Investigating the reassortment potential and pathogenicity of the S segment in Akabane virus using a reverse genetics system

Abstract

Background

Akabane virus (AKAV) is an arthropod-borne virus that causes congenital malformations and neuropathology in cattle and sheep. In South Korea, AKAVs are classified into two main genogroups: K0505 and AKAV-7 strains. The K0505 strain infects pregnant cattle, leading to fetal abnormalities, while the AKAV-7 strain induces encephalomyelitis in post-natal cattle. The pathogenicities of K0505 and AKAV-7 strains differ significantly; however, the specific gene in the AKAV-7 strain that drives its pathogenicity remains unidentified. In this study, changes in viral replication and pathogenicity were investigated, particularly when the S segment of AKAV-7 was mutated using a T7 RNA polymerase-based reverse genetics (RG) system.

Results

The rAKAV-7ΔNSs virus, with a deletion in the NSs protein of the wild-type AKAV-7 virus (wtAKAV-7), and the rAKAV-7(S-K0505) virus, where the S segment of wtAKAV-7 was reassorted with that from the wild type K0505 strain (wtK0505), were successfully rescued. The rAKAV-7ΔNSs virus demonstrated impaired replication in Vero cells and exhibited reduced mortality and RNA viral load in the organs of suckling mice compared to the wtAKAV-7. The rAKAV-7(S-K0505) virus displayed similar growth kinetics in Vero cells and showed no significant reduction in mortality rate in suckling mice compared to wtAKAV-7.

Conclusions

These observations suggest that the S segment, especially the NS protein, is associated with the pathogenicity of AKAV-7. Also, the results imply that the L and M segments might explain the differences in pathogenicity between the AKAV-7 and K0505 strains. Moreover, our findings indicate the potential for reassortment between distinct genogroups of AKAVs.

Background

Akabane virus (AKAV) is an arthropod-borne virus (arbovirus) known for its teratogenic and neuropathogenic effects on domestic livestock and wild animals [1]. Although AKAV infection is typically asymptomatic in adult cattle, its transplacental transmission can cause teratogenic effects such as stillbirth, polioencephalomyelitis, and microencephaly [2]. Moreover, highly virulent strains of AKAV are recognized for causing encephalomyelitis following postnatal infection [3].

AKAV belongs to the Orthobunyavirus genus of the Peribunyavidae family within the order Bunyavirales [4]. AKAV possesses a tripartite, single-stranded, negative-sense RNA genome, designated as L (large), M (medium), and S (small). The L segment encodes an RNA-dependent RNA polymerase (RdRp). The M segment encodes two surface glycoproteins, Gn and Gc, which facilitate viral attachment and neutralization, and a non-structural (NSm) protein, crucial for viral assembly and morphogenesis [5]. The S segment encodes a nucleoprotein (N) and a nonstructural (NSs) protein, which are significant in antagonizing alpha/beta-interferon (IFN) and regulating host-protein synthesis [6]. The N protein encapsidates the AKAV genome and interacts with the viral RdRp to form ribonucleoprotein complexes (RNPs) [7].

Phylogenetic analysis indicates that all AKAVs from East Asia are classified into two genogroups: I and II [8]. Genogroup I primarily causes bovine encephalomyelitis through post-natal infection in cattle and has been further subdivided into genogroup Ia and Ib, while genogroup II primarily causes abnormal births in pregnant cattle. The K0505 strain, which causes abortion, was isolated from a native Korean cow in 2005 and is categorized within genogroup II [9]. The AKAV-7 strain, isolated from central nervous system samples of cows displaying AKAV-induced encephalomyelitis in 2010, is part of genogroup Ia [5]. Furthermore, pathogenicity studies of the K0505 and AKAV-7 strains reveal significant differences in suckling mice; the K0505 strain is avirulent with a survival rate of 100%, while the AKAV-7 strain demonstrates greater neurovirulence, leading to a survival rate of 30% in suckling mice [5].

The establishment of a bunyavirus reverse genetics (RG) system has provided critical insights into the pathogenesis and life cycles of bunyavirus infections [10]. Since AKAV is a negative-sense RNA virus, full-length complementary DNAs (cDNA) copies of AKAV genomes must be introduced to the RNA polymerase promoter [11, 12]. RG systems for bunyaviruses utilize either cellular RNA polymerase I or bacteriophage T7 RNA polymerase [13]. The RG system for AKAV was initially based on an RNA polymerase I-based system using the Japanese OBE-1 strain, but due to its low efficiency, a more effective T7 RNA polymerase-based RG system was later developed [14, 15].

The M segment is a major determinant of virus virulence as it encodes glycoproteins [16]. Additionally, the NSs of the S segment plays a role in virus virulence [17]. However, the precise role of the S segment in determining pathogenicity remains unclear. To investigate this, a T7 RNA polymerase-based RG system for the AKAV-7 strain was developed. Using this RG system, a mutant AKAV-7 virus with a targeted deletion in the NSs gene was rescued, and a reassortant AKAV-7 virus incorporating the S segment from the K0505 strain was generated. Our objective is to elucidate the effects of modifications in the S segment on the pathogenicity of the AKAV-7 strain through comprehensive cell culture and animal model experiments.

Methods

Cells and viruses

Vero cells (African green monkey kidney cells; ATCC CCL-81) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% antibiotic/antimycotic solution (Gibco, USA) and 10% fetal bovine serum (FBS). BHK/T7-9 cells (BHK-21 derivative cells clone stably expressing T7 RNA polymerase; RCB4942; RIKEN BRC cell bank, Ibaraki, Japan) [18] were cultured in minimum essential medium (MEM) containing 1% antibiotic/antimycotic solution (Gibco), 5% FBS, and 10% tryptose phosphate broth (TPB; BD). The cell lines were maintained at 37 °C with 5% CO₂. The AKAV-7 strain (KVCC-VR1500001) and the K0505 strain (KVCC-VR1500047) were sourced from the Korea Veterinary Culture Collection (KVCC). The AKAV-7 and the K0505 strains were propagated in Vero cells through 3 passages in a biosafety level 2 (BSL-2) laboratory. A commercially available Akabane vaccine produced by a Korean animal vaccine company (ChoongAng Co.), Bobishot Akabane®, was purchased. The vaccine was inoculated into Vero cells in DMEM with 5% FBS and 1% antibiotic/antimycotic solution. The cells were incubated at 37 °C with 5% CO₂.

PCR amplification, sequencing and phylogenetic analysis

Viral RNA was extracted from the cell culture supernatant using the RNeasy Mini Kit (Qiagen, Germany). It was then transcribed using SuperScriptIII Reverse Transcriptase (Invitrogen, USA). The PCR primers were specifically designed based on sequences from NCBI: JQ308779, JQ308775, and JQ308771 for the AKAV-7 and FJ498800, FJ498796 for the K0505 strain (Table S1). To amplify the 5’ and 3' ends of the AKAV genome, rapid amplification of cDNA ends (RACE) was performed using the SMARTer® RACE 5'/3' Kit (Takara Bio Inc., Japan), following the manufacturer’s protocol. For the 3’ end, a A poly-A tail was added to the viral RNA using poly (A) polymerase (Takara Bio Inc., Japan) before cDNA synthesis. The genome end sequences were subsequently amplified using nested PCR with genome specific primers. The PCR products were electrophoresed on a 1.2% (w/v) agarose gel with 1X Tris–borate-EDTA (TBE) buffer and purified using the LaboPass Gel and PCR Clean-Up Kit (Cosmogenetech Co., South Korea). After purification, the samples were sent to Macrogen (Daejeon, South Korea) for Sanger sequencing. The sequences obtained were refined using Bioedit Sequence Alignment Editor version 7.0.5.3. Reference sequences were retrieved from the NCBI GenBank database. These sequences were aligned using Clustal Omega Ver. 1.2.4 [19]. Maximum likelihood (ML) phylogenetic trees were constructed with IQ-TREE software version 1.6.12 [20]. The best substitution models were identified using ModelFinder, and ultrafast bootstrap analysis was conducted with 1000 replicates in IQ-TREE [21, 22]. The phylogenetic trees were visualized in iTOL Ver. 6 [23] and Inkscape.

Plasmid construction

Viral RNA was extracted from virions in the supernatant of Vero cells infected with the AKAV-7 and K0505 strains, using the RNeasy Mini Kit (Qiagen, Germany). It was then transcribed by SuperScriptIII Reverse Transcriptase (Invitrogen, USA) with primers designed from AKAV sequences (Table S2). The viral antigenomic cDNAs were amplified using Invitrogen Platinum SuperFi II Green PCR Master Mix (Thermo Fisher Scientific, USA) with segment -specific primer sets (Table S2). To enhance the transcription reaction by T7 polymerase, an additional guanine (G) nucleotide was inserted downstream of the T7 promoter. The PCR products were purified on a 1.2% (w/v) agarose gel using the PureLink™ Quick Gel Extraction Kit (Invitrogen, USA).

The Plasmid TVT7R (0,0) (Addgene, plasmid #98,631), used for the rescue of AKAV, has been previously described and can undergo cleavage between the T7 promoter and the Hepatitis delta virus (HDV) antigenome ribozyme using the BbsI enzyme [12, 24]. To optimize rescue efficiency, helper vectors were constructed, and the plasmid pTM1-HB29L, used as a template vector, was generously provided by Benjamin Brennan from the MRC-University of Glasgow Centre for Virus Research.

The full-length cDNA fragments of the L, M, and S segments were cloned into the BbsI-linearized TVT7R (0,0) vector using the In-Fusion® HD Cloning Kit by Clontech (Takara Bio Inc., Japan). The resulting constructs were designated as pTVT7_AKAV-7_L, pTVT7_AKAV-7_M, pTVT7_AKAV-7_S, and pTVT7_K0505_S, respectively (Fig. 1). The ORFs of viral RdRp and N for AKAV-7 and K0505 were cloned into the pTM1 vector using the In-Fusion® HD Cloning Kit. These constructs were named pTM1_AKAV-7_RdRp, pTM1_AKAV-7_N, and pTM1_K0505_N respectively. The primers used are described in Table S2.

Fig. 1

Scheme for the reverse genetics (RG) system of AKAVs. For construction of the transcription plasmid, full-length cDNA fragments of the L, M, and S segments were cloned into the TVT7R vector, containing T7 promoter and HDV sequence. To construct the helper plasmids, ORFs of the RdRp and N were cloned into the pTM1 vector. Three transcription plasmids and two helper plasmids were co-transfected into BHK/T7-9 cells. The supernatants of BHK/T7-9 cells were passaged in Vero cells, and the rAKAV-7, rAKAV-7(S-K0505), and rAKAV-7ΔNSs viruses were rescued

The plasmid that abolished NSs expression without altering the amino acid sequence of N was constructed by previous research [14]. Briefly, to suppress NSs expression, the ATG start codon (nucleotides 59–61 of the S cDNA) was modified to ACG in the pTVT7_AKAV-7_S and pTM1_AKAV-7_N vectors using the In-Fusion® HD Cloning Kit. This modification resulted in the creation of pTVT7_AKAV-7_SΔNSs and pTM1_AKAV-7_NΔNSs. The primers were detailed in Table S2. All constructed plasmids were sequenced to confirm the absence of any unintended mutations.

Rescue of AKAVs

BHK/T7-9 cells were cultivated in six-well plates at 70% confluence. Each well was transfected with 1 μg of pTVT7_AKAV-7_L, 1 μg of pTVT7_AKAV-7_M, 1 μg of pTVT7_AKAV-7_S, 0.5 μg of pTM1_AKAV-7_RdRp, and 0.5 μg of pTM1_AKAV-7_N using 2 μL of Lipofectamine-3000 Transfection Reagent (Invitrogen, USA) per μg of DNA in 500 μL of Opti-MEM (Gibco, USA). After 24 h of incubation at 37 °C, the Opti-MEM was replaced with 500 μL of MEM containing 1% antibiotic/antimycotic solution (Gibco, USA), 3% FBS, and 10% TPB (BD, Difco, USA). At 5 days post-transfection (dpt), supernatants (passage 0, P0) were collected and passaged in Vero cells for 4 days (passage 1, P1). Blind passages were performed three times on Vero cells (to passage 3, P3). The rescued AKAV was designated rAKAV-7 to distinguish it from the wild type AKAV-7 virus (wtAKAV-7). To rescue the rAKAV-7ΔNSs virus with a deletion in the NSs protein of the wtAKAV-7 strain, 1 μg of pTVT7_AKAV-7_L, 1 μg of pTVT7_AKAV-7_M, 1 μg of pTVT7_AKAV-7_SΔNSs, 0.5 μg of pTM1_AKAV-7_RdRp, and 0.5 μg of pTM1_AKAV-7_NΔNSs were used for transfection. To generate the rAKAV-7(S-K0505) virus, in which the S segment of the wtAKAV-7 strain was reassorted with the S segment from the wild type K0505 strain (wtK0505), 1 μg of pTVT7_AKAV-7_L, 1 μg of pTVT7_AKAV-7_M, 1 μg of pTVT7_K0505_S, 0.5 μg of pTM1_AKAV-7_RdRp, and 0.5 μg of pTM1_K0505_N were used. The subsequent procedures were carried out as previously described. To confirm the AKAVs' rescue, an indirect immunofluorescence assay (IFA) was performed on the Vero cells. In addition, to verify the nucleotide identities of the wild type and rescued AKAVs, reverse transcriptase (RT)-PCR and Sanger sequencing were performed using primers listed in Table S1. The schematic organization of the reverse genetics system of AKAVs is depicted in Fig. 1.

Indirect immunofluorescence assay (IFA)

The Vero cells were plated in 6-well plates at 70–80% confluence and infected with AKAVs with a multiplicity of infection (MOI) of 0.01. After incubating at 37 °C for 18 h, the cells were fixed with 80% cold acetone for 5 min, allowed to air dry, and then treated with PBST (1 × PBS with 0.05% tween 20) for 5 min at room temperature (RT). Subsequently, the cells were incubated with an anti-AKAV nucleoprotein (N) monoclonal antibody, diluted at 1:200 (5B56, produced in-house, [25]), as the primary antibody at 37 °C for 1 h. After three PBST washes, the cells were stained with goat anti-mouse IgG heavy and light chain antibody FITC-conjugated (diluted 1:100, Bethyl, USA) as the secondary antibody at 37 °C for 1 h. The cells were then washed three times with PBST, and the nuclei were stained with the NucBlue Fixed Cell Stain Ready probes reagent (R37606, Invitrogen, USA) for 5 min in the dark at RT. The cells were observed under an EVOS M5000 microscope (Thermo Fisher Scientific, China).

Growth kinetics in cell culture

Sub-confluent monolayers of Vero cells were infected with both wild type and rescued AKAVs at a MOI of 0.01 for 1 h at 37 °C in 5% CO₂. After washing the infected cells three times with PBS, DMEM supplemented with 3% FBS was added. Supernatants were collected at various time points: 12, 24, 36, 48, 60 and 72 h post-infection (hpi), and were titrated for the 50% tissue culture infectious dose (TCID₅₀) using Vero cells.

Mouse experiment with suckling mouse

The BALB/c mice were purchased from Damool Science (Daejeon, Korea). Five- to six-day-old BALB/c suckling mice were inoculated via intraperitoneal (IP) routes with 10⁵ TCID₅₀/ml in a volume of 100 μL of the AKAVs. Each experimental group consists of suckling mice born to two pregnant dams. Seven, twelve, fourteen, and fifteen BALB/c mice in respective groups were inoculated with wtK0505, wtAKAV-7, rAKAV-7(S-K0505), and rAKAV-7ΔNSs virus. Six BALB/c mice were also inoculated with DMEM as the control group. Clinical signs were monitored three times daily, and body weights were recorded once daily throughout the experiment. Mice showing no clinical signs were sacrificed by bleeding under deep isoflurane (Hana Pharm Co., South Korea) anesthesia at 14 days post-infection (dpi). According to UC Berkeley Guidelines, mice were euthanized immediately upon the onset of paralysis. Since mice with severe paralysis sometimes did not survive tissue sample harvesting, paralysis was considered synonymous with death [26]. Thus, mice showing signs of paralysis were euthanized. Mice that lost 20% of their body weights were also sacrificed.

Quantification of viral loads in various tissue samples

To examine viral loads, tissues such as the brain, spinal cord, lung, liver, kidney, spleen, and small intestine were collected. The collected tissues were homogenized in PBS with 1% antibiotic/antimycotic solution (Gibco, USA). Viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. The RNA was reverse transcribed into cDNA using the WizScript™ cDNA Synthesis Kit (High Capacity) (Wizbio Solutions, Korea). Viral loads were determined using a real-time polymerase chain reaction (qPCR) with primers specific to the S segment of AKAV [27]. To quantify viral loads, standard curves were established through tenfold serial dilutions of cloned plasmids containing a 242 bp fragment of the AKAV N protein sequence, initially present at concentrations of 10⁸ copies/μL. The fragment was amplified by conventional PCR using the following primer set: Forward, 5'-GTAAGTATGGGCAGCAGCTC-3' and Reverse, 5'-CCTTGCACTGCTCAGCAACC-3'. It was then cloned into the RBC T&A Cloning vector (RBC Bioscience, Taiwan).

Histopathology and immunohistochemistry (IHC)

Five individual brain (cerebrum) and lumbar spinal cord samples from each group were randomly selected. The collected samples were immediately fixed in 10% neutral buffered formalin and sent to the Korea Pathology Technical Center (Cheongju, South Korea) for histopathological analysis. The tissues were embedded in paraffin, sectioned to a thickness of 4 μm, and stained with hematoxylin and eosin (H&E). Qualified pathologists examined the tissue sections using the Nonneoplastic Lesion Atlas (NNLA) as the diagnostic guideline. The severity of histopathological findings was evaluated and categorized into four grades: minimal ( ±), mild ( +), moderate (+ +), and marked (+ + +). The definitions were as follows: minimal with 1–2 foci, mild with 3–5 foci, moderate with 6–12 foci, and marked with more than 12 foci per examined sample.

For AKAV antigen detection, tissue sections were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide (H₂O₂) in methanol for 20 min. The sections underwent antigen retrieval in boiling citrate buffer for 30 min and were subsequently incubated with anti-AKAV nucleoprotein (N) monoclonal antibody from mouse, diluted at 1:100 (5B56, produced in-house, [25]) for 14 h at 4 °C. Thereafter, the sections were incubated with an ImmPRESS Universal Polymer kit, Peroxidase (horse anti-mouse/rabbit IgG) (MP-7500; Vector Laboratories, USA) for one hour at RT, and visualized using the ImmPACT DAB Substrate kit, Peroxidase (SK-4105, Vector Laboratories, USA). Finally, the slides were counterstained with Harris hematoxylin and mounted.

Data analysis

All statistical analyses were performed using GraphPad Prism software version 9.5.1. Differences between groups were examined using one-way and two-way analysis of variance (ANOVA) and the Tukey post hoc test. For comparisons within the same group, Student's t-test was used. Survival rates were analyzed using Kaplan–Meier estimates. A P value of less than 0.05 was considered statistically significant.

Results

Phylogenetic and genetic analysis of AKAVs

The nucleotide sequences obtained in this study have been submitted to the NCBI GenBank database and assigned the following accession numbers: PQ273264 to PQ273266 for the BoviShot Korean AKAV vaccine, PQ799179 to PQ799181 for AKAV-7, and PQ799177, PQ799178 and PQ497630 for the K0505 strains. Analyses in this study were conducted using these newly obtained sequences rather than the previously published sequences from GenBank, due to nucleotide sequence differences observed between the results obtained in this study and the published sequences. Phylogenetic trees were constructed using alignments of the complete coding regions from the L, M, and S segments of AKAV-7, K0505, BoviShot Korean AKAV vaccine strains, and currently deposited AKAV sequences in GenBank. The genogroups of AKAVs were categorized based on prior research [28]. Phylogenetic analysis showed that the L, M, and S segments of AKAV-7 grouped within genogroup Ia (East Asia A) (Fig. S1A-S1C). The L, M, and S segments of K0505 and BoviShot Korean AKAV vaccine were categorized similarly within genogroup II (East Asia B). The nucleotide and amino acid similarity of the open reading frames (ORFs) in AKAV-7, K0505, and BoviShot was analyzed (Table S3). The analysis revealed that K0505 and BoviShot, being in the same genogroup, exhibited high nucleotide and amino acid similarities. In contrast, K0505 and BoviShot showed low nucleotide and amino acid similarities to AKAV-7, particularly in the M segment. Within the S segment, there were 29 nucleotide sequence differences between AKAV-7 and K0505, while the amino acid sequence exhibited only one difference in the N protein. However, in the NSs protein of the S segment, 4 nucleotide sequence differences were observed, leading to 3 amino acid differences between AKAV-7 and K0505.

Rescue of mutant AKAVs

The inserted nucleotide sequences in the vector were confirmed through sequencing. Supernatant from BHK/T7-9 cells transfected with three transcription plasmids and two helper plasmids induced cytopathic effects (CPEs) in Vero cells within 48 h. The CPEs resulted in altered cell morphology, with Vero cells appearing rounded, wrinkled, and fragmented compared to uninfected cells. Additionally, green fluorescence signals were observed in Vero cells infected with both wild type and rescued AKAVs during IFA (Fig. 2). These results confirm the rescue of mutant AKAVs and sequencing analysis verified that no unintended nucleotide mutations were detected.

Fig. 2

IFA identification of wild type and rescued AKAVs in Vero cells. Vero cells were infected using a MOI of 0.01. At 18 h post infection, fluorescence of FITC (green) was observed, and nuclei were counterstained with NucBlue (blue). The DMEM (A), wtAKAV-7 (B), wtK0505 (C), rAKAV-7 (D), rAKAV-7(S-K0505) (E), and rAKAV-7ΔNSs (F) are displayed. Images were captured using an EVOS M5000 Imaging System. Scale bars represent 100 μm

Growth kinetics

The growth kinetics of wtK0505, wtAKAV-7, rAKAV-7, rAKAV-7(S-K0505), and rAKAV-7ΔNSs were evaluated in Vero cells. The viruses wtK0505, wtAKAV-7, rAKAV-7, and rAKAV-7(S-K0505) demonstrated growth kinetics similar to those of wtAKAV-7 (Fig. 3). The peak titer of wtK0505, wtAKAV-7, rAKAV-7, and rAKAV-7(S-K0505) reached 7.16, 7.49, 7.25, and 7.25 log TCID₅₀/ml, respectively. However, rAKAV-7ΔNSs showed significantly lower replication rates in Vero cells, with a final titer of 6.4 log TCID₅₀/ml at 36 and 48 hpi, approximately 1 log lower than the wtAKAV-7 (7.4 log TCID₅₀/ml).

Fig. 3

Multi-step growth curves of wild type and rescued AKAVs in Vero cells. Vero cells were infected using a MOI of 0.01. Supernatants from Vero cells were collected at 12, 24, 36, 48, 60, and 72 hpi. Dagger (†) and double dagger (‡) symbols indicate significant differences between AKAV-7ΔNSs and the other viruses (p < 0.05). Data points represent means, and error bars indicate standard deviation (SD). Statistical significance was determined by two-way ANOVA (Tukey's test)

Mortality and clinical signs of infected mice

As shown in Fig. 4A, all mice inoculated with DMEM and wtK0505 survived throughout the experiment. In contrast, mice infected with wtAKAV-7 experienced a 100% mortality rate at 6–12 dpi. Meanwhile, those infected with rAKAV-7(S-K0505) and rAKAV-7ΔNSs exhibited mortality rates of 85.7% and 20%, respectively. The suckling mice that succumbed to AKAV infection showed clinical signs including weight loss, inability to suckle, lethargy, unsteady gait, ataxia, and hind limb paralysis (Fig. 4B). However, the surviving suckling mice exhibited no clinical signs during the experiment.

Fig. 4

Body weight, body temperature, survival rate, and AKAV copy numbers of BALB/c suckling mice infected with wild type and rescued AKAVs were monitored over 14 days post-infection. Survival rate (A) and body weight (%) (B) were analyzed. Additionally, brain (C), spinal cord (D), lung (E), liver (F), spleen (G), kidney (H), and small intestine (I) were harvested for AKAV copy number analysis, which was conducted using qPCR. Data are presented as means ± SD. Viral RNA levels were analyzed using one-way ANOVA (Tukey’s test). The viral RNA load for euthanized or succumbed mice is highlighted with a red border. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Viral RNA copy number in different organs

To examine neurological impacts, brains and spinal cords were collected from suckling mice infected with AKAVs to verify if AKAV can replicate in the central nervous system (CNS) of these mice (Fig. 4C, D). Additionally, to assess AKAV replication in other organs, lungs, liver, spleen, kidneys, and small intestine were also harvested (Fig. 4E-4I). The results indicated that the viral RNA loads in the brain and spinal cord were higher than those in the lungs, liver, spleen, kidneys, and small intestine. Notably, brains and spinal cords from mice inoculated with the wtAKAV-7 virus had significantly higher RNA viral loads compared to other groups. In the rAKAV-7ΔNSs group, we conducted a statistical analysis (Student's t-test) to compare the viral RNA load between mice that were euthanized or succumbed and those that survived. The analysis revealed statistically significant differences in the brain and spinal cord (p < 0.05), while no significant differences were observed in other organs.

Histopathological and immunohistochemical findings

Histopathological examinations were conducted on the brains (cerebrum) and lumbar spinal cords due to neurological signs observed in suckling mice infected with AKAV (Table 1, Fig. 5). In those inoculated with DMEM and wtK0505, no pathological findings were noted (Fig. 5A, B, F, G). However, suckling mice that succumbed to wtAKAV-7 showed minimal gliosis, neutrophil infiltration, and neuronal necrosis in the brain (Fig. 5C). Moreover, spongiosis, mild gliosis, neutrophil infiltration, and minimal neuronal necrosis were noted in the spinal cords of these mice (Fig. 5H). Those that succumbed to rAKAV-7(S-K0505) exhibited minimal gliosis and minimal to mild neutrophil infiltration in the brain (Fig. 5D), and minimal gliosis and neutrophil infiltration in the spinal cord (Fig. 5I). Mice that succumbed to AKAV-7ΔNSs showed minimal gliosis and neutrophil infiltration in the brain (Fig. 5E), and minimal to mild gliosis, minimal perivascular inflammation, and neutrophil infiltration in the spinal cords (Fig. 5J).

Table 1 Histopathological grades of the brain (cerebrum) and lumbar spinal cord in suckling mice inoculated with DMEM, wild type, and rescued AKAVs (± , minimal; + , mild; -, without histopathological alterations)

Fig. 5

Histopathological findings in the brain (cerebrum) and lumbar spinal cord sections of mice infected with wild type and rescued AKAVs: DMEM (A, F); wtK0505 (B, G), wtAKAV-7 (C, H); rAKAV-7(S-K0505) (D, I) and rAKAV-7ΔNSs (E, J) (H&E). An arrow indicates neuronal necrosis, an arrowhead points to gliosis, asterisks denote inflammatory cell infiltration into perivascular and neuronal areas, and a crosshatch signifies spongiosis. Scale bars measure 100 μm. Immunohistochemistry (IHC) detected anti-AKAV N protein in brain (cerebrum) and lumbar spinal cord sections from infections with wild type and ruscued AKAVs; sections include: DMEM, brain (K); wtK0505, brain (L); wtAKAV-7, brain (M); rAKAV-7(S-K0505), spinal cord (N) and rAKAV-7ΔNSs, spinal cord (O). Positive staining appears brown in the cytoplasm. Scale bars measure 50 μm

Brains (cerebrum) and lumbar spinal cords were further examined using IHC staining (Fig. 5K-O). Virus antigens were detected in the cytoplasm of neurons in the brains and spinal cords infected with wtAKAV-7, rAKAV-7(S-K0505), and rAKAV-7ΔNSs (Fig. 5M-O). Particularly, a higher abundance of AKAV antigens was observed in the brains and spinal cords of mice inoculated with wtAKAV-7 compared to those with other AKAVs. No AKAV antigens were detected in the brains and spinal cords of mice infected with DMEM and wtK0505 (Fig. 5K, L).

Discussion

AKAV is an arbovirus transmitted by Culicoides biting midges and is prevalent in regions where vectors are distributed [29, 30]. The globalization of trade and climate changes are expanding vector habitats, potentially increasing the incidence of Akabane disease [31]. In South Korea, AKAV strains with different pathogenicity, K0505 and AKAV-7, have been documented [5, 32]. While the K0505 strain primarily causes abortion in cows, AKAV-7 strain is neuropathogenic, causing nonsuppurative encephalomyelitis characterized by meningitis, lymphohistiocytic perivascular cuffing (PCV), and gliosis as observed in histopathologic examinations [33]. However, the virulence factors contributing to AKAV pathogenicity have been poorly understood. The RG system is a valuable tool for studying virus pathogenesis and vaccine development [34]. In this study, wtAKAV-7 and mutant AKAV-7 viruses, namely rAKAV-7(S-K0505) and rAKAV-7ΔNSs, were successfully rescued using the T7 RNA polymerase-based RG system.

Previous research has demonstrated that the rescue of Orthobunyavirus was successfully achieved by transfecting only the three transcription plasmids, without the need for separate helper plasmids, and this approach has also been applied to AKAV [12, 15, 35, 36]. However, in this study, AKAV-7 could not be rescued without the use of helper plasmids. Therefore, helper plasmids expressing viral RdRp and N proteins were used. These results may be due to variations in experimental conditions, such as differences in the level of T7 polymerase expression in cells or the types of plasmids used.

Like the influenza virus, bunyaviruses are segmented, allowing reassortment to occur when two different bunyaviruses co-infect a host [37]. AKAV is also a segmented virus where genetic reassortment can take place, with reported genomic reassortment events in AKAV occurring naturally [38]. Theoretically, six potential reassortant viruses could be generated when two distinct bunyaviruses co-infect a host [37]. In this study, phylogenetic tree analysis revealed no reassortment between AKAV-7 and K0505. Subsequently, various reassortant combinations between AKAV-7 and K0505 strains were attempted using the reverse genetics system; however, only rAKAV-7(S-K0505) was successfully rescued. In nature, the L and S segments of bunyaviruses often originate from the same virus during reassortment, while the donor of the M segment frequently remains unidentified [37, 39]. Similarly, in the laboratory, not all possible reassortant bunyaviruses are generated when using the RG system to induce reassortment [40]. Two La Crosse viruses (LACVs) within a genetically similar lineage failed to produce all six reassortants [41]. Moreover, previous research demonstrated the potential for reassortment between Schmallenberg virus (SBV) and Oropouche virus (OROV), with successful reassortment achieved only for the M segment [42]. These observations may be due to the close association between the polymerase of the L segment and the N protein in the S segment during bunyavirus replication, rather than with the glycoprotein in the M segment [40]. Although reassortment of L and M segments between AKAV-7 and K0505 was unsuccessful, which possibly due to segment compatibility or replication efficiency, this study experimentally demonstrated the potential for reassortment between the S segments of AKAV-7 and K0505. Additionally, the S segment of the live attenuated vaccine BoviShot, used in South Korea, exhibited 100% nucleotide similarity with the S gene of K0505. This resemblance suggests the possibility of reassortment between the S segments of the two viruses when AKAV-7 infection occurs simultaneously with vaccinated cattle.

rAKAV-7 and rAKAV-7(S-K0505) exhibited growth kinetics in Vero cells similar to wtAKAV-7 and wtK0505. However, rAKAV-7ΔNSs demonstrated slower growth and lower maximal yield compared to wtAKAVs and rAKAV-7(S-K0505). The NSs protein in Bunyavirales is known to play a critical role in viral replication and pathogenicity by antagonizing IFN production and regulating apoptosis in infected cells [43, 44]. Consistent with previous studies, which showed that NSs deletion mutants produced by the RG system have a reduced viral replication rate [14, 15, 35], our findings suggest that the NSs protein of AKAV-7 contributes to efficient viral replication. While the exact functions of the AKAV-7 NSs protein remain unclear, further studies are needed to clarify its role in AKAV-7 replication and pathogenesis. Interestingly, rAKAV-7(S-K0505) does not influence AKAV replication, indicating that the S segment of K0505 did not affect the viral replication of AKAV-7.

The pathogenicity of wild type and rescued AKAVs was evaluated using a suckling mouse model of AKAV infection. In this model, rescued AKAVs demonstrated varying survival rates compared to wtAKAVs. The observed decrease in mortality rate and RNA viral load in organs relative to wtAKAVs indicates reduced pathogenicity in both rAKAV-7(S-K0505) and rAKAV-7ΔNSs viruses. Notably, many previous studies have revealed that the NSs protein is involved in the virulence of Orthobunyaviruses [14, 44,45,46]. Bunyaviruses with deleted NSs genes are currently being explored as live attenuated vaccine candidates [47, 48]. The MP-12 vaccine without NSs, employed in Rift Valley fever (RVF) virus, provided effective protection against RVF virus in mouse challenges [49]. Consequently, the AKAV-7ΔNSs virus could potentially serve as a novel AKAV vaccine candidate in the future.

In the phylogenetic tree analysis based on nucleotide sequences, the S segment of AKAV-7 and K0505 were classified into different genogroups. Despite the 29 nucleotide differences between the N protein coding region of AKAV-7 and K0505, only a single amino acid alteration was found. In suckling mice, although rAKAV-7(S-K0505) exhibited a lower organ RNA viral load compared to the wtAKAV-7, no significant reduction in mortality rate was observed. These findings indicate that amino acid differences, more so than nucleotide variations, might play a greater role in determining the virulence of AKAV. Moreover, the pathogenic differences between the AKAV-7 and K0505 strains might be attributable to other genomic segments.

No histopathological changes or AKAV antigens were noted in suckling mice infected with wtK0505 and DMEM. In contrast, histopathological changes and AKAV antigens were detected in the brains (cerebrum) and lumbar spinal cords of suckling mice that succumbed to wtAKAV-7, rAKAV-7(S-K0505), and rAKAV-7ΔNSs infection, correlating with the observed mortality rates and neurological symptoms in these groups. Notably, suckling mice exhibiting neurological symptoms also displayed either bilateral or unilateral hindlimb paralysis, indicating that AKAVs impact both the brain and the spinal cord of infected suckling mice. Consistent with previous research, our study also found distinct differences in pathogenicity between wtAKAV-7 and wtK0505, which belong to different genogroups [3, 5]. Despite mutations in the S segment potentially impairing the replication and infectivity in comparison to wtAKAV-7, suckling mice infected with rAKAV-7ΔNSs and rAKAV-7(S-K0505) did not achieve 100% survival rate observed in mice infected with wtK0505. Furthermore, in the succumbed mice, neurological signs and histopathological changes were observed. Thus, further research using the RG system on the L and M segments is necessary to determine which genes in AKAV contribute to pathogenicity differences among various genogroups.

Conclusion

In conclusion, the replication kinetics in cells and pathogenicity in suckling mice were investigated following mutations in the S segment of AKAV-7 using the RG system driven by T7 RNA polymerase. The results suggest that the S segment, particularly the NSs protein, contributes to pathogenicity of AKAV-7. However, further studies are required to explore how other segments influence pathogenicity of AKAV-7. Further research into the pathogenicity of novel reassortants containing the L and M segments of AKAV-7 would be beneficial.