Activity of BatIFIT5 in different species of healthy and naturally infected with rabies virus bats

Abstract

Bats are mammals with vital role played in numerous ecosystem services, however bats can be important reservoirs or hosts for several microorganisms. Rabies is a zoonosis caused by Rabies lyssavirus (RABV) that affects the central nervous system (CNS) of all mammals, including bats and humans. The action of IFN-stimulated genes (ISGs) could be responsible for inhibiting different stages of the viral replication cycle. A major family of ISGs are the Interferon-induced proteins with tetrapeptide repeats (IFITs) and your action against infections caused by viruses from different families was proven. This study describes the expression of BatIFIT5 by RT-qPCR in different species of healthy and naturally infected with RABV bats. A total, of 36 bats were analyzed (18 positive and 18 negative for rabies) and 16 (44.44%) were positive for BatIFIT5. Here we analyzed fourteen species of bats with different eating and behavioral habits. Seven genetics lineages of RABV were evaluated and included in these 14 species of bats, no cases of RABV spillover were identified. In addition, we did not verified relationship between the bat species expression of BatIFIT5 and RABV. Many points about immunology of bats are unknown and here we analyzed one of these points.

Introduction

Bats are mammals belonging to the Chiroptera order, traditionally divided based on morphology and behavior into the suborders Mega and Microchiroptera. However, recent cytogenetic and molecular evidence supports a new classification into two suborders, Yangochiroptera and Yinpterochiroptera. The first consists of twelve microbat families, while the second suborder includes megabats and four microbat families (Teeling et al. 2000, Teeling et al. 2002, Springer et al. 2002, Teeling et al. 2005,). The microbats are globally distributed, except in Antarctica (Nowak et al. 1999). In Latin America, only members of microbats are found, with more than 290 species (Bat Conservation International, 2021). Although the vital role played in numerous ecosystem services, bats can be important reservoirs or hosts for several microorganisms, mainly viruses. Numerous viruses detected in bats could potentially affect humans, posing a major public health threat worldwide. The most recent example of a spillover event is Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), the etiological agent of the current coronavirus disease 2019 (COVID-19) pandemic. On the other hand, rabies, an ancient fatal disease also maintained by bats, remains a serious public health concern in several countries (Kunz et al. 2011, Han et al. 2015, WHO, 2018, Bonilla-Aldana et al. 2021).

Rabies is a zoonosis caused by Rabies lyssavirus (RABV), a member of the Lyssavirus genus, that affects the central nervous system (CNS) of all mammals, including bats and humans (WHO, 2018, ICTV, 2018). Despite significant progress towards understanding RABV biology, the maintenance of the virus in nature is not completely elucidated. One of the most significant gaps is related to certain aspects of the pathogenesis of RABV infections in bats, the main reservoir of the virus. While RABV infects and causes death of some bats, previous studies have detected rabies antibodies in different species of these mammals, emphasizing that rabies is not always fatal in bats, unlike the other mammals (Jiang et al. 2010, O’shea et al. 2014, Costa et al. 2017, Almeida et al. 2019, Seetahal et al. 2020, Seidlova et al. 2020,).

The increase in viral load and efficient replication process that lead to a productive viral infection depends on intrinsic factors of the virus and the ability of the host immune system (IS) in controlling or not the disease. As viruses possess mechanisms to evade the IS, the host also has a coordinated defense network to prevent viral infection or at least make it less productive. The secretion of interferon (IFN) plays a critical role in the innate immune mechanism response to viral infections. There are different types of IFN, and their expression leads to activation of the JAK-STAT signalling cascate that culminates in expression of IFN-stimulated genes (ISGs), responsible for inhibiting different stages of the viral replication cycle. A major family of ISGs are the Interferon-induced proteins with tetrapeptide repeats (IFITs), a group of intracytoplasmic proteins without enzymatic activity with direct interaction capacity with viral RNAs. This family consists of four members in humans (IFIT1, IFIT2, IFIT3 and IFIT5), also expressed in other mammals (Stetson et al. 2006, Diamond et al. 2013, Goubau et al. 2013, Liu et al. 2013, Fensterl et al. 2015). The antiviral activity of IFIT5 in mammals demonstrated that IFIT5 levels were increased during viruses infections and lipopolysaccharides (Barber et al.2013, Li et al. 2020). The mechanism of this protein is attributed to interaction with nucleotides through 5’triphosphate portion from RNA (Feng et al. 2013, Santhakumar et al. 2018). The immune system of bats could have the same unknown particularities, therefore, studies about the underlying mechanism regarding antiviral activities could help to clarify important aspects in the maintenance of RABV in bats.

The action of IFITs against infections caused by viruses from different families was proven, nevertheless, studies regarding the role of these proteins during RABV infection are lacking in the scientific literature. Considering the significant role of ISGs against viral infection and the scarcity of studies related to the interaction between IFITs and RABV, in addition to the importance of bats as viruses reservoirs, this study describes the expression of BatIFIT5 in different species of healthy and naturally infected with RABV bats.

Materials and Methods

Bats

Thirty-six bats collected between 2015 and 2020 in different municipalities of São Paulo state, Brazil, sent for rabies diagnosis, were selected. Samples from the central nervous system (CNS) and spleen were collected and stored at -80 ºC until use. This work complies with Protocol No. 04/2020 issued by the Ethics Committee of the Pasteur Institute of São Paulo (Brazil).

Rabies diagnosis

Routine identification of RABV antigens was performed by the direct fluorescent antibody test (DFAT), following standard diagnostic procedures (Dean et al. 1996).

RNA extraction

Total RNA of CNS and spleen were extracted with Trizol® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Extracts from mice infected with the “fixed” strain Challenge Virus Standard (CVS-31) were included in all reactions as positive control and DEPC-treated water was included as negative control.

DNA extraction

Total DNA was extracted from the collected spleens using the Wizard® Genomic DNA Purification kit #TM050 (Promega Corporation) in accordance with the manufacturer’s instructions. Ultrapure DNase/RNase-free water was used as negative control.

Species identification

Positive bats for RABV were submitted to genetic and taxonomic identification (Vizzoto et al. 1973, Jones et al. 1976, Gregorin et al. 2002). Negatives bats for RABV were submitted only to genetic species identification. For genetic identification, total spleens DNA, extracted as described in 2.4 were submitted to amplification and sequencing of the mitochondrial cytochrome B and/or C (cytB or cytC) gene as described. The PCR and cytB sequencing were carried out with sense primer Bat 05A and antisense primer Bat 14A, for cytC was used sense primer LCO1490 and antisense primer HCO2198 (Table I).

Table. I. Primers used for PCR amplication and sequencing of cythcrome B (cytB) and C (cytC) for genetic identification of bat species.

Sequencing of RABV

The CNS of all positives bats were submitted to reverse transcription (RT) followed by polymerase chain reaction (RT-PCR), sequencing and phylogenetic analyses. For RT-PCR, extracted total RNA as described in 2.3 was used. The RT was carried out with primers that target the nucleoprotein gene (N) of RABV: sense primer Início_Posição1_F and antisense primer P784_Posição869_R (Table II). The reaction was performed in 50 volumes with aid of the SuperScript II reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. The reaction was incubated for 1 hour at 42 ºC followed by a final incubation at 72 ºC for 15 minutes. PCR was carried out as previously described (Carnieli et al. 2008), using the same primers. PCR amplicons were purified and sequenced with the same primers used for PCR. The RABV sequences were edited with the CHROMAS software (version 2.24 Copyright© 1998–2004 Technelysium Pty Ltd.). After editing, the output sequences were aligned with CLUSTAL/W using the BioEdit software (version 7.1.3.0). The putative amino acid (aa) sequences obtained were deduced with the BioEdit program (Hall, 1999). The phylogenetic tree was performed by the Maximum Likelihood method based on the Tamura 3-parameter model with Gamma distribution (Tamura et al. 1993), with MEGA 7 software (Tamura, 1992). A total of 591 nucleotides of the RABV N gene, nucleotide positions 71-592 in the PV strain of RABV (GenBank accession number: GB M13215.1), coding region aminoacids 1-174 were analyzed.

Table. II. Primers used for PCR amplification of partial nucleoprotein gene of rabies virus.

Expression of BatIFIT5

Total RNA extracted of collected spleens as described in 2.3 was used to synthesize first strand cDNA using Oligo dT (18) following the manufacturer’s instructions. To detect BatFIT5 gene expression, cDNA was amplified in a 20 μL reaction using a SYBR® Premix Ex Taq™ II kit (TaKaRa, Dalian, China) on an Applied Biosystems 7500 real-time PCR system (1 cycle at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s). The RT-qPCR was carried out with human IFIT5 sense and antisense primers (Table 3). All cDNA samples were tested in duplicate and the results were normalized to positive and negative controls. BatIFIT5 cloned into a pEFLink plasmid with FLAG peptide given by Dr. Muhammad Munir, from Lancaster University (UK) was used as a standard and positive control. Ultrapure DNase/RNase-free water was used as negative control.

Table. III. Primers used for PCR amplification of partial IFIT5 gene.

Results

Bats

Were collected in 17 municipalities from different regions of São Paulo State, southeast of Brazil. The exact location of each municipality is shown in Figure 1.

Figure. 1. Map of São Paulo, state of southeastern Brazil, showing the spatial distribution of the municipalities (dark gray) where the bats were collected.

Rabies diagnosis

A total of 18 bats were positive for RABV and 18 bats were negative by DFAT. The species of each bat and result for rabies are described in Table IV.

Table. IV. Identification of each bat collected, species identified, number of animals and rabies diagnosis.

Phylogenetic analysis of rabies virus

A phylogenetic tree was constructed utilizing the 18 samples of RABV sequenced in this work and additional 23 sequences of RABV recovered from GenBank (Figure 2). Eight clusters were formed: cluster 1 corresponds to sequences of viruses recovered from hematophagous bats Desmodus rotundus (DR); cluster 2 corresponds to sequences originated from bats Tadarida brasiliensis (TB); cluster 3 corresponds to sequences originated from bats Myotis sp. (Msp); cluster 4 corresponds to sequences originated from bats Eptesicus furinalis (EF); cluster 5 corresponds to sequences originated from bats Histiotus velatus (HV); cluster 6 corresponds to sequences originated from bats Nyctinomops laticaudatus (NL); cluster 7 corresponds to sequences originated from bats Lasiurus sp. (Lsp) from Brazil and cluster 8 corresponds to sequences originated from bats Lasiurus sp. (Lsp) from Americas. The species T.brasiliensis, Myotis sp., E. furinalis, H.velatus, N. laticaudatus and Lasiurus sp. were from non hematophagous bats.

Figure. 2. Phylogenetic analysis by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model [37]. The tree with the highest log likelihood (-2455.0703) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.2542)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 42 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 522 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Thirumal Kumar et al. 2019). Samples sequenced in this work are in red.

Detection of BatIFIT5

A total of 36 bats were analyzed (18 positive and 18 negative for rabies) and 16 (44.44%) analyzed bats were positive for BatIFIT5 in RT-qPCR. The description of detection of BatIFIT5 according to rabies diagnosis, number and bats species are presented in Table V.

Table. V. Detection of BatIFIT5, rabies diagnosis, number and percentage of bats and identified bats species.

Discussion and Conclusions

Currently, the majority of emerging infectious diseases are zoonoses originated from wildlife and bats constitute reservoirs of several high-impact viruses in nature (Jones et al. 2008, Wynne et al. 2013). Many biological aspects of these mammals are tough to be related to their capacity to carry several viral species, including longevity, capacity to fly and different eating and behavior habits. Here we investigated the transcript expression of BatIFIT5 in different species of healthy and infected with RABV bats.

The highest prevalence of the protein was detected in positive animals for RABV (66.67%). Despite some cases of cure in humans, the illness causes serious impairment of CNS and for this is considered 100% fatal (Jackson, 2002). The expression of BatIFIT5 in animals infected with RABV not scrap the antiviral activity of the protein and indicates activation of the innate response in presence of the virus. On the other hand, the virus can have mechanisms to counteract the action of BatIFIT5s, inhibiting therefore its clearance. Its should be consider, that all animals analyzed in this study were naturally infected and parameters as infectious dose, incubation period or previous immunological status are all unknown. Wang et al. 2015 developed a study that correlated the expression of duck IFIT5 (duIFIT5) in healthy ducks (Anas platyrhynchos domesticus) and ducks experimentally infected with Duck hepatitis virus type 1 (DHV-1). In their study different organs and the authors also identified expression of IFIT5 in healthy animals. In our study were tested only the spleens of the bats and the expression of BatIFIT5 was identified in a very low percentage of animals 4 (22.22%) not infected with RABV. It is important to emphasize that the animals were submitted only to rabies diagnosis, therefore these animals could be affected by other viruses or infectious pathogens as bacteria or fungi, which potentially induces IFITs.

Here we analyzed fourteen species of bats with different eating and behavioral habits. Seven genetics lineages of RABV were evaluated and included in these 14 species of bats, no cases of RABV spillover were identified (Oliveira et al. 2010). In addition, we did not verified relationship between the bat species expression of BatIFIT5 and RABV(except for the insectivorous bat N. laticaudatus). In fact expression of BatIFIT5 was not identified in any animal N. laticaudatus analyzed, despite the infection with RABV. For E. furinalis and A. lituratus species the same situation happened (Positive for rabies and negative for BatIFIT5) in only one sample of each specie. However, three and two animals belonging to E. furinalis and A. lituratus were positive for RABV and BatIFIT5 respectively. Therefore, the absence of BatIFIT5 expression is not an exclusivity of the species. . Despite the low number of samples analyzed, our results could indicate any difference in the immunological system of N. laticaudatus bats.

Many points about immunology of bats are unknown and here we analyzed one of these points but this is the first study relating BatIFIT5 and RABV in animals naturally infected and could help to better understand the relationship between viruses and reservoirs/hosts.

Conflicts of Interest

The authors declare no conflict of interest.

Acknowledgments

We would like to thank Dr. Muhammad Munir, from Lancaster University (UK) for providing pEFLink plasmid with FLAG peptide and Marcela Mello Zamudio from Universidade de São Paulo (USP-FFLCH) for producing the map.

Funding data

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo FAPESP (Grant. No. 2019/03146-2) and Health State Secretary of São Paulo. Part of this work was supported by FINEP project number 1.10.0783.00.