scholarly journals Evaluation of Viral RNA Recovery Methods in Vectors by Metagenomic Sequencing

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 562
Author(s):  
Joyce Odeke Akello ◽  
Stephen L. Leib ◽  
Olivier Engler ◽  
Christian Beuret
Keyword(s):  
Fold Increase ◽  
Extraction Methods ◽  
Viral Rna ◽  
Health Concern ◽  
Metagenomic Sequencing ◽  
Optimal Method ◽  
Viral Genomes ◽  
Tick Borne Encephalitis ◽  
Langat Virus ◽  
Tick Borne Encephalitis Virus

Identification and characterization of viral genomes in vectors including ticks and mosquitoes positive for pathogens of great public health concern using metagenomic next generation sequencing (mNGS) has challenges. One such challenge is the ability to efficiently recover viral RNA which is typically dependent on sample processing. We evaluated the quantitative effect of six different extraction methods in recovering viral RNA in vectors using negative tick homogenates spiked with serial dilutions of tick-borne encephalitis virus (TBEV) and surrogate Langat virus (LGTV). Evaluation was performed using qPCR and mNGS. Sensitivity and proof of concept of optimal method was tested using naturally positive TBEV tick homogenates and positive dengue, chikungunya, and Zika virus mosquito homogenates. The amount of observed viral genome copies, percentage of mapped reads, and genome coverage varied among different extractions methods. The developed Method 5 gave a 120.8-, 46-, 2.5-, 22.4-, and 9.9-fold increase in the number of viral reads mapping to the expected pathogen in comparison to Method 1, 2, 3, 4, and 6, respectively. Our developed Method 5 termed ROVIV (Recovery of Viruses in Vectors) greatly improved viral RNA recovery and identification in vectors using mNGS. Therefore, it may be a more sensitive method for use in arbovirus surveillance.

scholarly journals Computational and Rational Design of Single-Chain Antibody against Tick-Borne Encephalitis Virus for Modifying Its Specificity

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1494
Author(s):  
Ivan K. Baykov ◽  
Pavel Y. Desyukevich ◽  
Ekaterina E. Mikhaylova ◽  
Olga M. Kurchenko ◽  
Nina V. Tikunova
Keyword(s):  
Rational Design ◽  
Fold Increase ◽  
Encephalitis Virus ◽  
Chimeric Antibody ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Glycoprotein E ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Far Eastern

Tick-borne encephalitis virus (TBEV) causes 5−7 thousand cases of human meningitis and encephalitis annually. The neutralizing and protective antibody ch14D5 is a potential therapeutic agent. This antibody exhibits a high affinity for binding with the D3 domain of the glycoprotein E of the Far Eastern subtype of the virus, but a lower affinity for the D3 domains of the Siberian and European subtypes. In this study, a 2.2-fold increase in the affinity of single-chain antibody sc14D5 to D3 proteins of the Siberian and European subtypes of the virus was achieved using rational design and computational modeling. This improvement can be further enhanced in the case of the bivalent binding of the full-length chimeric antibody containing the identified mutation.

scholarly journals Optimization and Ev a Luation of Viral Metagenomic Amplification and Sequencing Methods Toward a Genome -le Vel Resolution of the Human Fecal DNA Virome

2021 ◽  
Author(s):  
Guangyang Wang ◽  
Shenghui Li ◽  
Qiulong Yan ◽  
Ruochun Guo ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Multiple Displacement Amplification ◽  
Pcr Amplification ◽  
Genomic Analysis ◽  
Optimal Number ◽  
Future Research ◽  
Metagenomic Sequencing ◽  
Optimal Method ◽  
Viral Genomes ◽  
A Genome ◽  
Long Read

Abstract Background: Viruses in the human gut have been linked to health and disease. Deciphering of the gut virome is dependent on metagenomic sequencing of the virus-like particles purified from the fecal specimens. A major limitation of conventional viral metagenomic sequencing is the low recoverability of viral genomes from the metagenomic dataset. Results: Herein, we developed an optimal method for viral amplification and metagenomic sequencing to maximize the recovery of viral genomes. Using 5 fecal specimens with multiple repetitions, we revealed the optimal number of PCR cycles of high-fidelity enzyme-based amplification and the reliability of multiple displacement amplification in virome DNA preparation, verified the reproducibility of the optimally whole viral metagenomic experimental process, and tested the capability of long-read sequencing for improving viral metagenomic assembly. Based on our optimized results, we generated 151 high-quality viruses using the data combined from short-read (15 cycles for PCR amplification) and long-read sequencing. Genomic analysis of these viruses found that most (60.3%) of them were previously unknown and showed a remarkable diversity of viral functions, especially the existence of 206 viral auxiliary metabolic genes. Finally, we compared the viral metagenomic and bulk metagenomic sequencing approaches and revealed significant differences in the efficiency and coverage of viral identification between them. Conclusions: Our study demonstrates the potential of optimized experiment and sequencing strategies in uncovering viral genomes from fecal specimens, which will facilitate future research about genome-level characterization of complex viral communities.

scholarly journals Comparison of whole genomes of tick-borne encephalitis virus from mountainous alpine regions and regions with a lower altitude

Virus Genes ◽  
2021 ◽  
Author(s):  
G. Lemhöfer ◽  
L. Chitimia-Dobler ◽  
G. Dobler ◽  
M. Bestehorn-Willmann
Keyword(s):  
Sea Level ◽  
Alpine Region ◽  
Northern Asia ◽  
Lower Altitude ◽  
Genetic Traits ◽  
Viral Genomes ◽  
Alpine Regions ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Favorable Conditions

AbstractTick-borne encephalitis (TBE) has been a notifiable disease in Germany since 2001. Its causative agent, the TBE virus (TBEV), is the most important arbovirus in Europe and Northern Asia. The illness, caused by the European Subtype usually displays flu-like symptoms, but can result in sequelae and, in 2 % of all cases, in death. Over the last few decades, the virus has spread into new habitats, such as higher altitudes in the Alpine region. For this study, it was hypothesized that the environmental challenges that the virus might be exposed to at such altitudes could lead to the selection of viral strains with a higher resilience to such environmental factors. To determine whether strains identified at higher altitudes possessed different genetic traits compared to viruses from lower altitudes, an analysis of viral genomes from higher Alpine altitudes (> 500 m above sea level) (n = 5) and lower altitudes (< 500 m above sea level) (n = 4) was performed. No common phylogenetic ancestry or shared amino acid substitutions could be identified that differentiated the alpine from the lowland viral strains. These findings support the idea of many individual introductions of TBEV into the alpine region and the establishment of foci due to non-viral specific factors such as favorable conditions for vector species and host animals due to climate change.

scholarly journals TRIM79α, an Interferon-Stimulated Gene Product, Restricts Tick-Borne Encephalitis Virus Replication by Degrading the Viral RNA Polymerase

2011 ◽  
Vol 10 (3) ◽  
pp. 185-196 ◽  
Author(s):  
R. Travis Taylor ◽  
Kirk J. Lubick ◽  
Shelly J. Robertson ◽  
James P. Broughton ◽  
Marshall E. Bloom ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Gene Product ◽  
Virus Replication ◽  
Encephalitis Virus ◽  
Viral Rna ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

Rapid detection of viruses of the tick-borne encephalitis virus complex by RT-PCR of viral RNA

1993 ◽  
Vol 45 (1) ◽  
pp. 103-114 ◽  
Author(s):  
J.E. Whitbya ◽  
H. Ni ◽  
H.E. Whitby ◽  
A.D. Jennings ◽  
L.M. Bradley ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Encephalitis Virus ◽  
Viral Rna ◽  
Rt Pcr ◽  
Tick Borne Encephalitis ◽  
Virus Complex ◽  
Tick Borne Encephalitis Virus

scholarly journals Comparative Neuropathogenesis and Neurovirulence of Attenuated Flaviviruses in Nonhuman Primates

2008 ◽  
Vol 82 (11) ◽  
pp. 5255-5268 ◽  
Author(s):  
Olga A. Maximova ◽  
Jerrold M. Ward ◽  
David M. Asher ◽  
Marisa St. Claire ◽  
Brad W. Finneyfrock ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Virus Vaccine ◽  
Nonhuman Primates ◽  
Vaccine Candidate ◽  
Tick Borne Encephalitis ◽  
Langat Virus ◽  
The Central Nervous System ◽  
Attenuated Viruses ◽  
Tick Borne Encephalitis Virus ◽  
Far Eastern

ABSTRACT Based on previous preclinical evaluation in mice and monkeys, the chimeric TBEV/DEN4Δ30 virus, carrying the prM and E protein genes from a highly virulent Far Eastern strain of tick-borne encephalitis virus (TBEV) on the backbone of a nonneuroinvasive dengue type 4 virus (DEN4), has been identified as a promising live attenuated virus vaccine candidate against disease caused by TBEV. However, prior to use of this vaccine candidate in humans, its neurovirulence in nonhuman primates needed to be evaluated. In the present study, we compared the neuropathogeneses of the chimeric TBEV/DEN4Δ30 virus; Langat virus (LGTV), a former live TBEV vaccine; and yellow fever 17D virus vaccine (YF 17D) in rhesus monkeys inoculated intracerebrally. TBEV/DEN4Δ30 and YF 17D demonstrated remarkably similar spatiotemporal profiles of virus replication and virus-associated histopathology in the central nervous system (CNS) that were high in cerebral hemispheres but progressively decreased toward the spinal cord. In contrast, the neurovirulence of LGTV exhibited the reverse profile, progressing from the site of inoculation toward the cerebellum and spinal cord. Analysis of the spatiotemporal distribution of viral antigens in the CNS of monkeys revealed a prominent neurotropism associated with all three attenuated viruses. Nevertheless, TBEV/DEN4Δ30 virus exhibited higher neurovirulence in monkeys than either LGTV or YF 17D, suggesting insufficient attenuation. These results provide insight into the neuropathogenesis associated with attenuated flaviviruses that may guide the design of safe vaccines.

scholarly journals Low-pathogenic virus induced immunity against TBEV protects mice from disease but not from virus entry into the CNS

2021 ◽  
Author(s):  
Monique Petry ◽  
Martin Palus ◽  
Eva Leitzen ◽  
Johanna Gracia Mitterreiter ◽  
Bei Huang ◽  
...  
Keyword(s):  
Histopathological Analysis ◽  
Neuronal Necrosis ◽  
Reduced Frequency ◽  
Tick Borne Encephalitis ◽  
Pathogenic Virus ◽  
Lethal Infection ◽  
Tbev Strain ◽  
Langat Virus ◽  
Vector Borne ◽  
Tick Borne Encephalitis Virus

AbstractTick-borne encephalitis virus (TBEV) is a leading cause of vector-borne viral encephalitis with expanding endemic regions across Europe. Although currently used inactivated whole virus vaccines are effective, vaccination breakthroughs have been reported for which the reasons are unclear. In this study we tested in mice the efficacy of pre-infection with a closely related low-virulent flavivirus, Langat virus (LGTV strain TP21), or a naturally avirulent TBEV strain (TBEV-280) in providing protection against lethal infection with the highly virulent TBEV strain TBEV-Hypr (referred to as TBEV-Hypr). LGTV has been evaluated as an experimental live vaccine against TBE, but further development was abandoned due to too high residual pathogenicity of a LGTV-based vaccine. Here we show that prior infection with TP21 or TBEV-280 is efficient in protecting mice from lethal TBEV-Hypr challenge. Histopathological analysis of brains from non-immunized control mice revealed neuronal TBEV infection and necrosis. Neuroinflammation, gliosis and neuronal necrosis was however also observed in some of the TP21 and TBEV-280 pre-infected mice although at reduced frequency as compared to the non-immunized TBEV-Hypr infected control mice. Interestingly, qPCR detected the presence of viral RNA in the brains and spinal cord of both TP21 and TBEV-280 immunized mice after TBEV-Hypr challenge, but significantly reduced compared to mock-immunized mice. Our results indicate that although TBEV-Hypr infection is effectively controlled in the periphery upon immunization with low-virulent LGTV or naturally avirulent TBEV-280, it may still enter the CNS of these animals. These findings improve our understanding of potential causes for vaccine failure in individuals vaccinated with TBE vaccines.

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