Detection and Molecular Characterization of Orientia tsutsugamushi from Suspected Scrub Typhus Patients in Mizoram, India

Serologic and molecular tests were performed for the diagnosis and to detect O. tsutsugamushi genotypes that are circulating in the state of Mizoram, India. Blood samples from scrub typhus-suspected patients were collected from Synod Hospital, Durtlang, Mizoram. Weil-Felix and immunochromatographic test (ICT) were performed from the serum samples. Nested PCR (nPCR) amplification of 47kDa outer membrane protein antigen gene and 56kDa type-specific antigen gene were done from the whole blood. 141/177 (79.66%) and 134/177 (75.7%) cases showed the presence of antibody against scrub typhus by Weil-Felix and ICT assays respectively. 76/177 (42.93%) patients showed the presence of 47kDa OMP antigen gene by nPCR while 55/177 (31.07%) showed the presence of 56kDa TSA gene by nPCR. Phylogenetic analysis of 56kDa TSA gene sequence revealed that Karp-related genotype was the most common genotype in the study area followed by Kato-related genotype. In this study, a high degree of diversity of O. tsutsugamushi was observed similar to the observations reported from other parts of India. Nested PCR of 47kDa OMP antigen gene showed higher sensitivity as compared to nPCR amplification of 56kDa TSA gene suggesting it as the assay of choice for diagnosis of scrub typhus disease.

Detection of Rickettsia lusitaniae Among Ornithodoros sawaii Soft Ticks Collected From Japanese Murrelet Seabird Nest Material From Gugul Island, Republic of Korea

In a follow-up to the investigations of soft ticks identified from seabird nest soil and litter collected from coastal islands of the Republic of Korea (ROK), Ornithodoros sawaii and Ornithodoros capensis were assessed for the presence and identification of rickettsiae. Ticks collected from samples of 50–100 g of nest litter and soil from seabird nests were identified individually by morphological techniques, and species confirmed by sequencing of the mt-rrs gene. Subsequently, tick DNA preparations were screened for the presence of rickettsiae using a genus-specific nested PCR (nPCR) assay targeting the 17 kDa antigen gene. The amplicons from the 17 kDa assay and two additional nPCR assays targeting the gltA and ompB gene fragments were sequenced and used to identify the rickettsiae. A total of 134 soft ticks belonging to two species, O. sawaii Kitaoka & Suzuki 1973 (n = 125) and O. capensis Neumann 1901 (n = 9), were collected. Rickettsia lusitaniae DNA was detected and identified among O. sawaii ticks (n = 11, 8.8%) collected from nest litter and soil of the Japanese murrelet (Synthliboramphus wumizusume Temminck 1836) at Gugul Island along the western coastal area of the ROK. This study confirmed for the first time the presence of R. lusitaniae associated with O. sawaii collected from migratory seabird nests in the ROK.

CRISPR/Cas9 targeting of MCPyV T antigen in Merkel tumors

ABSTRACTMerkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCPyV) T antigen gene expression, post integration (∼80% cases), or by UV mediated DNA damage. Viral-positive Merkel tumors are not only caused by but also oncogenically addicted to tumor antigen expression. In this study we used CRISPR-Cas9 based gene-editing to develop a potential therapeutic tool for MCPyV positive MCC. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a genome editing technology whereby a guide RNA (gRNA) molecule, targets a Cas endonuclease to a specific genomic site, using sequence homology, and induces a double strand break. To target MCPyV T antigens, we designed a strategy using 2 gRNAs targeting the T antigen genomic region that would cut off a substantial portion of the gene thereby rendering it dysfunctional. We validated the MCPYV T antigen targeting efficiency of our gRNAs, both individually and together by in vitro cleavage assays. Finally, to translate this finding, we delivered this CRISPR system in patient-derived MCC cell lines and show reduction in T antigen gene expression. Our proof-of-concept study shows that 2 MCPyV targeting CRISPR/Cas gRNAs in combination can knock out MCPyV T antigen, thus, being of therapeutic importance. We hope that this CRISPR system can be potentially delivered in vivo for advancing MCPyV positive MCC treatment in the future.