iGEM 2023 — Team pekinghsc

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

May

June

July

August

September

October

### May

From **May 1st to May 10th**, we decided that the topic of iGEM was "Influenza virus displayed by neuraminidase targeting tumors with high expression of transferrin receptor 1", and designed the initial experimental plan.

From **May 10th to May 15th**, we attempted to construct the A6-Ins15 plasmid through homologous recombination. While becoming familiar with the procedure, our aim was to create the first plasmid. Unfortunately, all the results turned out to be false positives, indicating unsuccessful mutation. As a result, we redesigned the homologous recombination primers and designed primers for point mutation. We attempted to insert these 45 bases through point mutation. Simultaneously, we also designed the primers required for constructing the A6-Ins30 plasmid.

From **May 15th to May 21st**, we successfully reconstructed the A6-Ins15 plasmid using a combination of homologous recombination and point mutation. Additionally, we constructed the A6-Ins30 plasmid through homologous recombination. The sequencing results confirmed the successful insertion of all the sequences.

![](https://static.igem.wiki/teams/4638/wiki/notebook/may1.png)
![](https://static.igem.wiki/teams/4638/wiki/notebook/may2.png)

Fig. 1. Sequencing results of 1.A6-Ins15 and A6-Ins30. P15 represents point mutation insertion of 15 amino acids, S15 represents homologous recombination insertion of 15 amino acids, S30 represents homologous recombination insertion of 30 amino acids, and the sequencing primer is Na-F.

From **May 22nd to May 31st**, we extracted a small amount of plasmids from the successfully constructed A6-Ins15 and A6-Ins30 plasmids in preparation for future virus rescue experiments. Additionally, we designed primers for constructing A6-HAps and A6-NAps plasmids to exchange packaging signals. Plasmids requiring packaging signal exchange needed DNA fragments to be inserted at both the 5' and 3' ends, requiring two consecutive homologous recombination steps for successful construction. On May 30th, we obtained the results of the first round of testing, some of which indicated successful insertion of the 5' end packaging signal.

### June

From **June 1st to June 11th**, we extracted plasmids from the previously tested samples and performed a second round of 3' end packaging signal replacement. On June 11th, we received the sequencing results, and some samples showed successful insertion of the 3'-end packaging signal. During this time, we carefully compared the sequences and discovered that we missed an initial codon in the NA packaging signal at the 5' end of the A4-NAps plasmid. Additionally, a base was inserted at the end of the HA packaging signal at the 5' end of the A6-HAps plasmid. To address these issues, we designed a point mutation primer to improve these parts of the plasmids.

From **June 12th to June 15th**, we successfully performed the point mutation and obtained the sequencing results on June 14th. All the samples achieved the desired point mutation. As a result, we successfully constructed the A4-NAps plasmid and A6-HAps plasmid. We also extracted a small amount of plasmids for subsequent virus rescue experiments. During this time, we attempted the rescue of wild-type, S15, and S30 influenza viruses for the first time. After 3 days, we collected the culture medium and removed cell debris through centrifugation. However, the harvested virus supernatant did not show hemagglutination inhibition of chicken red blood cells in the hemagglutination inhibition experiment, including the wild-type virus. This may be attributed to unskilled operation.

From **June 15th to June 20th**, we attempted to rescue the wild-type, S15, S30, and PS mutant viruses again. After 3 days, we collected the culture medium and removed cell debris through centrifugation. Unfortunately, the harvested virus supernatant still did not show hemagglutination inhibition of chicken red blood cells in the hemagglutination inhibition experiment (Figure 2). We suspected that the virus titer was too low. To investigate further, we directly used the virus supernatant to infect HEK293T cells. After 4 hours, we extracted RNA, performed reverse transcription into cDNA, and attempted to amplify the NA protein and HA protein genes through PCR to determine if the corresponding genes were obtained. Unfortunately, we were unable to amplify the NA protein and HA protein genes (Figure 3).

![](https://static.igem.wiki/teams/4638/wiki/notebook/june1.jpg)

Fig. 2. Hemagglutination inhibition test of chicken red blood cells. From bottom to bottom are PS, S30, S15, WT and non-virus.

![](https://static.igem.wiki/teams/4638/wiki/notebook/june2.png)

Fig. 3. agarose gel electrophoresis of 3.NA gene and HA gene.

During the final period from **June 20th to June 30th**, the experimenters needed to prepare for exams and suspended the experiment.

### July

From **July 1st to July 12th**, the experimenters needed to prepare for graduate student interviews and participate in the 10th iGEMer exchange meeting in CCiC China, and suspended the experiment.

From **July 13th to July 28th**, we repeated the operations of virus rescue, virus infection, RNA extraction, and PCR. We made constant adjustments to the virus infection time, annealing temperature, and PCR cycle times. During this period, we successfully detected the target band several times through PCR, but all the sequences turned out to be wild-type after being tested (Figure 4). Suspecting an issue with the plasmid, we performed plasmid analysis. Upon comparing the sequencing results, we found no obvious problems with the plasmid.

![](https://static.igem.wiki/teams/4638/wiki/notebook/july1.png)

Fig. 4. agarose gel electrophoresis of virus gene amplification on July 20. CDNA indicates its primer amplification coding region, vRNA indicates its primer amplification of the whole virus RNA, and 4h and 12h indicate the virus attack time.

### August

From **July 29th to August 5th**, the experimenter went out to participate in a social practice activity and suspended the experiment.

From **August 6 to August 12**, we repeated the previous processes: virus rescue, virus infection, RNA extraction, PCR, etc. However, we still did not obtain any positive results. Suspecting that the issue may lie with the design itself, I decided to make changes. For the neuraminidase extension, we found a new paper on length modification of neuraminidase based on the WSN strain. Based on this paper, we designed related primers for SD24, SD9, SI14, and SI28. As for the packaging signal exchange, we planned to carry out synonymous mutations on the remaining packaging signals in the coding region. However, due to the lack of specific instructions in the literature regarding the procedure for synonymous mutation, we temporarily put that on hold.

From **August 12th to August 20th**, we repeated the processes of virus rescue, virus infection, RNA extraction, and PCR for S15, S30, and PS mutant viruses. Fortunately, the sequencing results showed successful insertion for S15 (Figure 5). However, it did not effectively cause a cytopathic effect (CPE) in HEK293T cells (Figure 6). During this time, we also constructed the plasmids A6-SD24, A6-SD9, A6-SI14, and A6-SI28. The sequencing results confirmed the successful construction of these four plasmids.

![](https://static.igem.wiki/teams/4638/wiki/notebook/august1.png)

Fig. 5 results of viral neuraminidase RNA sequencing

![](https://static.igem.wiki/teams/4638/wiki/notebook/august2.png)

Fig. 6 CPE effect results of wild-type influenza virus, Ins15 influenza virus and Ins30 influenza virus.

From **August 20th to August 31st**, we conducted virus rescue experiments for SD24, SD9, SI14, and SI28. After harvesting the virus supernatant, we performed the hemagglutination inhibition experiment using chicken red blood cells. Unfortunately, the results showed that none of the viruses effectively inhibited the agglutination of chicken red blood cells (Figure 7). Nonetheless, we proceeded with the virus infection experiment, extracted RNA for testing, and observed the cytopathic effect (CPE) in cells. Fortunately, both SD9 and SI14 showed successful insertion in the sequencing results, and obvious CPE effects were observed in the cells (Figure 9). Consequently, we prepared a large quantity of plasmids for the twelve-plasmid system, including A6-SD9 and A6-SI14 plasmids, for the large-scale production of viruses. Meanwhile, we decided to discontinue the rescue attempts for the S15 and S30 viruses. However, we persisted in our efforts to rescue the PS mutant virus, but unfortunately, we did not achieve any positive results.

![](https://static.igem.wiki/teams/4638/wiki/notebook/august3.jpg)

Fig. 7. Chicken red blood cell hemagglutination inhibition test (8.25). From top to bottom, they are non-virus, WT, S15, S30, SD9, SD24, SI14, SI28 and PS.

![](https://static.igem.wiki/teams/4638/wiki/notebook/august4.png)

Fig. 9 CPE effect results of wild-type influenza virus, SD9 and SI14 influenza virus.

### September

From **September 1st to September 10th**, we prepared a large quantity of wild-type influenza viruses along with SD9 and SI14 mutant viruses. We collected 100 ml of wild-type virus supernatant and 150 ml of SD9 and SI14 virus supernatant. Subsequently, we concentrated all three viruses to 2 ml using ultracentrifugation. The concentrated virus solution exhibited significant hemagglutination inhibition of chicken red blood cells (Figure 10). We then performed absolute quantification of these three viruses (Figure 11) and discovered that the wild-type virus had the lowest titer, possibly due to premature harvest. Additionally, after discussing with my colleague in the laboratory, we decided to carry out synonymous mutations on the remaining packaging signals in the coding regions of the A6-HAps and A4-NAps plasmids for the PS mutant virus. The specific method involved humanizing these sequences using a codon optimization website. We synthesized the corresponding gene fragments and designed corresponding primers, which were then sent to our company for synthesis.Moreover, we also tried to construct a cell model with high TfR1 expression on CHO cells by spreading it in a 12-well plate.Cells were added with 1 ml of 1ml 2%FBS DMEM medium. After 24 hours, 1 ug pCMV3-TfR1 plasmid was transfected, and 6 hours after transfection, 1 ml of 2% FBS DMEM medium was transposed. Two days later, the protein was extracted for Western blotting to see the expression of TfR1. The results showed that we successfully constructed CHO cells with high TFR1 expression (Figure 12).

![](https://static.igem.wiki/teams/4638/wiki/notebook/september1.jpg)

![](https://static.igem.wiki/teams/4638/wiki/notebook/september2.jpg)

Fig. 10 WT, SD9 and SI14 chicken erythrocyte hemagglutination inhibition experiment. The left picture is a front view, with WT, SD9 and SI14 from top to bottom, and the virus concentration decreases from left to right. The picture on the right shows the negative view, which is SI14, SD9 and WT from top to bottom, and the virus concentration decreases from left to right.

![](https://static.igem.wiki/teams/4638/wiki/notebook/september33.png)

Fig. 11. Virus titer of the first batch of viruses prepared in large quantities. From left to right are WT, SD9 and SI14.

![](https://static.igem.wiki/teams/4638/wiki/notebook/september4.png)

Fig. 12 results of Western blotting experiment after transfection of TfR1 plasmid by CHO for 2 days.

From **September 10th to September 20th**, we successfully performed synonymous mutations on the residual packaging signals at the 5' and 3' ends of the coding regions of the HA protein and NA protein in the A6-HAps and A4-NAps plasmids. We constructed A6-HAps(mut) and A4-NAps(mut) plasmids and extracted a small amount of plasmids for the rescue of the PS(mut) mutant virus. In a 6-well plate, we successfully rescued SD24 and SI28 mutant influenza viruses. The results indicated that we successfully deleted 24 amino acids/inserted 28 amino acids at the gene level. Additionally, to provide characterization, we included agarose gel electrophoresis images of the PCR-derived genes (Figure 13). Following the success of SD24 and SI28, we promptly prepared a large quantity of A6-SD24 plasmids and A6-SI28 plasmids for virus preparation. Furthermore, we also evaluated the targeting of the previously prepared large quantities of SD9 and SI14. Due to the low titer of the wild-type influenza virus, our initial batch of large-scale prepared influenza viruses only underwent one time of targeting evaluation. The remaining small amount of virus was utilized in Western blotting experiments. By exploring different conditions, we determined the appropriate electrophoresis conditions and verified the shortening and lengthening of the neuraminidase protein at the protein level (Figure 14).

![](https://static.igem.wiki/teams/4638/wiki/notebook/september5.png)

Fig. 13. agarose gel electrophoresis of PCR products of viral RNA.

![](https://static.igem.wiki/teams/4638/wiki/notebook/september6.jpg)

Fig. 14. Western blotting color diagrams of SD9, WT and SI14.

From **September 21st to September 30th**, we made extensive preparations for the second and third batches of viruses. The second batch included preparations of WT, SD9, SI14, and SI28, while the third batch consisted of SD24 and SI28. In the second batch of viruses, the titer of SI28 was low, and it was consumed after two targeted experiments. Therefore, the third batch of viruses was prepared together with SD24. After the preparation of the third batch of viruses, we collected all the influenza viruses with different lengths of neuraminidase that we designed. Consequently, we conducted an experiment to inhibit the agglutination of chicken red blood cells (Figure 15). The results revealed that SD24 and SI28 did not effectively inhibit the agglutination of chicken red blood cells, presumably due to their low virus titer. Consequently, we performed absolute quantification of these viruses and confirmed that the virus titers of SD24 and SI28 were indeed significantly lower than those of WT, SD9, and SI28. The low titer of SI28 contradicted the original literature and made us realize that there was an issue with our design. Upon investigation, we discovered that the wrong sequence had been inserted into our SI28, prompting us to redesign the related primers. Additionally, we conducted a small-scale test on PS(mut) in a 6-well plate, but unfortunately, it was not successful this time. Furthermore, we evaluated the targeting of a large number of prepared viruses. However, due to the low virus titer of SD24, we only evaluated the targeting of the other three mutant viruses and the wild-type viruses, excluding SD24.

![](https://static.igem.wiki/teams/4638/wiki/notebook/september7.jpg)
![](https://static.igem.wiki/teams/4638/wiki/notebook/september8.jpg)

Fig. 15 results of hemagglutination test of chicken red blood cells

The following figure shows the reverse view of the above figure, which is SI28, SI14, SD9, SD24 and WT from top to bottom.

![](https://static.igem.wiki/teams/4638/wiki/notebook/september99.png)

Fig. 16 virus titer result chart

### October

From **October 1st to October 10th**, we summarized and analyzed the data from the previous targeted experiments (Figure 17). Simultaneously, we reconstructed the A6-SI28-new plasmid and conducted virus rescue experiments with two mutant viruses, SI28-new and PS. However, we were still unable to successfully rescue the PS mutant virus. Upon examining the A4-NAps(mut) plasmid, we discovered that the start codon remained within its 5' packaging signal. Consequently, we performed a point mutation on the plasmid.

![](https://static.igem.wiki/teams/4638/wiki/notebook/notebook-final.jpg)

Fig. 17 results of virus targeting evaluation. Horizontal axis: virus type; Vertical axis: virus infection efficiency (%,normalized by wild-type influenza virus infected cells in blank plasmid group). After transferring to TfR1, the virus infection efficiency was improved, among which, the wild type increased by 1.930 times, SD9 by 1.237 times, SI14 by 1.854 times and SI28 by 1.935 times.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

Introduction

Extend

Exchange

Explore

Reference

# Introduction

In order to design and construct the neuraminidase-boosted influenza virus, we utilized engineering principles. This engineering design cycle comprises four stages: design, construction, testing, and learning.(Figure 1).

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1.png)

Figure 1. Engineering schematic diagram. A cycle includes design, build, test, and learning.The image used in this content has been sourced from <https://2022.igem.wiki/peking/engineering>.

# Rescued the virus whose neuraminidase was extended.

Overview: In our project, we aim to construct a neuraminidase-boosted influenza virus. We have employed two methods to modify the influenza virus. One of these methods involves inserting amino acid fragments from other influenza viruses into the stalk region of neuraminidase, thereby extending neuraminidase and making it more prominent on the surface of the influenza virus. Throughout this process, we have undergone three rounds of engineering rounds.

### First round

1.**Design**: Based on the A/PR/8 (H1N1) influenza virus strain, Broecker et al. inserted amino acid fragments from the A/California/04/2009 (Cal09) virus and the A/New York/61/2012 (H3N2) virus into the stalk region of the PR8 neuraminidase and constructed Ins15 virus with 15 amino acids inserted and Ins30 virus with 30 amino acids inserted (Figure 1.1). This successful modification induced a better immune response against the neuraminidase in mice [[1]](#reference). Since the influenza virus we utilized was the A/WSN/1933 (H1N1) strain, which shares a strong affinity with the PR8 influenza virus, we intended to insert the same sequence into the neuraminidase of the WSN strain. Referring to the comparison of neuraminidase protein sequences between the WSN strain and the PR8 strain by Hiti AL et al. (Fig. 1.2) [[2]](#reference), we also inserted the same 15 amino acids and 30 amino acids at similar sites in the WSN strain.

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-1.png)

Figure 1.1 Influenza virus designed to extend the neuraminidase stack domain
[[1]](#reference)

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-2.png)

Figure 1.2 Sequence comparison of neuraminidase between PR8 strain and WSN strain
[[2]](#reference).The position marked by the red line in the figure is the insertion point

2.**Construction**: The virus rescue system we utilized was the 12-plasmid system provided by Academician Gao Fu. The A1-A8 plasmids were employed for the transcription of viral RNA, while the A9-A12 plasmids were used to express the PA, PB1, PB2, and NP proteins to simulate the state of virus-infected cells. Within the virus rescue system, the A6 plasmid was utilized for the transcription of RNA corresponding to the viral neuraminidase. We aimed to insert the 15 amino acids and 30 amino acids, as shown in Figure 1, at the 61st position of the neuraminidase. Consequently, we constructed the A6-Ins15 plasmid and the A6-Ins30 plasmid through homologous recombination, based on the A6 plasmid. Sequencing results confirmed the successful insertion of the target fragment (Figure 1.3).

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-3.png)

Fig. 1.3 Sequencing Results of A6-Is15 Plasmid and A6-Is30 Plasmid

3.**Test**: Once the mutant plasmid was successfully constructed, we proceeded to rescue the virus using the mutant plasmid. HEK293T cells were transfected with the mutant plasmid using PEI transfection reagent. When approximately 90% of the cells had died, we collected the supernatant containing the virus and removed cell debris through centrifugation. Subsequently, we introduced a small amount of the virus solution into a fresh batch of HEK293T cells and harvested the total RNA after 4 hours for reverse transcription to obtain cDNA. The cDNA was then amplified through PCR to target the NA protein. The amplified cDNA was sent to a sequencing company for mutation detection. The sequencing results revealed the successful rescue of the wild-type influenza virus and the Ins15 mutant virus, but not the Ins30 mutant virus (Figure 1.4). However, the Ins15 mutant virus did not effectively induce cytopathic effects (CPE) in cells (Fig. 1.5). In other words, directly introducing the same mutations from the PR8 strain into the WSN strain to extend the neuraminidase is not a feasible approach.

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-4.png)

Figure 1.4 Results of viral neuraminidase RNA sequencing

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-5.png)

Figure 1.5 CPE Effect Results of Wild Influenza Virus, Ins15 Influenza Virus and Ins30 Influenza Virus

4.**Learning**: Upon reviewing the entire design, we discovered significant differences in the sequence of the stalk region between the WSN and PR8 strains, particularly at our insertion position. This difference may be the reason for our failure. Consequently, we conducted further literature research and came across studies on the modification of the neuraminidase stalk region in the WSN strain. In their investigation of the biological activity of the influenza virus neuraminidase stalk length, Castrucci MR et al. constructed influenza viruses with varying neuraminidase stalk lengths based on the WSN strain. These modifications included deleting 24 amino acids (SD0), deleting 9 amino acids (SD9), inserting 14 amino acids (SA14), and inserting 28 amino acids (SA28) (Figure 1.6). The researchers observed that the stalk length had an impact on virus replication efficiency: the longer the stalk, the better the replication effect[[3]](#reference).

Figure 1.5 CPE Effect Results of Wild Influenza Virus, Ins15 Influenza Virus and Ins30 Influenza Virus

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-6.png)

Figure 1.6 Design of neuraminidase stack regions with different lengths
[[3]](#reference)

### Second round

1.**Design**:Drawing from the research conducted by Castrucci MR et al., we attempted to rescue four mutant viruses they had constructed in HEK293T cells.

2.**Construction**: Similar to the initial construction process, our focus remained on modifying the A6 plasmid within the twelve-plasmid system. We performed deletions and insertions to generate the following mutants: A6-SD24, which involved the deletion of amino acids 38-61; A6-SD9, which involved the deletion of amino acids 43-51; A6-SI14, which entailed the insertion of 14 amino acids from A/Tokyo/67 (H2N2) at position 42; and A6-SI28, which involved the insertion of 28 amino acids from A/Tokyo/67 (H2N2) and A/Tern/Australia/G70C/75 (H11N9) at position 42. Through point mutation or homologous recombination, we successfully constructed these plasmids. Sequencing results confirmed the successful deletion or insertion of the target fragments (Figure 1.7).

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-7-1.png)
![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-7-2.png)

Figure 1.7 Comparison Diagram of Mutant Plasmid Sequencing Results

3.**Test**: Once the mutant plasmids were successfully constructed, we attempted to rescue the viruses using these mutants. HEK293T cells were transfected with the mutant plasmids using PEI transfection reagent. After observing cell death in approximately 90% of the cells, we collected the supernatant containing the viruses and removed cell debris through centrifugation. Subsequently, we added a small amount of the virus solution to a fresh batch of HEK293T cells and extracted total RNA 4 hours later for reverse transcription to obtain cDNA. The cDNA was then amplified through PCR to target the NA protein, and the resulting PCR products were sent to a sequencing company to detect any mutation sequences. The sequencing results confirmed the successful rescue of the wild-type influenza virus and the four mutant viruses. Additionally, the mutant viruses effectively induced cytopathic effects (CPE) in cells (Fig. 1.8 and 1.9). Encouraged by these results, we attempted to generate a larger quantity of mutant viruses and performed a hemagglutination inhibition assay using chicken red blood cells to obtain a rough quantification of the viruses. We observed significant inhibitory effects on chicken red blood cell agglutination for both the SD9 and SI14 mutant viruses, while SD24 and SI28 did not exhibit such effects (Figure 1.9). Furthermore, upon absolute quantification of the virus RNA, we discovered that the virus titers of SD24 and SI28 were much lower than those of the wild-type, SD9, and SI14 viruses (Figure 1.10). According to the literature, we were aware that SD24 was a highly defective virus, and its low viral titer was expected. However, SI28, which was reported as the most efficient virus in the literature, displayed a low virus titer, indicating potential issues with our design.

In this round, we successfully generated four mutant viruses: SD24, SD9, SI14, and SI28, although SI28 did not meet our expectations.

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-8.png)

Figure 1.8 CPE Effect Results of Wild Influenza Virus, SD9 and SI14 Influenza Virus

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-9.png)

Figure 1.9 CPE effect of SI28 and SD24 mutant influenza viruses

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-10-1.jpg)
![](https://static.igem.wiki/teams/4638/wiki/attributions/fig1-10-2.jpg)

Figure 1.10 Results of chicken red blood cell hemagglutination test.

The figure below is the opposite of the figure above, SI28, SI14, SD9, SD24 and WT from top to bottom.

![](https://static.igem.wiki/teams/4638/wiki/notebook/september99.png)

Figure 1.11 Results of virus titer

4.**Learning**: Upon rechecking the sequence, we identified the problem. During the design of the SI28 mutant plasmid, we inadvertently copied the base sequence of 14 amino acids from A/Tern/Australia/G70C/75 (H11N9) incorrectly. Instead of copying amino acids at positions 58-71, we mistakenly copied amino acids at positions 52-65. As there was no coding shift mutation, we initially failed to detect this error.

### Third round

1.**Design**: Considering the previous realization, we proceeded to redesign A6-SI28-new to rectify the errors identified.

2.**Construction**: We obtained the corresponding primers and performed a re-synthesis. By employing homologous recombination, we successfully constructed the aforementioned plasmid. Sequencing results confirmed the successful insertion of the target fragment.

![](https://static.igem.wiki/teams/4638/wiki/engineering/fig1-12.jpg)

Figure 1.12 Comparison Diagram of SI28 new Sequencing Results

3.**Test**: After successfully constructing the mutant plasmid, we proceeded to rescue the virus using this mutant construct. HEK293T cells were transfected with the mutant plasmid using PEI transfection reagent. Following the observation of cell death in approximately 90% of the cells, we collected the virus-containing supernatant and removed cell debris through centrifugation. Subsequently, we added a small amount of the virus solution to a fresh batch of HEK293T cells. After 4 hours, we extracted total RNA and performed reverse transcription to obtain cDNA. We then amplified the cDNA corresponding to the NA protein through PCR and sent it to a sequencing company to detect any mutation sequences. The sequencing results confirmed the successful rescue of the SI28-new mutant virus. However, due to the virus packaging round (requiring at least one week), we were unable to complete the large-scale preparation of the SI28-new mutant virus before the submission of the wiki. Therefore, we were unable to confirm the successful rescue of the mutant virus at the protein level. We plan to continue this experiment before the jamboree to address this limitation.

4.**Learning**: During the plasmid construction process, it is crucial to exercise caution to avoid mistakenly inserting fewer or additional fragments. Multiple checks and confirmations should be conducted before proceeding with the experiment to prevent unnecessary time wastage. This mistake also highlighted the highly variable nature of the neuraminidase stalk region.

# Rescued the virus whose packaging signals of hemagglutinin and neuraminidase were swapped.

Overview: Our project focuses on constructing a neuraminidase-boosted influenza virus. We have employed two methods to modify the influenza virus, one of which involves reversing the expression levels of neuraminidase and hemagglutinin by exchanging the packaging signals of their vRNA segments. This approach aims to enhance the display of neuraminidase on the surface of the influenza virus. Throughout the project, we have undergone three rounds of engineering rounds.

### First round

1.**Design**: Allen Zheng et al., based on A/PR/8 (H1N1) influenza virus strain, rescued the influenza virus expressing more neuraminidase and less hemagglutinin by exchanging the packaging signals at the 5 'and 3' ends of the hemagglutinin and neuraminidase vRNA segments. The vaccine prepared by this influenza virus triggered a stronger immune response against neuraminidase in mice (Figure 2.1)[[4]](#reference).

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig2-1.png)

Figure 2.1 Exchange HA and NA packaging signal design
[[4]](#reference)

Drawing on the ideas of this article, we also hoped to build a WSN mutant virus that exchanges HA and NA packaging signals. To accomplish this, we conducted an extensive literature review to identify the specific sequences of the HA and NA packaging signals[[5][6]](#reference) (Figure 2.2, Figure 2.3).

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig2-2-1.png)

Figure 2.2 HA Packaging Signal
[[5]](#reference).The part marked with yellow in the figure indicates the length of packaging signal 5 'end packaging signal: 33nt non coding area+9nt coding area; 3 'packaging signal: 45nt non coding area+80nt coding area

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig2-2-2.png)

Figure 2.2 NA Packaging Signal
[[6]](#reference).The part marked with yellow in the figure indicates the length of packaging signal
5 'end packaging signal: 19nt non coding area+183nt coding area; 3 'packaging signal: 28nt non coding area+157nt coding area

2.**Construction**:In the twelve-plasmid system, HA corresponds to the A4 plasmid, and NA corresponds to the A6 plasmid. Based on the aforementioned research, we designed multiple pairs of primers to obtain the packaging signal from the A4 and A6 plasmids. Using the homologous recombination method, we added the packaging signal from each other on both sides of the coding region of HA and NA. This allowed us to construct the A4-NAps plasmid and A6-HAps plasmid, and we also mutated the ATG in the coding region of the packaging signal to TTG. The sequencing results confirmed the successful insertion of the target fragment.

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig2-3-1.png)

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig2-3-2.png)

Figure 2.3 Schematic Diagram of A4 NAPs and A6 HAps

3.**Test**:Following the successful construction of the mutant plasmid, we attempted to rescue the virus using this modified construct. HEK293T cells were transfected with the mutant plasmid using PEI transfection reagent. Upon observing approximately 90% cell death, we collected the virus-containing supernatant and removed cell debris through centrifugation. Subsequently, we added a small amount of the virus solution to a fresh batch of HEK293T cells. After 4 hours, we extracted total RNA for reverse transcription to obtain cDNA. We then performed PCR amplification using vRNA primers specific to the NA and HA proteins. The amplified cDNA samples were sent to a sequencing company to detect any mutation sequences. Unfortunately, the sequencing results from the samples we submitted all indicated wild-type sequences.Furthermore, due to the shorter vRNA sequence of NA (1409bp) compared to HA (1775bp), the results of cDNA amplification using the corresponding vRNA primers also revealed the lack of successful mutations (Figure 2.4). Moreover, after exposing HEK293T cells to the virus supernatant, no noticeable cytopathic effects (CPE) were observed (Figure 2.5).

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig2-4.png)

Figure 2.4 Agarose gel electrophoresis of exchange packaging signal virus vRNA PCR products

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig2-5.png)

Figure 2.5 CPE effect diagram of exchange packaging signal virus

4.**Learning**:Further consideration of the literature led us to recognize that the packaging signal can be divided into two components: the non-coding region and the partial coding region. After exchanging the packaging signals, we neglected to remove the packaging signals in the coding regions of the HA and NA proteins. This oversight may have had a negative impact on the successful rescue of the virus.

### Second round

1.**Design**: Based on the first round of analysis, we used the codon optimization website (<https://www.vectorbuilder.cn/tool/codon-optimization.html>)1. to introduce synonymous mutations in the packaging signals of the coding regions of the HA protein and NA protein.

2.**Construction**: Using the results of the synonymous mutation analysis, we designed a series of primers and modified the A4-NAps plasmid and A6-HAps plasmid through homologous recombination. This allowed us to successfully introduce synonymous mutations in the packaging signal of the coding region, resulting in the construction of the A4-NAps(mut) plasmid and A6-HAps(mut) plasmid. The sequencing results confirmed the successful introduction of synonymous mutations.

3.**Testing**: After successfully constructing the mutant plasmids, we attempted to rescue the virus using these plasmids. HEK293T cells were transfected with the mutant plasmids using PEI transfection reagent. When approximately 90% of the cells died, we collected the virus-containing supernatant and removed cell debris through centrifugation. Subsequently, a small amount of the virus solution was added to another batch of HEK293T cells, and the total RNA was extracted after 4 hours for reverse transcription to obtain cDNA. PCR was performed to amplify the vRNA of the NA and HA proteins, and the amplified products were sent to a sequencing company for mutation analysis. The sequencing results revealed that we were unable to successfully rescue the mutant virus with exchanged packaging signals. Instead, the wild-type influenza virus was still present.

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig2-6.png)

Figure 2.6 Agarose gel electrophoresis of PS(mut) virus vRNA PCR products

4.**Learning**: We reexamined the A4-NAps(mut) and A6-HAps(mut) plasmids and identified the cause. In the A4-NAps(mut) plasmid, we had mutated the two initiation codons ATG in the 5'-end packaging signal. However, upon further review, we discovered two additional frameshift initiation codons, ATG. These two ATGs may be the cause of the failure.

### Third round

1.**Design**: We made point mutations in the two initiation codons mentioned above.

2.**Construction**: Two pairs of point mutation primers were designed, and the remaining two ATGs were successfully mutated to TTG in A4-NAps(mut). The sequencing results confirmed the successful introduction of the point mutation.

3.**Testing**: Due to a lack of time, we did not test the new plasmids before the wiki submission. We will continue this part of the experiment before the jamboree.

4.**Learning**: There is always a reason why experiments don't meet expectations, and by carefully examining them, we can discover interesting aspects.

# Exploration of Western blotting conditions

Overview: Western blotting can separate proteins with different molecular weights and semi-quantify them. We hoped to characterize the extended neuraminidase by this method. In the process of exploring suitable conditions, we adopted the principle of engineering. In this process, we went through two rounds.

### First round

1.**Design**: When the virus was prepared in large quantities and concentrated, we extracted the total RNA by attacking the virus and obtained the cDNA by reverse transcription. After PCR, the mutation was confirmed at the gene level, and it was also necessary to confirm the mutation at the protein level. Through literature research, we used Western blotting to characterize protein.

2.**Construction**: We tried using the methods and parameters commonly used in the laboratory. For the characterization of neuraminidase with different lengths, because it does not need accurate quantification, we directly use the stock solutions of different viruses to prepare samples ,which need to be diluted to the same order of magnitude according to the results of chicken red blood cell hemagglutination experiment. After boiling the sample at 100℃ for 10 min, 12% preformed glue was used for electrophoresis at 140V for 60min. After electrophoresis, transfer membrane at 100V for 60min. Incubate the primary antibody and secondary antibody, and then expose it.

3.**Test**: After exposure, we did not distinguish neuraminidase with different lengths obviously (Figure 3.1).

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig3-1.png)

Fig. 3.1 Western blotting chromogram of neuraminidase with different lengths.

4.**Learning**: The average molecular weight of amino acids is calculated, 1kD is equivalent to about 8 amino acids. If the wild-type neuraminidase is taken as a reference, the neuraminidase of SD24 is about 3kd smaller, that of SD9 is about 1kd smaller, that of SI14 is 1.7kd larger and that of SI28 is 3.5kD larger. Among them, SD9 and SI14 are not easy to distinguish from wild type. Western blotting can be roughly divided into the following steps: sample preparation, SDS-PAGE electrophoresis, membrane transfer, antibody incubation and exposure. Among them, the step of determining whether protein with different lengths can be distinguished is mainly SDS-PAGE electrophoresis. So, we tried to explore the different conditions of SDS-PAGE electrophoresis.

### Second round

1.**Design**: Fortunately, according to the experiment in the first round, we determined the approximate position of neuraminidase (between 55 KD and 70 KD), so we can adjust the electrophoresis conditions by the distance between marker bands near neuraminidase. We designed different SDS-PAGE electrophoresis conditions, and the main transformation parameters were voltage and time.

2.**Construction**: We tried different electrophoresis conditions with 12% preformed glue.

3.**Testing**: After electrophoresis, membrane transfer, antibody incubation and exposure were carried out under uniform conditions. The following are the results of each condition. You can see that the bands gradually separate with the exploration of conditions.

![](https://static.igem.wiki/teams/4638/wiki/attributions/fig3-2.png)

Fig. 3.2 Western blotting chromogram under different electrophoresis conditions

4.**Learning**: 4.Adjusting the experimental parameters according to the experimental purpose is a necessary ability in scientific research, and it is forbidden to mechanically copy the experiment.

# Reference

**[1]**Broecker F, Zheng A, Suntronwong N, et al. Extending the Stalk Enhances Immunogenicity of the Influenza Virus Neuraminidase. J Virol. 2019;93(18):e00840-19. Published 2019 Aug 28. doi:10.1128/JVI.00840-19

**[2]**Hiti AL, Nayak DP. Complete nucleotide sequence of the neuraminidase gene of human influenza virus A/WSN/33. J Virol. 1982;41(2):730-734. doi:10.1128/JVI.41.2.730-734.1982

**[3]**Castrucci MR, Kawaoka Y. Biologic importance of neuraminidase stalk length in influenza A virus. J Virol. 1993;67(2):759-764. doi:10.1128/JVI.67.2.759-764.1993

**[4]**Zheng A, Sun W, Xiong X, et al. Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals. J Virol. 2020;94(16):e00742-20. Published 2020 Jul 30. doi:10.1128/JVI.00742-20

**[5]**Watanabe T, Watanabe S, Noda T, Fujii Y, Kawaoka Y. Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes. J Virol. 2003;77(19):10575-10583. doi:10.1128/jvi.77.19.10575-10583.2003

**[6]**Fujii Y, Goto H, Watanabe T, Yoshida T, Kawaoka Y. Selective incorporation of influenza virus RNA segments into virions. Proc Natl Acad Sci U S A. 2003;100(4):2002-2007. doi:10.1073/pnas.0437772100

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

Extend

Exchange

Cell model

NS1 truncated

Target verification

Summary

Reference

NA-boost IAV aims to transform the influenza virus through synthetic biological methods so that the neuraminidase (NA) can be better displayed on the surface of the influenza virus. This, in turn, improves the targeting of tumor cells with high TfR1 expression. This page will show the results obtained by our team in the competition cycle.

### Rescuing and characterization of extended neuraminidase virus

Rescuing and characterization of extended neuraminidase viruses. By inserting amino acid segments from other influenza viruses into the stalk region of neuraminidase, we have achieved the purpose of extending neuraminidase and making it more prominent on the surface of the influenza virus. Referring to the method described by Castrucci MR et al.[[1]](#reference), we constructed four mutant influenza viruses with neuraminidase of different stalk lengths. In SD24, amino acids 38-61 of neuraminidase were deleted. In SD9, amino acids 43-51 were deleted. In SI14, 14 amino acids (43-56) from A/Tokyo/67(H2N2) were inserted at position 42. Building upon SI14, SI28 contains an additional 14 amino acids (positions 58-71) from A/Tern/Australia/G70C/75(H11N9).

![](https://static.igem.wiki/teams/4638/wiki/results/fig1-1.png)

Fig. 1.1 stalk region design of neuraminidase with different lengths
[[1]](#reference)

By point mutation or homologous recombination, based on the A6 plasmid in the 12-plasmid rescue system of the influenza virus, we successfully constructed four mutant plasmids: A6-SD24, A6-SD9, A6-SI14, and A6-SI28. We also successfully rescued the mutant virus in HEK293T cells.

Initially, we used a 6-well plate for virus rescue. After harvesting the virus, we infected 293T cells with the virus, extracted the viral RNA, and reverse transcribed it into cDNA. The cDNA was then amplified by PCR and sent to a sequencing company for testing. The sequencing results confirmed the introduction of mutations at the gene level (Figure 1.2). Additionally, we observed cytopathic effects (CPE) in HEK293T cells 1-2 days after viral infection. The results demonstrated that all four mutant viruses effectively induced pathological changes in the cells (Figures 1.3 and 1.4).

![](https://static.igem.wiki/teams/4638/wiki/results/fig1-2-1.png)
![](https://static.igem.wiki/teams/4638/wiki/results/fig1-2-2.png)

Figure 1.2 Comparison of Sequencing Results of Mutant Plasmids

![](https://static.igem.wiki/teams/4638/wiki/results/fig1-3.png)

Fig. 1.3 CPE effect results of wild-type influenza virus, SD9 and SI14 influenza virus.

![](https://static.igem.wiki/teams/4638/wiki/results/fig1-4.png)

Fig. 1.4 CPE effect results of SI28 and SD24 mutant influenza viruses.

Subsequently, we used a 10 cm dish to generate a large quantity of viruses. After harvesting the virus, we concentrated it through ultracentrifugation. The concentrated virus was subjected to testing using the hemagglutination assay of chicken red blood cells and Western blotting. The former test serves to further confirm the successful rescue of the virus, while the latter verifies the successful introduction of the mutation at the protein level. In the hemagglutination assay of chicken red blood cells, the SD9 and SI14 mutant viruses exhibited significant inhibition of chicken red blood cell agglutination, whereas SD24 and SI28 did not display such a phenomenon (Figure 1.5). This observation suggests that the SD24 and SI28 virus titer was too low, which was subsequently confirmed when we later quantified the virus. In the Western blotting experiment, we observed distinct NA proteins of varying lengths, including SD9, SI14 and the wild type. This observation provides conclusive evidence of the successful introduction of the mutation at the protein level (Figure 1.6).

![](https://static.igem.wiki/teams/4638/wiki/results/fig1-5-1.jpg)
![](https://static.igem.wiki/teams/4638/wiki/results/fig1-5-2.jpg)

Fig. 1.5 Results of hemagglutination test of chicken red blood cells.

The following figure shows the reverse view of the above figure, which is SI28, SI14, SD9, SD24 and WT from top to bottom.

![](https://static.igem.wiki/teams/4638/wiki/results/fig1-6.png)

Fig. 1.6 results of western blotting experiment.

In the process of preparing a large quantity of viruses, we discovered that although the SI28 virus was successfully rescued, its viral titer was unexpectedly low, which contradicted the findings reported in the original literature. Therefore, upon careful examination, we identified an error in the insertion of the base sequence of 14 amino acids from A/Tern/Australia/G70C/75(H11N9). Instead of copying amino acids 58-71, we mistakenly inserted amino acids 52-65. Subsequently, we redesigned the packaged plasmid and successfully validated it using a 6-well plate. Unfortunately, we were unable to complete the bulk virus preparation before the submission deadline for the wiki. However, we will continue this part of the experiment before the jamboree begins.

### Rescuing and characterization of mutant virus with exchange packaging signal

By exchanging the packaging signals of the neuraminidase and hemagglutinin RNA segments, we reversed the expression levels of neuraminidase and hemagglutinin, thereby achieving the objective of better displaying neuraminidase on the surface of the influenza virus. Following the method outlined by Allen Zheng et al.[[2]](#reference), we performed an exchange of the packaging signals between the HA protein gene segment and NA protein gene segment of the WSN virus. Additionally, we mutated the initiation codon ATG within the 5'-end packaging signal to TTG.

Through homologous recombination and point mutation, we successfully constructed the A6-HAps plasmid and A4-NAps plasmid. However, upon replacing the A6 plasmid and A4 plasmid within the 12-plasmid system with these two plasmids, we were unable to effectively rescue the mutant virus.

![](https://static.igem.wiki/teams/4638/wiki/results/fig2-1.png)

Figure 2.1 Design drawing of mutant virus with exchange packaging signal

We suspected that we were unable to effectively rescue the virus due to the presence of residual packaging signals in the coding regions of the HA protein and NA protein. Therefore, we introduced synonymous mutations in this region to avoid the influence of the remaining packaging signal. Through homologous recombination, we successfully constructed the A6-HAps(mut) plasmid and A4-NAps(mut) plasmid. However, we still did not succeed in rescuing the PS mutant virus in the 6-well plate virus rescue assay.

We carefully examined the A6-HAps(mut) plasmid and A4-NAps(mut) plasmid and found that there were two initiation codons (ATG) remaining within the packaging signal at the 5' end of the A4-NAps(mut) plasmid, which might be the cause of this issue. Therefore, we made point mutations in the A4-NAps(mut) plasmid and successfully rectified the plasmid. However, due to a lack of time, we did not complete the virus rescue before the wiki submission deadline. We will continue this part of the experiment before the jamboree begins.

### Construction and characterization of a cell model with high TfR1 expression

The original intention of packaging the NA boost influenza virus was to target tumor cells with high TfR1 expression. Therefore, using Chinese hamster ovary (CHO) cells as a basis, we constructed cell models with different levels of TfR1 expression through plasmid transfection in order to evaluate the infectivity of the mutant virus. CHO cells are not susceptible to influenza virus, making them suitable for comparing differences in virus infection ability.

We used a 12-well plate to construct the model, transfected 1ug of pCMV3-TfR1 plasmid into each well, and changed the solution after transfection for 6 hours. Two days later, we extracted total protein from the cells and performed a Western blotting experiment to determine the level of TfR1 expression. The results of the Western blotting experiment indicated successful construction of CHO cells with high expression of TfR1 (Figure 3.1).

![](https://static.igem.wiki/teams/4638/wiki/results/fig3-1.png)

Fig. 3.1 results of Western blotting experiment after transfection of TfR1 plasmid by CHO for 2 days.

### Rescuing the NS1-truncated Mutant Virus

In the 'human practice' aspect of our project, experts in the field raised concerns about the safety of oncolytic viruses. They suggested that we should consider the safety issue while improving the virus's targeting ability towards tumors. Through literature research, we discovered that oncolytic viruses based on the influenza virus primarily focus on the NS1 protein. Adolfo García-Sastre et al. found that an influenza virus lacking the NS1 protein can only replicate and survive in cells lacking interferon[[3]](#reference). Subsequently, numerous studies have emerged on virus oncolysis through cutting NS1 protein and arming cytokines[[4]](#reference).

![](https://static.igem.wiki/teams/4638/wiki/results/fig4-1.png)

Figure 4.1 Common design 4.1 NS1 truncated influenza virus
[[4]](#reference)

We consider that truncating the NS1 protein of the influenza virus prevents its replication in normal cells, ensuring the safety of the virus. Furthermore, the truncation of NS1 does not conflict with our modification of the surface proteins of the influenza virus in theory. This led us to the idea of attempting to rescue the NS1-truncated influenza virus. Using the A8 plasmid in the 12-plasmid system, we successfully deleted the NS1 protein and retained only the NS2 protein. Unfortunately, despite several attempts, we were unable to rescue the NS1-deleted influenza virus in HEK293T cells. Due to time constraints, we could not continue further experimentation in this area. Our plan is to wait for positive results from our original design before exploring suitable conditions for rescuing the NS1-deleted influenza virus.

### Target verification of mutant virus

After rescuing the NA-boosted influenza virus and constructing the corresponding cell model with high TfR1 expression, our aim is to verify the targeting ability of the mutant virus.

First, we need to quantify the virus accurately. Eight hours after infecting CHO cells with the virus, we extracted the viral RNA and reverse transcribed it into cDNA. Using the A7 plasmid (containing transcribed M1 protein RNA) in the twelve-plasmid system, we prepared a series of concentration gradients and obtained a standard curve through real-time fluorescence quantitative PCR. Next, we calculated the virus titers based on the standard curve and the Cq value of the virus cDNA. We measured the virus titers of SD24, SD9, Wild Type, SI14, and SI28 (Figure 5.1). The results showed that the virus titer of the wild-type influenza virus was the highest. The titers of SI14 and SD9 viruses were lower than that of the wild-type, while the titers of SD24 and SI28 were significantly lower than that of the wild-type.

![](https://static.igem.wiki/teams/4638/wiki/results/fig5-1.png)

Fig. 5.1 virus titers of different kinds of viruses.

We constructed three cell models to assess the infection efficiency of the virus. One model was transformed with the pCMV3-TfR1 plasmid, another with the pCMV3 plasmid, and the third model was not transfected with any plasmid. Two days after transfecting CHO cells with the respective plasmids, we conducted the virus attack experiment. The amount of virus used for the attack was determined through absolute quantification, ensuring consistent titers across different virus types.

After an 8-hour virus challenge, we extracted virus RNA from the cells and reverse transcribed it into cDNA. We then evaluated the virus infection ability using real-time fluorescence quantitative PCR, with GAPDH selected as the internal reference protein. Due to the low titer of the SD24 virus, we were unable to ensure consistent added amounts with the other viruses, even if we used up all the available virus. Therefore, we did not perform a targeted evaluation of SD24, but focused on evaluating the other three mutant viruses and wild types.

The results (Figure 5.2) demonstrate that the infection efficiency of the wild-type influenza virus with high TfR1 expression is doubled compared to the blank plasmid group, confirming the crucial role of TfR1 in influenza virus infection.

The insertion of 14 amino acids moderately improved the virus's infection efficiency, as described in the literature. However, it did not enhance the targeting of TfR1 high-expression cells. This suggests that the insertion of 14 amino acids may not effectively display NA on the surface of the influenza virus.

The deletion of nine amino acids reduced the virus's infection efficiency to some extent, consistent with the literature. Furthermore, the infection efficiency of the SD9 mutant influenza virus with high TfR1 expression was only 20% higher than that of the blank plasmid group. This finding confirms the important role of neuraminidase in the interaction between the influenza virus and TfR1, providing validation for the concept of our project from a different perspective.

The insertion of 28 amino acids significantly weakened the virus's infection efficiency, which contradicts the literature. Upon sequence comparison, we discovered that the reason for this discrepancy was an incorrect insertion of the extended sequence. We plan to conduct a new round of evaluation after expressing the correct SI28 to rectify this issue.

![](https://static.igem.wiki/teams/4638/wiki/notebook/notebook-final.jpg)

Fig. 5.2 results of virus targeting evaluation. Horizontal axis: virus type; Vertical axis: virus infection efficiency (%,normalized by wild-type influenza virus infected cells in blank plasmid group). After transferring to TfR1, the virus infection efficiency was improved, among which, the wild type increased by 1.930 times, SD9 by 1.237 times, SI14 by 1.854 times and SI28 by 1.935 times.

### Summary

In this iGEM project, we attempted to rescue eight mutant influenza viruses, namely S15, S30, SD24, SD9, SI24, SI28, SI28-new, and PS, in order to modify the display state of neuraminidase on the surface of influenza viruses. We successfully rescued six mutant viruses (S15, SD24, SD9, SI24, SI28, SI28-new) and evaluated the TfR1 targeting of three mutant viruses (SD9, SI24, SI28). Unfortunately, due to time constraints, we were unable to rescue the PS mutant virus before the wiki submission. However, after three rounds of engineering cycles, we have identified the reason for the failure and plan to make another attempt in the future.

Additionally, we were unable to complete the targeted evaluation of the SI28-new mutant viruses before the wiki submission. Furthermore, we did not evaluate the targeting of SD24 due to its low viral titer. Nevertheless, the results obtained for the wild-type virus and SD9 mutant virus are promising. Based on these findings, we have demonstrated that cells with high TfR1 expression are more susceptible to influenza virus infection, and the length of the neuraminidase protein plays a crucial role in determining this susceptibility. In our future work, we aim to rescue more influenza viruses with different neuraminidase display states and conduct targeted evaluations to identify influenza viruses that exhibit enhanced targeting towards cells with high TfR1 expression.

### Reference

**[1]**Castrucci MR, Kawaoka Y. Biologic importance of neuraminidase stalk length in influenza A virus. J Virol. 1993;67(2):759-764. doi:10.1128/JVI.67.2.759-764.1993

**[2]**Zheng A, Sun W, Xiong X, et al. Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals. J Virol. 2020;94(16):e00742-20. Published 2020 Jul 30. doi:10.1128/JVI.00742-20

**[3]**García-Sastre A, Egorov A, Matassov D, et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology. 1998;252(2):324-330. doi:10.1006/viro.1998.9508

**[4]**Kabiljo J, Laengle J, Bergmann M. From threat to cure: understanding of virus-induced cell death leads to highly immunogenic oncolytic influenza viruses. Cell Death Discov. 2020;6:48. Published 2020 Jun 11. doi:10.1038/s41420-020-0284-1

{% endblock %}