Borne Encephalitis Virus Strains and Their Pathogenic Properties
Total Page：16
File Type：pdf, Size：1020Kb
The Relationship between the Structure of the Tick- Borne Encephalitis Virus Strains and Their Pathogenic Properties Sergei I. Belikov1*, Ilya G. Kondratov1, Ulyana V. Potapova1, Galina N. Leonova2 1 Limnological Institute, Siberian Branch, Russian Academy of Sciences, Irkutsk, Russia, 2 Research Institute of Epidemiology and Microbiology, Siberian Branch, Russian Academy of Medical Sciences, Vladivostok, Russia Abstract Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates. Citation: Belikov SI, Kondratov IG, Potapova UV, Leonova GN (2014) The Relationship between the Structure of the Tick-Borne Encephalitis Virus Strains and Their Pathogenic Properties. PLoS ONE 9(4): e94946. doi:10.1371/journal.pone.0094946 Editor: Ulrike Gertrud Munderloh, University of Minnesota, United States of America Received December 2, 2013; Accepted March 20, 2014; Published April 16, 2014 Copyright: ß 2014 Belikov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by International Scientific Technical Center Project No. 4006, Interdisciplinary Integration Projects of the Siberian Branch of the Russian Academy of Sciences No. 63 and 141 and State Contract No. P389. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction Far-Eastern (FE), Siberian (SIB), and (Western) European (WE) subtypes [9]. They were named according to their predominant Tick-borne encephalitis virus (TBEV) is one of the most dangerous distribution in Eurasia [4,5,10,11,12]. neuroviral infections in humans; transmission occurs via tick bites. Its The clinical course of TBE in the Far East is recognized to be circulation areas are in the forested zones of many European more severe than in other Eurasian areas [13]. The dominant countries and northern Japan, China, and Mongolia. In Russia, the encephalitic form of the disease is characterized by extreme natural foci of the infection are spread across Kaliningrad Oblast in severity and lethality. The main characteristic of FE TBE is the the west to Sakhalin Island in the east [1]. During the period 1973– rapid manifestation of overall lesions in the central nervous system, 2003, there was a 400% increase in the incidence of tick-borne causing focal or diffuse meningoencephalitis involving the brain encephalitis (TBE) in Europe [2,3]. In the 1990s, when the morbidity stem and spinal cord. Until the 1990s, mortality sometimes rate was high, TBE was the cause of at least 11,000 cases of disease in reached 30% [14]. Since then, the transformation of clinical Russia and approximately 3,000 in the rest of Europe [4,5,6,7]. In manifestations of infection in the Far East has been reported to the 2000s, the incidence of TBE gradually reduced; according to reduce the mortality to 13% and increase the ratio of non-focal official statistics, Russia recorded only 1,088 cases in 2009 (Federal TBE forms [13]. This is probably due to the occurrence and Service for Supervision of Consumer Rights Protection and Human spread in the area of new TBEV variants with mutations inducing Welfare (http://rospotrebnadzor.ru/documents/10156/a0a5f062- the subclinical forms of the disease [15]. 7498-4af7-b3f6-0a0d7d302c05). The exact genetic basis of these differences in the form and TBEV is a member of the virus genus Flavivirus, of the family severity of the disease is unclear. Previously, the variable severity Flaviviridae. This genus includes approximately 80 species, many was associated with the virus subtype [16]. Later, it became clear of which are pathogenic in humans, such as West Nile virus, that there is no direct relationship between the TBEV subtype and dengue, yellow fever, Japanese encephalitis, etc. [8]. The severity of the disease. The discrepancy between the virus subtype International Classification of flaviviruses divides TBEV into and the severity of clinical manifestations of the disease is PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e94946 TBEV Mutation and Pathogenicity particularly observed in the Russian Far East, where the FE of the complete sequenced genomes compared to the previously subtype prevails in the absence of SIB and WE TBEV. However, reported genome sequences of the FE subtype and prototype SIB different forms of the disease are registered there, from encephalic (Vasilchenko) and WE (Neudoerfl) subtypes of TBEV (Figure 1). forms, which often result in a fatal outcome, to asymptotic and The analysis indicates that all of the sequenced strains are related subclinical forms [13]. Previously, many researchers have shown to the FE subtype of the virus and are clearly different from the that the pathogenic markers of flaviviruses are localized in the strains of the SIB and WE subtypes. gene of the envelope protein E [17,18,19,20]. Additionally, The pathogenic Efd strains are divided into two branches, mutations located in other parts of the viral genome can change which may be due to the geographical isolation of the speciation the pathogenicity of the virus [21]. areas of the ancestral forms of the virus. Indeed, the strains of As a rule, in order to determine the virulence and pathogenicity cluster II include those related to the Sofjin strain isolated in of viral strains, their neuroinvasiveness and neurovirulence in mice Russia, Primorye, and the strains of cluster III include those or changes in the growth characteristics of cell culture have been related to the Senzhang strain isolated in northern China. The Sfd studied. However, there is no clear correlation between the virulence of strains in mice and strain pathogenicity in humans [22,23,24,25]. Therefore, this requires the definition and analysis of the complete nucleotide sequences of strains isolated from patients with variable severity of the disease for the detection of mutations in the TBEV genome that affect the pathogenicity of strains in humans. The comparison of complete genomes can make it possible to determine previously unknown genome regions that might be correlated with pathogenesis. In our previous studies, we compared the genomes of several strains isolated from patients with subclinical forms of the disease and indicated specific mutations in strains with different patho- genicity [15]. Nevertheless, it is necessary to analyze more strains to increase the reliability of these results and to exclude single spontaneous mutations that emerged during the evolution of the virus. In this work, we analyzed 34 complete genomes of FE- TBEV strains, which induced variable severity disease in humans. The analysis included the complete genomes of 11 human pathogenic strains isolated from patients with the encephalitic form of the disease (Efd), 19 strains from patients with the subclinical form of the disease (Sfd), 4 Ffd strains with intermediate characteristics, which caused febrile disease in comparison with previously published genomes of the Efd strains Sofjin, Glubinnoe and Senzhang, and a low-virulence strain Oshima 5-10, which was isolated in Japan. We examined mutations in different groups of human Efd and Sfd strains and attempted describe the influence of individual amino acid substitutions in proteins of the virus on the variability of the pathogenicity of TBEV strains. Results Comparison of nucleotide sequences The length of the nucleotide sequences of the genomes ranged from 10,405 to 11,103 nucleotides (the conserved region contained 10,376 nucleotides, and the differences were due to the variable length of the 39 untranslated region (UTR)). Moreover, 8,131 positions were constant in all of the analyzed TBEV strains. Mutations were most often located in the third codon position and were found at 2,245 positions of the genome (21.6%). The aligned sequences of the genomes, 39 UTR, and polyproteins are not shown, but can be provided upon request. Figure S1 schematically represents the aligned 39 UTR sequences. A comparison of the complete genome and amino acid sequences of individual viral proteins from all of the sequenced samples and previously reported strains with certain types of human pathogenicity is given in the form of a matrix (Figure S2).