Identification and epidemiological study of an uncultured flavivirus from ticks using viral metagenomics and pseudoinfectious viral particles.

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

A Regional Network Organization for Thrombectomy for Acute Ischemic Stroke in the Anterior Circulation; Timing, Safety, and Effectiveness.

BACKGROUND: Mechanical thrombectomy (MT) in association with intravenous thrombolysis is recommended for treatment of acute ischemic stroke (AIS), with large vessel occlusion (LVO) in the anterior circulation. Because MT is only available in comprehensive stroke centers (CSC), the challenge of stroke organization is to ensure equitable access to the fastest endovascular suite. Our aim was to evaluate the feasibility, efficacy, and safety of MT in patients initially managed in 1 CSC (mothership), compared with patients first managed in primary stroke center (PSC), and then transferred to the CSC for MT (drip-and-ship). METHODS: We retrospectively analyzed 179 consecutive patients (93 in the mothership group and 86 in the drip-and-ship group), with AIS secondary to LVO in the anterior cerebral circulation and a clinical-radiological mismatch (NIHSS ≥ 8 and DWI-ASPECT score ≥5), up to 6 hours after symptoms onset. We evaluated 3-month functional modified Rankin scale (mRS), periprocedural time management, mortality, and symptomatic intracranial haemorrhage (sICH). RESULTS: Despite significant longer process time in the drip-and-ship group, mRS ≤ 2 at 3 months (39.8% versus 44.1%, P = .562), Thrombolysis in cerebral infarction 2b-3 (85% versus 78%, P = .256), and sICH (7.0% versus 9.7%, P = .515) were similar in both group regardless of baseline clinical or radiological characteristics. After multivariate logistic regression, the predictive factors for favorable outcome were age (odds ratio [OR] -5years= 1.32, P < .001), initial NIHSS (OR -5points = 1.59, P = .010), absence of diabetes (OR = 3.35, P = .075), and the delay magnetic resonance imagining-puncture (OR -30min = 1.16, P = .048). CONCLUSIONS: Our study showed encouraging results from a regional protocol of MT comparing patients transferred from PSC or brought directly in CSC.

A Reverse Mutation E143K within the PrM Protein of Zika Virus Asian Lineage Natal RGN Strain Increases Infectivity and Cytopathicity.

Zika virus (ZIKV) is a positive-sense single-stranded RNA virus in the Flaviviridae, which is classified into two different lineages Asian and African. The outbreak of ZIKV Asian lineage isolates in 2015-2016 is associated with the increase in cases with prenatal microcephaly and Guillain-Barré syndrome, and has sparked attention throughout the world. Genome sequence alignment and the analysis of Asian and African lineage isolates indicate that amino acid changes, particular in positively charged amino acid substitutions in the pr region of prM protein might involve a phenotypic change that links with the global outbreak of ZIKV Asian-lineage. The study generated and characterized the virological properties of wild type and mutants of single-round infectious particles (SRIPs) and infectious clones (i.c.s) of ZIKV Asian-lineage Natal RGN strain, and then identified the function of amino acid substitutions at the positions 139 [Asn139→Ser139 (N139S)] and 143 [Glu143→Lys143 (E143K)] in ZIKV polyproteins (located within the pr region of prM protein) in the infectivity and cytopathogenicity. The E143K SRIP and i.c. of Natal RGN strain exhibited relatively higher levels of cytopathic effect, EGFP reporter, viral RNA and protein synthesis, and virus yield in three types of human cell lines, TE617, SF268 and HMC3, compared to wild type (WT), N139S SRIPs and i.c.s, which displayed more efficiency in replication kinetics. Additionally, E143K Natal RGN i.c. had greater activities of virus attachment and entry, yielded higher titers of intracellular and extracellular virions, and assembled the E proteins near to the plasma membrane in infected cells than the other i.c.s. The results indicate that the positively charged amino acid residue Lys143, a conserved residue in the pr region of prM of ZIKV African lineages, plays a crucial role in viral replication kinetics, including viral attachment, entry, assembly and egress. Thus, the negatively charged amino acid residue Glu143 within the pr region of prM leads to an alteration of the phenotypes, in particular, a lower replication efficiency of ZIKV Asian-lineage isolates with the attenuation of infectivity and cytopathicity.

Behavioural response of mosquito vectors Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus to synthetic pyrethroid and organophosphorus-based slow-release insecticidal paint.

BACKGROUND: The direct toxicological impact of insecticides on vector mosquitoes has been well emphasized; however, behavioural responses such as excito-repellency and physical avoidance as a result of insecticide exposure have not been much studied. We have demonstrated the excito-repellency and behavioural avoidance in certain vector mosquito species on exposure to a slow-release insecticidal paint (SRIP) formulation in addition to direct toxicity. METHODS: A SRIP formulation developed by the Defence Research and Development Establishment, Gwalior, contains chlorpyriphos, deltamethrin and pyriproxyfen as active insecticides. Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti mosquitoes were used to study the excito-repellency response of the formulation. The experiments were performed in a specially designed dual-choice exposure and escape chamber made of transparent polymethyl methacrylate. For the experiments, the SRIP formulation was applied undiluted at a rate of 8 m2 per kg on 15 cm2 metallic surfaces. Mosquitoes were introduced into the exposure chamber, and observations of the movement of mosquitoes into the escape chamber through the exit portal were taken at 1-min intervals for up to 30 min. RESULTS: The evaluated formulation displayed strong excito-repellency against all three tested vector mosquito species. Results showed that the ET50 (escape time 50%) for Ae. aegypti, An. stephensi and Cx. quinquefasciatus was 20.9 min, 14.5 min and 17.9 min for contact exposure (CE) respectively. Altogether in CE, the escape rates were stronger in An. stephensi mosquitoes at different time intervals compared to Ae. aegypti and Cx. quinquefasciatus mosquitoes. The probit analysis revealed that the determined ET did not deviate from linearity for both non-contact exposure (NCE) and placebo exposure (PE) (χ2 ≤ 7.9; p = 1.0) for Ae. aegypti mosquitoes and for NCE (χ2 = 8.3; p = 1.0) and PE (χ2 = 1.7; p = 1.0) treatments in Cx. quinquefasciatus. Mortality (24 h) was found to be statistically higher (F = 6.4; p = 0.02) in An. stephensi for CE but did not vary for NCE (p ≥ 0.3) and PE (p = 0.6) treatments among the tested mosquito species. Survival probability response suggested that all the three tested species displayed similar survival responses for similar exposures (χ2 ≤ 2.3; p ≥ 0.1). CONCLUSION: The study demonstrates the toxicity and strong behavioural avoidance in known vector mosquito species on exposure to an insecticide-based paint formulation. The combination of insecticides in the present formulation will broaden the overall impact spectrum for protecting users from mosquito bites. The efficacy data generated in the study provide crucial information on the effectiveness of the tested formulation and could be useful in reducing the transmission intensity and disease risk in endemic countries.