Abstract
Human endogenous retrovirus W family envelope protein (HERV-W env) is associated with several neurological and psychiatric disorders, including multiple sclerosis (MS) and schizophrenia. Clinical studies have demonstrated a common link between inflammatory abnormalities and HERV-W env in neuropsychiatric diseases. Nonetheless, the molecular mechanisms by which HERV-W env mediates neuroinflammation are still unclear. In this study, we found that HERV-W env significantly increased the mRNA and protein levels of TNF-α and IL-10 in U251 and A172 cells. HERV-W env also induced a notable increase in Toll-like receptor 4 (TLR4). Knockdown of TLR4 impaired the expressions of TNF-α and IL-10 induced by HERV-W env. Overexpression of HERV-W env led to the upregulation of MyD88 but caused a decrease in MyD88s. MyD88s overexpression suppressed the expressions of TNF-α and IL-10 induced by HERV-W env. These findings indicate that HERV-W env upregulates the expressions of IL-10 and TNF-α by inhibiting the production of MyD88s in glial cells. This work sheds light on the immune pathogenesis of HERV-W env in neuropsychiatric disorders.
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Introduction
Human endogenous retroviruses (HERVs) account for 8% of the human genome, and 3173 HERV sequences have been identified. Of these, 1214 are segregated into 39 canonical clades (groups), belonging to class I (gamma- and epsilon-like), class II (beta-like), and class III (spuma-like). The other 1959 noncanonical HERVs include 31 less distinct groups [1, 2]. Most HERVs are defective, containing major deletions or nonsense mutations. Only a few of HERVs have intact open reading frames (ORFs). These HERVs encode functional proteins, some of which play pivotal roles in placentation and gene regulation [3, 4]. HERV-W, one of the HERV groups, was initially discovered in multiple sclerosis (MS) patients [5]. HERV-W env, located on chromosome 7, encodes the HERV-W envelope protein syncytin-1. Previous investigations have shown that various environmental factors, including viruses and caffeine, regulate HERV-W env mRNA and protein expression [6, 7]. Clinical studies have revealed an increase in the amount of HERV-W env in the serums of schizophrenia and multiple sclerosis (MS) patients [8, 9]. Moreover, recent research findings suggest that HERV-W env has potential immunomodulatory properties in the nervous system [10, 11] and participates in the development of several neurological and psychiatric disorders [12, 13]. Expression of pro-inflammatory cytokines such as interleukin (IL)-1β and IL-6 can be induced by HERV-W env in peripheral blood mononuclear cells (PBMCs) and microglial cells [14, 15]. Nevertheless, the role of HERV-W env in neuropsychiatric diseases is still unclear.
Inflammation is a biological response to harmful stimuli [16, 17]. Chronic inflammation contributes to various diseases, such as atherosclerosis [18], rheumatoid arthritis [19], cancer [20, 21], and even schizophrenia [22]. Neuroinflammation is chronic inflammation, together with abnormal expression of inflammation-related cytokines in the nervous system [22,23,24]. The IL-10 gene is a candidate gene for susceptibility to schizophrenia, and IL-10 is a cytokine with pleiotropic effects in inflammation [25, 26]. Tumour necrosis factor-alpha (TNF-α) is one of the inflammatory markers that is elevated in patients with schizophrenia [27, 28]. Nevertheless, it is still unknown whether HERV-W env is involved in the regulation of IL-10 and TNF-α.
TLR4 (Toll-like receptor 4), a pattern-recognition receptor (PRR), plays a vital role in the innate immune system. TLR4 is a pivotal player in the inflammatory response, initiating an intracellular signal cascade via the NF-κB-dependent pathway [29]. A recent study showed that HERV-W env regulates the inflammatory effect via TLR4 [30]. Accumulating evidence indicates that HERV-W env induces TLR4 activation [31]. Meanwhile, neutralizing antibodies against TLR4 were shown to impair HERV-W-env-induced inflammation in human monocytes [15, 22]. Myeloid differentiation primary response 88 (MyD88) is a universal adapter protein for TLR4. It triggers the activation of the NF-κB pathway and the subsequent production of pro-inflammatory cytokines [32]. MyD88s, a splice variant of MyD88, lacks the small intermediate domain (ID) separating the N-terminal death domain (DD) and the C-terminal Toll/interleukin-1 receptor (TIR) domain [33]. MyD88s blocks TLR4-mediated NF-κB activation and negatively regulates the inflammatory response [34].
In this study, we demonstrated that HERV-W env induced the production of the inflammation-related cytokines IL-10 and TNF-α in glial cells. Moreover, we found that MyD88s was capable of inhibiting the production of inflammation-related cytokines induced by HERV-W env. These findings suggested a potential regulatory role of HERV-W env in neuroinflammation. We also discovered a MyD88s-dependent mechanism by which inflammation is regulated by HERV-W env.
Materials and methods
Plasmid construction
The HERV-W env plasmid was constructed as described previously [35]. The pCMV-Myc-MyD88s plasmid was constructed as follows: The upstream fragment of the MyD88 gene (nt 110-557) was amplified using the primers 5’-GGG AAT TCT CTC GGA AAG CGA AAG CC-3’ and 5’-CCA AGC TTA TGC TGG GTC CCA GCT CC-3’. The downstream fragment (nt 692-1243) was amplified using the primers 5’-CCA AGC TTG CGT TTC GAT GCC TTC AT-3’ and 5’-CGG GTA CCA GAG CAC AGA TTC CTC CTA C-3’. The two fragments were then linked and cloned into the mammalian expression vector pCMV-Myc, which contains an N-terminal c-Myc epitope tag and a CMV promoter. Specific siRNAs against human TLR4 (siTLR4) and a negative control siRNA (siNC) were purchased from the Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The target sequence of siTLR4 was GTGCAATTTGACCATTGAA.
Cell culture and transfection
Two human glioma cell lines, U251 and A172, were purchased from the American Type Culture Collection (ATCC) and maintained in DMEM (GIBCO, California, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, California, USA) and 100 U of penicillin/streptomycin per ml at 37°C with 5% CO2.
Transient transfection was carried out using Lipofectamine® LTX and PLUS™ (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Briefly, 1 μg of plasmid DNA was mixed with 1 μl of PLUS™ Reagent in Opti-MEM medium in a 12-well format, and subsequently incubated with 3 μl of Lipofectamine LTX reagent for 30 min. Next, the DNA-lipid complex was added dropwise to the cells. Cells were harvested 48 hours after transfection for further study.
For cotransfection, 0.5 μg of pCMV-env plasmid and 0.5 μg of pCMV-Myc-MyD88s plasmid were used with Lipofectamine® LTX and PLUS™ Reagent to transfect cells in a 12-well format. Cells were harvested 48 hours after transfection for further study. Untransfected cells and the cells that were cotransfected with plasmids pCMV-env and pEGFP were used as controls.
Cells were also cotransfected with the plasmid pCMV-env and siTLR4. Briefly, 0.5 μg of pCMV-env plasmid and 10 nM specific siTLR4 were co-transfected using Lipofectamine® LTX and PLUS™ Reagent in a 12-well format. Cells were harvested 48 hours after transfection for further study. Untransfected cells and the cells that were cotransfected with plasmid pCMV-env and siNC were used as controls.
Analysis of mRNA expression
Total RNA was isolated from cultured cells using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and treated with DNase I (Fermentas, Massachusetts, USA) to remove genomic DNA. Reverse transcription was carried out using the MMLV reverse transcriptase kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Quantitative real-time PCR (Q-PCR) was performed with SYBR® Select Master Mix (Invitrogen, Carlsbad, CA, USA) using an iCycler System (Bio-Rad, Hercules, CA, USA). The 2-ΔΔCT method was used to analyze the relative difference in gene expression. β-actin was used as an internal control to normalize the expression of mRNA levels between different samples. The sequences of this primer that were used are shown in Table S1.
ELISA assay
The culture supernatants were collected from the treated cells. The intracellular contents were obtained by lysing the cells with Cell Lysis Buffer P0013 (Beyotime Institute of Biotechnology, Shanghai, China). The levels of IL-10 and TNF-α in culture supernatants and cell lysates were determined using an OptEIA ELISA kit (Becton, Dickinson and Company, New Jersey, USA) according to the manufacturer’s instructions.
Western blot
Western blot analysis was performed as described previously [36] with slight modifications. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose filter (NC) membranes (Millipore, Billerica, MA). The membranes were then blocked with 5% skim milk and incubated with specific anti-TNF-α (ab183218) or anti-IL-10 (ab52909) antibodies (Abcam, Cambridge, MA) at a dilution of 1:1000. The expression levels of the targeted proteins were quantitated relative to β-actin in the same sample and normalized to the respective control group, for which the value of the band density was arbitrarily set to 1.
Statistical analysis
All results are shown as the mean ± SD (standard deviation) from at least three independent experiments. One-way analysis of variance and Student’s t-test were used to analyze the data. The mRNA and protein levels were calculated as the fold change relative to the control group. A p-value less than 0.05 was considered to represent a significant difference.
Results
HERV-W env increases the TNF-α/IL-10 ratio in U251 and A172 cells
We first investigated whether HERV-W env could regulate the expressions of the inflammation-related cytokines TNF-α and IL-10 in glial cells. We used the human glioma cell lines U251 and A172, which are the most widely used in neuroinflammation research [37, 38]. We transfected the U251 and A172 cells with the plasmid encoding HERV-W env, and the transfection efficiency proved to be robust (Fig. S1). Using quantitative PCR, we found that TNF-α mRNA levels were elevated in HERV-W-overexpressing U251 and A172 cells, by 1.7- and 5.7-fold, respectively (Fig. 1A and D). Meanwhile, western blot showed that the overexpression of HERV-W env increased the protein levels of TNF-α in U251 and A172 cells (Fig. 1B, C, E, and F). ELISA assay confirmed that the level of TNF-α protein in the culture supernatant of U251 cells overexpressing HERV-W env was significantly higher than in the control (17.6 pg/mL and 4.0 pg/mL, respectively) (Fig. 1G, p < 0.01). HERV-W env overexpression also led to a high protein concentration of 12.5 pg/mL of TNF-α in the culture supernatant of A172 cells, compared to 3.9 pg/mL in the control (Fig. 1I, p < 0.01). Similarly, HERV-W env also increased the amount of intracellular TNF-α in U251 and A172 cells (Fig. 1H and J).
The mRNA levels of IL-10 also increased by 1.8- and 1.7-fold, respectively, in U251 and A172 cells transfected with HERV-W env (Fig. 2A and D). Consistently, the protein levels of IL-10 also increased after overexpression of HERV-W env in glial cells (Fig. 2B, C, E, and F). The western blot results were corroborated by ELISA, showing that HERV-W env promoted IL-10 expression, with protein concentrations of 335.8 and 274.7 pg/mL in the culture supernatant of U251 and A172 cells, respectively, and 224.7 and 158.1 pg/mL in that of pCMV-transfected U251 and A172 cells, respectively (Fig. 2G and I, p < 0.05). Similar increases in IL-10 in the intracellular fluid were observed in U251 and A172 cells overexpressing HERV-W env (Fig. 2H and J). A time-course analysis showed that HERV-W env overexpression elevated the protein levels of TNF-α and IL-10 at different time points in U251 and A172 cells, which peaked at 36 hours after transfection (Fig. S2). These findings suggested that HERV-W env upregulated the expressions of the inflammation-related cytokines TNF-α and IL-10 in glial cells.
Because the TNF-α/IL-10 ratio reflects the balance of pro- and anti-inflammatory cytokines [39, 40], we measured the TNF-α/IL-10 ratio in the supernatants of glial cells after overexpression of HERV-W env. The results indicated that overexpression of HERV-W env significantly increased the TNF-α/IL-10 ratio when compared to the control (Fig. 2K and L), suggesting that HERV-W env might trigger a pro-inflammatory response.
HERV-W env increases the expression of TLR4 in U251 and A172 cells
As the TLR4/MyD88 pathway predominantly mediates the production of inflammation-related cytokines, including IL-10 and TNF-α [41, 42], we explored whether the TLR4/MyD88 signal pathway contributed to the release of increased TNF-α and IL-10 induced by HERV-W env. The results showed that HERV-W env significantly increased the mRNA levels of TLR4 in U251 and A172 cells, by 1.6- and 1.8-fold, respectively (Fig. 3A and D). HERV-W env expression also resulted in increased protein levels of TLR4 in U251 and A172 cells (Fig. 3B, C, E, and F). Furthermore, knockdown of TLR4 decreased the mRNA levels of TNF-α and IL-10 induced by HERV-W env, but transfection with negative control siRNAs did not affect these genes (Fig. 3G-J). Meanwhile, ELISA assay showed that the knockdown of TLR4 also suppressed the secretions of TNF-α and IL-10 into the culture supernatants (Fig. 3K-N) and lysates of HERV-W-env-overexpressing cells (Fig. S3). These findings suggested that TLR4 might mediate the regulatory effect of HERV-W env on TNF-α and IL-10.
HERV-W env increases the expression of MyD88 but inhibits the expression of MyD88s in U251 and A172 cells
We then investigated the effect of HERV-W env on the downstream signaling pathway of TLR4. The results indicated that HERV-W env increased the mRNA levels of MyD88, a downstream target of TLR4, by 4.4- and 1.8-fold in U251 and A172 cells, respectively (Fig. 4A and B). MyD88s, a splice variant of MyD88, functions as a negative regulator of the TLR4 signal [43]. The data showed that the mRNA level of MyD88s decreased by 72% when HERV-W env was overexpressed in U251 cells (Fig. 4C). Consistently, the mRNA level of MyD88s also decreased by 70% in A172 cells after overexpression of HERV-W env (Fig. 4D).
MyD88s suppresses the expression of TNF-α and IL-10 induced by HERV-W env in U251 and 1127 cells
To further investigate the role of MyD88s in HERV-W-env-induced inflammation, U251 and A172 cells were transfected with the plasmid pCMV-Myc-MyD88s. The over-expression of MyD88s is shown in Fig. S4. Compared with pCMV-env-transfected cells, Myd88s decreased the mRNA levels of HERV-W-env-induced TNF-α by 85% and 90%, respectively, in U251 and A172 cells (Fig. 4E and F). Meanwhile, Myd88s also decreased the mRNA levels of IL-10 induced by HERV-W env to 21% and 42% in U251 and A172 cells, respectively (Fig. 5A and B). Consistent with this, ELISA results showed that MyD88s overexpression reduced the protein levels of TNF-α in the culture supernatant and lysates of U251 and A172 cells (Fig. 4G-J). Similarly, MyD88s also decreased the secretion of IL-10 in these cells (Fig. 5C-F). The significant difference between cells transfected with pCMV-env + pEGFP and cells transfected with pCMV-env + MyD88s suggested a specific regulatory role of MyD88s in HERV-W-env-induced TNF-α and IL-10 expression. These results indicated that MyD88s might downregulate the expressions of IL-10 and TNF-α induced by HERV-W env.
In summary, the results of this study suggest that HERV-W env promotes the production of TNF-α and IL-10 through the TLR4/MyD88 cascade by inhibiting MyD88s.
Discussion
Neuroinflammation is a crucial pathogenic mechanism contributing to several neuropsychiatric disorders, such as MS and schizophrenia. Accumulating evidence suggests that HERV-W env is a potential pathogenic factor in neuroinflammation. Our previous studies have shown that HERV-W env can boost nitric oxide levels [8] and elicit a strong cytotoxic T lymphocyte response [11]. We have also discovered that HERV-W env can elevate C-reactive protein (CRP) expression, leading to an inflammatory response in the CNS [44, 45]. In this study, we showed that HERV-W env overexpression increased the level of TNF-α, one of the inflammatory cytokines, in glial cells. Interestingly, we also found that HERV-W env increased the level of IL-10. IL-10 is an essential cytokine with anti-inflammatory properties that suppresses the production of several pro-inflammatory cytokines [25, 26]. This suggests that HERV-W env affects the inflammatory balance in glial cells. IL-10 is a potential pathogenic gene involved in several neuropsychiatric disorders, such as bipolar disorder and schizophrenia [25, 26]. Hence, the study of HERV-W-env-induced IL-10 might indicate a possible pathogenic mechanism of HERV-W env in neuropsychiatric disorders.
Increasing evidence shows that TNF-α is an essential regulatory target of IL-10. In monocytes, macrophages, and neutrophilic granulocytes, IL-10 can effectively inhibit TNF-α production [46,47,48]. Although IL-10 is considered an anti-inflammatory cytokine and can inhibit the secretion of TNF-α, several studies have identified other functions of IL-10 [48,49,50]. For example, IL-10 stimulates the cytotoxic activity of NK cells and enhances the IL-2-induced production of TNF-α in these cells [51]. IL-10 can cooperate with TNF-α to activate HIV-1 from latently and acutely infected monocyte/macrophage cells and T cells [52,53,54]. Furthermore, clinical studies have demonstrated simultaneous increases in IL-10 and TNF-α in neuropsychiatric disorders [25]. Here, we found that HERV-W env enhanced TNF-α expression and concurrently upregulated TNF-α and IL-10 in glial cells. Thus, IL-10 might cooperate with TNF-α to mediate the pathogenic role of HERV-W env in neuropsychiatric disorders.
TNF-α and IL-10 maintain the intracellular balance of pro- and anti-inflammatory responses. The TNF-α/IL-10 ratio is a vital inflammatory marker that reflects the level of systemic inflammation [39, 40]. Clinical studies have shown an increased plasma TNF-α/IL-10 ratio in periodontitis patients [55]. Our results suggested that HERV-W env might disrupt the pro-inflammatory/anti-inflammatory balance in the nervous system. The high ratio of secreted TNF-α/IL-10 might also contribute to a systemic pro-inflammatory state in the circulating peripheral blood.
TLRs are a type of PRRs that play a pivotal role in the production of inflammation-related cytokines [56]. In recent years, it has been established that TLRs may be a vital mediator of the inflammatory reaction induced by HERV-W env in the nervous system [57, 58]. In our previous work, we discover a HERV-W env/TLR3/CRP signaling pathway in glial cells, in which TLR3 functions as a receptor of HERV-W env [44]. Other studies have suggested that HERV-W env is also a highly potent TLR4 agonist of endogenous origin and induces TLR4-dependent pro-inflammatory stimulation of immune cells in vitro and in vivo [14, 15, 30]. As expected, in this work, we also found a robust regulatory effect of HERV-W env on TLR4 at both the mRNA and protein levels in human glial cells, further supporting this assumption. We also found that TLR4 deficiency effectively impaired the expressions of the HERV-W-env-induced cytokines TNF-α and IL-10, suggesting that TLR4 might be a pivotal molecule in the regulation of TNF-α and IL-10 by HERV-W env. TLR3 and TLR4 may be essential factors in the molecular mechanisms by which HERV-W env regulates inflammation in neuropsychological diseases, triggering an innate immune reaction. Therefore, different TLRs may respond to HERV-W env in various tissues or fluids, functioning as mediators to induce inflammation.
Previous findings have suggested a potential role of TLR4 in the inflammatory effect of HERV-W env. However, to the best of our knowledge, no specific downstream pathways induced by HERV-W env through TLR4 have been reported. The MyD88-dependent mechanism is one of the most critical signal pathways downstream of TLR4. The TLR4 cascade occurs via the adaptor molecule MyD88 and evokes a pro-inflammatory cytokine response by regulating the activation of transcription factor NF-κB [59, 60]. Indeed, in this study, we found that HERV-W env elevated the expression of MyD88 in human glioma cells, suggesting that MyD88 was a potential downstream target in the HERV-W env-TLR4 signal cascade.
In contrast, MyD88s, a splice variant of MyD88 that lacks the INT domain, acts as a dominant-negative form of MyD88 [61]. MyD88s can compete with full-length MyD88 for receptor binding and inhibit the downstream signaling pathway of MyD88 [62]. In monocytes, MyD88s inhibits the induction of TNF-α [43, 63]. More interestingly, we found that HERV-W env could downregulate the expression of MyD88s in U251 and A172 cells. Furthermore, MyD88s overexpression suppressed the mRNA and protein expressions of IL-10 and TNF-α induced by HERV-W env. These results indicated that inhibiting the negative regulator MyD88s might be a possible mechanism for HERV-W env to activate the TLR4/MyD88 downstream pathway. Collectively, our findings suggested the existence of a HERV-W env-TLR4-Myd88 cascade signaling pathway and a Myd88s-dependent negative regulatory mechanism for inducing the secretions of the cytokines TNF-α and IL-10 in glial cells. However, more work still needs to be done to explore how HERV-W env impairs the generation of MyD88s.
Recent studies have suggested that HERV-W env is a potential pathogenic factor in neuropsychological diseases, but the specific molecular mechanisms involved are still unclear. Our results provided evidence of the pro-inflammatory potential of HERV-W env in the nervous system. We also found that HERV-W env induced TNF-α and IL-10 expressions simultaneously and that the TLR4/MyD88 pathway played a vital role in triggering neuroinflammation. These findings will lead to a better understanding of neuroinflammation, a common thread in neurological diseases.
In conclusion, we observed a regulatory effect of HERV-W env on the expressions of the inflammation-related cytokines IL-10 and TNF-α in glial cells that involves the downregulation of MyD88s. These data strengthen the evidence that HERV-W env has an inflammation-regulatory function and provide further evidence for the immunomodulatory properties of HERV-W env in the nervous system.
Change history
20 January 2021
ORCHID of author Fan ZHU corrected.
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Acknowledgements
This work was supported by grants from the National Natural Sciences Foundation of China (grant nos. 81971943, 81772196, 31470264, 81271820, 30870789, and 30300117) and the Stanley Foundation from the Stanley Medical Research Institute (SMRI), United States (grant no. 06R-1366) for Dr. F Zhu, and the Medical Science Advancement Program (Basic Medical Sciences) of Wuhan University (grant no. TFJC 2018002).
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Overexpression of HERV-W env in U251 and A172 cells. (A) Protein expression level of HERV-W env after overexpression of HERV-W env in U251 cells. (B) Protein expression level of HERV-W env after overexpression of HERV-W env in A172 cells (JPEG 124 kb)
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Time-course analysis of TNF-α and IL-10 by ELISA in U251 and A172 cells. (A and B) Expression levels of TNF-α in the culture supernatant and lysates of U251 cells at 12, 24, 36, and 48 hours after transfection, measured by ELISA. (C and D) Expression levels of TNF-α in the culture supernatant and lysates of A172 cells at 12, 24, 36, and 48 hours after transfection, measured by ELISA. (E and F) Expression levels of IL-10 in the culture supernatant and lysates of U251 cells at 12, 24, 36, and 48 hours after transfection, measured by ELISA. (G and H) Expression levels of IL-10 in the culture supernatant and lysates of A172 cells at 12, 24, 36, and 48 hours after transfection, measured by ELISA (JPEG 770 kb)
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Knockdown of TLR4 suppresses the expression of TNF-α and IL-10 in U251 and A172 cells. (A and B) Expression levels of TNF-α in lysates of U251 and A172 cells transfected with HERV-W env and siTLR4, measured by ELISA assay. (C and D) Expression levels of IL-10 in lysates of U251 and A172 cells transfected with HERV-W env and siTLR4, measured by ELISA. Data represent the mean ± standard deviation of three independent experiments. *, P < 0.05 compared to pCMV, pCMV-env, or pCMV-env + siTLR4 (JPEG 440 kb)
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Overexpression of MyD88s in U251 and A172 cells. (A) Protein expression level of MyD88s after overexpression of MyD88s in U251 cells. (B) Protein expression level of MyD88s after overexpression of MyD88s in A172 cells (JPEG 112 kb)
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Wang, X., Wu, X., Huang, J. et al. Human endogenous retrovirus W family envelope protein (HERV-W env) facilitates the production of TNF-α and IL-10 by inhibiting MyD88s in glial cells. Arch Virol 166, 1035–1045 (2021). https://doi.org/10.1007/s00705-020-04933-8
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DOI: https://doi.org/10.1007/s00705-020-04933-8