Adaptation of tick-borne encephalitis virus from human brain to different cell cultures induces multiple genomic substitutions

The clinical presentation of tick-borne encephalitis virus (TBEV) infection varies from asymptomatic to severe meningoencephalitis or meningoencephalomyelitis. The TBEV subtype has been suggested as one of the most important risk factors for disease severity, but TBEV genetic characterization is difficult. Infection is usually diagnosed in the post-viremic phase, and so relevant clinical samples of TBEV are extremely rare and, when present, are associated with low viral loads. To date, only two complete TBEV genomes sequenced directly from patient clinical samples are publicly available. The aim of this study was to develop novel protocols for the direct sequencing of the TBEV genome, enabling studies of viral genetic determinants that influence disease severity. We developed a novel oligonucleotide primer scheme for amplification of the complete TBEV genome. The primer set was tested on 21 clinical samples with various viral loads and collected over a 15-year period using the two most common sequencing platforms. The amplicon-based strategy was compared to direct shotgun sequencing. Using the novel primer set, we successfully obtained nearly complete TBEV genomes (>90% of genome) from all clinical samples, including those with extremely low viral loads. Comparison of consensus sequences of the TBEV genome generated using the novel amplicon-based strategy and shotgun sequencing showed no difference. We conclude that the novel primer set is a powerful tool for future studies on genetic determinants of TBEV that influence disease severity and will lead to a better understanding of TBE pathogenesis.

Section: Discussionmentioning

confidence: 99%

Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing

Zakotnik

Knap

Bogovič

et al. 2022

“…Interestingly, this observation correlates with pathogenicity in mammals. For instance, the Hypr strain lacks most of its 3′VR and is considered the most virulent strain within TBEV-Eu, and TBEV strains isolated from brain tissue of patients with severe cases of encephalitis consistently display deletions in the 3′VR [ 67 , 69 , 88 , 93 ]. The mortality rate across the three subtypes—TBEV-FE, TBEV-Si, and TBEV-Eu—correlates with the length of the 3′VR among the subtypes as well, where members of TBEV-Eu contain the longest 3′VRs overall compared to those of TBEV-FE strains [ 23 , 25 , 75 , 94 ].…”

Section: Effects Of the Untranslated Regions (Utrs) On Virulencementioning

confidence: 99%

Viral Determinants of Virulence in Tick-Borne Flaviviruses

Kellman¹,

Offerdahl²,

Melik

et al. 2018

Tick-borne flaviviruses have a global distribution and cause significant human disease, including encephalitis and hemorrhagic fever, and often result in neurologic sequelae. There are two distinct properties that determine the neuropathogenesis of a virus. The ability to invade the central nervous system (CNS) is referred to as the neuroinvasiveness of the agent, while the ability to infect and damage cells within the CNS is referred to as its neurovirulence. Examination of laboratory variants, cDNA clones, natural isolates with varying pathogenicity, and virally encoded immune evasion strategies have contributed extensively to our understanding of these properties. Here we will review the major viral determinants of virulence that contribute to pathogenesis and influence both neuroinvasiveness and neurovirulence properties of tick-borne flaviviruses, focusing particularly on the envelope protein (E), nonstructural protein 5 (NS5), and the 3′ untranslated region (UTR).

“…Cell lines of neural origin were found to be highly susceptible to TBEV infection, providing 100- to 10,000-fold higher virus titer than cells of non-neural origin [ 38 ]. Experimental studies show that virus adaptation to a specific cell line necessitates serial passages for structural rearrangements that favor viral load increasing to detectable levels [ 39 ]. As a result, compatible viral host and cell line origin may have played a significant role in high virus isolation success.…”

Section: Discussionmentioning

confidence: 99%

The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania

Simkute,

Pautienius,

Grigas

et al. 2024

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7–81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.