Foot-and-mouth disease regularly causes serious outbreaks in livestock. The virus that causes the disease can infect cattle, pigs, sheep, goats, and many species of wild animals; the disease is also highly contagious and spreads very quickly and easily. To control the spread of foot-and-mouth disease, farmers must often kill entire herds of animals that have been exposed. Wild animals that can spread the virus may also be killed in an effort to stop the spread of the disease.

Vaccines that protect against foot-and-mouth disease are available and are often used to help prevent the spread of the disease. However, once an outbreak of foot-and-mouth disease begins it may be too late for vaccines to stop its spread. This is because the vaccines can take about a week to provide protection, and by that time an exposed animal may already be very ill.

Previous work conducted in 2010 reported that mice could be genetically engineered to produce short stretches of RNA molecules that can switch off genes from the foot-and-mouth disease virus. Compared with normal mice infected with the foot-and-mouth disease virus, the genetically engineered mice showed little sign of the disease in their bodies. Now, Hu, Qiao, Fu et al.—including some of the researchers involved in the 2010 work—have genetically engineered pigs in the same way. The experiments show that when cells from these pigs are exposed to the foot-and-mouth disease virus in the laboratory, the virus grows much less than normal.

Next, Hu, Qiao, Fu et al. injected genetically engineered pigs and non-genetically engineered pigs with the virus. All of the normal pigs developed severe symptoms very quickly, including the disease's characteristic mouth and foot sores. Additionally, examinations of these pigs' cells showed signs of the disease. But the genetically engineered pigs did not become seriously ill and their cells showed little sign of the disease. Some of the genetically engineered pigs developed a few sores but these sores appeared much later than normal. So far, the results suggest that it may be possible to develop pigs that are resistant to foot-and-mouth disease. Hu, Qiao, Fu et al. will next determine whether or not the genetically engineered pigs can pass the foot-and-mouth virus on to other pigs and livestock.

Foot-and-mouth disease (FMD) is an acute and highly contagious disease of cloven-hoofed animals, including cattle, pigs, sheep and goats and more than 70 wildlife species, and is devastating especially in young animals (Grubman and Baxt, 2004). The etiological agent of FMD is foot-and-mouth disease virus (FMDV), which belongs to the genus aphthovirus of the family Picornaviridae (Bachrach, 1968). Some control strategies including eradication, vaccination, selective test and slaughter have been widely used for preventing FMDV infection (Leforban, 1999; Barnett and Carabin, 2002), but diseases caused by FMDV remain prevalent in pigs and cattle all over the world owing to the absence of reciprocal protection among several FMDV serotypes (Haydon et al., 2001).

RNA interference (RNAi) is a post-transcriptional process initiated by double-stranded RNA (dsRNA) homologous to a target gene sequence (Meister and Tuschl, 2004). Specific gene silencing can also be triggered in mammalian cells by using synthetic short interfering RNA (siRNA), and plasmid or virus-mediated short hairpin RNA (shRNA) (Elbashir et al., 2001; Hammond et al., 2001; Paul et al., 2002; Michel et al., 2005; Kim and Rossi, 2007). The shRNA was proposed as a therapy for suppressing the infection of FMDV in vitro and in vivo (Chen et al., 2006; Kim et al., 2008). Recently, we extended that finding by producing genetically engineered mice integrating shRNA targeting FMDV (Pengyan et al., 2008, 2010). The majority of transgenic (TG) mice infected with FMDV were resistant to infection and showed only slightly abnormal pathology compared with controls. Now, we report that TG pigs expressing siRNA against FMDV are resistant to viral challenge.

We constructed a total of 10 shRNA expression vectors (Figure 1A) targeting viral structural protein VP1 of FMDV type O and determined the efficacy of shRNAs for inhibiting FMDV replication in BHK cells by real-time RT-PCR. The V3 shRNA reduced the expression of viral RNA by 96.8% compared to scrambled control (Figure 1B). The V3 shRNA expression vector was used to generate TG pigs by somatic cell nuclear transfer. A total of 42 pigs were born alive, eight of which survived at least 6 months. TG pigs contained the stably integrated transgene as evidenced by PCR (Figure 2A) and green fluorescent protein (GFP) expression (Figure 2B and Figure 2—figure supplement 1). The copy numbers of transgene were measured by real-time PCR. The copy numbers of the inserted vector were calculated to be 3–11 (Figure 2C). Expression of siRNA in fibroblast cells isolated from TG pigs was examined by custom TaqMan small RNA assays (Figure 2D). The siRNA expression in TG 11, 19, 69 and 101 was 10–30-fold that from TG 24, 45, 49 and 78. After necropsy of TG 69 and 101, expression of siRNA was detected in various tissues, including heart, lung, spleen, liver, kidney and muscle, although the siRNA levels were diverse among different tissues (Figure 2E).

Figure 1

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Figure 2 with 1 supplement see all

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Next, we tested shRNA transgene resistance to FMDV infection in fibroblast cells isolated from high-siRNA TG (11, 19, 69 and 101), low-siRNA TG (24, 45, 49 and 78) and non-TG pigs. Compared to non-TG cells, viral RNA expression was reduced by 30-fold in high-siRNA TG and 12-fold in low-siRNA TG cells at 36 hr after virus challenge (Figure 3A). Inhibition of FMDV infection was a positive correlation with siRNA expression in fibroblast cells (Figure 2B and Figure 3A). Moreover, TG fibroblasts visibly reduced FMDV-induced cytopathogenic effects as compared with non-TG fibroblasts (Figure 3B).

Figure 3

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The resistance of TG pigs to FMDV infection was further tested by intramuscular injection of O serotypes of FMDV. The challenged animals included high-siRNA TG (11 and 19), low-siRNA TG (24, 49 and 78) and non-TG pigs (n=5). Prior to the day of infection, no animal tested was positive for FMDV. After FMDV challenge, all non-TG pigs developed high fever within 72 hr of challenge and severe clinical signs of FMD, the appearance of vesicles on the feet and nose (Figure 4A and Figure 4—figure supplement 1). All non-TG pigs became deteriorated and the lesion score reached 24 at 5 d after challenge (Figure 4A and Figure 4—figure supplement 1). Some smaller vesicles in low-siRNA TG (24, 49 and 78) pigs were also observed until 7 d after challenge, as shown in Figure 4A. However, the body temperature of high-siRNA TG (11 and 19) pigs remained normal throughout the experiment (Figure 4A and Figure 4—figure supplement 1). TG pigs 11 and 19 developed one small vesicle at day 9 of challenge, but TG pig 11 recovered soon on the next day (Figure 4A). We subsequently quantified the viral genome RNA in the serum of the infected animals. Consistent with the clinical signs data, the viral load in the serum of the high-siRNA TG and low-siRNA TG pigs was lower than that in the non-TG pigs (Figure 4B). The viral RNA expression in serum was 42-fold lower in the high-siRNA TG group than that in the non-TG pigs at day 10 of challenge.

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All animals were killed on the 10th day post-infection, and major tissues including lesions were collected for levels of virus RNA and histopathology analysis. Viral RNA was not detected in the heart, lung, spleen, liver, kidney and muscle in the challenged TG pigs, but viral RNA still maintained high levels of expression in lymph and lesions in the non-TG pigs, except heart, lung, spleen and liver (Figure 4C). No viral RNA in the non-TG heart, lung, spleen and liver showed clearance of viral RNA from the tissues, consistent with prior findings (Zhang and Alexandersen, 2004; Chen et al., 2006). Furthermore, lesions, as a potential source of virus transmission by aerosol, were well known to be the predominant tissue site of FMDV infection and amplification (Zhang and Bashiruddin, 2009; Dillon, 2011). Levels of viral RNA in foot lesions of TG pigs were much lower than those in the non-TG pigs (Figure 4C), suggesting an encouraging result for blocking transmission.

Haematoxilin/eosin (HE) staining of major tissues revealed that non-TG pigs had severe abnormal pathology compared to TG pigs. In particular, non-TG pigs showed multifocal necrosis in the liver, and interstitial pneumonia and severe congestion in the lung (Figure 4D). None of the TG pigs showed typical histopathological changes, except one case of interstitial pneumonia.

Recently, Lyall et al. (2011) in Science reported that onward transmission of avian influenza in TG shRNA chickens was prevented, although the TG chickens succumbed to the initial direct challenge, leading to a strategy for controlling avian influenza outbreaks. Our results showed that the TG pigs exhibited a marked resistence to FMDV infection after direct challenge. As encouraging as these results are, an onward transmission experiment will be performed in the future when producing enough high-siRNA TG pigs.

The most important threat caused by FMDV is the high speed of viral replication, short incubation time, and high contagiousness. Although protective immune responses from vaccination against FMDV can be efficacious, the rapidity of virus replication and spread can outpace the development of immune defenses and overrun the immune system (Summerfield et al., 2009). Current FMDV vaccines do not induce a protective response until 7 d post-vaccination (Barnett et al., 2002; Doel, 2003). FMD signs in high-siRNA TG pigs in our study were delayed for at least 8 d after FMDV infection (Figure 4A). siRNA expressed in TG pigs can also play a role as co-agent to induce rapid resistance before routine vaccination can evoke protective immunity. TG siRNA pigs immunized with current vaccines may achieve complete protection for an FMDV outbreak, which provides a novel strategy for preventing FMD in a disease-free country.

The shRNA-based transgene strategy has substantial benefits over vaccination by offering potential sub-serotype protection when using multiple-shRNA expression systems targeting different viruses (Cong et al., 2010). Our findings demonstrate that RNAi technology combining animal cloning offers the possibility to produce TG animal with improved resistance to viral infection.

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Thank you for sending your work entitled “Transgenic shRNA pigs resist foot and mouth disease virus infection” for consideration at eLife. Your article has been favorably evaluated by Charles Sawyers (Senior editor) and two reviewers, one of whom is a member of our Board of Reviewing Editors.

The Reviewing editor and the other reviewer discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

We reviewers were in near perfect agreement that your paper is exciting but had some deficiencies that need to be addressed.

Most important is the need for further analysis of the status of the virus in the experimental and control animals. These include the expression of the siRNAs in tissues; DNA copy number of the transgene; the virus titers/viremia from plasma and perhaps in nasal secretions; and issues of strains of virus. Please try to address as many of these issues as possible in a revised draft. We appreciate that there are severe technical limitations in working with large animal models, but more data on the course of the disease is important for the paper.

Reviewer #1:

This short paper reports the generation of a set of transgenic pig lines that express an shRNA targeting the FMDV RNA, and documents the resistance of some of these lines to pathology upon challenge with high doses of FMDV. Both high-level shRNA expressors and low-level expressors were obtained and showed resistance. This is an exciting development with important implications for the management of this troublesome and economically significant virus. Enormous efforts have been invested in the production of vaccines, with only limited success. This approach offers significant potential benefits to the livestock industry.

The paper lacks much detail about the gene expression and virus inhibition. Some of the information that one would like to see:

1) How many copies of the transgene are present in each of the eight lines? What tissues (beyond fibroblasts) express the transgene, and at what levels? Is it not surprising that both the high-expressing and low-expressing lines show substantial blocks to virus replication?

2) We are given body temperature and vesicle count, but little more about the course of disease (or lack thereof). We have serum levels of virus RNA, but where does virus replicate most extensively (if at all)? Are pathology sections completely free of viral RNA or just indicating lower levels of virus RNA? Is virus shed? (This readout should be very sensitive.) When is it cleared? Does virus persist (for as long as the animals are maintained?) Given the importance of blocking transmission, simple tests of virus shedding would be important to include here.

Reviewer #2:

As a possible additional approach to control FMD Hu and colleagues have produced transgenic pigs that constitutively expressed FMDV specific siRNA. They demonstrated that FMDV infection of fibroblasts derived from these pigs resulted in reduced viral RNA synthesis as compared to fibroblasts from wild-type pigs. They then infected transgenic pigs with FMDV and found that these pigs had much reduced viral RNA synthesis and developed significantly reduced clinical signs as compared to wild-type pigs. As far as I am aware this is the first demonstration of the production of transgenic animals resistant to FMDV using animals naturally susceptible to FMD.

Comments:

1) Since all transgenic pigs developed some clinical signs of FMDV I suggest that the title should be changed to reflect this, e.g.: “Transgenic shRNA pigs have reduced susceptibility to foot and mouth disease virus infection”. Perhaps if the authors use 2 shRNAs to produce transgenic pigs the replication of FMDV could be almost completely blocked.

2) At the end of the third paragraph of the Main text, the authors state that they presumed that FMDV specific shRNA is expressed in various tissues of transgenic pigs (data not shown). It would be valuable to show expression of the shRNA particularly in tissues susceptible to FMDV such as the lungs, skin, etc.

3) It would have been useful to assay for viremia in addition to viral RNA. Hopefully the authors saved serum to perform this assay.

4) Since a concern after infection with a highly infectious virus such as FMDV is the transmission of virus to naïve animals it would have been worthwhile to at least examine the viral RNA load and/or virus titer in nasal secretions. This is an indication of virus transmission by aerosol.

5) The authors indicated that they selected conserved sequences of VP1 from serotype A, O and Asia 1 by gene alignment. How conserved are these sequences across the 3 serotypes? How many strains of each serotype were compared?

6) In their previous manuscript (Pengyan et al., 2010) the authors produced transgenic mice expressing siRNAs targeted to the conserved regions of viral proteins 3D and 2B. These viral nonstructural proteins are more conserved proteins than the viral structural proteins. Why did the authors target the viral structural proteins in this manuscript?

7) The authors indicate that the TG pigs were infected with FMDV serotype O, but they do not indicate what FMDV serotype(s) were used to infect TG and WT fibroblasts. Presumably they also used serotype O. It would have been informative if the authors had infected TG fibroblasts with the other FMDV serotypes, i.e., types A and Asia 1. The authors should be able to infect TG and WT fibroblasts with other FMDV serotypes.

8. How old were the transgenic pigs at the time of FMDV challenge?

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled “Transgenic shRNA pigs reduce susceptibility to foot and mouth disease virus infection” for further consideration at eLife. Your revised article has been favorably evaluated by Charles Sawyers (Senior editor), a Reviewing editor, and one reviewer. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The additional required revisions are detailed below by our reviewer. These mostly seem straightforward to make. It seems to him important to add lesion scores for all 5 control animals if available; if not, some comment would have to be made to justify the absence. The issue of housing of the animals should be clarified, and there might need to be some change in the comments as to whether the non-TG animals were infected or not (though no problem with the fact that they were protected). It seems desirable to retain Figure 4B from the original version. Finally there is a set of small points that should be readily addressed. The manuscript seems likely to move forward after these issues are dealt with.

Reviewer #2:

Hu and colleagues have addressed most of my concerns. Of course it's unfortunate that there was no serum available to assay for viremia or nasal swabs to assay for virus transmission. However, I still have a few suggestions. In Figure 4A and Figure 4–figure supplement 1 the data for the 5 non-TG pigs is presented as if representing 1 animal. The authors especially need to show the lesion scores for all 5 non-TG animals in Figure 4A. This is important to demonstrate that the challenge was severe for all the non-TG animals.

A second extremely important point is regarding the authors response to my comment #3 (Reviewer #2). The authors suggest that the reason there is viral RNA in the serum of both TG and non-TG pigs at all times postinfection is probably due to contamination by aerosol transmission of virus. In the Materials and methods section the authors don't indicate if the TG and non-TG animals were kept in separate rooms. From their answer to this comment it would appear that all animals were kept together in one room. The authors need to clarify this. If the two groups were kept in separate rooms it is difficult to conclude that the presence of viral RNA in the serum of TG animals was due to aerosol contamination and this strongly suggests that the TG animals did in fact get infected. Certainly the TG animals are more protected from disease than the non-TG animals, e.g., see the clinical scores in Figure 4A. I believe that it is important to show the data from the first manuscript (Figure 4B) that indicates the level of viral RNA in the serum of TG vs non-TG animals over the course of the challenge. It clearly shows much higher levels of viral RNA in the non-TG pigs as compared to the TG pigs. This is in support of the clinical score data. Therefore, I would not eliminate this panel as the authors have done. Clearly this is a very important point. However, I would definitely suggest that in future experiments the control and experimental groups be kept in separate rooms.

Additional data files and statistical comments:

As I stated in my comments in the General assessment section I believe that the authors should include Figure 4B from the original manuscript in this revised manuscript.

Reviewer #1:

1) How many copies of the transgene are present in each of the eight lines? What tissues (beyond fibroblasts) express the transgene, and at what levels? Is it not surprising that both the high-expressing and low-expressing lines show substantial blocks to virus replication?

We have added data about transgene copies in each of the eight lines. The new data is presented in Figure 2C and additional text describing this experiment is provided (in the third paragraph of the Main text).

Because we needed to keep some TG pigs alive for FMDV infection, only TG 69 and 101 were slaughtered for analysis of the siRNA expression in various organs. Expression of siRNA was detected in all organs tested, including heart, lung, spleen, liver, kidney and muscle, although the siRNA levels were diverse among different tissues (Figure 2E).

Yes, it is surprising that both the high-siRNA TG and low-siRNA TG pigs show substantial blocks to virus replication. However, we note that similar results have been reported in prior studies (Lyall et al., 2011; Daniel-Carlier et al., 2013). Lyall et al. reported that the siRNA expression levels were unstable and low (below the limit of detection of Northern blot analysis), but transgenic RNAi chickens against influenza prevented virus transmission. The cause of low-expressing lines capable of inhibiting virus infection need be further investigated.

2) We are given body temperature and vesicle count, but little more about the course of disease (or lack thereof). We have serum levels of virus RNA, but where does virus replicate most extensively (if at all)? Are pathology sections completely free of viral RNA or just indicating lower levels of virus RNA? Is virus shed? (This readout should be very sensitive.) When is it cleared? Does virus persist (for as long as the animals are maintained?) Given the importance of blocking transmission, simple tests of virus shedding would be important to include here.

We agree that more information about the course of disease is important for the paper. We have added analysis of virus RNA levels in various tissues from all challenged animals at 10th day post-infection (Figure 4B). Viral RNA was not detected in heart, lung, spleen, liver, kidney and muscle in the challenged TG pigs, but viral RNA still kept high levels of expression in lymph and lesion in the Non-TG pigs, except heart, lung, spleen and liver (Figure 4B). No viral RNA in the Non-TG heart, lung, spleen and liver showed clearance of viral RNA from the tissues, consistent with prior findings (Zhang et al., 2004; Chen et al., 2006). Furthermore, lesion, as a potential source of virus transmission by aerosol, was well known to be the predominant tissue sites of FMDV infection and amplification (Dillon et al., 2011). Levels of viral RNA in foot lesion of TG pigs was much lower than that in the Non-TG pigs (Figure 4B), suggesting an encouraged result for blocking transmission.

In the initial manuscript, we showed that levels of virus RNA in serums of both Non-TG and TG pigs increased continuously and immediately after infection until last collecting for serum (tenth day postinfection). However, FMDV generally can be cleared from serum and most of tissues in 7 days postinfection (Zhang et al., 2004; Zhang et al., 2009). We cannot give a reasonable explanation about the results. It is most likely that serums were contaminated by aerosol transmission of virus excreted by those sick animals when blood was collected or any other time. We think the data from contaminated serum is invalid. Thus we remove the data about serum virus RNA from the initial manuscript. We apologize for this.

Reviewer #2:

1) Since all transgenic pigs developed some clinical signs of FMDV I suggest that the title should be changed to reflect this, e.g.: “Transgenic shRNA pigs have reduced susceptibility to foot and mouth disease virus infection”. Perhaps if the authors use 2 shRNAs to produce transgenic pigs the replication of FMDV could be almost completely blocked.

We have changed the title to “Transgenic shRNA pigs reduce susceptibility to foot and mouth disease virus infection”.

2) At the end of the third paragraph of the Main text, the authors state that they presumed that FMDV specific shRNA is expressed in various tissues of transgenic pigs (data not shown). It would be valuable to show expression of the shRNA particularly in tissues susceptible to FMDV such as the lungs, skin, etc.

We think your comment is important. We have added analysis of the siRNA expression in various organs. Expression of siRNA was detected in all organs tested, including heart, lung, spleen, liver, kidney and muscle, although the siRNA levels were diverse among different tissues. The new data is presented in Figure 2E and additional text describing this experiment is provided (in the third paragraph of the Main text).

3) It would have been useful to assay for viremia in addition to viral RNA. Hopefully the authors saved serum to perform this assay.

Because not enough serum was kept, we could not perform this assay. To show more information about challenge experiment, we have added analysis of virus RNA levels in various tissues from all challenged animals at 10th day post-infection (Figure 4B). Viral RNA was not detected in heart, lung, spleen, liver, kidney and muscle in the challenged TG pigs, but viral RNA still kept high levels of expression in lymph and lesion in the Non-TG pigs, except heart, lung, spleen and liver (Figure 4B). No viral RNA in the Non-TG heart, lung, spleen and liver showed clearance of viral RNA from the tissues, consistent with prior findings (Zhang et al., 2004; Chen et al., 2006).

In the initial manuscript, we showed that levels of virus RNA in serums of both Non-TG and TG pigs increased continuously and immediately after infection until last collecting for serum (tenth day postinfection). However, FMDV generally can be cleared from serum and most of tissues in 7 days postinfection (Zhang et al., 2004; Zhang et al., 2009). We cannot give a reasonable explanation about the results. It is most likely that serums were contaminated by aerosol transmission of virus excreted by those sick animals when blood was collected or any other time. We think the data from contaminated serum is invalid. Thus we remove the data about serum virus RNA from the initial manuscript. We apologize for this.

4) Since a concern after infection with a highly infectious virus such as FMDV is the transmission of virus to naïve animals it would have been worthwhile to at least examine the viral RNA load and/or virus titer in nasal secretions. This is an indication of virus transmission by aerosol.

We have added analysis of viral RNA in lesion of the challenged animals. Lesion, as a potential source of virus transmission by aerosol, was well known to be the predominant tissue sites of FMDV infection and amplification (Dillon et al., 2011). Levels of viral RNA in foot lesion of TG pigs was much lower than that in the Non-TG pigs (Figure 4B), suggesting an encouraged result for blocking transmission.

5) The authors indicated that they selected conserved sequences of VP1 from serotype A, O and Asia 1 by gene alignment. How conserved are these sequences across the 3 serotypes? How many strains of each serotype were compared?

We apologize for our unclear description in the initial manuscript. Because conserved sequences as siRNA target sites were an alternative strategy preventing escape mutants of virus (Dave et al., 2004), our suppose is to prevent escape mutants of virus by choosing conserved sequences as siRNA target sites in our research. The conserved sequences of VP1 have 95-100% similarity in almost all strains of serotype O of FMDV deposited in GenBank, simultaneously showing 74-100% similarity in 3-5 strains of serotype A and Asia 1. Our aim is not to target serotype A, O and Asia 1 simultaneously. We have added some texts to make it clearer (in the last paragraph of the Main text).

6) In their previous manuscript (Pengyan et al. 2010) the authors produced transgenic mice expressing siRNAs targeted to the conserved regions of viral proteins 3D and 2B. These viral nonstructural proteins are more conserved proteins than the viral structural proteins. Why did the authors target the viral structural proteins in this manuscript?

We used the siRNA targeting viral 3D and 2B to produce transgenic pigs; unfortunately cloned pigs could not be obtained efficiently. Many cloned fetus showed developmental abnormalities (mummies or stillborn). The cause has been investigated, and will be published separately.

7) The authors indicate that the TG pigs were infected with FMDV serotype O, but they do not indicate what FMDV serotype(s) were used to infect TG and WT fibroblasts. Presumably they also used serotype O. It would have been informative if the authors had infected TG fibroblasts with the other FMDV serotypes, i.e., types A and Asia 1. The authors should be able to infect TG and WT fibroblasts with other FMDV serotypes.

Yes, O serotype of FMDV was used to infect TG and WT fibroblasts. We have indicated it in the revised manuscript. Again we apologize for our unclear description. Because our aim is not to target serotype A, O and Asia 1 simultaneously, and in view of similarity between siRNA and serotype A or Asia 1. We think it is unnecessary to test infection of TG fibroblasts with other FMDV serotypes.

8. How old were the transgenic pigs at the time of FMDV challenge?

The challenged pigs were 10 to 13 months old. We have specified it in the revised manuscript.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Reviewer #2:

Hu and colleagues have addressed most of my concerns. Of course it's unfortunate that there was no serum available to assay for viremia or nasal swabs to assay for virus transmission. However, I still have a few suggestions. In Figure 4A and Figure 4–figure supplement 1 the data for the 5 non-TG pigs is presented as if representing 1 animal. The authors especially need to show the lesion scores for all 5 non-TG animals in Figure 4A. This is important to demonstrate that the challenge was severe for all the non-TG animals.

Thanks for your comments. We have added the data of all 5 Non-TG pigs in Figure 4A and Figure 4–figure supplement 1 in the revised manuscript.

A second extremely important point is regarding the authors response to my comment #3 (Reviewer #2). The authors suggest that the reason there is viral RNA in the serum of both TG and non-TG pigs at all times postinfection is probably due to contamination by aerosol transmission of virus. In the Materials and methods section the authors don't indicate if the TG and non-TG animals were kept in separate rooms. From their answer to this comment it would appear that all animals were kept together in one room. The authors need to clarify this. If the two groups were kept in separate rooms it is difficult to conclude that the presence of viral RNA in the serum of TG animals was due to aerosol contamination and this strongly suggests that the TG animals did in fact get infected. Certainly the TG animals are more protected from disease than the non-TG animals, e.g., see the clinical scores in Figure 4A. I believe that it is important to show the data from the first manuscript (Figure 4B) that indicates the level of viral RNA in the serum of TG vs non-TG animals over the course of the challenge. It clearly shows much higher levels of viral RNA in the non-TG pigs as compared to the TG pigs. This is in support of the clinical score data. Therefore, I would not eliminate this panel as the authors have done. Clearly this is a very important point. However, I would definitely suggest that in future experiments the control and experimental groups be kept in separate rooms.

In our viral challenge assay, all animals were housed in one room. We have indicated it in the Materials and methods in the revised manuscript. We have included Figure 4B from the original manuscript in this revised manuscript.

Additional data files and statistical comments:

As I stated in my comments in the General assessment section I believe that the authors should include Figure 4B from the original manuscript in this revised manuscript.

We have included Figure 4B from the original manuscript in this revised manuscript.

Author details

1. Shengwei Hu

   1. College of Life Sciences, Shihezi University, Shihezi, China
   2. College of Animal Sciences, Shihezi University, Shihezi, China

   Contribution

   SH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Jun Qiao and Qiang Fu

   Competing interests

   The authors declare that no competing interests exist.

2. Jun Qiao

   College of Life Sciences, Shihezi University, Shihezi, China

   Contribution

   JQ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Shengwei Hu and Qiang Fu

   Competing interests

   The authors declare that no competing interests exist.

3. Qiang Fu

   College of Life Sciences, Shihezi University, Shihezi, China

   Contribution

   QF, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Shengwei Hu and Jun Qiao

   Competing interests

   The authors declare that no competing interests exist.

4. Chuangfu Chen

   College of Life Sciences, Shihezi University, Shihezi, China

   Contribution

   CC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   chencf1962@yahoo.com

   Competing interests

   The authors declare that no competing interests exist.

5. Wei Ni

   College of Animal Sciences, Shihezi University, Shihezi, China

   Contribution

   WN, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   niweiwonderful@sina.com

   Competing interests

   The authors declare that no competing interests exist.

6. Sai Wujiafu

   College of Life Sciences, Shihezi University, Shihezi, China

   Contribution

   SW, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Shiwei Ma

   College of Life Sciences, Shihezi University, Shihezi, China

   Contribution

   SM, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Hui Zhang

   College of Life Sciences, Shihezi University, Shihezi, China

   Contribution

   HZ, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

9. Jingliang Sheng

   College of Life Sciences, Shihezi University, Shihezi, China

   Contribution

   JS, Conception and design, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

10. Pengyan Wang

    College of Life Sciences, Shihezi University, Shihezi, China

    Contribution

    PW, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

11. Dawei Wang

    College of Life Sciences, Shihezi University, Shihezi, China

    Contribution

    DW, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

12. Jiong Huang

    Institute of Veterinary Medicine, Xinjiang Academy of Animal Science, Urumqi, China

    Contribution

    JH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

13. Lijuan Cao

    College of Life Sciences, Shihezi University, Shihezi, China

    Contribution

    LC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

14. Hongsheng Ouyang

    College of Animal Science and Veterinary Medicine, Jilin University, Changchun, China

    Contribution

    HO, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

    For correspondence

    28021883@qq.com

    Competing interests

    The authors declare that no competing interests exist.

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