Functional mutations in spike glycoprotein of Zaire ebolavirus associated with an increase in infection efficiency

Ebola virus (EBOV) is extremely virulent, and its glycoprotein is necessary for viral entry. EBOV may adapt to its new host humans during outbreaks by acquiring mutations especially in glycoprotein, which allows EBOV to spread more efficiently. To identify these evolutionary selected mutations and examine their effects on viral infectivity, we used experimental–phylogenetic–structural interdisciplinary approaches. In evolutionary analysis of all available Zaire ebolavirus glycoprotein sequences, we detected two codon sites under positive selection, which are located near/within the region critical for the host‐viral membrane fusion, namely alanine‐to‐valine and threonine‐to‐isoleucine mutations at 82 (A82V) and 544 (T544I), respectively. The fine‐scale transmission dynamics of EBOV Makona variants that caused the 2014–2015 outbreak showed that A82V mutant was fixed in the population, whereas T544I was not. Furthermore, pseudotype assays for the Makona glycoprotein showed that the A82V mutation caused a small increase in viral infectivity compared with the T544I mutation. These findings suggest that mutation fixation in EBOV glycoprotein may be associated with their increased infectivity levels; the mutant with a moderate increase in infectivity will fix. Our findings showed that a driving force for Ebola virus evolution via glycoprotein may be a balance between costs and benefits of its virulence

Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea.

Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure

Sequences of LAMP primers for EBOV.

<p>Sequences of LAMP primers for EBOV.</p

Diagnostic accuracy of the RT-LAMP test for EBOV compared with the reference rRT-PCR test.

<p>Diagnostic accuracy of the RT-LAMP test for EBOV compared with the reference rRT-PCR test.</p

Sensitivity and detection time of the RT-LAMP assay.

<p>Sensitivity and detection time of the RT-LAMP assay.</p

Detection time of EBOV RNA from the clinical samples with the RT-LAMP assay.

<p>(A) Tp of respective samples determined by RT-LAMP test with primers for trailer and NP were plotted against C_t value determined with the reference rRT-PCR test. The virus load of each sample determined by C_t value is indicated on the lower x-axis. (B) Mean times of detection of each group with indicated virus load were determined. White and black bars indicate the results of RT-LAMP with primers specific for trailer and NP, respectively (mean ± standard deviation).</p

Clinical characteristics of the samples tested in this study.

<p>Clinical characteristics of the samples tested in this study.</p

2012年から2016年にかけてナイジェリア南東部で検出されたラッサウイルスの遺伝学的解析

Lassa virus (LASV) is endemic in parts of West Africa where it causes Lassa fever (LF), a viral hemorrhagic fever with frequent fatal outcomes. The diverse LASV strains are grouped into six major lineages based on the geographical location of the isolated strains. In this study, we have focused on the lineage II strains from southern Nigeria. We determined the viral sequences from positive cases of LF reported at tertiary hospitals in Ebonyi and Enugu between 2012 and 2016. Reverse transcription-polymerase chain reaction (RT-PCR) showed that 29 out of 123 suspected cases were positive for the virus among which 11 viral gene sequences were determined. Phylogenetic analysis of the complete coding sequences of the four viral proteins revealed that lineage II strains are broadly divided into two genetic clades that diverged from a common ancestor 195 years ago. One clade, consisting of strains from Ebonyi and Enugu, was more conserved than the other from Irrua, although the four viral proteins were evolving at similar rates in both clades. These results suggested that the viruses of these clades have been distinctively evolving in geographically separate parts of southern Nigeria. Furthermore, the epidemiological data of the 2014 outbreak highlighted the role of human-to-human transmission in this outbreak, which was supported by phylogenetic analysis showing that 13 of the 16 sequences clustered together. These results provide new insights into the evolution of LASV in southern Nigeria and have important implications for vaccine development, diagnostic assay design, and LF outbreak management.長崎大学学位論文 学位記番号:博(医歯薬)甲第1107号 学位授与年月日:平成30年12月5日Author: Olamide K. Oloniniyi, Uche S. Unigwe, Sayaka Okada, Mayuko Kimura, Shota Koyano, Yukiko Miyazaki, Michael O. Iroezindu, Nnenna A. Ajayi, Chinedu M. Chukwubike, Nneka M. Chika-Igwenyi, Anne C. Ndu, Damian U. Nwidi, Haruka Abe, Shuzo Urata, Yohei Kurosaki, Jiro YasudaCitation: PLoS Neglected Tropical Diseases, 12(11), e0006971; 2018Nagasaki University (長崎大学)課程博