Naturally occurring genotype 2b/1a hepatitis C virus in the United States

<p>Abstract</p> <p>Background</p> <p>Hepatitis C Virus (HCV) infected patients are frequently repeatedly exposed to the virus, but very few recombinants between two genotypes have been reported.</p> <p>Findings</p> <p>We describe the discovery of an HCV recombinant using a method developed in a United States clinical lab for HCV genotyping that employs sequencing of both 5' and 3' portions of the HCV genome. Over twelve months, 133 consecutive isolates were analyzed, and a virus from one patient was found with discordant 5' and 3' sequences suggesting it was a genotype 2b/1a recombinant. We ruled out a mixed infection and mapped a recombination point near the NS2/3 cleavage site.</p> <p>Conclusions</p> <p>This unique HCV recombinant virus described shares some features with other recombinant viruses although it is the only reported recombinant of a genotype 2 with a subtype 1a. This recombinant represents a conundrum for current clinical treatment guidelines, including treatment with protease inhibitors. This recombinant is also challenging to detect by the most commonly employed methods of genotyping that are directed primarily at the 5' structural portion of the HCV genome.</p

Functional relevance of novel p300-mediated lysine 314 and 315 acetylation of RelA/p65

Nuclear factor kappaB (NF-κB) plays an important role in the transcriptional regulation of genes involved in immunity and cell survival. We show here in vitro and in vivo acetylation of RelA/p65 by p300 on lysine 314 and 315, two novel acetylation sites. Additionally, we confirmed the acetylation on lysine 310 shown previously. Genetic complementation of RelA/p65−/− cells with wild type and non-acetylatable mutants of RelA/p65 (K314R and K315R) revealed that neither shuttling, DNA binding nor the induction of anti-apoptotic genes by tumor necrosis factor α was affected by acetylation on these residues. Microarray analysis of these cells treated with TNFα identified specific sets of genes differently regulated by wild type or acetylation-deficient mutants of RelA/p65. Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type. These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-κB dependent gene expression

Molecular typing of major histocompatibility complex class I alleles in the Indian rhesus macaque which restrict SIV CD8+ T cell epitopes

The utility of the rhesus macaque as an animal model in both HIV vaccine development and pathogenesis studies necessitates the development of accurate and efficient major histocompatibility complex (MHC) genotyping technologies. In this paper, we describe the development and application of allele-specific polymerase chain reaction (PCR) amplification for the simultaneous detection of eight MHC class I alleles from the rhesus macaque (Macaca mulatta) of Indian descent. These alleles were selected, as they have been implicated in the restriction of CD8+ T cell epitopes of simian immunodeficiency virus (SIV). Molecular typing of Mamu-A*01, Mamu-A*02, Mamu-A*08, Mamu-A*11, Mamu-B*01, Mamu-B*03, Mamu-B*04, and Mamu-B*17 was conducted in a high throughput fashion using genomic DNA. Our amplification strategy included a conserved internal control target to minimize false negative results and can be completed in less than 5 h. We have genotyped over 4,000 animals to establish allele frequencies from colonies all over the western hemisphere. The ability to identify MHC-defined rhesus macaques will greatly enhance investigation of the immune responses, which are responsible for the control of viral replication. Furthermore, application of this technically simple and accurate typing method should facilitate selection, utilization, and breeding of rhesus macaques for AIDS virus pathogenesis and vaccine studies

An atypical case of Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC)-associated renal cell carcinoma identified by next-generation sequencing

Germline mutations in the fumarate hydratase (FH) gene classically lead to Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) syndrome. Patients with HLRCC typically exhibit multiple cutaneous and uterine leiomyomas at a young age. They also display a 20–30% lifetime risk for renal carcinomas, which commonly present before 40 years of age, have a distinct papillary morphology, and an aggressive phenotype. However, the clinical presentation of HLRCC and the morphology of HLRCC-associated renal cell carcinomas (RCCs) can be variable and thereby evade diagnosis. Here, we present two cases of HLRCC-associated RCC to emphasize this point. The first case is typical of HLRCC, involving a 29-year-old man with multiple cutaneous leiomyomas and a renal tumor with characteristic papillary morphology. Next, we describe a 48-year-old man presenting with metastatic cancer of unknown primary origin and no skin findings. Interestingly, next-generation sequencing of his metastatic tumor identified two unique FH mutations. In both cases, FH mutations were confirmed as germline. These cases highlight the variable presentations of HLRCC-associated RCC and underscore the importance of screening tumors of unknown origin for FH mutations using next-generation sequencing. Keywords: Renal cell carcinoma, HLRCC, Fumarate hydratase, Next-generation sequencin

Expression of the Major Histocompatibility Complex Class I Molecule Mamu-A*01 Is Associated with Control of Simian Immunodeficiency Virus SIV(mac)239 Replication

Several HLA alleles are associated with attenuated human immunodeficiency virus disease progression. We explored the relationship between the expression of particular major histocompatibility complex (MHC) class I alleles and viremia in simian immunodeficiency virus SIV(mac)239-infected macaques. Of the common MHC class I alleles, animals that expressed Mamu-A*01 exhibited the best control of viral replication

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples.

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/μl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained

The high frequency Indian rhesus macaque MHC class I molecule, Mamu-B01, does not appear to be involved in CD8+ T lymphocyte responses to SIVmac239

Although the SIV-infected Indian rhesus macaque (Macaca mulatta) is the animal model most widely used for studying HIV infection, our current understanding of the functional macaque MHC class I molecules is limited. To date, SIV-derived CD8+ T lymphocyte epitopes from only three high frequency macaque MHC class I molecules have been extensively characterized. In this study, we defined the peptide-binding properties of the high frequency Indian rhesus macaque class I molecule, Mamu-B*01 ( approximately 26%). We first identified a preliminary binding motif by eluting and sequencing endogenously bound Mamu-B*01 ligands. We further characterized the peptide-binding characteristics using panels of single amino acid substitution analogs. Using this detailed motif, 507 peptides derived from SIV(mac)239 were identified and tested for their Mamu-B*01 binding capacity. Surprisingly, only 11 (2.2%) of these motif-containing peptides bound with IC50 values < or =500 nM. We assessed the immunogenicity of these peptides using freshly isolated PBMC from ten Mamu-B*01+ SIV-infected rhesus macaques in IFN-gamma ELISPOT and IFN-gamma/TNF-alpha intracellular cytokine staining assays. Lymphocytes from these SIV-infected macaques responded to none of these peptides. Furthermore, there was no sequence variation indicative of escape in the regions of the virus that encoded these peptides. Additionally, we could not confirm previous reports of SIV-derived Mamu-B*01-restricted epitopes in the Env and Gag proteins. Our results suggest that the high frequency MHC class I molecule, Mamu-B*01, is not involved in SIV-specific CD8+ T lymphocyte responses