Accidental acquisition of a JEV rescued virus with introns unspliced in the viral genome using intron-based stabilization approach

doi:10.21203/rs.3.rs-1958012/v2

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Research Article

Accidental acquisition of a JEV rescued virus with introns unspliced in the viral genome using intron-based stabilization approach

https://doi.org/10.21203/rs.3.rs-1958012/v2

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published 11 Jan, 2023

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The intron-based stabilization approach is a very useful strategy for construction of stable flavivirus infectious clones. SA₁₄-14-2 is a highly attenuated Japanese encephalitis (JE) live vaccine strain and widely used since 1989 in China. To develop safe and effective recombinant vaccines with SA₁₄-14-2 as a backbone vector, we constructed the DNA-based infectious clone pCMW-JEV of SA₁₄-14-2 using the intron-based stabilization approach, and acquired the rescued virus rDJEV which retained the identical biological properties of the parental virus. Unexpectedly, a rescued virus strain, designated rHV-DJEV, with altered virulence was accidentally acquired in one of the transfection experiments. rHV-DJEV showed up to 10⁵-fold increased neurovirulence compared with SA₁₄-14-2 parental strain. Genome sequencing found that the inserted introns still existed in the genome of rHV-DJEV. Therefore, we think that the intron-based stabilization approach should be used prudently in vaccine development and direct iDNA immunization.

Japanese encephalitis

DNA-based infectious clone

rescued virus

altered virulence

Flaviviruses are a group of single-stranded, positive-sense RNA viruses that cause worldwide epidemics with thousands of deaths annually, bringing great human and veterinary health burdens [1]. Construction of flavivirus infectious clones enables direct genetic manipulation of viral genomes and is a powerful tool which can be used in many aspects of virus research. However, some flavivirus infectious clones are very difficult to maintain in E. coli due to their predisposition to spontaneous rearrangements and/or frequent mutations. Various approaches, such as circular polymerase extension reaction, Gibson assembly, intron-based stabilization approach, have been employed to overcome the instability problems of flavivirus and construct the full length of cDNA clones successfully [2].

Japanese encephalitis is a disease of the central nervous system in humans caused by Japanese encephalitis virus (JEV), which is a member of the family Flaviviridae. The disease remains a pressing public health problem in the Asia-Pacific region [3]. JE attenuated live vaccine SA₁₄-14-2 was developed by Prof Yu Yongxin research group in NIFDC and licensed in 1989 in China. After the introduction of SA₁₄-14-2 vaccine into the national immunization program, a decline in JE morbidity was evident since then [4]. The vaccine was proven to have long-term immunogenicity, an ideal protective efficacy, and minor side effects after two doses. In recent years, research on novel flavivirus vaccines has targeted the production of chimeric vaccines using the attenuated viruses or live vaccine as backbone vectors. [5–7]; Under this background, we also attempted to explore SA₁₄-14-2 as a vaccine backbone vector to deliver the structural proteins (prM and E) of other flaviviruses and obtain chimeric live-attenuated vaccine candidates.

In this study, we constructed a DNA-based infectious clone of the SA₁₄-14-2 vaccine strain using the intron-based stabilization approach [8]. Upon direct transfection into cells, the infectious clone, pCMW-JEV, produced the rescued virus, rDJEV, which retained the same biological properties of the parental virus as expected. However, we also found a rescued virus strain (designated rHV-DJEV) with altered virulence in one of the repeated transfection experiments. The neurovirulence test showed that rHV-DJEV had a more than 10⁵-fold increased virulence compared with SA₁₄-14-2. Sequencing the full-length genome of rHV-DJEV, we found the inserted introns still existed in the genome. The extra introns in the genome of the rescued virus might be responsible for the increased neurovirulence of rHV-DJEV for mice. For the sake of safety, we recommended that the intron-based stabilization method to develop engineered viruses and direct iDNA immunization in vaccine preparation should be utilized with caution.

Cells, viruses, and animals

Vero cells were grown in minimal essential medium (MEM) supplemented with antibiotics and 10% fetal calf serum (FCS). BHK-21 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2–10% FCS. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Both the Vero and BHK-21 cells, the vaccine strain SA₁₄-14-2 and high-virulence JEV strain P3 were obtained from NIFDC.

Specific pathogen free Kunming weanling mice, and guinea pigs were obtained from Beijing Laboratory Animal Center (Beijing, China). All animal experiments were carried out in accordance with the guidelines of the Laboratory Animal Care and Use Committee of the Beijing Institute of Biotechnology.

Design And Assembly Of A Dna-based Infectious Clone Of SA₁₄-14-2

The construction of the DNA-based ICs of JEV was designed on the basis of JEV RNA-based infectious clone pMW-JEV and DNA-based replicon pCMW-2M constructed previously [9]. The JEV prM-E genes were amplified from pMW-JEV by PCR, and inserted into the Apa I and BspE I enzyme sites of pCMW-2M to construct the DNA-based ICs. However, the initial attempts to assemble the full-length cDNA clone in one step failed due to the instability of the plasmids in E.coli (Fig. 1A).

To avoid the instability problems that occurred widely in construction of flavivirus cDNA clones, we employed the intron-based stabilization approach, which was reported by Yamshchikov [8]. As shown in Fig. 1B, seven PCR-amplified cDNA fragments by using pMW-JEV as the template were sequentially inserted into the low copy plasmid pMW-118 (Nikkon, Japan) to construct the SA₁₄-14-2 DNA-based full-length infectious clone (designated pCMW-JEV). During the subcloning steps, two artificial introns were inserted into the JEV genome at pos. 354 and pos.2217, and two silent mutations were introduced at position 9392–9403 of the JEV genome, creating a Kpn I restriction endonuclease site, thus making it a genetic marker. The human cytomegalovirus (CMV) promoter was designed to fuse with the beginning of SA₁₄-14-2 genome, and the BGHTT signal sequence was inserted immediately downstream of the last nucleotide of the genome to mediate termination of JEV transcripts at the natural 3′ end. All resultant subclones and the final ICs pCMW-JEV were sequenced to monitor and correct nucleotide mutations.

Infectivity Of The Idnas In Vero Cells

Transfection of Vero cells with pCMW-JEV was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) transfection reagent according to the manufacturer’s protocol. The cells in 6-well plate were transfected with 2 µg of DNAs per well. After 3–4 days of incubation, the supernatants were collected and clarified by centrifugation at 1000 × g for 20 min to obtain passage 1 (P1) progeny virus. Working stocks of P2 viruses were prepared by infecting Vero cells with the P1 virus at an MOI = 0.1 and harvested between 4 and 5 days postinfection with a moderate CPE. The resulting P2 viruses were harvested and amplified once to produce P3 viruses, and so on. As more additional passages were performed, the viruses were harvested and stored at -80 °C. Titers of the rescued viruses at different passages were determined on BHK-21 cells using plaque assays.

Virus Neutralization (Vn) Assay

To identify the biological properties of the cDNA-derived viruses, the virus neutralization assay was performed as follows. Virus stock was prepared in ten-fold dilutions ranging from undiluted to 10^− 5 using sterilized PBS. The virus dilutions were mixed with an equal volume of national standard of JEV antiserum at known concentrations, with non-related national standard of WNV antiserum as negative controls. The viruses-serum mixtures were incubated at 37 °C for 90 minutes, and then added to triplicate wells of 6-well plates containing confluent monolayers of BHK-21 cells with a volume of 200 µL per well. Following 2 h of adsorption with gentle shaking every 15 min, the cells were overlaid with 5 ml of overlay medium containing methylcellulose. Five days later, plaques were visualized after 0.5% crystal violet staining and plaque numbers were counted. The neutralizing index (NI) was calculated as: NI= (Tn-Tp + 3)¹⁰, where Tn and Tp represent the remaining virus titers of 10^− 1 dilutions of viruses mixed with positive serum and 10^− 4 dilutions of viruses mixed with negative serum, respectively. The virus of which NI value exceeded 1000 was determined as the antiserum-related virus.

Immunological Identification Tests

The P8 virus-infected cell culture fluid was 1000-fold diluted with maintenance medium and 100 µl of it was inoculated into confluent BHK-21 cell monolayers cultured in 6-well plates for 12 or 48 h. For immunohistochemical staining (IHC), the cells were fixed in 100% methanol for 10 min at room temperature, and then reacted sequentially with antiJEV E mouse monoclonal antibody 2G2 (Uncover, Beijing, China), anti-mouse IgG (Zhongshan, Beijing, China), peroxidaseantiperoxidase (PAP) complex, and 3, 3'-diaminobenzidine (DAB) solution.

For immunofluorescence analysis (IFA), the cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After blocking with 2% BSA, the cells were incubated with mAb 2G2 as the primary antibody and a fluorescein isothiocyanate (FITC)conjugated anti-mouse IgG as the secondary antibody (Santa Cruz, CA, USA). Cells were stained with Evans blue. The images were viewed and recorded by using a confocal microscope under blue light at 450–480 nm.

Western blotting was also used to identify the expression of the E protein. The P8 virus-infected cells were cultivated for 48 h in serum-free medium. After 34 repeated freeze-thaw cycles, supernatants were collected and clarified by centrifugation at 1000 × g for 20 min, then the supernatants were 10-fold concentrated through centrifugal filter units (Millipore, Billerica, MA, USA). Samples were separated by SDS-PAGE under reducing and non-reducing conditions, transferred to polyvinylidene difluoride membranes, incubated with mAb 2G2 followed by incubation with HRP-conjugated anti-mouse IgG antibody (Zhongshan, Beijing, China). The bound peroxidase was visualized using the Millipore Immobilon Western blot detection system (Invitrogen, Carlsbad, CA, USA).

Growth Curves Of The Rescued Viruses

The growth characteristics of clone-derived viruses were examined on Vero and BHK-21 cells. Subconfluent cells were infected at an MOI = 0.01 with the 8th passage of rescued viruses or SA₁₄-14-2 as the control. Samples were taken for each virus at approximately 24 h intervals postinfection. Viral progeny were harvested and frozen until all samples were collected. Virus yields at different time point were quantitated using plaque titration in BHK-21 cells.

Detection Of Viremia In Mice And Guinea Pigs

Groups of four 10–12 g mice were inoculated subcutaneously with 10³ pfu of rHVDJEV, rDJEV, SA₁₄-14-2 vaccine strain, and virulent JEV P3 strain as controls. Two 250–300 g Hartley guinea pigs per group were inoculated intraperitoneally with 2.5 × 10⁴ pfu of the four viruses mentioned above. Blood samples for the determination of viremia were collected from the orbital sinus of the mice or heart of the guinea pigs daily for 7 days post-infection. Undiluted or diluted (1:10, 1:100) pooled sera of each group were titered by plaque assays on BHK-21 cells. Assays were performed in triplicate per dilution.

Comparison Of The Virulence Of The Parental And Rescued Viruses

Assessment of neuroinvasiveness of the rescued viruses was performed in Kunming weanling mice. Groups of four or six 10–12 g mice were inoculated by the s.c. route with 10-fold dilutions of rescued viral stocks at passage 8 or parental SA₁₄-14-2 virus. The inoculums were back-titered immediately after the injection procedure. One group of mice were injected with PBS only as a negative control. The LD₅₀ values for neuroinvasiveness were calculated using the Reed and Muench method after 3 weeks of observation for the development of encephalitis and mortality.

Neurovirulence of the P1–P10 viruses was assessed in four or six 12–-14 g Kunming weanling mice per group, which were inoculated i.c. with 30 µl of the virus, which was 10-fold diluted in PBS. The virus inoculums were immediately retitered. The mice were observed for 2 weeks for signs of encephalitis and death, and then the LD₅₀ values were calculated.

Genome Sequencing

Sequence analysis of full genome of the rHV-DJEV was conducted, which provided the clues to the virulence-related residues. The whole genome of rHV-DJEV(P8), including the cDNA fragments of 5′ and 3′ termini of the RNA, using SMARTer™ RACE cDNA amplification method (Clontech, Mountain View, CA, USA), was wequenced. To investigate whether nucleotide mutations occurred during serial passage of rHV-DJEV in Vero cells, virion RNAs of rHV-DJEV at different passages were extracted, and then used for subsequent sequencing. The genomic sequence of rHV-DJEV was compared with the plasmid DNA pCMW-JEV sequences and the SA₁₄-14-2 genome sequence.

Recovery of clone-derived viruses on Vero cells

To explore SA₁₄-14-2 as a novel vaccine vector and facilitate the selection of viable chimeric viruses as vaccine candidates of other flaviviruses. JEV DNA-based infectious clone pCMW-JEV was constructed first. Due to the instability of infectious clone constructs in E.coli, it is hard to obtain the full-length cDNA clones (Fig. 1A) till two artificial introns were inserted into the JEV genome at pos. 354 and pos.2217 using intron-based stabilization approach (Fig. 1B).

As has been reported elsewhere [10, 11], the introns in flavivirus cDNA clones were in vivo spliced after transfection into susceptive cell lines, resulting in a rescue virus genome that is identical to the original virus. The rescued virus rDJEV retained the biological properties of the parental virus well in almost all of the repeated transfection experiments (data not shown). However, we also found a rescued virus rHV-DJEV with changed virulence by chance. Rescued virions rHV-DJEV were cultured and harvested on Vero cells. On day 3 posttransfection, cytopathic effects (CPE) was observed and developed progressively from day 4 to 6. After more additional passages in Vero cells, the titers of the P2-P12 rHV-DJEV were in the range of 4.37–7.34 log pfu/ml. The plaque displayed needle tip-like shape, similar to that of the parental SA₁₄-14-2. As Fig. 1C showed, there is no difference in plaque size and morphology between the rescued viruses rDJEV, rHV-DJEV and the parental viruses. Plaques produced by virus recovered from RNA-based clone pMW-JEV exhibited size difference, indicating synthetic JEV RNA from in vitro transcription were not homogeneous and produced heterogenous populations of rescued virus.

Immunological Identification Of rHV-DJEV

The standard virus neutralization assays are always used to identify unknown viruses. In this study, the assay was performed to identify the cDNA-derived viruses using JEV antiserum, with the WNV antiserum as the control. As shown in Table 1, rDJEV and rHV-DJEV reacted with the JEV antiserum with NIs exceeding 1000, while it hardly reacted with the WNV antiserum. The results showed that rHV-DJEV belong to JEV. However, the NIs of the vaccine strain SA₁₄-14-2 and rDJEV with JEV antiserum exceed 200000, and are significantly higher than that of rHV-DJEV, indicating that there exist difference between rHV-DJEV and SA₁₄-14-2.

Table 1

Serological identification of the clone-derived virus
Virus	Neutralization index with
Virus	National standard of WNV antiserum		National standard of JEV antiserum
SA₁₄-14-2		85	≥ 251189
rDJEV		57	≥ 225893
rHV-JEV		33	6457

Virus at 8th passages were re-infected into BHK-21 cells. At 12 h postinfection, positive signals were detected in rHV-DJEV-infected cells by IHC assay (Fig. 2A). The viruses could easily infect the cells and form multiple-cell foci on BHK-21 cells. Meanwhile, IFA was performed to detect rHV-DJEV, together with its parental virus. As shown in Fig. 2B, the cells infected with JE virions at 48 h exhibited obvious fluorescence, and there was no significant difference between the density of the positive signals obtained for rHV-DJEV and SA₁₄-14-2.

Western blotting was further used to analyze the JEV E protein expression from SA₁₄-14-2 and rHV-DJEV under reducing and non-reducing conditions. When using a reduced virion sample for SDS-PAGE, the antibodies did not recognize the E protein. Under non-reducing conditions, the samples from supernatants of SA₁₄-14-2 or rDJEV-infected cells identified a molecular weight 55 kDa band presumed to be the E protein, while samples from supernatants of rHV-DJEV infected cells showed several clear positive bands with molecular masses between 35 and 70 kDa. The results significantly showed that the envelope protein of rHV-DJEV had changed, and was no longer the same E protein of the original virus SA₁₄-14-2.

Growth Properties Of rHV-DJEV and SA₁₄-14-2

To assess the growth characteristics of rHV-DJEV compared with the parental virus SA₁₄-14-2, experiments were carried out on BHK-21 or Vero cells with viruses at a low multiplicity of infection (MOI). Both in the BHK-21 and Vero cells (Fig. 3), significant differences were observed between the viruses in terms of the maximal yield and duration of virus production. The virus yield reached its peak at day 2 postinfection for rHV-DJEV in BHK-21 cells, while the yield of SA₁₄-14-2 reached the peak titer at day 4. However, the maximal virus yield of SA₁₄-14-2 (7.95 logPFU/ml) was 20-fold higher than that of the rescued virus. In Vero cells, rHV-DJEV also showed different growth kinetics compared with the parental virus. rHV-DJEV accumulated rapidly and reached its high peak at day 5. However, there was a significant delay in the replication of SA₁₄-14-2 viruses, which lasted for two weeks in Vero cells without a vigorous CPE. The titers of SA₁₄-14-2 increased slowly and reached their peak at day 13.

Viremia In Mice And Guinea Pigs

To assess the peripheral replication of rHV-DJEV, levels and durations of viremia were measured in the sera of mice and guinea pigs infected with wild-type and clone-derived viruses. Table 2 shows the pattern of viremia in mice after inoculation with different viruses. Transient viremia was observed in the SA₁₄-14-2-infected and rDJEV-infected mice for only 1 day. rHV-DJEV developed low viremia on day 2, then caused increasing virus levels in the blood, peaking at day 4, and clearance occurring 1–2 days later. In comparison, the JEV virulent strain P3 produced vigorous viremia. Virus titers in the serum reached 40 PFU/ml postinfection and peaked around day 5 (3700 PFU/ml). All mice in P3-injected groups developed neurological symptoms at day 3–4 and succumbed to death at day 6 and 7, consistent with the increased levels of viremia.

Table 2

Viremia of wild-type and clone-derived viruses in mice
Virus^a	Virus titers in serum (PFU/ml ) ^b
Virus^a	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
JEV P3	40	105	150	330	3700	ND	ND
SA₁₄-14-2	0	7	0	0	0	0	0
rDJEV	0	9	0	0	0	0	0
rHV-DJEV	0	18	28	30	0	0	0
a Mice at 10–12 g were inoculated subcutaneously with 10³ plaque forming units (pfu) of rHV-DJEV, rDJEV, SA₁₄-14-2 vaccine strain and highly virulent JEV P3 strain.
b Pooled sera of every group were collected and titered by plaque assays on BHK-21 cells.
ND: not determined.

The levels and duration of viremia were also detected in guinea pigs. No viruses were detected in the sera of guinea pigs injected with SA₁₄-14-2 and rHV-DJEV during the observation period of 10 days. By contrast, the guinea pigs injected with P3 virulent strains developed a slight viremia, with virus titers of 106 PFU/ml on day 1 postinfection. The duration of viremia lasted for 2–3 days, with a peak titer of 1000 PFU/ml. The amount of virus in sera decreased dramatically at 4 days postinfection until no virus was detected. Guinea pigs in the P3 group also survived infection with no significant symptoms.

Mouse Neurovirulence And Neuroinvasiveness Testing

The neuroinvasiveness of rHV-DJEV(P8) was tested by subcutaneous (s.c.) inoculation of Kunming weanling mice and compared with that of SA₁₄-14-2. The cumulative results are given in Table 3. All mice survived s.c. inoculation of SA₁₄-14-2 and rHV-DJEV, even at the highest titers. This showed that rHV-DJEV was non-neuroinvasive in weanling mice and resembled its parental virus SA₁₄-14-2.

Table 3

Neuroinvasiveness and neurovirulence of rescued viruses in Kunming weanling mice
Virus	Neurovirulence (i.c.) ^a			Neuroinvasiveness (s.c.) ^b
Virus	Dose (pfu)	D/T	LD₅₀		Dose (pfu)		D/T	LD₅₀
rHV-DJEV	9.49×10³	4/4	16.8			1.04×10⁵	0/6	> 1.04×10⁵
	9.49×10²	4/4				1.04×10⁴	0/6
	94.9	3/4				1.04×10³	0/6
	9.49	2/4				1.04×10²	0/6
	0.949	0/4				10.4	0/6
SA₁₄-14-2	1.1×10⁵	0/6	> 1.1×10⁵			3.65×10⁵	0/6	> 3.65×10⁵
	1.1×10⁴	0/6				3.65×10⁴	0/6
	1.1×10³	0/6				3.65×10³	0/6
a Actual dose delivered intracerebrally (i.c.) with 0.03ml viral solution in 12–14 g Kunming weanling mice.
b Actual dose delivered subcutaneously (s.c). with 0.1ml viral solution in 10–12 g Kunming weanling mice.
D/T: Death/Total

Neurovirulence tests were assessed in 12–14 g Kunming mice, which were inoculated i.c with 10-fold dilutions of viral stocks and observed for 2 weeks. As shown in Table 3, rHV-DJEV caused 100% mortality at the dose of 9.49 × 10² pfu after i.c. inoculation, and showed a high neurovirulence, with an LD₅₀ of 16.8 pfu. These results are not consistent with our expectations. The neurovirulence of the rescued virus rHV-DJEV increased up to 10⁵-fold compared with that of SA₁₄-14-2.

To assess whether the neurovirulence of rHV-DJEV at P8 changed or not during the amplification of the virus on Vero cells, groups of four to six 12–14 g mice were inoculated i.c. with serial dilutions of P1–P10 viruses. The LD₅₀ value for rHV-DJEV at different passages was calculated. Like rHV-DJEV at P8, viruses at other passages also exhibited a relatively high neurovirulence, with the LD₅₀ in the range of 16.5 to 59.9 pfu.

Nucleotide Sequence Analysis Of The Rescued Viruses

SA₁₄-14-2 virion genome contains 10,977 nucleotides. To identify the nucleotide differences between the rescued virus and the parental virus, virion genome of rHV-DJEV were sequenced using nine overlapping RT-PCR products. The two terminal fragments were also detected using the rapid amplification of cDNA end (RACE) method. Analysis of the genome of rHV-DJEV(P8) identified 3 main differences from the consensus sequence of SA₁₄-14-2: i) The two introns inserted into the plasmid pCMW-JEV were unexpectedly present in the viral genome of rHV-DJEV; ii) a genetic marker with two silent mutations existed in the viral genome at position 9392–9403 of rHV-DJEV; iii) two spontaneous nucleotide mutations resulted in amino acid substitutions, one located in prM (prM82: Thr-Ala) and the other in NS4A (NS4A190: Ala-Asp).

The two residues (prM82, NS4A190) were then determined by sequence analysis in the genome of rHV-DJEV virus at P1. It was found that both sites were consistent with the parental virus SA₁₄-14-2, indicating that the mutations happened during virus amplification in Vero cells. The neurovirulence of the P1 virus was tested and confirmed to be also high, indicating that the two mutations had little influence on the increased virulence of rHV-DJEV.

In previous studies, we reported the construction of an RNA-based IC of SA₁₄-14-2 named pMW-JEV [9]. JEV RNAs were in vitro transcribed from pMW-JEV, transfected into BHK-21 cells, and then the virus rRJEV was rescued as expected. However, the RNA-based IC had the limitation that a heterogeneous RNA population is always produced in the in vitro transcription procedure, and plaques of different sizes were observed in the detection of the rescued virus by plaque assay (Fig. 1C).

To solve the impurity problem and simplify the procedure, the RNA-based clone pMW-JEV was converted to the DNA-based format. The construction of DNA-based ICs designated pCMW-JEV was accomplished by using the intron-based stabilization approach with two artificial introns inserted into the SA₁₄-14-2 genome at positions 354 and 2217.

Upon transfection of the cDNA to Vero cells, the added introns were spliced from the precursor nuclear messenger RNA (pre-mRNA) in the nucleus, resulting in a true copy of the viral RNA which transported to the cytoplasm. Like other rescued viruses which has been reported elsewhere [10–14], our rescued virus rDJEV retained the biological properties of the parental virus well. However, we also accidentally found a rescued virus, termed rHV-DJEV, with altered virulence identified in the neurovirulence test.

Neutralization assays were performed to identify rHV-DJEV using JEV antiserum. rHV-DJEV reacted with the JEV antiserum with neutralization indexes (NIs) of 6457. More immunological identification tests were also used to assess the biological properties of rHV-DJEV. IHC and IFA results showed that rHV-DJEV could react with the anti-JEV E mAb, but could not distinguish its binding site difference with mAb 2G2 from SA₁₄-14-2. Western blotting further showed that the JEV E antigens of rHV-DJEV and SA₁₄-14-2 displayed different molecular size, indicating that JEV E antigens of rHV-DJEV might be a mixture of E proteins with different molecular weights.

Viremia in susceptible animal models is a key predictor for the severity of the virus disease [15]. In general, tansient viremia was observed in the SA₁₄-14-2-infected mice for only 1 day. In this study, a slight and transient viremia was observed in the rHV-DJEV-infected mice. The in vivo blood clearance kinetics of rHV-DJEV was rapid, similar to that of the parental virus. No viruses were detected in the sera of guinea pigs injected with SA₁₄-14-2 and rHV-DJEV during the observation period of 10 days.

Assessment of neuroinvasiveness and neurovirulence of the rescued viruses was performed in Kunming weanling mice. It showed that rHV-DJEV resembled SA₁₄-14-2 with non-neuroinvasive in weanling mice. A surprising finding was the 10⁵-fold increased neurovirulence of rHV-DJEV relative to the parental virus SA₁₄-14-2. As shown in Table 3, SA₁₄-14-2 is avirulent with a high LD₅₀ as reported previously [4]. While rHV-DJEV caused 100% mortality at the dose of 9.49 × 10² pfu after i.c. inoculation, and showed a high neurovirulence with an LD₅₀ of only16.8 pfu.

Sequence analysis of the genome of the rescued virus at P8 identified a few differences including two Vero cell-adaption nucleotide mutations that were not present in the plasmid pCMW-JEV. Earlier, Pletnev reported the construction of an IC of TBEV [16], providing evidence that mutations occurred during amplification of the rescued virus in Vero cells. The spontaneous mutations might increase the virus replication efficiency and allow it to become more adaptive to the culture media; However, sometimes the mutations caused phenotype changes, including the growth characteristics and virulence [17]. Liu also demonstrated that the genetic stability in the infectious cDNA clone-derived dengue type 4 virus was hardly maintained during serial passage in Vero cells. The virus neurovirulence increased significantly following passage in Vero cells [18].

Subsequent tests verified that the two Vero cell-adaption mutations had little influence on the increased neurovirulence of rHV-DJEV, because the rescued virus at P1, without mutations at prM82 and NS4A190, also had high neurovirulence, with an LD50 of 42.4 pfu. Additional experiments were done to assess the virulence of the resulting rescued virus rDJEV. Similar to SA₁₄-14-2, rDJEV showed no virulence for weanling mice after i.c. inoculation, even at high titers (LD₅₀ > 6.25×10⁶ pfu). The complete genome of rDJEV was sequenced and compared with the rHV-DJEV (P1) genome, and only one difference existed in that rDJEV that it had no introns. All the above results showed that the introns inserted into the genome of rHV-DJEV might be responsible for the increased neurovirulence of rHV-DJEV for mice.

As earlier reported by González [19], they extracted cytoplasmic and nuclear RNAs and amplified the region covering the intron insertion site by RT-PCR after the transfection of a full-length ICs of transmissible gastroenteritis coronavirus with an intron. Bands of two different sizes were obtained in the cases of nonspliced and spliced RNAs, indicating the efficiency of intron removal in viral RNAs transcribed in vivo from the intron-containing full-length cDNAs was not 100%. We speculated that a small number of RNA transcripts were unspliced during nuclear transcription of pCMW-JEV. The unspliced products entered the cytoplasm through the nuclear pore for translation. Although the translated product was not identical to the correct viral protein, it happened to not affect the assembly of the virus and the infectious rescue virus was obtained. During subsequent virus cultivation and amplification, the virus entered the cells and released the nucleocapsid into the cytoplasm. Virion RNA containing the introns entered the next replicative cycle, producing the progeny viruses which still contain the introns in the genome.

It is obvious that it is hard to avoid obtaining unexpected rescued genetic engineering viruses, and whether the biological properties of rescued virions with unspliced introns altered was unkonwn. Therefore, we should use the reverse genetic system technology such as the intron-based stabilization approach more carefully in the development of engineered viruses for vaccine development and direct iDNA immunization

Author Contributions

Y.Y.X., and Z.X.Y., designed and conceived the experiments. Y.H., H.S.X., S.L., J.S.L., Y.P., and L.L.J., performed the experiments. Z.X.Y., and Y.H.L., contributed to the acquisition, analysis, and interpretation of data. Y.H., and R.W., wrote, edited, and reviewed the manuscript.

Funding

This work was supported by grants from the National Key Research and Development Program of China (grant no. 2018YFA0900800).

Declarations Conflict of interest

The authors declare no confict of interest.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript.

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