BioNanoSci. DOI 10.1007/s12668-016-0338-2
Genetic Characterization of Small (s)-Segment Genome Puumala Virus Strain Kazan E. Kabwe 1 & N. Y. Davidyuk 1 & S. P. Morzunov 2 & V. G. Shakirova 3 & V. A Anokhin 4 & G. S. Isaeva 5 & R. K. Ismagilova 1 & S. F. Khaiboullina 1,2 & A. A. Rizvanov 1
# Springer Science+Business Media New York 2016
Abstract The Republic of Tatarstan is one of the most active endemic regions for nephropathia epidemica (NE) in the Russian Federation. Annually, over 1000 cases of NE are registered, with an average mortality rate of 0.43 %. NE is a zoonosis where human become infected by inhaling a hantavirus-contaminated aerosol. Puumala virus is commonly detected in NE cases. Although NE cases have been registered in the region since 1958, little is known about the genetic variability of Puumala virus circulating in Tatarstan. We conducted phylogenetic analysis of the full length (1828 nt) small (S) segment genome sequence of Puumala virus RNA isolated from four bank voles (Myodes glareolus) captured during the 2014 outbreak. These virus sequences were compared to known sequences of Puumala viruses from nearby regions such as Samara and the Republic of Bashkortostan, as well as to European isolates. We found that there was over 89 % nucleotide identity between Puumala virus sequences isolated from the bank voles captured in Tatarstan. Sequence identity between Puumala viruses from the Tatarstan bank voles and the sequences from Republic of Bashkortostan and the Samara region were 90–95 %. Less similarity was found between * S. P. Morzunov
[email protected]
1
Kazan Federal University, Kazan, Republic of Tatarstan, Russian Federation
2
University of Nevada, Reno, NV, USA
3
Kazan State Medical Academy, Kazan, Republic of Tatarstan, Russian Federation
4
Kazan State Medical University, Kazan, Republic of Tatarstan, Russian Federation
5
Kazan Research Institute of Epidemiology and Microbiology, Kazan, Republic of Tatarstan, Russian Federation
Tatarstan sequences and Puumala strains circulating in Europe (79.7–87.7 %). Taken together, these data suggest that Puumala viruses circulating within the bank vole population in the Republic of Tatarstan are phylogenetically closer to the viruses circulating in neighboring regions of Russia. Keywords S-segment . Puumala virus . Hantaviruses . Bunyaviridae . Nephropathia Epidemica (NE) . Hemorrhagic fever with renal syndrome (HFRS)
1 Introduction The mild form of hemorrhagic fever with renal syndrome (HFRS) often referred to as nephropathia epidemica (NE), is endemic in the Republic of Tatarstan [1–4]. HFRS and NE are the most prevalent zoonoses in the Russian Federation, comprising up to 47.3 % of all registered zoonotic infections. In the Volga Federal District (which includes the Republic of Tatarstan, the Republic of Bashkortostan, Udmurtia, Mordovia and the Samara region), 83.3 % of all NE cases are registered in the Republic of Tatarstan making it one of the most active endemic regions, followed by Udmurtia, Republic of Bashkortostan and Mordovia. Puumala virus (PUUV) has been reported as the main cause of NE in the European part of Russia, which includes the Republic of Tatarstan [1, 2, 5]. PUUV is a segmented negative-sense RNA virus belonging to the genus Hantavirus, a member of Bunyaviridae family. In nature, PUUV circulates in small rodents causing a lifelong asymptomatic infection [1, 6]. Human infection occurs via inhaling a virus-contaminated aerosol. The disease has an acute onset with flu-like symptoms. As the disease progresses symptoms of impaired renal function and disturbed hemodynamics develop. The mortality rate is 0.1–0.3 %.
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Although the first NE case was diagnosed in 1958, little is known about genetic diversity of the PUUV strains circulating in the Republic of Tatarstan. Therefore, the goal of this study was to analyze the diversity of the PUUV strains circulating in the bank vole population in the Republic of Tatarstan. The complete nucleotide sequence of the PUUV S genome segment (1828 nucleotides) from four bank voles with high anti-hantavirus titer, captured in 2014, was determined. These S-segment sequences were compared to known PUUV strains from the nearby regions of Samara and the Republic of Bashkortostan as well as selected strains from Europe. Sequence analysis identified a new PUUV strain circulating in bank voles in the Republic of Tatarstan.
2 Materials and Methods Fifty rodents were captured in the north-west part of the Republic of Tatarstan. The presence of anti-hantavirus antibodies in the lungs was determined using the Hantagnost kit (Vektor, Russia). Lung tissue from four animals with high antibody titer was used for total RNA extraction using Trizol (Invitrogen, CA). After cDNA synthesis, 1070- and 911-bp fragments of the S-segment RNA were amplified by PCR. Amplified products were separated by gel electrophoresis and sequenced, using ABI PRISM 310 big Dye Terminator 3.1 sequencing kit. Lasergene package [7] was used to analyze sequences and for phylogenetic analysis.
3 Results Sequences from four rodent lung samples were named PUUV/3/ Kazan2014/KFU, PUUV/4/Kazan2014/KFU, PUUV/32/ Kazan2014/KFU and PUUV/37/Kazan2014/KFU. The length of the S segment RNA was 1828 nucleotides in all four samples. Phylogenetic analysis of the sequences was conducted including known sequences from GenBank such as Udmurtia/894Cg/91, Puu/Kazan, CG17/Baskiria/2001, Samara/49/CG/2005 Munga/ Mg16/05, Baltic/49Cg/00, PUUV/Mg23/HungaryTR17/00, and Umea/hu. Sequence analysis of the S segment coding region (CDS) revealed 27 mutations that were found consistently in three of the isolated sequences (PUUV/3/Kazan2014/KFU, PUUV/4/Kazan2014/KFU and PUUV/37/Kazan2014/KFU) when compared to strains from the Volga Region of Russia (Udmurtia/894Cg/91, Puu/Kazan, CG17/Baskiria/2001, and Samara/49/CG/2005). All 27 mutations were in the third position of the codon; therefore, we conclude that these mutations do not lead to changes in the amino acid sequence. In addition to these 27 mutations, PUUV/3/Kazan2014/KFU had a unique mutation A → U (transversion) at position 1035 which leads to a change in amino acid sequence Gly331Asp. Additionally, the sequence PUUV/32/Kazan2014/KFU differed from the remaining three sequences isolated from bank voles in that it
contained only 16 out of 27 nucleotide substitutions. Furthermore, sequence PUUV/32/Kazan2014/KFU contained 24 additional nucleotide substitutions which matched the strains Baltic/49Cg/00, PUUV/Mg23/HungaryTR17/00, Munga/ Mg16/05, and Umea/hu. Sequences PUUV/3/Kazan2014/KFU, PUUV/4/Kazan2014/ KFU, and PUUV/37/Kazan2014/KFU were closely related to each other (98.6–99.4 % nucleotide identity). Also, these sequences were closely related to strains from the Volga Region such as DTK/Ufa/97, Udmurtia/894Cg/91, CG17/Baskiria/2001 and Samara/49/CG/2005, with similarities of 93.0–94.3 % (Table 1). The identity between the isolated sequences and PUUV strains from Central and Northern Europe (Baltic/49Cg/ 00, PUUV/Mg23/HungaryTR17/00, Munga/Mg16/05 and Umea/hu) was lower, 80.3–87.7 %. The sequence PUUV/32/ Kazan2014/KFU showed the lowest nucleotide identity (89.3– 93.4 %) to the other three PUU/Kazan/KFU sequences and had 79.9–86.3 % identity with the GenBank sequences Munga/ Mg16/05-GQ339487.1, Baltic/49Cg/00-AJ314598.1, PUUV/Mg23/HungaryTR17/00-FN377822.1, and Umea/huAY526219. A phylogenetic tree was generated on the basis of the nucleotide sequences of the complete S segment PUUV RNA. The lengths of the horizontal branches represent numbers of substitutions per site between corresponding taxa (see scale bar). Vertical branches are for visual clarity only. Three samples, PUUV/3/Kazan2014/KFU, PUUV/4/Kazan2014/KFU, and PUUV/37/Kazan2014/KFU, grouped together on one branch and sample PUUV/32/Kazan2014/KFU sequence formed an intermediate between the strains circulating in Volga Region of Russia (DTK/Ufa/97, Udmurtia/894Cg/91, Puu/Kazan, CG17/Baskiria/2001, Samara/49/CG/2005), and East and North Europe (PUUV/Mg23/HungaryTR17/00, Umea/hu, Baltic/49Cg/00, and Munga/Mg16/05) (Fig 1).
4 Discussion Four PUUV sequences from bank voles captured in the Republic of Tatarstan during 2014 were analyzed. The full length PUUV S-segment sequences of each sample were 1828 nucleotides, and they were 79.9 to 87.7 % similar to PUUV strains from Europe such as Munga/Mg16/05GQ339487.1, Baltic/49Cg/00-AJ314598.1, PUUV/Mg23/ HungaryTR17/00-FN377822.1, and Umea/hu-AY526219.1, and 90.1 to 94.3 % similar to the other sequences used for analysis, Udmurtia/894Cg/91-Z21497.1, Puu/Kazan-Z84204.1, CG17/Baskiria/2001-AF442613.1, DTK/Ufa/97- AB297665.2 and Samara/49/CG/2005-AB433843.2. Comparative analysis of the samples revealed a lower number of nucleotide substitutions in CDS (2 %) indicating noticeable genetic variability and presumably a slow rate of molecular evolution of PUUV in the Republic of Tatarstan. These results agree with observations made in Northern Finland, where significant differences in
93.6 90.5
89.3 100
100
94.0
98.7
93.4
93.4
100
98.6
93.4
99.4
90.7
94.0
94.1
93.8
90.1
94.1
93.8
93.7
Samara/ PUUV/32/ PUUV/37/ DTK/ CG17/ PUUV/3/ PUUV/4/ Kazan2014/ Kazan2014/ Kazan2014/ Kazan2014/ Ufa/ Baskiria/ 49/CG/ 2005 2001 97 KFU KFU KFU KFU
% Similarities
90.9
93.0
94.1
93.4
90.5
93.1
93.8
93.1
90.8
93.2
94.3
93.6
86.3
87.7
87.3
86.6
81.4
84.0
82.1
81.5
79.9
84.1
80.9
80.3
81.5
84.7
81.9
81.1
68.6
72.1
69.5
69.2
57.9
58.0
57.4
57.4
Udmurtia/ Udmurtia/ Puu/ Baltic/ Munga/ PUUV/Mg23/ Umea/ Tula.Moravia/ Dobrava/ 5302 Ma/94 Belgrade 458Cg/88 894Cg/91 Kazan 49Cg/ Mg16/ HungaryTR17/ hu 00 05 00
Comparison of the nucleotide sequences of the complete S segment of PUUV recovered from bank voles in Republic of Tatarstan in 2014
PUUV/3/Kazan 100 2014/KFU PUUV/4/Kazan 2014/KFU PUUV/32/Kazan 2014/KFU PUUV/37/Kazan 2014/KFU
Viruses
Table 1
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identified to be circulating in the Republic of Tatarstan. Interestingly, one PUUV sequence, PUUV/32/Kazan2014/ KFU, appears to be intermediate between strains from the Volga Region and Europe.
Acknowledgments This work was supported by the Russian Science Foundation grant 15-14-00016. The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University and subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities. Some of the experiments were conducted using the equipment of Interdisciplinary Center for Collective Use of Kazan Federal University supported by the Ministry of Education of Russian Federation (ID RFMEFI59414X0003) and the Pharmaceutical Research and Education Center of Kazan (Volga Region) Federal University, Kazan, Russian Federation. Fig. 1 Phylogenetic relationships between PUUV strains recovered from bank voles in the Republic of Tatarstan and viruses circulating in the Volga Region of Russia and Europe Compliance with Ethical Standards
PUUV nucleotide sequences were detected [8, 9]. Sequence identity between PUUV/32/Kazan2014/KFU and the remaining three sequences isolated from the bank voles was 89.3–93.4 %. Genetic diversity among the three samples was lower (0.6– 1.4 %) while the genetic diversity of PUUV/32/Kazan2014/ KFU compared to the three samples indicated a higher diversity level (6.9–11.6 %), and compared to the strains from the Volga region appeared to be 9.5–10.3 %. Previously, data obtained in Northern Finland showed genetic diversity between strains belonging to same lineage of 0.2–1.1 %, and between strains belonging to different lineages there was a higher percentage diversity, 18.3–19.4 % [8]. Further, comparison of our results with the Finnish data suggests that three strains, PUUV/3/ Kazan2014/KFU, PUUV/4/Kazan2014/KFU, and PUUV/37/ Kazan2014/KFU, belong to the same genetic lineage, while strain PUUV/32/Kazan2014/KFU belongs to the other lineage. Additionally, we can suggest that sample PUUV/32/ Kazan2014/KFU probably originated from the European lineage [8]. This data indicates co-circulation of at least two distinct genetic lineages of PUUV in the territory of the Republic of Tatarstan. The possibility of such co-circulation in the bank vole population within the limited area was previously suggested in Finland [8, 9]. Our data suggest that PUUV strains circulating in the Volga Region of Russia and in Europe originated from a common ancestor. We suggest that migration of virus-carrying small rodents may be the reason for the observed distribution of PUUV across Volga Region of Russia.
5 Conclusion Based on the analysis of PUUV complete S segment sequence, two genetically distinct lineages of PUUV were
Conflict of Interest The authors declare that they have no conflict of interest.
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