Sensitivity of Pipistrellus Nathusii Kidney Diploid Cell Strain to Viruses of Rhabdoviridae, Reoviridae, Bunyaviridae and Paramixoviridae Families

The discovery of a signi�cant number of viral pathogens in bat tissue samples and excrement point to a potential prominent role of chiropters in the maintenance and spread of human and animal diseases. It also indicates the potential sensitivity of bat cells to a broad spectrum of viruses. The migratory pipistrelle bat, Pipistrellus nathusii, inhabits northeastern Europe and typically migrates to the southwest. Our study revealed the sensitivity (susceptibility) of a diploid cell line, derived from the kidney of P. nathusii to several transmissible animal disease causative agents such as Epizootic hemorrhagic disease, Akabane disease, Vesicular stomatitis virus and Peste des petit ruminants. High sensitivity of the P. nathusii kidney diploid cell line to viruses from various taxonomic groups allows them to be recommend for extensive use in virological studies.


Introduction
Currently, members of the order Chiroptera are considered to be one of the most important reservoirs and carriers of causative agents of particularly dangerous human and animal diseases.[1][2][3].Genomes of viruses from different taxonomic groups have been detected in tissue and excrement samples taken sampled from bats [4].Identi cation of these pathogens in bats has been carried out mainly using molecular genetics.Studying the biological properties of virus isolates which transmit from Chiropterans to target animal species is hampered by the absence of vivaria for bats, as well as the particular immune response of this mammalian species [5].Furthermore, the most frequently used ("traditional") cell cultures are not effective enough for the isolation of viruses persisting in bats.
Therefore, obtaining cell cultures from tissues and organs of different subspecies of bats is relevant and solves problems regarding primary isolation and subsequent investigation into the biological properties of viruses.Additionally, it helps the assessment of the possible role of tissue-donor bats in the formation of natural foci of infection.Earlier, our group developed a stable epithelial cell line from the kidneys of P. nathusii (PKDCL) [9].In another study, this diploid cell line demonstrated the ability to maintain reproductions of Bluetongue virus, Rift Valley fever, Lumpy skin disease virus and Myxoma virus [10].
This study aimed at further exploring the range of susceptibility of diploid cell lines, derived from bat tissues to viruses from different taxonomic groups.Including those posing a risk of being introduced to, and spreading in, European countries.

Results And Discussion
Morphologically, PKDCL is characterized by epithelial-like polygonal cells containing oviform or ellipsoidal karyons with 1-3 (sometimes more) rounded nucleoli varying in size.The nuclear matrix was uniform (Fig. 1).
Virological tests carried out between passages 14-43 showed commonality between, and active proliferation of, the cells in the culture.
In this period, with the replanting coe cient 1:2 − 1:3, the con uent monolayer developed within 48 hours and persisted without changing the medium for 45 days (the period of observation).
Earlier, we demonstrated the sensitivity (permissivity) of this diploid cell strain to bluetongue virus, lumpy skin disease virus and myxoma virus [10].In this study, we assessed its sensitivity to RNA-viruses belonging to different families: Rhabdoviridae, Bunyaviridae, Reoviridae and Paramixoviridae.For this study, viruses causing transmissible animal infections were selected, including the Peste des petits ruminants virus, which is currently posing a risk of being introduced to, and spreading in Russia.
The reproduction of VSV, with a multiplicity of infection (MOI) of 0.1 − 0.01 TCD 50 /cell in PKDCL, revealed early signs of CPE in the form of slight rounding of cells on the second day post-inoculation.On the third day, the delamination of infected cells from the substrate, followed by lysis and destruction of the monolayer, was observed in the cell culture.The accumulation of the virus in PKDCL reached 7,5 ± 0,25 lg TCID 50 /см 3 .In the control cell culture (L-929), this parameter was 6,75 ± 0,25 lg TCID 50 /см 3 .
EHDV with MOI of 0,1 − 0,01 TCD 50 /cell, caused slight CPE on the second day post-infection which was expressed by a partial lysis of the monolayer.Cell destruction was noted, in addition to elongated cells along the substrate forming strands.Infectious activity of EHDV in P. natussii kidney cell culture was 5,0 ± 0,25 lg ТЦД 50 /cm 3 .In the control culture CV-1, its activity was 6,25 ± 0,25 lg ТЦД 50 /cm 3 .
Migratory routes of bats cover territories of Russia with a high density of small ruminants.Furthermore, causative agents of disease affecting animals belonging to the subfamily Caprine, including PPRV, may potentially be circulating in these territories, as well.Consequently, we assessed the sensitivity of PKDCL to this virus.
It was found that in PKDCL, the PPR virus with a MOI rate of 0,1 − 0,01 TCD 50 /cell caused rounding and detachment of infected cells from the substrate accompanying with lysis and destruction of the monolayer on the fourth day post virus introduction.The virus accumulated in PKDCL cells and reached levels of 7.75 ± 0.25 lg TCID 50 /см 3 .That is much higher than in the control infected sensitive cell culture -GT (4,25 ± 0,25 lg TCID 50 /cm 3 ).
In parallel, to compare the dynamics of CEP development, we used cultures sensitive to each virus (CV-1, L-929 or GT).The summarized results of virological studies are presented in Table 1.In the experiments testing the susceptibility of DCLPK cells to the viruses, the control (uninfected) DCLPK remained its typical morphology, and no CPE was observed.
The characteristics of CPE caused by the viruses in DCLPK cells is presented in Fig. 2.
Analysis of the results of the virological studies has shown that the subcultures of these cells are sensitive to VSV, EHDV and PPRV, and can be considered as a promising cell substrate for primary isolation and further study of these viral pathogens.
With respect to the high infectious activities of VSV and PPRV in DCLPK, exceeding their activities in the control sensitive cell cultures, we carried out a control of their authenticity using molecular methods.
For this purpose, amplicons of VS and PPR viruses were produced, and nucleotide sequencing was carried out to determine which branch they belong to.Obtained sequences were compared with sequences from the GenBank database by using "Blast" software (https://blast.ncbi.nlm.nih.gov/Blast.cgi).It was con rmed that the obtained culture substrates contain VSV and PPRV.
Additionally, the authenticity of CPE caused by PPRV was con rmed by two serological methods: neutralization test (NT) and ELISA.To perform the NT we used 2-day cell monolayer on the 40th passage grown in 96-wells templates.
The reaction was evaluated based on the dynamics/manifestation of CPE, and by comparing the values of the Reed-Muench virus titrations between normal and speci c sera.The calculations revealed a neutralization index (NI) of 10 3,25 or 3,25 lg.
Antibodies against PPRV were detected in serum samples from sheep immunized by PPRV and infected cultural material (see material and methods).
Thus, the results of NT and ELISA tests con rm the authenticity of the PPR virus cultivated in the diploid P. nathusii kidney cell line.

Conclusions
The results of the evaluation of the sensitivity (susceptibility, permittivity) of the diploid cell line derived from the kidney of P. nathusii, demonstrated a high level of its permittivity to causative agents of Vesicular stomatitis, Epizootic hemorrhagic disease and Peste des petits ruminants.
Reproduction of the above-mentioned viral agents in the cell culture was accompanied by expressed cytopathic effect beginning from the very rst passage without preliminary adaptation of the viruses to cells.
P. nathusii inhabits and migrates throughout the area from southern and middle Europe to the middle east.Considering the sensitivity of the cell culture derived from the kidney of this species to BTV, RVFV, LSDV and MV, demonstrated earlier [10], and to VSV, EHDV and PPRV, described in this paper, we can conclude that the diploid cell line derived from P. nathusii kidney has a broad spectrum of susceptibility to viruses from different taxonomic groups.It allows us to recommend using them for primary virus isolation and other virological studies, as well as to consider this species as a possible source, carrier (transmitter) and reservoir for the transmissible viral diseases mentioned above.
To assess the reproduction and dynamics of the development of the cytopathogenic effect (CPE), we used the following susceptible cell cultures as controls for each virus: CV-1 line (African Green Monkey Kidney Fibroblast continuous cell line); L-929 (clone strain L cell culture from subcutaneous tissue of mouse), GT (primary goat kid testicle cell subculture) obtained from The Cell Cultures Collection of the Federal Research Center for Virology and Microbiology [11].
The viruses received from The State Collection of Microorganisms Causing Dangerous or Highly Dangerous Diseases, Including Zooanthroponoses, and Exotic Diseaseshttp://ckp-rf.ru/ckp/441429/
When changing the medium, we dispersed the cell monolayer using a mixture of 0.25% trypsin solution and 0.02% Versene solution 1:3 pre-warmed to 37 0 C.
The cell cultures were maintained by a conventional method of serial passages at 37 ± 0,5 °С in an atmosphere containing elevated gaseous carbon dioxide levels, up to 5%, and a relative humidity of 95%.

Methods
The sensitivity (permissivity) of the P. nathusii kidney diploid cell line (PKDCL) was tested by inoculation with a corresponding viral material, with a multiplicity of infection (MOI) of 0.1 − 0.01 TCD 50 /cell (50% tissue culture infectious dose), into a culture ask containing a con uent cell monolayer.Exposition lasted 60 min.After that, we added a supporting medium containing 2% FBS and incubated the infected culture at 37,0 ± 0,5°C until the CPE of the virus became visible.
The infectious activity of a virus was determined by titration in a virus-sensitive (susceptible) cell culture, using the time of onset of CPE as an index.The virus titer was determined using the Reed and Muench method.
The authenticity of PPRV and VSV was con rmed using molecular genetic methods.For the detection of the PPRV genome, we used ID Gene™ Peste des Petits Ruminants Duplex kit (IDvet, France) and primers as described by Kwiatek et al. [12].To detect the VSV genome, we used primers proposed by Rodriguez et.al [13] followed by sequencing the obtained PCR product.
Puri cation of PCR products was performed with a Cleanup Standard kit (Evrogen, Russia) followed by Sanger sequencing with BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, Austin, TX, U.S.A.) on Applied Biosystem Genetic Analyser 3130 XL (USA).
For additional con rmation of the PPRV authenticity, we performed a neutralization test according to a convenient scheme [15-18] and ELISA test (ID Screen® PPR Competition test kit, France).For this purpose, we tested serum samples taken from three PPRV immunized sheep ("zero-serum") and speci c sera collected from animals on the 14th day post-inoculation of the viral material obtained from a titration of a virus in the P. nathusii kidney diploid cell line from wells of 7th dilution after the development of a pronounced CPE.
The animal experimental protocols were approved by the Ethics Committee of the Federal Research Center for virology and Microbiology (Protocol № 1 from February 1, 2021) and were conducted according to Russian legislation and international (ARRIVE) guidelines in animal biosecurity facilities (77.99.03.001.K.000702.0405) of the Federal Research Center for Virology and Microbiology (FRCVM), Volginskiy, Russia.

Declarations Figures
Con uent monolayer of PKDCL at the twelfth passage level (magni cation×150) Studies on the susceptibility of bat cells have only been carried out on a few viral pathogens; most relating to causative agents of human disease.I. Eckerle et al. immortalized trachea cells lines from Carollia perspicillata (Seba's short-tailed bat) and Eidolon helvum (Straw-colored fruit bat) of the suborders Yangochiroptera and Yinpterochiroptera, respectively.These cell lines were characterized by susceptibility to viral infection with Rift-Valley fever virus and Vesicular stomatitis virus (Indiana strain) [6].G. Vrameri et al. obtained cell lines from twenty different organs of Black ying foxes (Pteropus alecto) which were sensitive to Hendra and Nipah viruses [7].A new bat cell line (NIV-BtEPC), taken/harvested from the embryo of a Plocamopherus ceylonicus, developed by D.T. Mourya and colleagues was susceptible to Chandipura (CHPV) and novel adenovirus (BtAdv-RLM) [8].

Table 1
Sensitivity (susceptibility) of the diploid cell line P. nathusii kidney to viruses n = 3