microbiomes. We found non-G-72nt-duplicated RSV-A genotypes were more frequent in male infants, whereas G-72nt-duplicated RSV-A strains (ON1 lineage) were seen equally in both males and females. Microbiome analysis show Haemophilus was significantly more abundant in infants with RSV-A compared to infants with RSV-B infection and the microbial clustering of the abundant genera was associated with infant sex. Overall, we show sex differences in RSV genotype-nasopharyngeal microbiome, suggesting an interaction host genetics-virus-microbiome interaction.

Molecular characterization of respiratory syncytial viruses circulating in a paediatric cohort in Amman, Jordan.

Respiratory syncytial viruses (RSVs) are an important cause of mortality worldwide and a major cause of respiratory tract infections in children, driving development of vaccine candidates. However, there are large gaps in our knowledge of the local evolutionary and transmission dynamics of RSVs, particularly in understudied regions such as the Middle East. To address this gap, we sequenced the complete genomes of 58 RSVA and 27 RSVB samples collected in a paediatric cohort in Amman, Jordan, between 2010 and 2013. RSVA and RSVB co-circulated during each winter epidemic of RSV in Amman, and each epidemic comprised multiple independent viral introductions of RSVA and RSVB. However, RSVA and RSVB alternated in dominance across years, potential evidence of immunological interactions. Children infected with RSVA tended to be older than RSVB-infected children [30 months versus 22.4 months, respectively (P value = 0.02)], and tended to developed bronchopneumonia less frequently than those with RSVB, although the difference was not statistically significant (P value = 0.06). Differences in spatial patterns were investigated, and RSVA lineages were often identified in multiple regions in Amman, whereas RSVB introductions did not spread beyond a single region of the city, although these findings were based on small sample sizes. Multiple RSVA genotypes were identified in Amman, including GA2 viruses as well as three viruses from the ON1 sub-genotype that emerged in 2009 and are now the dominant genotype circulating worldwide. As vaccine development advances, further sequencing of RSV is needed to understand viral ecology and transmission, particularly in under-studied locations.

A Review of Skin Banking Guidelines and Standards Worldwide: Towards the Harmonization of Guidelines for Skin Banking in Therapeutic Applications for the Regions under the Asia Pacific Burn Association (APBA).

Currently, there are no harmonized guidelines which govern skin banking in the Asia Pacific region. Therefore, skin banks are either unregulated or rely on their nation's legislation or international accreditation to uphold their quality standards. A new set of skin banking guidelines was developed through a comprehensive review and collation of best international practices for the Asia Pacific Burn Association (APBA) members, from donor screening and testing, to skin recovery, processing, storage and distribution, and quality assurance. National regulatory requirements reviewed include the European directives, Australia's Therapeutic Goods Administration and Singapore's tissue banking standards. Further technical and quality management recommendations are referenced from the American Association of Tissue Banks (AATB), the United States Food and Drug Administration standards and guidance documents, various relevant European guides, Japanese Society of Tissue Transplantation guidelines and the Asia Pacific Association of Surgical Tissue Banking. Adapted mainly from the AATB standards, the new Asia Pacific Burn Association Guidelines for Skin Banking in Therapeutic Applications offer a comprehensive manual, addressing: governance and contracts; staff responsibilities; quality management; facilities, equipment and supplies management; donor consent and testing; and recommendations of good practices pertaining to skin recovery, processing, storage and distribution. Besides complementing current generic regulations, they provide technical specifications of major aspects unaddressed in most legislations. This inaugural set of new regional skin banking guidelines would be a start for regional members of the APBA to adopt, and will hopefully culminate in a set of standards so that, in the long run, skin allografts from this region can be of similar quality, which can simplify import process and facilitate the exchange of allografts between members.

A broadly neutralizing germline-like human monoclonal antibody against dengue virus envelope domain III.

Dengue is the most widespread vector-borne viral disease caused by dengue virus (DENV) for which there are no safe, effective drugs approved for clinical use. Here, by using sequential antigen panning of a yeast antibody library derived from healthy donors against the DENV envelop protein domain III (DIII) combined with depletion by an entry defective DIII mutant, we identified a cross-reactive human monoclonal antibody (mAb), m366.6, which bound with high affinity to DENV DIII from all four DENV serotypes. Immunogenetic analysis indicated that m366.6 is a germline-like mAb with very few somatic mutations from the closest VH and Vλ germline genes. Importantly, we demonstrated that it potently neutralized DENV both in vitro and in the mouse models of DENV infection without detectable antibody-dependent enhancement (ADE) effect. The epitope of m366.6 was mapped to the highly conserved regions on DIII, which may guide the design of effective dengue vaccine immunogens. Furthermore, as the first germline-like mAb derived from a naïve antibody library that could neutralize all four DENV serotypes, the m366.6 can be a tool for exploring mechanisms of DENV infection, and is a promising therapeutic candidate.

Childhood Burns Leading to Skeletal and Soft Tissue Deformities: A Case Report.

Postburn contracture is an undesirable sequel of burn injury which affects patients in many ways. A secondary deformity of the growing skeleton in case of pediatric burn patients is a challenging problem to reconstructive surgeons, and careful planning and staged correction is required for such patients. Here we discuss a case of burn injury sustained in childhood and its consequent deformities and their management.

Optimization of qRT-PCR assay for zika virus detection in human serum and urine.

Zika Virus (ZIKV) is a mosquito-borne flavivirus that the World Health Organization (WHO) declared a global concern due to the severity of infection. This study focuses on determining the level of detection of ZIKV RNA in human serum and urine. Known amounts of Zika virus were added to uninfected human serum and urine samples. Different reverse transcriptases were compared to select the optimal enzyme for this application. Zika RNA in these samples was then quantified with qRT-PCR to determine the lower limit of detection in these fluids and to construct a standard curve. Student's t-test of paired samples was used in order to identify statistical differences. The SuperScript III enzyme was able to produce more ZIKV cDNA when compared to PrimeScript. Zika virus RNA was found to be detectable at lower levels (2.5 PFU/mL) in urine than in serum (250 PFU/mL) when using SuperScript III. This study demonstrates how the selection of both the human clinical specimen, and the reverse transcriptase enzyme involved in the molecular detection of ZIKV by quantitative real-time polymerase chain reaction (qRT-PCR), play an important role in enabling improved detection of the virus.

The scourge of burn contractures: Who will bell the cat?

INTRODUCTION: Majority of burn injuries occur in developing world. There have been many advances in burn care management. But this advance comes at a price of ever increasing burn sequel of scarring. Burn scar contractures, in spite of being preventable, continue to be a common reality in our world. This paper aims to evaluate the lacunae in burn care leading to formation of contractures, to evaluate common sites of contractures, practice of advising splintage and anti-deformity positioning. PROCEDURE: This retrospective observational study was conducted over a period of six years (January 2010-December 2015) at a tertiary Burn unit in Mumbai (India). The records of burn contracture patients were scrutinized to obtain the data regarding patient's socio-demographic profile, details of burn injury, splinting and exercises advised and details of surgeries required. Data was tabulated and analysed. RESULTS: There was a significantly lower prevalence of advice regarding splinting, mobilization exercises and pressure garments. The prevalence of early surgery was also found to be low. CONCLUSIONS: Attempt has been made to identify the factors affecting the prevalence of burn scar contractures. Authors recommend that efforts be made for improving the knowledge of the treating doctors. Also, efforts should be made to increase awareness of burn prevention and emergent management.

Whole genome sequencing, variant analysis, phylogenetics, and deep sequencing of Zika virus strains.

The recent emergence of Zika virus (ZIKV) has been concentrated in the Caribbean, Southeastern United States, and South- and Central America; resulting in travel-based cases being reported around the globe. As multi-disciplinary collaborations are combatting the ZIKV outbreak, the need to validate the sequence of existing strains has become apparent. Here, we report high-quality sequence data for multiple ZIKV strains made publicly available through the National Institutes of Health- (NIH) funded biorepository, BEI Resources (www.beiresources.org). Next-generation sequencing, 3' rapid amplification of cDNA ends (RACE), and viral genome annotation pipelines generated GenBank sequence records for 16 BEI Resources strains. Minor variants, consensus mutations, and consensus insertions/deletions were identified within the viral stocks using next-generation sequencing (NGS) and consensus changes were confirmed with Sanger sequencing. Bioinformatics analyses of the sequencing results confirm that the virus stocks available to the scientific research community through BEI Resources adequately represent the viral population diversity of ZIKV.

Differing epidemiological dynamics of Chikungunya virus in the Americas during the 2014-2015 epidemic.

Chikungunya virus (CHIKV) has been detected sporadically since the 1950s and includes three distinct co-circulating genotypes. In late 2013, the Asian genotype of CHIKV was responsible for the Caribbean outbreak (CO) that rapidly became an epidemic throughout the Americas. There is a limited understanding of the molecular evolution of CHIKV in the Americas during this epidemic. We sequenced 185 complete CHIKV genomes collected mainly from Nicaragua in Central America and Florida in the United States during the 2014-2015 Caribbean/Americas epidemic. Our comprehensive phylogenetic analyses estimated the epidemic history of the Asian genotype and the recent Caribbean outbreak (CO) clade, revealed considerable genetic diversity within the CO clade, and described different epidemiological dynamics of CHIKV in the Americas. Specifically, we identified multiple introductions in both Nicaragua and Florida, with rapid local spread of viruses in Nicaragua but limited autochthonous transmission in Florida in the US. Our phylogenetic analysis also showed phylogeographic clustering of the CO clade. In addition, we identified the significant amino acid substitutions that were observed across the entire Asian genotype during its evolution and examined amino acid changes that were specific to the CO clade. Deep sequencing analysis identified specific minor variants present in clinical specimens below-consensus levels. Finally, we investigated the association between viral phylogeny and geographic/clinical metadata in Nicaragua. To date, this study represents the largest single collection of CHIKV complete genomes during the Caribbean/Americas epidemic and significantly expands our understanding of the emergence and evolution of CHIKV CO clade in the Americas.

Sequences of Zika Virus Genomes from a Pediatric Cohort in Nicaragua.

We report here the whole-genome sequence of 11 Zika virus (ZIKV) samples from six pediatric patients in Nicaragua. Serum samples were collected, and ZIKV was isolated in tissue culture. Both serum and virus isolates were sequenced. The consensus ZIKV genomes are greater than 99% identical to each other.

Large-Scale Complete-Genome Sequencing and Phylodynamic Analysis of Eastern Equine Encephalitis Virus Reveals Source-Sink Transmission Dynamics in the United States.

Eastern equine encephalitis virus (EEEV) has a high case-fatality rate in horses and humans, and Florida has been hypothesized to be the source of EEEV epidemics for the northeastern United States. To test this hypothesis, we sequenced complete genomes of 433 EEEV strains collected within the United States from 1934 to 2014. Phylogenetic analysis suggested EEEV evolves relatively slowly and that transmission is enzootic in Florida, characterized by higher genetic diversity and long-term local persistence. In contrast, EEEV strains in New York and Massachusetts were characterized by lower genetic diversity, multiple introductions, and shorter local persistence. Our phylogeographic analysis supported a source-sink model in which Florida is the major source of EEEV compared to the other localities sampled. In sum, this study revealed the complex epidemiological dynamics of EEEV in different geographic regions in the United States and provided general insights into the evolution and transmission of other avian mosquito-borne viruses in this region.IMPORTANCE Eastern equine encephalitis virus (EEEV) infections are severe in horses and humans on the east coast of the United States with a >90% mortality rate in horses, an ∼33% mortality rate in humans, and significant brain damage in most human survivors. However, little is known about the evolutionary characteristics of EEEV due to the lack of genome sequences. By generating large collection of publicly available complete genome sequences, this study comprehensively determined the evolution of the virus, described the epidemiological dynamics of EEEV in different states in the United States, and identified Florida as one of the major sources. These results may have important implications for the control and prevention of other mosquito-borne viruses in the Americas.

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.

Whole-Genome Sequences of Zika Virus FLR Strains after Passage in Vero or C6/36 Cells.

We report 26 complete genomes of Zika virus (ZIKV) isolated after passaging the Zika virus strain FLR in mosquito (C6/36) and mammalian (Vero) cell lines. The consensus ZIKV genomes we recovered show greater than 99% nucleotide identify with each other and with the FLR strain used as input.

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.

Identification of Dezidougou Virus in a DAK AR 41524 Zika Virus Stock.

We report here the complete genome of a Dezidougou virus (DEZV) isolated from a passaged culture of the Zika virus strain DAK AR 41524. The consensus DEZV sequence we recovered shows 99% nucleotide similarity using BLASTN to a previously reported DEZV (accession no. JQ675604.1). The current sequence has additional repeat regions as well as a deleted repeat region, which we confirmed by Sanger sequencing, that were not present in the originally published sequence, JQ675604.1.

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

Ebola virus and Marburg virus are members of the Filovirdae 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.