Genomic and In Vitro Phenotypic Comparisons of Epidemic and Non-Epidemic Getah Virus Strains

Getah virus is an emerging mosquito-borne animal pathogen. Four phylogenetic groups of GETV, Group I (GI), GII, GIII and GIV, were identified. However, only the GETV GIII was associated with disease epidemics suggesting possible virulence difference in this virus group. Here, we compared the genetic and in vitro phenotypic characteristics between the epidemic and non-epidemic GETV. Our complete coding genome sequence analyses revealed several amino acid substitutions unique to the GETV GIII and GIV groups, which were found mainly in the hypervariable domain of nsP3 and E2 proteins. Replication kinetics of the epidemic (GIII MI-110 and GIII 14-I-605) and non-epidemic GETV strains (prototype GI MM2021 and GIV B254) were compared in mammalian Vero cells and mosquito C6/36 and U4.4 cells. In all cells used, both epidemic GETV GIII MI-110 and GIII 14-I-605 strains showed replication rates and mean maximum titers at least 2.7-fold and 2.3-fold higher than those of GIV B254, respectively (Bonferroni posttest, p < 0.01). In Vero cells, the epidemic GETV strains caused more pronounced cytopathic effects in comparison to the GIV B254. Our findings suggest that higher virus replication competency that produces higher virus titers during infection may be the main determinant of virulence and epidemic potential of GETV.


Introduction
Getah virus (GETV) is a mosquito-borne virus that belongs to the genus Alphavirus in the family of Togaviridae [1]. It is enveloped and spherical with a diameter of approximately 70 nm [2] and contains a single-stranded positive-sense RNA genome of 11-12 kb in length. The genome consists of two open reading frames that encode four non-structural proteins (nsP1, nsP2, nsP3 and nsP4), which are responsible for viral RNA transcription and replication, and five structural proteins (capsid protein C, glycoproteins E3, E2, E1, and 6K), which are responsible for viral binding and entry into host cells during infection [1,3].
The first GETV strain, MM2021, was isolated in Malaysia in 1955 from Culex gelidus [4]. Currently, GETV is present throughout the East and Southeast Asia, as well as in Northern Australia [5][6][7]. Mosquitoes of Culex and Aedes species are the main vectors for the transmission of GETV [6,8]. Serological evidence of GETV infection has been reported in a wide range of vertebrate hosts including birds, reptiles, and mammals, and humans [9].
GETV has become one of the emerging animal pathogens that poses increased health threat to racehorses and pigs. Several outbreaks of epizootic diseases have been reported in these animals in Japan, China and India causing great economic losses [2,[10][11][12][13][14][15]. The disease in horses is generally self-limiting, present with fever, anorexia, hind limb edema and stiff gaits [13]. The GETV infection, on the other hand, caused severe and fatal diseases in young piglets, and reproductive failure in pregnant sows that lead to stillbirths and fetal deaths [15]. Recently, GETV infection has also been associated with neurological symptoms Japan Racing Association, Tochigi, Japan. The MM2021 and B254 represent GI and GIV, respectively [18,19], while both MI-110 and 14-I-605 represent GIII GETVs [18].

Sequence Comparison of GETV Strains
Whole genome sequences of all GETVs available in GenBank were downloaded and aligned using Clustal X version 2.0 software. A sequence alignment based on the complete coding region was generated using GeneDoc version 2.7 software [21] and subjected to nucleotide and amino acid sequence analyses using the GeneDoc and BioEdit version 7.2.5 [22] software.

Infection of Cells with Different GETV Strains
All three cell lines, Vero, C6/36, and U4.4, were seeded in a 96-well plate at a concentration of 2 × 10 4 cells/100 µL/well in the maintenance media. Cells were incubated overnight at appropriate culture temperature and CO 2 conditions, as mentioned previously in Section 2.1, for cell attachment. Each cell line was then infected with different GETV strains at multiplicity of infection (MOI) of 0.1. Infection was performed in triplicates. Cells were incubated at room temperature for 1 h with gentle rocking before the inoculum was replaced with maintenance media. Infected cell culture supernatants were harvested at 0, 8,24,48,72, and 96 hours post infection (hpi). One hundred and forty microliters of the cell culture supernatants were subjected to viral RNA extraction using QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. The viral RNA was eluted in 60 µL of RNase-free water and kept at −80 • C until used.

Viral RNA Quantitation Using TaqMan ® Probe-Based qRT-PCR
The GETV RNA titer was quantitated using an in-house established TaqMan ® probebased quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay as previously described [23]. The qRT-PCR was performed in a total of 10 µL in a reaction containing 5.0 µL of 2× SensiFAST Probe Hi-ROX One-Step Mix, 0.22 µL of probe/primer, 0.1 µL of Reverse Transcriptase, 0.2 µL of RiboSafe RNase Inhibitor, 1.33 µL of RNA, and 3.15 µL of DEPC-treated water. The qRT-PCR was performed using an Applied Biosystem StepOne Real-Time PCR System (Thermo Fisher Scientific" Waltham, MA, USA) with a thermal profile as follows: 45 • C for 10 min; 95 • C for 2 min; and 40 cycles of 95 • C for 5 s and 60 • C for 20 s. Titers of GETV in the supernatants were determined based on a standard curve generated using serial dilutions of the GETV RNA standard that ranged from 10 7 -10 1 RNA copies/µL.

Plaque Assay
Vero cells were seeded in a 24-well plate at a concentration of 2 × 10 5 /500 µL/well in DMEM supplemented with 10% FBS, 2 mM of L-glutamine, and 0.1 mM of 1× NEAA, and incubated overnight. The medium was removed from each well and replaced with 200 µL of viral inoculum mixed with serum-free media in a 1/10 dilution. Plates were left to rock for 1 h at room temperature. The inoculum was discarded and replaced with 1 mL of carboxy methyl cellulose (CMC) in DMEM containing 2% FBS, 2 mM of L-glutamine, and 0.1 mM of 1× NEAA. The plates were incubated at 37 • C in 5% CO 2 for 3 days before cell fixing and staining with 4% paraformaldehyde (PFA) and 1% crystal violet mixed in 20% EtOH, respectively.

Statistical Analyses
The replication growth curves of the GETV strains in respective cell lines were plotted and analyzed with two-way ANOVA and linear regression analyses. The replication rate of GETV was estimated by determining the slope of the linear regression curve. The Bonferroni posttest was performed to determine significant differences of the mean titers attained between GETV strains. All statistical analyses were performed using Graph Pad Prism 5 (Graph Pad Software Inc., San Diego, CA, USA).

Sequence Analyses of Different GETV Groups
Multiple sequence alignments comprised of the complete coding sequences of GETV strains were generated and a phylogenetic tree constructed for the different GETV groups (Supplementary Data: Figure S1). The nucleotide and amino acid sequences of the different GETV groups (GI, GII, GIIIa-GIIIe, GIV) were analyzed using the GeneDoc version 2.7 [21] and BioEdit version 7.2.5 [22] software. Comparisons of the amino acid sequences at the non-structural and structural proteins of GETV GI, GIII, and GIV viruses against the GETV Sagiyama revealed amino acid substitutions exclusive to the GIII and GIV viruses. Distinct amino acid substitutions in the nsP1, nsP2, nsP3, C, E2, and E1 genes were noted within the GETV GIII ( Figure 1 and Table 1). Two non-conservative amino acid substitutions, T461P and G467E, were found in the hypervariable carboxyl-terminal (C-terminal) of nsP3 of the GIII viruses. On the other hand, amino acid substitutions specific to the GIV Malaysian GETV B254, China GETV YN12031, and GETV/SW/Thailand/2017 were observed in the nsP2, nsP3, C, E3 and E2 proteins. In contrast to the GIII viruses, these GIV strains accumulated more non-conservative amino acid substitutions (n = 7), which were H374Y (nsP2), D386A (nsP3), P466R (nsP3), W501Q (nsP3), T505I (nsP3), D109M (E2) and S205N (E2), when compared against the GII Sagiyama strain. The GIV Russia LEIV16275 Mag, on the other hand, showed rather different amino acid substitutions in comparison to the other GIV strains.
The replication growth curves of the GETV strains in respective cell lines were plotted and analyzed with two-way ANOVA and linear regression analyses. The replication rate of GETV was estimated by determining the slope of the linear regression curve. The Bonferroni posttest was performed to determine significant differences of the mean titers attained between GETV strains. All statistical analyses were performed using Graph Pad Prism 5 (Graph Pad Software Inc., San Diego, CA, USA).

Sequence Analyses of Different GETV Groups
Multiple sequence alignments comprised of the complete coding sequences of GETV strains were generated and a phylogenetic tree constructed for the different GETV groups (Supplementary Data: Figure S1). The nucleotide and amino acid sequences of the different GETV groups (GI, GII, GIIIa-GIIIe, GIV) were analyzed using the GeneDoc version 2.7 [21] and BioEdit version 7.2.5 [22] software. Comparisons of the amino acid sequences at the non-structural and structural proteins of GETV GI, GIII, and GIV viruses against the GETV Sagiyama revealed amino acid substitutions exclusive to the GIII and GIV viruses. Distinct amino acid substitutions in the nsP1, nsP2, nsP3, C, E2, and E1 genes were noted within the GETV GIII ( Figure 1 and Table 1). Two non-conservative amino acid substitutions, T461P and G467E, were found in the hypervariable carboxyl-terminal (C-terminal) of nsP3 of the GIII viruses. On the other hand, amino acid substitutions specific to the GIV Malaysian GETV B254, China GETV YN12031, and GETV/SW/Thailand/2017 were observed in the nsP2, nsP3, C, E3 and E2 proteins. In contrast to the GIII viruses, these GIV strains accumulated more non-conservative amino acid substitutions (n = 7), which were H374Y (nsP2), D386A (nsP3), P466R (nsP3), W501Q (nsP3), T505I (nsP3), D109M (E2) and S205N (E2), when compared against the GII Sagiyama strain. The GIV Russia LEIV16275 Mag, on the other hand, showed rather different amino acid substitutions in comparison to the other GIV strains.

Plaque Morphology of Different GETV Strains
In this study, four GETV strains, GI MM2021, GIII MI-110, GIII 14-I-605, and GIV B254 were used and compared for their in vitro replications. The virus inoculums were prepared using C6/36 cells and virus titers were determined by plaque assays using Vero cells. The plaque sizes were measured across four independent experiments and t-test was used to compare means. All GETV strains used produced distinct plaques of heterogenous sizes ( Figure 2). In general, the sizes of the plaques formed by GIV B254, ranging between 9.3-28.2 mm, were significantly smaller than those of GI MM2021 (24.8-50.1 mm) (p < 0.01), GIII MI-110 (11.6-49. 8 mm) (p < 0.05), and GIII 14-I-605 (28.7-63.1 mm) (p < 0.01).
In order to validate the infectivity of the extracellular viral samples, a plaque assay was performed to measure the infectious virus titers for selected time points during the exponential phase of infections (Supplementary Data: Figure S2). Overall, all GETVs showed increase in the infectious titers in all infected cells. Consistently, the GIV B254 showed lower infectious virus titers than those of GI MM2021, GIII MI-110, and GIII 14-I-605 at 48 hpi in the infected Vero and C6/36 cells, and at 24 hpi in the U4.4 cells (Supplementary Data: Figure S2).

Discussion
Of the four major phylogenetic groups of GETV, GIII and GIV were the most recent circulating and geographically expanding virus groups. However, to date, the GETV GIII has been the sole lineage that was associated with manifestation of diseases in animals

Discussion
Of the four major phylogenetic groups of GETV, GIII and GIV were the most recent circulating and geographically expanding virus groups. However, to date, the GETV GIII has been the sole lineage that was associated with manifestation of diseases in animals [5,10,13]. In this study, we examined and compared the genomic and in vitro phenotypic characteristics between the epidemic and non-epidemic GETV strains. While both epidemic GETV GIII strains consistently replicated at higher rates and produced higher virus titers in all cell lines, the non-epidemic GETV GIV strain showed the lowest replication rate and virus titer during infection. Our findings suggest that the phenotypic differences between the different GETV groups could be attributed to the genotypic variations unique to their respective groups, particularly those resulting in the non-conservative amino acid substitutions in the nsP3 and E2 proteins.
The Japanese GETV MI-110 was among the first strains of GIII lineage that emerged and caused an outbreak of infection in horses in 1978, at Miho Training Centre, Ibaraki Prefecture, Eastern Japan [5]. In 2014, a recurrent outbreak caused by the GETV GIII 14-I-605 strain occurred among vaccinated racehorses at the same training center [13]. Sequence analyses between these two virus strains suggested the potential importance of the amino acid substitutions in the hypervariable domain (HVD) region of nsP3, which includes the T416P reported in this study, on the virological properties of the virus [5,24,25]. The nsP3 protein has two conserved domains and a HVD region; the latter is crucial for the interactions with host factors and plays an essential role in virus replication in the mosquito vectors and vertebrate hosts [26,27]. Thus, the genetic variations in this gene region may probably influence the virus replication competency in a particular host. In this study, both GIII GETV MI-110 and 14-I-605 strains exhibited higher replication rates and produced higher virus titers than the non-GIII strains in the mosquito and mammalian cells. This suggests that the GIII GETV undergoes an infection cycle more rapidly, thus infecting a greater number of cells and causing more CPE within the same period of infection, compared to the non-GIII strains. A virulence characteristic allowing the virus to replicate to a sufficient virus load before the onset of robust host immune response could be an important key advantage for the GIII GETV strains. This may also suggest the higher competency of the GIII strains in spreading from the initial infection site to other target tissues and organs where pathogenicity was observed in the infected hosts.
The GETV GIV B254 strain is a new virus strain recently isolated from Culex fuscocephalus in Malaysia, since the first virus isolation in 1955 [19]. It is phylogenetically distinct from the old Malaysian GETV MM2021, but similar to other GIV strains, where it shared the closest relationship with the China YN12031 strain isolated in 2012 [19,28]. It has been hypothesized that the GETV GIII and GIV viruses evolved from the GII Sagiyama strain. However, in comparison to the GIII viruses, the GIV viruses showed excessive amino acid substitutions not only in the nsP3 but also in the structural genes. This suggests that the GIV lineage may be under a different selection pressure potentially caused by differences in hosts.
So far, both Malaysian GETV MM2021 and B254 have not been associated with any disease outbreaks in animals or humans. In our study, the GETV B254 demonstrated a relatively lower replication competence in all the cell lines used, as shown by the slower rate of replication and lower virus titers produced, compared to those of the GIII GETV strains. Relatively lesser CPE and dead cells were observed in the GETV B254-infected Vero and C6/36 cells through microscopic examination; however, further experiments are desired in future to quantitatively determine the degree of CPE caused by different GETV strains for better comparisons. Nevertheless, like the other strains with reduced virulence, GETV B254 formed plaques of much smaller sizes [29][30][31][32]. Evidently, these phenotypes suggest that the GIV B254 undergo a longer delay for virus replication and release, and consistent with a longer elapsed time between the successive infection cycles. As such, the GIV B254 strain is unable to effectively infect a large number of tissue cells and cause CPE that result in manifestation of disease. This also means that the GIV viruses could be transmitted between the mosquitoes and vertebrate hosts in nature without being detected due to the absence of disease. In relation, the GETV/SW/Thailand/2017 belonging to the GIV group was isolated from pig serum during a sero-surveillance in Thailand, where no disease was reported [20]. It is worth noting that the pig-origin GIV strain, in comparison to the mosquito-origin GIV viruses, showed an amino acid substitution at the E2 (L269V), which was exclusively associated with GIII lineage and was found to be the sole positive selection site in the structural genes ( Table 1). As the E2 of alphaviruses has been associated with host range and pathogenicity [33], the substitution in this gene could possibly mark an adjustment towards acquisition of epidemic potential of the GIV virus strains, possibly resembling that of the A226V substitution in Chikungunya virus which resulted in a pandemic [34].
The first discovered Malaysian GETV MM2021 (1955) was of the GI lineage [4]. Between 1960s to 1970s, GETV was associated with large domestic animals in Malaysia, where the carabaos, horses and pigs showed the highest serological prevalence of infection [35,36]. The virus infections in these animals, however, were mostly inapparent. Isolation of several Malaysian GETVs from various mosquito species was reported during the same period. These viruses, of which the molecular characters were unknown, could be the other strains of the GI lineage which may be associated with mild or asymptomatic infections in the vertebrate hosts. In this study, the GETV MM2021 prototype strain showed replication efficiency comparable to the epidemic GETV GIII strains, although there were no common mutations between these viruses to explain this. Nevertheless, this could be caused by the in vitro adaptation of MM2021 strain to the cell culture after repeated and prolonged culture in the laboratory. This may lead to enhanced virus replication to produce higher virus titer, as previously seen in several other viruses [37][38][39].
The mammalian Vero cells and the Aedes albopictus C6/36 mosquito cells are the common susceptible cell lines used for arbovirus propagation and replication studies. The alphaviruses, such as CHIKV and SINV, have been shown to cause acute, lytic infection in the mammalian cells leading to strong CPE and apoptosis, while inducing persistent infection accompanied by lower virus titers in the mosquito cells [40,41]. These different infection dynamics were probably attributed to the spatial and temporal differences of virus replication and assembly process in the different types of cells [42,43]. Similarly, in our study, the GETV replicated to higher virus titers and caused more pronounced CPE in Vero cells than in the Aedes albopictus-origin C6/36 and U4.4 cells. While the C6/36 cells are lacking an intact RNA interference (RNAi) defense mechanism [44], the U4.4 cells are RNAi competent, thus, making it a better cell model for a more accurate presentation of the alphavirus infection in nature. Our findings showed an early declining titer of all GETV strains during infection in the U4.4 cells, indicating the virus growth restriction most likely by the RNAi response. Nevertheless, the epidemic GETV GIII strains have consistently exhibited higher replication competence even in this cell, in comparison to the non-epidemic GETV strains. Further investigations in mosquitoes are needed to better characterize the in vivo vector competence of the different GETV strains.
In summary, we compared the genetic and in vitro phenotypic characteristics between the epidemic and non-epidemic GETV. Several amino acid substitutions specific to the GETV GIII and GIV viruses in the nsP3 and E2 genes were identified. These amino acid substitutions may play a role in the higher replication rates, higher virus titers, and more pronounced CPE of the epidemic GIII viruses, compared to the non-epidemic viruses of GI and GIV groups. This further suggests that the higher virus replication competency to produce high virus titer during an infection may be the crucial determinants of virulence and epidemic potential of GETV. An in vivo study using a suitable animal model would be desired to further confirm the pathogenicity differences between the epidemic and non-epidemic GETV strains.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v14050942/s1, Figure S1: Maximum likelihood phylogenetic analysis of GETV based on the complete coding sequences. Figure