Phylogenetic analysis of a newfound bat-borne hantavirus supports a laurasiatherian host association for ancestral mammalian hantaviruses

Until recently, hantaviruses (family Bunyaviridae) were believed to originate from rodent reservoirs. However, genetically distinct hantaviruses were lately found in shrews and moles, as well as in bats from Africa and Asia. Bats (order Chiroptera) are considered important reservoir hosts for emerging human pathogens. Here, we report on the identification of a novel hantavirus, provisionally named Makokou virus (MAKV), in Noack's Roundleaf Bat (Hipposideros ruber) in Gabon, Central Africa. Phylogenetic analysis of the genomic L-segment showed that MAKV was the most closely related to other bat-borne hantaviruses and shared a most recent common ancestor with the Asian hantaviruses Xuan Son and Laibin. Breakdown of the virus load in a bat animal showed that MAKV resembles rodent-borne hantaviruses in its organ distribution in that it predominantly occurred in the spleen and kidney; this provides a first insight into the infection pattern of bat-borne hantaviruses. Ancestral state reconstruction based on a tree of L gene sequences of all relevant hantavirus lineages was combined with phylogenetic fossil host hypothesis testing, leading to a statistically significant rejection of the mammalian superorder Euarchontoglires (including rodents) but not the superorder Laurasiatheria (including shrews, moles, and bats) as potential hosts of ancestral hantaviruses at most basal tree nodes. Our data supports the emerging concept of bats as previously overlooked hantavirus reservoir hosts. (C) 2016 Elsevier B.V. All rights reserved

Chimaeric virus growth kinetics in cell culture.

<p>Viruses are specified as shown in the text. PS cells were infected with TBEV at a multiplicity of 1 pfu per cell in at least 4 replicates. Samples of cell culture media were collected every 4 hours during the first day pi and then once a day. Virus infectivity was established by plaque assay. A-C) HyprIC and Hypr-based recombinant viruses; D-F) VsIC and Vs-based recombinant viruses. Error bars reflect 95% confidence intervals.</p

TBEV replication in mice following transmission from ticks.

<p>Mice infected via tick bites during the NVT experiments were observed for 21 days and cumulative numbers of challenged and moribund mice were analysed. A) Survival dynamics of mice infected with Vs-based (green lines/bars) chimaeras; B) Survival dynamics of mice infected with Hypr-based (red lines/bars) chimaeras. The probabilities of survival were estimated using Kaplan-Meier method; C) Correlation between mouse morbidity and infectious dose of TBEV in adult tick salivary glands following the termination of co-feeding. The data are arranged in descending order of mouse morbidity. The trend lines were drawn using the order 4 polynomial method (R² ≥ 0.95). The (r) value in the top right corner of the chart reflects the Pearson’s coefficient of correlation between datasets.</p

Vs and Hypr chimaeric viruses.

<p>A) The polyproteins, with individual proteins of Hypr virus (grey bars) and Vs virus (white bars) are flanked with 3`UTR (thick and thin lines for Hypr and Vs respectively). B) Schematic representation of the 3`UTRs., Numbers specify nucleotide positions in 3’UTRs following the stop codon. Internal deletions present in the Hypr and Vs genomes are identified by the numbers corresponding to those of the Hypr-long 3’UTR. Intermediate plasmids, constructed to recover Hypr, Vs and chimaeric Hypr/Vs infectious virus, are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158105#pone.0158105.s001" target="_blank">S1 Fig</a>.</p

Virion stability.

<p>Identical aliquots of virus suspension were treated as indicated in chart legend (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158105#sec013" target="_blank">Materials and Methods</a>) with virus titres determined by plaque infectivity assay. Error bars show 95% confidence intervals from the mean across three replicates.</p

Impact of genetic background on NVT of TBEV.

<p>A) Correlation of non-viratemic transmission (NVT) rates of different TBEV chimaeras with reproduction rate in different life stages of ticks. The trend lines are estimated by the polynomial method. The Pearson`s correlation coefficients between NVT rate (solid trend line) and titres in ticks (dashed lines) are shown by brackets with indicated r values. B) Correlation of TBEV gene replacements with NVT rate. The solid and dashed trend-lines reflect the changes in Hypr and Vs NVT rate efficiency respectively, in correspondence with size of exchanged gene fragments. Trend-lines were drawn using order 3 polynomial method with R-square value shown at the right end of the corresponding trend line. C) Correlation between NVT rate and the amino acid substitutions. The amino acid differences between C-prM-E region of polyprotein of control and chimaeric viruses are plotted on the X-axis as number of amino acid substitutions per site in comparison to VsIC sequence. The NVT rates are plotted on the Y-axis. The linear regression is calculated using equation [Y = 914.53*X + 4.77] and shown as solid line (goodness of fit R² = 0.58). The Pearson’s correlation coefficient (r) is shown in top left corner of the panel.</p

Efficiency of non-viraemic transmission of SIB-TBEV (Vs) and EU-TBEV (Hypr) between co-feeding <i>I</i>. <i>ricinus</i>.

<p>Two infected female ticks were placed in close proximity to 15 uninfected nymphs on the same skin site of laboratory mice. Following three days of co-feeding, nymphs were removed and virus titres determined by plaque titration. The mean transmission rate is expressed as percentage of positive nymphs in which TBEV was detected.</p

TBEV transmission and replication in <i>I</i>. <i>ricinus</i> ticks.

<p>Each adult tick female was injected with 500 pfu of virus (as specified). After 14 days two adult females were co-fed with 15 nymphs for three days and virus titres in females and nymphs were determined by plaque infectivity assay. Left and right panels illustrate biological effects of gene replacements in Vs-based and Hypr-based chimaeras respectively. Mean values are shown with error bars indicating 95% confidence intervals. A) Transmission rate of TBEV between co-feeding ticks is presented as a proportion of nymphs that acquired TBEV infection after co-feeding with infected female ticks. Depending on levels of statistically significant differences (Mann-Whitney test) between chimaeric and control viruses, results are labelled with double (p < 0.01) or single asterisks (p < 0.05). B) Virus titres of TBEV in nymphs and females of <i>I</i>. <i>ricinus</i>. Vs- and Hypr-based chimaeras are identified as green and red bars respectively; the control viruses are shown as empty bars. Highly significant differences (p < 0.05, unpaired two-tailed t-test) between chimaeric and control viruses are labelled with an asterisk.</p

Morbidity and average survival time (AST) of mice, infected with control and chimaeric TBEV during the feeding of female <i>I</i>. <i>ricinus</i>.

<p>Morbidity and average survival time (AST) of mice, infected with control and chimaeric TBEV during the feeding of female <i>I</i>. <i>ricinus</i>.</p

TBEV cytopathic effect in porcine kidney cells.

<p>Confluent monolayers of PS cells were infected at an moi of 1, in at least 4 replicates and incubated for 96 hours followed by staining with crystal violet and quantification of viable cells (grey blocks) as described in Materials and methods. Error bars reflect 95% confidence intervals.</p