Acute Influenza A virus outbreak in an enzootic infected sow herd: Impact on viral dynamics, genetic and antigenic variability and effect of maternally derived antibodies and vaccination.

Influenza A virus (IAV) is a highly contagious pathogen in pigs. Swine IAV (swIAV) infection causes respiratory disease and is thereby a challenge for animal health, animal welfare and the production economy. In Europe, the most widespread strategy for controlling swIAV is implementation of sow vaccination programs, to secure delivery of protective maternally derived antibodies (MDAs) to the newborn piglets. In this study we report a unique case, where a persistently swIAV (A/sw/Denmark/P5U4/2016(H1N1)) infected herd experienced an acute outbreak with a new swIAV subtype (A/sw/Denmark/HB4280U1/2017(H1N2)) and subsequently decided to implement a mass sow vaccination program. Clinical registrations, nasal swabs and blood samples were collected from four different batches of pigs before and after vaccination. Virus isolation, sequencing of the virus strain and hemagglutinin inhibition (HI) tests were performed on samples collected before and during the outbreak and after implementation of mass sow vaccination. After implementation of the sow mass vaccination, the time of infection was delayed and the viral load significantly decreased. An increased number of pigs, however, tested positive at two consecutive sampling times indicating prolonged shedding. In addition, a significantly smaller proportion of the 10-12 weeks old pigs were seropositive by the end of the study, indicating an impaired induction of antibodies against swIAV in the presence of MDAs. Sequencing of the herd strains revealed major differences in the hemagglutinin gene of the strain isolated before- and during the acute outbreak despite that, the two strains belonged to the same HA lineage. The HI tests confirmed a limited degree of cross-reaction between the two strains. Furthermore, the sequencing results of the hemagglutinin gene obtained before and after implementation of mass sow vaccination revealed an increased substitution rate and an increase in positively selected sites in the globular head of the hemagglutinin after vaccination.

Introduction

Influenza A virus (IAV) in swine (swIAV) is an enzootic virus of swine herds globally. SwIAV infects the cells of the respiratory tract, inducing clinical signs of respiratory disease and fever [1-3]. Additionally, IAV impairs the immune system, making the infected pig more susceptible to other pathogens [4-6]. During the last 10–20 years, the pig industry has undergone profound structural changes resulting in a significant increase in herd size and a continuous movement of pigs between production units. These changes has altered the dynamics of swIAV from an epizootic disease that resolved in a few weeks to a more enzootic situation with persistent circulation of the virus in same herds for years due to the continuous exposure of naïve piglets [7-15]. These changes have emphasized that there is a need for effective control measures at the herd level, and have resulted in a marked increase in the sales of IAV vaccines. One of the most used vaccines on the European market is Respiporc FLU3, which is an inactivated, adjuvanted, whole virus trivalent vaccine including the subtypes; Bakum/IDT1769/2003 (H3N2), Haselünne/IDT2617/2003 (H1N1) and Bakum/1832/2000 (H1N2) [16]. This vaccine is currently used in Danish herds to control infections with H1av- like viruses including H1, which is the most prevalent subtype found in Denmark [17]. In general, the H1av-like viruses circulating in Denmark share a high level of genetic identity to the H1av-like viruses circulating in the rest of Europe [18].

As pigs have an impermeable epitheliochorial placenta, newborn piglets depend on immunoglobulins from the sow colostrum for protection against infections during the first weeks of life [19]. Sow vaccination is therefore a widely used strategy for the prevention of many porcine pathogens. However, recent reports indicate that the impact of MDAs may be more complex than previously perceived [8,20-25]. Described examples of “unwanted" effects of MDAs include impaired/delayed development of immunity, prolonged shedding periods, an increased risk of enzootic IAV infection at the herd level and vaccine associated enhanced respiratory disease (VAERD) [8,20,21,25-28].

The high mutation rate of RNA viruses enable them to rapidly evolve variants with a better fitness and/or modified antigenicity, capable of evading the immune system [29,30]. The surface protein hemagglutinin (HA) of IAV is more variable than the other viral proteins, consistent with the fact that it is the major target for neutralizing antibodies [31-34]. Especially mutations in the globular head of the hemagglutinin protein, which includes the receptor binding site for host cell entry and five specific antigenic sites/epitopes (Sa, Sb, Ca1, Ca2 and Cb) have been shown to modify the binding of neutralizing antibodies [35-39].

The continuous circulation of a huge variety of antigenically distinct variants of swIAV provides a significant challenge for the control of swIAV, because herd immunity may be compromised by introduction of new strains and/or by emergence of antigenically different variants within the herd. There is, however, a lack of controlled field studies on the swIAV dynamics in these herds.

A Danish sow herd that had been persistently infected with a swIAV of the H1avN1 subtype for years suddenly experienced an acute outbreak involving an H1avN2sw strain. By a co-incidence, this herd was included in another project and therefore we were able to perform a prospective study including observations and samplings both prior to, during, and after the outbreak. The aim of the study was to examine the clinical impact, the viral dynamics, as well as the genetic and antigenic variability of circulating strains prior to, during and after the acute outbreak. Following the acute outbreak, the herd decided to start a mass sow vaccination program, and therefore the study was extended to include exploration of the effects of a mass sow vaccination initiated during an acute outbreak.

Materials and methods

Ethical statement

The study was carried out in strict accordance with the guidelines of the Good Experimental Practices (GEP) standard adopted by the European Union, and all experimental procedures were performed in accordance with the recommendations provided by the National Veterinary Institute of Denmark. All samples were collected by a trained veterinarian and with the farmers consent. The Danish authorities do not require a specific license to obtain diagnostic samples in field settings according to the legislation LBK no. 474 of 15/05/2014.

Herd

The herd was located in the northwestern part of Jutland, Denmark and had 600 sows with a main production of 30 kilos pigs and a small production of finisher pigs. The herd had a known health status according to the Danish Specific Pathogen Free program [40] termed “Blue SPF + AP2 + PRRS Type 1”, indicating that the herd was serologically positive for Actinobacillus pleuropneumoniae type 2 and PRRSv type 1. However, both of these pathogens were under control. In addition, the health status specified that herd was declared free from Mycoplasma hyopneumoniae, Brachyspira hyodysenteriae, Pasteurella multocida, Haematopinus suis and Sarcoptes scabiei var. suis. The herd bought all new gilts from an external source, which had an identical health status. The replacement rate of the sows was approx. 50%/year. All piglets were weaned batch-wise at four weeks of age and placed in empty nursery stables where they were allocated to pens according to size. The herd had four farrowing stables and ten nursery stables. All stables were washed with high pressure and disinfected using hydrated lime between batches. No strict “all in all out” flow of pigs was maintained in any of the stables.

Study design and sampling

As it was the plan to include the herd in another IAV study [41], it was screened for the presence of swIAV in December 2016. At the screening, 30 nasal swabs were collected which included five nasal swabs obtained from one-week-old piglets (farrowing unit), five nasal swabs of three-week-old piglets (farrowing unit), 10 nasal swabs from five-week-old weaners (nursery) and 10 nasal swabs from 9-week-old weaners (nursery).

In February 2017, the herd veterinarian reported an increase in respiratory problems in the farrowing unit, and increased secondary bacterial infections in nursery pigs. The 1st round of sampling was carried out from March to June 2017. The sampling round included four batches of sows with farrowing dates one-week apart. At farrowing, five piglets born from each sow were ear-tagged. Nasal swabs were collected from the ear-tagged pigs at week 1, 3, 5 and 10–12 and blood samples were collected at week 3 and at weeks 10–12. The exact same study design was conducted for the 2nd round of sampling, which was carried out from May to August 2017 after implementation of mass sow vaccination with Respiporc Flu®3 (IDT Biologika GmbH, Dessau-Rosslau, Germany) (Fig 1A and 1B). All sows with farrowing dates from May and onwards were vaccinated for the first time in the third week of March, and for the second time three weeks later. To avoid a mix of unvaccinated and vaccinated sows in the farrowing unit for the 2nd sampling, the first batch of sows included farrowed the last week of May, five weeks after their 2nd vaccination. The following sow batches were thereby six, seven and eight weeks post 2nd vaccination. All sows of the 1st and 2nd sampling round were randomly selected. A timeline showing the different sampling rounds in relation to IAV occurrence and vaccination is presented in Fig 1A.

Fig 1

Overview on the timeline of the study in relation to IAV occurrence and vaccination (1A) and the study design (1B).

Blood samples and nasal swabs were collected from the sows and the piglets at different time points according to Fig 1B. Between two-five ml of blood were collected, using a vacutainer serum tube (Becton Dickinson, Denmark), from sows and piglets in vena jugularis and vena cava cranialis respectively. The blood samples were kept at 5°C for a maximum of 2 days. Subsequently, the samples were centrifuged at 3000rpm for 10 minutes, and the serum stored at -20 °C until test.

The nasal swabs were collected with a small or large rayon swab (Medical Wire, UK) according to the size of the animal. The swab was inserted and turned 360 degrees in both nostrils of each pig, and then immersed into the Sigma Virocult media (MWE, England). The samples were kept at 5° C for a maximum of 2 days until pooling and RNA extraction. Extracted RNA was kept at -80 °C until use.

Clinical registrations

The clinical registrations were performed as previously described [41]. Briefly, a coughing index for the pen including minimum one ear tagged pig was calculated and individual clinical signs including dyspnea, lacrimation, nasal discharge, conjunctivitis, fecal soiling, body condition, limping and hernia were recorded for ear-tagged pigs.

Pooling of nasal swabs, RNA extraction and quantitative real time RT-PCR

The pooling, RNA extraction and quantitative real time RT-PCR was performed as previously described [41]. Briefly, the nasal swabs were pooled litter-wise for the piglets and batch-wise for the sows. RNA was extracted from the pools using the RNeasy mini kit automated on the QIAcube (QIAGEN) using the large sample protocol version 2. For each extraction swIAV positive and negative controls were included. Following extraction, five μL of RNA was used as a template in a previously published quantitative real time RT-PCR assay targeting the matrix gene influenza A [42], for determining if a pool was positive for IAV. Samples with a Ct value <36 was considered positive. If a pool was positive, the RNA was extracted from the individual samples and then tested by quantitative real time RT-PCR as described above. The positive individual samples with a Ct value <31 were tested in a previously described multiplex quantitative real time RT-PCR [41], to determine the IAV subtype. Samples with a Ct value <31 was considered positive in the given HA and NA assay included in the multiplex real time RT-PCR.

Viral isolation and sequencing

At least one positive individual sample of each batch with a Ct value < 31 were chosen for PCR amplification of the HA and NA genes followed by Sanger sequencing as previously described [43]. In addition, the sample with the lowest Ct value from the initial screening and five samples with the lowest Ct value from the 1st and 2nd sampling round were chosen for isolation in Madin-Darby Canine Kidney (MDCK) cells and sequenced on the Illumina Miseq platform as previously described [43].

Analyses of the viral sequences

The generation of consensus nucleotide and amino acid sequences of the Sanger’s sequencing data and the Illumina sequence data was performed as previously described [43] using the program CLC Main Workbench version 8 for the Sanger sequencing reads and CLC Genomics Workbench version 11.0.1 for the Illumina reads. All sequences are available in NCBI Genbank with the following accession numbers: MN410726-MN410785 (Sanger sequences) and MN410796-MN410883 (Illumina sequences).

The consensus nucleotide- and amino acid-sequence of each gene (HA, NA, M, NP, NS, PB1, PB2 and PA) derived from the Sanger and Illumina sequencing were aligned using the MUSCLE algorithm [44] in CLC Main Workbench version 8. The subtype(s) of the IAV strain circulating in the herd were determined by constructing a phylogenetic tree (using neighbor joining) that included both contemporary HA and NA sequences, obtained in the Danish annual swine IAV surveillance, and also the HA and NA sequences from the present study, aligned using MUSCLE. Thereafter, the location of known antigenic sites (Sa, Sb, Ca1, Ca2 and Cb) of the H1 gene [35,37,38,45,46] were identified in the HA amino acid alignment of the present study, and examined manually for differences/mutations. For the remaining genes a BLAST analysis was performed against NCBI Genbank [47] to determine the closest sequence match and thereby the origin (avian or pandemic). Finally, the sequences were subjected to pairwise comparison, to reveal the overall sequence identity among sequences of the different samplings (screening and 1st and 2nd sampling rounds) and to the HA protein of the vaccine strain representing the lineage circulating in the herd.

Viral evolution of the HA gene

We used programs from the software package BEAST2 version 2.5.2 [48] to estimate the substitution rate for the HA gene both before vaccination (during the 1st sampling round) and after mass sow vaccination (during the 2nd sampling round). Specifically, the substitution model was specified to be HKY with gamma distributed rates over sites, with a strict clock model, and using tip dates (sampling dates). The following priors were specified: The tree model was set to “Birth Death Skyline Serial”, which is used when lineages are sampled sequentially through time. The reproduction number was set to be between 0 and 10 with a log normal distrubution. The “BecomeUninfectiousRate” was estimated to be approximately 52 per year (corresponding to an average time being infectious of 1 week) with a log normal distribution and CI95% = [44.4–224]. The clock rate was set as a log normal distribution with a mean value of 0.001 and, which is estimated to be substitution rate of RNA viruses, with a CI95% = [3.95 x 10−5–0.005]. The gamma shape prior and the kappa prior were left at the default values. A gamma distribution of the “origin prior” was chosen with an alpha value of 0.5 and a beta value of 2. Lastly, the sampling proportion prior was set to a log normal distribution with a mean value of 0.001 and CI95% = [3.95 x 10−5–0.005]. The chain length was set to 10.000.000 with a log every 1000, and the MCMC was run twice. The program BEAUti2 [48] was used to set up the analysis, and Tracer version 1.7.1 [49] was used to inspect the results and check for convergence of the MCMC runs.

The program CODEML in the program package PAML [50] was used to investigate whether there were positively selected sites in the two datasets. Specifically, we did this by comparing the fits of CODEML’s Model 1a (M1a) and Model 2a (M2a) (NSsites = 1 and 2). In these models, selection is quantified using the dN/dS ratio (the ratio between the rate of non-silent substitutions per non-silent site and the rate of synonymous substitutions per synonymous site). A dN/dS ratio larger than 1 indicates the presence of positive selection (there are more amino-acid changing substitutions than expected for random reasons). M1a is a two-parameter model, which assumes two classes of codons, one class with negatively selected sites (dN/dS < 1) and one with neutral sites (dN/dS = 1), whereas M2a is a three-parameter model, which includes an additional class of positively selected sites (dN/dS > 1) [51]. If M2a fits the data significantly better than M1a (given the extra parameters in the model), then this is statistical evidence for the presence of positive selection in some codons. The Bayes Empirical Bayes (BEB) procedure [52] implemented in CODEML, was used to identify which sites that were positively selected. Model fits were compared using the Akaike Information Criterion (AIC), and Akaike weights, and also using likelihood ratio tests [53,54]. Moreover, an additional CODEML analysis, was used to determine the average global dN/dS (ω) value for the HA genes (NSsites = 0)[51,55].

We also used the program MrBayes [56] to estimate both clock rates and the presence of positively selected sites simultaneously. Specifically the codon model with gamma distributed rates was specified as: lset nucmodel = codon omegavar = ny98 rates = gamma, and report possel = yes site omega = yes. Node Dating was specified using the function “calibrate” to add a fixed sampling time to each sequence. The following priors were set for each data set: prset brlenspr = clock:uniform clockratepr = normal treeagepr = truncatednormal nodeagepr = calibrated. The data analysis was performed using two parallel runs for 3.000.000 generations with a sample frequency of 600. The phylogenetic tree was inferred in a Bayesian framework and with MCMC sampling of posterior probabilities. Tracer version 1.7.1 [49] was used to inspect results and check for convergence of the two MCMC runs. Tree visualization was performed using FigTree version 1.4.4 [57]

Influenza ELISA

All blood samples were tested for antibodies against all Influenza A types using a commercially available blocking ELISA (IDEXX; Influenza A Ab Test; IDEXX Laboratories, Inc.). This test targets a conserved epitope in the nucleoprotein (NP) of influenza A virus. The OD values of the samples were divided by the OD value of the negative control to determine the S/N ratios. Samples were regarded as positive if they had an S/N ratio <0.6 and negative if it had an S/N ration ≥0.6.

Hemagglutinin inhibition (HI)-test

Following analysis of the full genome sequences obtained from the viral isolates, one viral isolate from each sampling (screening, 1st sampling and 2nd sampling), were selected for the use in the HI-tests. The HI-tests were performed to determine the specific antibody titers of the sows included in the 1st and 2nd sampling rounds, against the three different viral strains of the study: the enzootic IAV strain found at the screening test “P5-U4” (A/sw/Denmark/P5U4/2016(H1N1): accession no. for HA; MN410806) and two different variants of the epizootic IAV strain–“HB4” (A/sw/Denmark/HB4280U1/2017(H1N2): accession no. for HA; MN410800) isolated before and “VB4” (A/sw/Denmark/VB4379U3/2017(H1N2): accession no. for HA; MN410805) isolated after the implementation of mass sow vaccination. To test if the vaccinated sows had indeed been vaccinated, an additional HI-test was performed including an H3N2 isolate, similar to the H3N2sw included in the vaccine. Immune sera raised against Respiporc FLU 3 and against the H1N1 component of the vaccine was used as controls. First, a hemagglutination (HA) test was performed to determine the HA titer of each viral isolate, and four HA-units (HAU) of the viral isolates were used as antigen for the HI-test. The sera were inactivated at 56°C for 30 minutes and then treated with receptor-destroying enzyme (RDE). Then, the sera were mixed with 50% erythrocytes to remove specific inhibitors of haemagglutination and agglutination factors. Two-fold serum dilutions were tested against the four isolates, starting at a dilution of 1:20 followed by the addition of 0.6% guinea pig red blood cells. The titers were expressed as the reciprocal of the highest dilution of serum inhibiting the four HAU, and subjected to log2 transformation for statistical analysis. The average mean log2 values were subsequently converted back to average HI-titers. An HI-titer <20 were considered negative.

Statistics

For each sampling round, a statistical analysis was performed comparing the prevalence of IAV-positive and IAV-negative individuals at each sampling time (weeks 1, 3, 5 and 10–12) with the presence of one of the clinical signs registered at the individual level using a Pearson’s Chi squared Test. The same test was performed for comparing a difference in prevalence of seropositive and seronegative pigs at week 3 and week 10–12 between the two sampling rounds.

For an overall statistical comparison of means from the normally distributed data (CI, HI-titer, substitution rate and omega values etc.) a Student’s t-Test was performed comparing both IAV positive and negative pigs and comparing the results of the 1st and 2nd sampling round [58].

All statistical analysis and graphs were completed using GraphPad Software [58] and Microsoft Excel. P-values below 0.05 were considered statistically significant.

Results

The initial plan was to include the herd in another study describing the swIAV dynamics in enzootic infected herds, and therefore screening samples were obtained in December 2016. The collection of screening samples provided us with the enzootic H1avN1 strain for comparison to the epizootic H1N2sw strain, which was introduced in the herd around February 2017. In total, 30 screening samples were collected from two different age groups in the farrowing and nursery unit, respectively. At the 1st and 2nd sampling round 16 sows and 80 ear-tagged pigs were included, respectively. The number of ear-tagged pigs varied slightly between samplings due to mortality or ability to locate the ear-tagged pigs (Table 1).

Table 1

Number of pigs testing positive for IAV in nasal swabs in the different batches at week 1, 3, 5 and 10–12 during the 1st and 2nd sampling.

	Batch 1		Batch 2		Batch 3		Batch 4		Total
Sampling:	1st	2nd	1st	2nd	1st	2nd	1st	2nd	1st	2nd
Week 1	16/20	4/19	12/19	0/20	9/18	3/19	19/19	0/17	56/76	7/75
Week 3	0/18	4/18	0/19	15/20	0/17	6/18	1/19	7/15	1/73	32/71
Week 5	0/18	8/18	0/19	4/20	0/18	14/19	0/19	14/17	0/74	40/74
Week 10–12	0/17	1/18	0/18	3/19	0/14	2/19	0/18	5/16	0/67	11/72

The values are given as the number of pigs testing positive of IAV in nasal swabs out of the total number of pigs sampled at the given sampling time.

IAV at the screening test

At the screening test in December 2016, the three- and five-weeks old pigs tested positive for IAV in nasal swabs, while the one-week old piglets and nine-week old weaners were negative.

IAV and IAV antibodies– 1st sampling round (before mass sow vaccination)

Test of serum for antibodies against IAV by ELISA revealed that all of the sows of the four batches were seropositive two weeks before farrowing (Fig 2A). In total, 81% of the three-week old piglets were seropositive, whereas the number of seropositive piglets decreased to 31% at week 10–12 (Fig 2A).

Fig 2

The percentage of seropositive sows and pigs and the summed percentage of the number of pigs testing positive for IAV in nasal swabs at the 1st (2A) and 2nd (2B) sampling round.

The columns shows the percentage of seropositive sow and pigs. The blood samples were collected 2 weeks before farrowing from the sows and at week 3 and week 10–12 from the pigs born from the sampled sows. The red line show the summed percentage of pigs at each sampling time (week 1, 3, 5 and 10–12) testing positive for IAV in nasal swabs. “2A” presents the results of the 1st sampling round (before mass sow vaccination), and “2B” presents the results of the 2nd sampling (after mass sow vaccination).

At week 1, 73.7% of all the piglets tested positive for IAV in nasal swabs (Fig 2A). One of these piglets also tested positive at week 3, but the remaining pigs were negative for IAV at weeks 3, 5 or 10–12 (Table 1). Detailed results on viral shedding and antibody status of each individual sow and ear-tagged pig are available in S1 table.

IAV and IAV antibodies—2nd sampling round (after mass sow vaccination)

Test of serum for antibodies against IAV by ELISA revealed that all of the sows in the four batches were seropositive two weeks before farrowing and that 82.3% of the three-week old piglets were seropositive. Conversely, a significantly lower (p = 0.006) number of 10–12 week-old-pigs were seropositive in the 2nd sampling round compared the 1st sampling round (31%). After vaccination, the number of pigs that tested positive for IAV in nasal swabs at week 1 was significantly reduced (p <0.001) as only 9.3% were positive (Fig 2B). However, compared to the 1st sampling round, a significant increased (p <0.001) number of IAV positive pigs at the subsequent three samplings were observed (week 3, 5 and 10–12), since 45.1%, 54% and 15.3% of the pigs tested positive for IAV in nasal swabs at week 3, 5 and 10–12, respectively (Fig 2B and Table 1). Detailed results of viral shedding and antibody status of each individual sow and ear-tagged pig are available in S1 table.

Differences in viral shedding between the 1st and 2nd sampling rounds

The comparisons of the total number of individual pigs that were infected at least once during the study period, the number of pigs that tested positive at two consecutive sampling times, which we defined as “prolonged shedders” and the average Ct value at the different sampling times are shown in Table 2. No statistical significant difference was observed in the total percentage of pigs being infected at least once during the study period between the 1st and 2nd sampling rounds. However, a statistical significant increased number of “prolonged shedders” (p <0.001) was observed at the 2nd sampling round after the implementation of mass sow vaccination. Moreover, a marked significant difference (p <0.0001) was identified in the average Ct values between the two sampling rounds, indicating a significant decrease in viral shedding after implementation of the mass sow vaccination program.

Table 2

Prolonged shedders, total number of infected individuals and Ct values of the 1st and 2nd sampling.

	1st sampling	2nd sampling	P-value
No. of prolonged shedders	1.8% (1/56)	28.3% (17/60)	<0.001
No. of infected individuals	73.7% (56/76)	80% (60/75)	0.467
Average Ct value:
Week 1	16.7–34.2 (25.4)	28.9–34.5 (31.3)	0.0014
Week 3	20.92*	19.1–35.8 (30.7)	-*
Week 5	-	21.9–35.5 (31.9)	-
Week 10–12	-	23.7–34.4 (32)	-
Total:	16.7–34.2 (25.11)	19.1–35.8 (31.3)	<0.0001

The percentage of prolonged shedders is calculated based on the total number IAV positive pigs during the study. The total percentage of infected pigs during the study is calculated compared to the number of pigs at the beginning of the study. The range of Ct values obtained from the individual pigs is given for each sampling time, and the mean Ct value for each sampling time is provided in the parentheses.

*In the 1st sampling round only one pig was positive at week 3 and therefore no range of Ct values are available and no p-value for the differences between the 1st and 2nd sampling could be estimated.

Clinical signs

The average coughing index (CI) for the IAV positive and negative litters/pens at the different sampling times and the presence of nasal discharge at the different sampling times compared to the number of pigs testing positive or negative for IAV are shown for the 1st and 2nd sampling rounds in Tables 3 and 4, respectively. No correlation was observed between the CI and the number of pigs with nasal discharge and the presence of IAV at the 1st sampling. Conversely, a significant correlation was seen between the presence of IAV and an increased coughing index at week 1, week 5 and over all samplings in the 2nd sampling round. Moreover, a significant correlation between the presence of IAV and nasal discharge was seen at week 1 and over all samplings in the 2nd sampling round. No significant correlations between IAV and the presence of dyspnea, lacrimation, conjunctivitis, fecal soiling, body condition, limping and hernia were revealed in any of the sampling rounds.

Table 3

Differences in mean coughing index between the 1st and 2nd sampling round.

	Week 1		Week 3		Week 5		Week 10–12		Total
Sampling round:	1st	2nd	1st	2nd	1st	2nd	1st	2nd	1st	2nd
Virus positive:	0.14	0.13	1.14*	0.37	-	0.23	-	0.02	0.2	0.23
SD:	0.1	0.11	0	0.19		0.2		0.01		0.21
Virus negative:	0.12	0.07	0.6	0.47	0.17	0.08	0.05	0.03	0.27	0.09
SD	0	0.06	0.38	0.4		0.1		0.03		0.18
p-value	0.77	0.06	n/a	0.50	-	0.05	-	0.51	0.49	0.0008

The p-value describes if a significant difference in the average coughing index was observed between virus positive and virus negative litters during the first and second sampling respectively.

*only one registration as only one litter was positive and therefore no p-value has been included for this sampling.

Table 4

Differences in the number of IAV positive and negative animals with nasal discharge between the 1st and 2nd sampling.

	Week 1		Week 3		Week 5		Total*
Sampling round:	1st	2nd	1st	2nd	1st	2nd	1st	2nd
Virus positive:	60.7% (34/56)	85.7% (6/7)	100% (1/1)	71.9% (23/32)	0% (0/0)	85% (34/40)	61.4% (35/57)	79.7% (63/79)
Virus negative:	80% (16/20)	39.7% (27/68)	66% (48/72)	74.4% (29/39)	66% (49/74)	67.5% (23/34)	68% (113/166)	56% (79/141)
p-value	0.2	0.05	0.71	0.97	-	0.14	0.45	0.001

The parentages gives the number of pigs with nasal discharge out of the total number of positive or negative pigs.

*Week 10–12 were not included as nasal discharge was difficult to evaluate when using a nasal wire to restrain the pigs.

Subtyping and strain-characterization—Screening test (the enzootic IAV)

The results of the multiplex real time RT-PCR tests revealed that all the samples obtained at the time of the screening test were the H1avN1 subtype. This was consistent with the phylogenetic analyses of the HA and NA consensus sequences. The BLAST results revealed that all the internal genes were of avian-like H1Nx origin. The same subtype had been identified in the herd earlier trough diagnostic samples obtained by the herd veterinarian (personal communication).

Subtyping and strain-characterization—1st sampling round (before mass sow vaccination)

The multiplex PCR tests revealed that all the samples obtained during the 1st sampling round, prior to mass-vaccination, belonged to the H1avN2sw subtype. This was in accordance with the HA and NA consensus sequences derived from the Illumina and Sanger’s sequencing. The BLAST analysis of the internal genes revealed that the M, NP, PB1, PB2 and PA gene originated from the H1N1pdm09 subtype, whereas the NS gene were of avian-like H1Nx origin. A pairwise comparison of all HA sequences (n = 18) obtained from pigs sampled before vaccination revealed a close identity with 0–6 nucleotide differences corresponding to a sequence identity of 99.62–100%. The comparison also included two HA consensus sequences derived from the same pigs at two different sampling times (weeks 1 and 3), indicating that the pigs tested positive for the same strain for minimum two weeks. The full length of the HA protein was not obtained from all of the HA consensus sequences and therefore 21 nucleotides corresponding to seven amino acids were removed from the 5’end the all of the HA consensus sequences before further analysis. The HA proteins obtained from the 1st sampling round were aligned with the avian HA protein of the vaccine strain (Haselünne/IDT2617/2003, accession number: GQ161124), and the pairwise comparison revealed 91% amino acid identity, with several amino acid differences in antigenic sites (Fig 3).

Fig 3

Alignment of HA proteins obtained at screening, the 1st and 2nd sampling round and from the vaccine strain (Haselünne/IDT2617/2003 (H1N1)).

Dots indicate identical amino acids among the HA proteins, whereas a green background indicates differences between at least one of the four HA proteins. The colored underlines represent the five antigenic sites Sa (purple), Sb (green), Ca1 (blue), Ca2 (orange) and Cb (red).

Subtyping and strain-characterization—2nd sampling round (after mass sow vaccination)

The multiplex PCR revealed that all the positive samples obtained from the 2nd sampling round were of the H1avN2sw subtype. This result was consistent with the HA and NA consensus sequences derived from the Illumina and Sanger’s sequencing. Equal to the 1st sampling, the BLAST analysis of the internal genes revealed that the M, NP, PB1, PB2 and PA gene were of H1NXpdm09 origin, whereas the NS gene were of avian-like H1Nx origin. A pairwise comparison of all HA sequences (n = 19) from pigs sampled after vaccination revealed a close identity with 0–15 nucleotide differences corresponding to a sequence identity of 99–100%. The comparison also included seven HA consensus sequences derived from the same three pigs at two-three different sampling times, revealing that these pigs were positive for the same strain for two weeks and in some cases (pig 326 and 361) up to seven weeks (S1 table). To ensure an equal length of the HA sequences 21 nucleotides corresponding to seven amino acids were trimmed from the 5’end before further analysis.

Comparison of HA consensus sequences obtained at the screening test and during the 1st sampling round

The avian-like HA genes from isolates collected at the screening test and at the 1st sampling round revealed a sequence identity of 87% at the nucleotide level and 89% at the amino acid level, which equaled 59–61 amino acid differences. The majority of the differences were present between amino acid Nos. 74–290 (numbering from DTIC), which includes the HA1 part of the HA gene, that encodes the globular head and contains the receptor binding site for host-cell-entry. Thirteen of the 54 differenes were present in specific antigenic sites (Sa, Sb, Cb, Ca1 and Ca2), including S74N, P124S, D125N, P137S, N142S/R, R155G, R162S, K163R, T164S, K169Q, G170E, S186R, N195D and E202A (numbering from DTIC). An alignment of HA proteins representing each sampling (screening and 1st and 2nd sampling round) can be visualized in Fig 3 along with the HA protein of the avian H1N1 vaccine strain (Haselünne/IDT2617/2003, accession number: GQ161124) covering the H1av component.

Comparison of the HA consensus sequences obtained at the 1st and 2nd sampling rounds (before and after mass sow vaccination)

Only minor differences were observed between the consensus sequences obtained at the 1st and 2nd sampling rounds (before and after the implementation of mass sow vaccination). On the nucleotide level, the HA genes were between 99–100% identical on nucleotide level and 98–100% identical on the amino acid level, corresponding to 0–8 amino acid changes. The amino acid HA sequences of both the 1st and 2nd sampling round contained a deletion at position 127 and therefore amino acid numbers above 127 would have 1 added to them to correspond to the current H1 numbering, from the DTIC. Some of the amino acid differences between the sequences obtained from the 1st and 2nd sampling rounds, were in specific antigenic sites (Fig 3) and included S142R (Ca2), which were present in 8/19 sequences from the 2nd sampling, T190A/S/N (Sb) present in 6/19 sequences form the 2nd sampling round and Q193H (Sb) present in 1/19 sequences from the 2nd sampling round.

Comparisons of NA and the internal genes obtained at the 1st and 2nd sampling rounds (before and after mass sow vaccination)

The NA gene sequences of the H1avN2sw strain before and after vaccination were between 99–100% identical corresponding to up to 14 nucleotide differences. Amino acid differences between isolates from the 1st and 2nd sampling rounds were identified in ten positions, however, the majority of differences were only found in one or two sequences. The similarity of the remaining genes between the 1st and 2nd sampling rounds varied among genes but was generally high with a maximum of ten nucleotide differences. Only one amino acid difference in the PB1 gene (V724I) were consistent in all the sequences of the 2nd sampling round compared to the sequences of the isolates from the 1st sampling round. The number of nucleotide differences and the location of the amino acid differences in the NA- and internal genes are listed in S2 table.

Viral evolution of the HA gene—1st sampling round (before mass sow vaccination)

Based on analysis using BEAST, the substitution rate for the HA gene before vaccination was estimated to be 0.00316 substitutions per site per year (SEM = 0.000032) corresponding to an average of 5.04 nucleotide substitutions per year for the entire gene (1596 nucleotides long in this dataset).

CODEML analysis of the 18 HA sequences derived from pigs of the 1st sampling round (before mass sow vaccination) suggested that positive selection was mostly absent. Thus, likelihood ratio testing indicated that M2a did not fit the data significantly better than M1a (p > 0.05). In addition, the Akaike weights for M1a and M2a were 0.63 and 0.37 respectively, suggesting somewhat higher support for the model without selection. Under model M2a (which was only weakly supported) there was some evidence for positive selection site at position 203 (probability of being positively selected, Pr+, estimated to be 0.81). The global dN/dS (ω) value was estimated to be 0.31, i.e., on average the gene is under medium strong negative selection (the tendency is to conserve the sequence).

The CODEML analysis makes no assumption about the substitution rate being clock-like (rates are instead free to vary on each branch of the phylogeny). However, since there is evidence that influenza sequences typically do evolve according to a molecular clock, it can be advantageous to use a model that explicitly makes that assumption (and which will then use much fewer parameters). We therefore also analyzed the data using MrBayes, and a model including both a strict clock rate, and a codon-based substitution model for estimating dN/dS rates (S1 Fig). In agreement with the CODEML analysis, there is weak evidence for positive selection on position186 (Pr+ = 0.54), with an estimated dN/dS = 1.5. The average dN/dS rates for the negatively and positively selected sites were estimated to be 0.29 (negatively selected) and 1.81 (positively selected). Table 5 list estimated dN/dS and Pr+, and also whether the codon is a known antigenic site, for the codons with most support for being positively selected.

Table 5

Identification of positively selected sites of the HA gene by MrBayes and CODEML.

1st sampling round (before vaccination)				2nd sampling round (after vaccination)
Amino acid change:	ω valueMb/CO	Pr+:Mb/Co	Antigenic site:	Amino acid change:	ω valueMb/CO	Pr+Mb/CO	Antigenic site:
I5V	1.1672	0.3905	-	N-5S	2.1589	0.4676	-
S142R	1.1689	0.3913	Ca2	K-2T	3.2259/7.241	0.7100/0.821	-
R186S	1.4519/4.709	0.5427/0.806	Sb	A-1D	2.5500/4.835	0.5521/0.53	-
A224E	1.1772	0.3952	-	T2A	2.5533/4.99	0.5527/0.548	-
T368P	1.1782	0.3957	-	D84N	2.2352	0.4841	-*
L407M	1.1743	0.3938	-	S142R	4.1941/8.612	0.9715/0.999	Ca2
				R186S	3.0122/5.604	0.6562/0.631	Sb
				T190N/S/A	4.1222/8.57	0.9431/0.992	Sb
				Q193H	2.5254/5.47	0.5469/0.605	Sb
				D346N	2.2431	0.4858	-
				W348G	2.6210/5.643	0.5674/0.625	-
				G360D	2.5860/4.778	0.5600/0.523	-
				R403W	2.5194/5.023	0.5456/0.552	-^
				R454K	2.4940/4.98	0.5401/0.547	-^

Numbering is based on the HA protein without the signal peptide, thereby initiating the numbering from the amino acids “DTIC”. Due to the deletion at position 144, values above have been added one. Amino acid positions with a “-”indicates that the change occurred in the sequence prior to “DTIC”. “ω value” gives the dN/dS ratio for the positive selected sites. “Pr+” gives the probability of the codon being positive selected. “Mb” gives the results of the MrBases analysis. “CO” gives the results of the CODEML analysis. Antigenic sites were defined as the previously published Sa, Sb, Ca1, Ca2 and Cb sites of H1 [35,36,38,45,46].The codon highlighted in bold are the codon positions, which were defined as positive selected sites in the both analysis.

* located in a B-cell epitope identified in H1N1pdm09 [59].

^ located in a T-cell epitope identified in human H1[60].

Viral evolution of the HA gene—2nd sampling (after mass sow vaccination)

Using BEAST, the substitution rate for the HA sequences after vaccination was estimated to be 0.00357 substitutions per site per year (SEM = 0.0000176), corresponding to 5.7 nucleotide substitutions per year for the entire HA gene. This is 12% higher than the rate estimated before mass sow vaccination (significantly different with p <0.001).

CODEML-based analysis of the 19 HA sequences derived from pigs after vaccination strongly supported the presence of positive selection. Specifically, the Akaike weight for M1a and M2a was 0.00035 and 0.9996 respectively, and M2a (which includes a class of positively selected sites) thus has much higher support than M1a. Under model M2a, positions 159 and 207 showed very strong evidence (>99%) of being positively selected. An additional 9 sites were identified as being positively selected with a lower probability (<95%) (Table 5). In agreement with these results, the average dN/dS (global ω) value for the entire HA gene was estimated to be 0.35, and thereby slightly higher than that of the HA sequences before vaccination (0.31).

Analysis using MrBayes (where the model includes both a constant, clock-like substitution rate and a codon-based substitution model for estimating dN/dS) supported the conclusions from the CODEML-based analysis (Table 5 and S2 Fig). Thus, several sites now have support for being positively selected, with substantially higher estimated dN/dS rates. Specifically, the estimated dN/dS rates were 0.05 for negatively selected sites and 4.23 for positively selected sites. The dN/dS value for positive selection was significantly increased (p-value <0.0001) compared to the dN/dS value identified among the sequences before vaccination. The positively selected sites are presented and compared to the CODEML analysis in Table 5. Six of the sites were located in previously known antigenic sites or in B-cell or T-cell epitopes, and included the two codons (159 and 207) which were identified through both analyses, as having the highest probability of being positively selected.

Hemagglutinin inhibition test (HI-test)

The results of the HI-test of the sow sera from the unvaccinated sows during the 1st sampling and HI-titers of the vaccinated sows of the 2nd sampling are shown in Fig 4 and S1 table. The sera was tested against three different virus isolates: P5-U4, which were isolated from one of the screening samples, HB4 which was isolated from one of the nasal swabs from the 1st sampling, and VB4 isolated from the 2nd sampling round. The VB4 isolate had three amino acid changes (S159R, T207S and Q210H) compared to the HB4 isolate. For the HB4 isolate, serum dilutions were only made until 1:640 due to a lack of viral isolate.

Fig 4

Results of hemagglutination inhibition (HI) test of sow sera collected during the 1st (left) and 2nd (right) sampling round.

Each sow sera was tested against three different virus: P5-U4 (A/sw/Denmark/P5U4/2016(H1N1)) isolated from a pig sampled during the screening; HB4 (A/sw/Denmark/HB4280U1/2017(H1N2)) sampled from a pig during the 1st sampling round and before vaccination and VB4 (A/sw/Denmark/VB4379U3/2017(H1N2)) collected during the 2nd sampling round after start of mass sow vaccination. Negative samples were samples with a HI titer below 20.

The results revealed that all of the sows from the 1st sampling round had antibodies that reacted with the enzootic H1avN1sw strain (P5-U4) (mean log2 = 8.05; mean titer: 266). However, only six of the fifteen sows had antibodies towards the epizootic H1avN2sw strain (HB4) (mean log2 = 7.98; mean titer: 254) isolated before start of mass vaccination and, similarly, only four of the fifteen sows had antibodies towards the epizootic H1avN2sw strain (VB4) (mean log2 = 8.57; mean titer: 381) isolated after mass vaccination. All the vaccinated sows of the 2nd sampling round reacted with the enzootic H1avN1 (P5-U4) strain, and with a significant (p-value 0.025) higher titer (mean log2 = 9.26/mean titer: 612) than the sows of the 1st sampling round. Furthermore, an increase in the number of vaccinated sows (11/15) with antibodies reacting against the epizootic strain isolated prior to vaccination (HB2) was observed along with a higher, but not statistically significant, average titer of 364 (mean log2 = 8.5) (p = 0.209). Interestingly, only 9 out of 15 of the vaccinated sows reacted against the strain isolated after start of vaccination (VB4) albeit those that reacted had a high mean titer of 275 (mean log2 = 8.1). The sow sera of the vaccinated sows were also tested against an H3N2sw strain with high level of genetic similarity to the vaccine strain. The results of this test revealed that all vaccinated sows reacted against the H3N2 and with very high titers >1280, indicating that the sows had indeed been vaccinated.

Discussion

In this study, the effect of mass sow vaccination in relation to an outbreak with a new swIAV strain in a previously persistently infected herd was investigated. A unique dataset was collected before, during and after the outbreak. Samples collected during the acute outbreak revealed that the infections with IAV solely occurred in the farrowing unit, with the vast majority of piglets being infected at week 1. This infection pattern clearly changed during the 2nd sampling round, which was conducted after the implementation of mass sow vaccination. A clear delay in onset of infection was observed, as very few piglets were infected at week 1. However, at week 3, almost 50% of the piglets were positive for IAV in nasal swabs, resulting in a high number of IAV positive piglets at weaning. At weaning (week 4), the piglets were mixed into the nursery, providing new naïve individuals for infection, which was most likely the reason for the peak of infection observed at week 5. The presence of IAV circulation in the nursery unit resulted in the presence of IAV positive pigs at the end of the nursery period, which increased the risk of IAV being transferred into the finisher unit. It should be noted that as the RNA concentration was not measured prior to performing the real time RT-PCR, and there was a risk of pigs testing false negative for IAV. However, the marked difference in the infection pattern between the 1st and 2nd sampling rounds would still be evident. The delayed infection time and the significantly lower viral load observed in the pigs after mass sow vaccination, suggested that the vaccine provided some level of protection through the transfer of MDAs to the piglets. However, as previously described, the presence of MDA at the time of infection can increase the individual shedding time [8,21,61]. Indeed, a marked increase in numbers of prolonged shedders, after the use of mass sow vaccination, was observed in this study. This increase in prolonged shedders and the delay in infection time resulted in a higher number of IAV positive pigs being moved around the production system, consequently spreading the IAV to all age groups present in the herd. This observation supports the modeling performed by Cador et al. [20] who concluded that the presence of MDA would extend the IAV persistence within the herd. Another consequence of the presence of MDA at the time of IAV infection can be a suppressed active immune response, which has been described by several studies both in regards to neutralizing antibodies, IgG, IgA and the T-cell responses [21,25-27]. In this study, it was observed that significantly fewer pigs were seropositive at weeks 10–12 from the 2nd sampling (after mass sow vaccination) compared to the 1st sampling (before mass sow vaccination), suggesting that the presence of MDAs, also in this study, impaired the development of antibodies. The study did not provide data to elucidate if these seronegative pigs are protected against subsequent infection with the same IAV strain despite the lack of measurable antibodies or if they have developed a sufficient memory response. Re-infection with the same subtype after the MDA has declined has only been shown in one study [21] and needs to be investigated further. It should be noted that the MDAs present during the 2nd sampling round were probably stimulated both by the vaccine and by natural exposure to the epizootic strain. Thereby, the study mainly highlights the effects of having heterologous antibodies (1st sampling round) and homologous antibodies (2nd sampling round) towards the epizootic strain. Moreover, if the sows included in the second sampling had seroconverted towards the epizootic strain prior to vaccination, interference between pre-existing antibodies and the vaccine strain could be expected, possibly lowering the effect of the vaccine. However, mass sow vaccination in swIAV positive herds is extensively practiced in the field, and there is a need for further studies investigating the effect of pre-existing immunity on swIAV vaccination efficacy.

The vaccine used in this study is approved for use in sows and pigs older than 56 days. The specific product characteristics (SPC) recommend a basic immunization of two doses applied with three weeks interval to obtain between four-six month immunity according to the age of the pig at the time of vaccination. However, when gestating sows are boosted two weeks before farrowing, the SPC of the vaccine claims to protect piglets against clinical signs of disease the first 33 days of life through transfer of MDAs [62]. In this study, mass sow vaccination was performed, meaning that while the sows included in the 2nd sampling all had received two vaccinations, they did not receive a booster two weeks before farrowing. However, this vaccination strategy is widely used in Denmark, and a protection of the piglets is expected. Clinical registrations were obtained, to reveal a possible clinical protection of the piglets, as a result of mass sow vaccination. Since different age groups became infected during the 1st (week 1) and 2nd (weeks 3–12) sampling rounds it is difficult to compare the results. However, a higher coughing index and the presence of nasal discharge was correlated with the presence of IAV in nasal swabs during the 2nd sampling round, indicating that vaccination of sows did not provide protection against upper respiratory tract infections. Since Denmark is almost free of swIAV of the H3NX subtypes, antibodies against this subtype can be used as a marker of vaccination with quite high sensitivity, and therefore we could confirm that the sows of the 2nd sampling round had indeed been vaccinated.

The results of this study indicated that mass sow vaccination during an acute outbreak might not be an optimal control strategy if the goal is to protect piglets against infection. However, it should be emphasized that the study was only performed in one herd. Furthermore, it is also important not to undermine the effect the vaccine might have had on protection of the sows, both during the gestation and when entering the farrowing unit, were circulation of IAV thrived [41]. In general, vaccination of naïve herds without IAV circulation is warranted, as an IAV introduction probably will lead to a major outbreak until the herd immunity has been built up. On the other hand, before initiating mass sow vaccination it is important to consider the impacts of the MDAs on the transmission dynamics.

Another important aspect with regard to protection achieved through antibodies was demonstrated in this study, as the herd, which was persistently infected with an H1avN1 strain, had a massive outbreak by an H1avN2sw strain. These two IAV strains have the same avian-like H1 gene, and thereby some level of cross protection was expected. However, it was clear from the 1st sampling round that most of the piglets became infected in week 1, despite most of them likely receiving MDAs from their seropositive mothers. Characterisation of the genetic differences of the enzootic strain found at the screening test and the epizootic strain found during the 1st sampling revealed major differences, suggesting that the outbreak strain was introduced into the herd from an external source. The influx of external gilts into the herd could be a possible route of novel swIAV introductions. The HA genes, while both being of avian-like H1 origin, were only approx. 88% identical, with several amino acid differences. A large proportion of these differences were located in parts of the HA gene encoding the globular head, and in locations corresponding to specific antigenic sites. Furthermore, the HI-results revealed that there was a weak cross-protection between the two strains, as 100% of the sows of the 1st sampling round showed a reaction against the enzootic strain H1avN1 as opposed to only 40% showing a reaction against the epizootic strain H1avN2sw. The results indicated that some swIAV strains, belonging to the avian-like H1 clade, have undergone antigenic drift to a degree that almost has abolished serological cross-reaction. The fact that the majority of the amino acid differences were located in the globular head of the HA protein, suggests that changes in these sites could increase viral survival by avoiding antibody binding. In addition, differences in the other genes might also have an impact on the level of cross-protection between the two strains. This also raises the question if the avian-like H1 strain included in commercially available vaccines, provides cross-protection to all the different variants of the H1 avian-like subtypes. Indeed, a German study has investigated the genetic and antigenic diversity of the avian-like H1Nx viruses in several European countries, and documented an extensive diversity within this subtype, revealing an antigenic difference of up to ten antigenic units (AU) (personal communication Professor Tim Harder, FLI, Germany). In humans, the seasonal IAV vaccines are updated when the new strain differs four AU from the vaccine strain. Thus, this clearly suggests that a more regular update of strains in IAV vaccines intended for use in swine could be beneficial, but unfortunately, the European Medical Authority (EMA) does not allow for updates of veterinary IAV vaccines without going through a new licencing. The vaccine strains included in vaccine used in the herd of this study, contains IAV strains that are 15–19 years old, and from the alignment it is observed that several amino acid differences are observed between the vaccine strain and the herd strains, including differences in antigenic sites. Genetic differences between herd strain and vaccine strain might impact the vaccine efficacy. However, antigenic cartography or experiment animal trials are needed to investigate this further.

Differences in evolutionary dynamics were also observed between the viral sequences obtained from the 1st sampling round (before vaccination) and the 2nd sampling round (after mass sow vaccination). The molecular clock-based analysis, which takes sampling dates into account, revealed a significant increase in the nucleotide substitution rate in sequences obtained from pigs originating from vaccinated sows, compared to the ones originating from non-vaccinated sows. Furthermore, analysis using CODEML and MrBayes showed a substantial increase in positive selection (both the number of sites, and the strength of selection) after vaccination. Interestingly, several of the positively selected sites found in the sequences were located in the globular head of HA and in known antigenic sites. An isolate containing mutations in three sites (positions 159, 203, and 207) with strong support for being positively selected, was included in the HI-test. The results of the HI test showed that the number of sows from both the 1st and 2nd sampling that developed antibodies that recognized this isolate was decreased compared to the number of sows that reacted with the isolate without these three mutations. Though not significant, this suggested that the mutations had an impact on antibody binding. In conclusion, the evolutionary analysis clearly showed that an increase in the general substitution rate of the HA gene and positive selection in codons encoding antigenic sites occurred between the 1st and 2nd sampling round and that this had a negative impact on antibody binding. It is known that the global population immunity against human seasonal IAV strains leads to selection of escape mutants [35,38,63,64], but to our knowledge, this has not been described for IAV infection in swine under field conditions. One explanation for the expanded diversity and the positive selection of escape mutants could be that the increase in the infection time of the individual pig we observed after mass sow vaccination increased the likelihood of mutations through viral drift. In the present study, it is not possible to conclude if this change in viral diversity were due to the use of the vaccine or if it was driven by the immunity raised against the circulating field strain. Further studies are needed to explore the impact of herd immunity, vaccination and the generation of escape mutants in swine.

Conclusion

The results of this study provided unique data on a case, where a previously persistently infected herd experienced an outbreak with a new subtype of IAV and it was thereafter decided to start mass sow vaccination. The genetic analysis revealed that differences within the same IAV subtype can lead to a lack of cross-protection toward a similar strain and consequently to an acute outbreak. The presence of homologous MDAs in piglets during infection resulted in a lower viral load, but also in an increased shedding time and an impaired active immune response to infection. The increased shedding time along with the presence of maternal derived antibodies could be factors driving the positive selection of the HA gene, which in time might lead to the generation of escape mutants.

Bayesian strict molecular clock tree of the HA sequences of the 1st sampling.

The x-axis represents time in years. Node labels represent posterior probabilities. The sequences are named as follows: HB indicates that the sequence was obtained in the first sampling round, and the following cipher gives the batch-number. The next three ciphers gives the ear tag number of the pig and “U1”, “U3”, “U5” and “U10” indicates the sampling time according to week 1, 3, 5 and 10–12. “HA” indicates that the sequences encode the hemagglutinin gene. The name of the sequences of the phylogenetic tree corresponds to the specific sequence ID “X” of the sequences uploaded in NCBI Genbank (A/sw/Denmark/X/2017(H1N2).

(DOCX)

Click here for additional data file.

Bayesian strict molecular clock tree of the HA sequences of the 2nd sampling.

The x-axis represents time in years. Node labels represent posterior probabilities. The sequences are named as follows: VB indicates that the sequence was obtained in the second sampling round, and the following cipher gives the batch-number. The next three ciphers gives the ear tag number of the pig and “U1”, “U3”, “U5” and “U10” indicates the sampling time according to week 1, 3, 5 and 10–12. “HA” indicates that the sequences encode the hemagglutinin gene. The name of the sequences of the phylogenetic tree corresponds to the specific sequence ID “X” of the sequences uploaded in NCBI Genbank (A/sw/Denmark/X/2017(H1N2).

(DOCX)

Click here for additional data file.

Detailed table of the viral shedding and the antibody status (ELISA and HI-test results) of the sows and ear-tagged pigs at the different sampling times.

The table presents the four different batches of sows and their respective piglets at the different sampling times during the 1st and 2nd sampling round. The pigs of the 1st and 2nd sampling round were ear-tagged with numbers ranging from 200–282 and 300–380 respectively, whereas the sows were number from one-16. The green color indicates that the sow/pig tested positive in the antibody ELISA, whereas the red color indicates that the sow/pig tested negative in the antibody ELISA. “+IAV” indicates the nasal swab of the individual pigs or sows tested positive in the real time RT-PCR targeting the matrix gene of IAV. If a box is empty it either indicates that the ear tagged pig is dead or not sampled. The HI-test results of the sow sera is highlighted in yellow, and represents the HI-titers towards the three different swIAV strain found in the herd; P5-U4 (A/sw/Denmark/P5U4/2016(H1N1)), HB4 (A/sw/Denmark/HB4280U1/2017(H1N2)) and VB4 (A/sw/Denmark/VB4379U3/2017(H1N2)).

(DOCX)

Click here for additional data file.

Nucleotide and amino acid differences among NA and the internal genes of the sequences derived from the pigs of the 1st and 2nd sampling.

The first columns describes the different genes. The second column describes the results of the pairwise comparison performed on the nucleotide consensus sequences. The third column describes the differences in amino acids according to the IUPAC codes. The forth column gives the position according to numbering from the first Methionine. The fifth column gives the number of sequences which had the given mutation compared to total number of sequences obtained from the samplings; 1st = 1st sampling and 2nd = 2nd sampling.

(DOCX)

Click here for additional data file.

3 Oct 2019

PONE-D-19-25024

Acute Influenza A virus outbreak in an enzootic infected sow herd: Impact on viral dynamics, genetic and antigenic variability and effect of maternally derived antibodies and vaccination

PLOS ONE

Dear Mrs. Ryt-Hansen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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This study describes the dynamics and variation of swine Influenza viruses (SIV) in a swineherd in Denmark before and after vaccination, and how maternally derived antibodies alter the viral diversity and immune response. Three age groups of pigs (piglets, weanlings and sows) were examined. Viruses were isolated from nasal swabs and viral genomes sequenced using Sanger and Illumina sequencing and compared to the circulating viruses. The study shows that immunization did select for key HA epitopes in the progeny viruses. Testing HA-specific antibody titers pre- and post-immunization revealed that pigs responded to both the pre-vaccination enzootic strain as well as the novel outbreak strain. Vaccination provided little benefit. They observed prolonged viral shedding in piglets. The reviewers indicate their concerns, but please particularly address:

The notion that the 2017-outbreak virus ‘evolved’ from the 2016 virus and why this is the case based on the number of differences in the HA1 amino acid sequence. Describe other evidence for this.

As the vaccination appears to provide limited protection, as all the pigs are already seropositive please clarify the pre- vs. post-vaccination results (1st vs. 2nd sampling).

Please address comments from all reviewers, and clarify responses to comments particularly to reviewer 2.

==============================

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Additional Editor Comments (if provided):

This study describes the dynamics and variation of swine Influenza viruses (SIV) in a swineherd in Denmark before and after vaccination, and how maternally derived antibodies alter the viral diversity and immune response. Three age groups of pigs (piglets, weanlings and sows) were examined. Viruses were isolated from nasal swabs and viral genomes sequenced using Sanger and Illumina sequencing and compared to the circulating viruses. The study shows that immunization did select for key HA epitopes in the progeny viruses. Testing HA-specific antibody titers pre- and post-immunization revealed that pigs responded to both the pre-vaccination enzootic strain as well as the novel outbreak strain. Vaccination provided little benefit. They observed prolonged viral shedding in piglets. The reviewers indicate their concerns, but please particularly address:

• The notion that the 2017-outbreak virus ‘evolved’ from the 2016 virus and why this is the case based on the number of differences in the HA1 amino acid sequence. Describe other evidence for this.

• As the vaccination appears to provide limited protection, as all the pigs are already seropositive, please clarify the pre- vs. post-vaccination results (1st vs. 2nd sampling).

• Please address general comments from all reviewers, and clarify responses to comments, particularly to reviewer 2.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Summary-

The ability to monitor “real world” swine influenza outbreaks within large-scale production systems can provide invaluable information. Elucidating the impact and timing of maternal antibodies in accordance with vaccination in an ongoing outbreak in pigs can offer insight into future vaccine mitigation strategies. Taking into consideration that these types of studies are difficult to recapitulate due to cost, complexity, and happening in “real time” in a BSL-2 animal facility, there are some fundamental issues that the authors need to resolve so the reviewer and ultimately the reader can interpret the data appropriately. As a reviewer I’m left awfully confused as to the nature of the virus that was detected initially at the screening process and how that relates to the virus detected a few months later during the outbreak that was circulating before/after vaccination. As it appears that the herd was persistently infected with H1avN1 strain, had a massive outbreak of H1avN2sw in Feb. 2017 (likely from a 2nd different clade that didn’t evolve from the first in in late-2016)- to what extent vaccination would help afterwards with the pigs all being sero-positive from infection before vaccination (Fig. 2A) adds to the confusion as it relates to maternal antibody protection. Furthermore before vaccination all your sows have antibodies to the 2016 virus (Fig.3, P5-U4) while half seem to have antbodies to the 2017 virus whereas half do not have antibodies (Fig. 3, HB4) this difference is not parsed out in the data. Lastly, the data tables and figures is presented in a summary form and therefore makes it hard as to how this provides insight to other researchers as to best practices in the farm-setting. Regrettably the title of the paper suggests that there this manuscript provides advances in the dynamics, genetic/antigenic variability, maternal antibodies and vaccination- but you have not teased apart these processes but simply provided a summary of a complex outbreak on a farm with 2 antigenically distinct HA1av strains in which you tried to vaccinate to mitigate the impact that was minimally impactful due to pre-existing immunity already present. Most importantly the impression is given that the 2017 virus in the outbreak “evolved” from the 2016 virus and that simply is not the case based on the number of differences in the HA1 amino acid sequence.

General comments are noted in the hopes to improve the manuscript so that a reviewer can provide a better critique of the manuscript.

General Comments-

1. The purpose of describing the screening process is unclear. Line 239, what is the purpose of the 30 screening samples and what is the result? You mention the screening process in the study design on lines 98-100, but is this simply to obtain a 2016 virus for comparison? Please clarify.

2. It appears that all sows were sero-positive by ELISA (Fig. 2A). It appears all sows are sero-positive to the 2016 strain (Fig. 3, left) while half are to the 2017 strain and half are not (Fig, 3 left). How does vaccinating an already flu sero-positive make a difference? It seems vaccination provides no benefit.

3. As shown in Figure 1B, do you know which sow gave birth to each ear-tagged piglet? This is imperative in deciphering maternal antibody impacts. Showing the piglet data based on the mother sow serology data (Fig 3), and not simply barn location is important.

4. Figure 2 in connection with Table 1, is awfully hard to decipher, and just showing %positive does not do the data justice. Could they have been exposed to the virus a week or two earlier and were now recovered and that is why they were not detected in nasal swabs at week 1? These and other questions are not answered, we are left guessing with a simple %summary of the data. Because of this it makes the data minimally impactful, these pigs have different immune-exposure histories, it is not appropriate to lump the data like this.

5. Table 3- coughing index, this is rarely shown in the swine influenza literature as a measure of infection as pigs can be asymptomatic to an active infection depending on the strain. Some pigs don’t cough at all during infection. Since you only have 1 of 73 pigs actively shedding virus after week one in the first sampling and you found no real differences this does not justify a stand-alone table. This table can be summarized in one sentence instead that “while there was nasal discharge indicative of infection (Table 1), there were no discernable differences in coughing.” Alternatively this data should be shown in bar graph form for one to even interpret.

6. This ties into #1-2. For instance, you show in figure 2A that all the pigs in the first round have ELISA antibodies. How did they become sero-positive and to what extent? From the 2016 or 2017 outbreak? This isn’t really answered until Fig. 3. Also we have no idea if maternal antibodies for the piglets are derived from an infection or vaccination. It appears that vaccination was minimally effective since all your pigs were sero-positive to begin with so this make interpreting and vaccination data in 1st sampling round vs. 2nd sampling round difficult.

7. The impression is given (lines 340-348) that the outbreak virus H1 in 2017 somehow evolved from the circulating strain a few months earlier in 2016 because you use the word “mutation”. This is not known, as you very well could have had two co-circulating viruses on the farm and then through reassortment they share the other 7 segments besides the HA. Also in Table 5 data I find it very difficult to believe that immune pressure lead by vaccination lead to this many changes in the HA. Please provide evidence that this many HA1 can change due to vaccination pressure in this short of a time frame. A more plausible explanation is you have 2 co-circulating strains on the farm and the vaccine preferentially prevented on type while the other was allowed to persist. While I am not an evolutionary biologist I very rarely see dN/dS ratios presented in flu studies in which viruses are circulating in less than a year’s duration, these types of analysis are done to look at evolution over decades.

8. You state the amino acid numbering scheme is based on the first methionine. In the flu literature H1 numbering occurs after the polypeptide sequence is cleaved, are you using this numbering scheme?

9. The data would be highly strengthened if we were shown an amino acid alignment of the 2016 strain, the 2017 strain and the viruses sequences with difference in amino acids in the H1, if there are duplicate sequences they can be lumped, however Table 5 does not suffice for depicting the evolution. There isn’t even the known amino acid changes shown in “codon” in table 5.

10. The strains used in the vaccine are known, please state them and speak to them and if the amino acid sequence is known, maybe even include it in the alignment (suggested in #8).

11. All relevant viral segments for at least one represenative strain circulating on the farm in 2016 and in 2017 needs to be deposited into a proper publicly accessible database (NCBI) and referenced in the manuscript (A/Sw/xxx/2016 for example), this doesn’t need to be done with every isolate but virologists need a reference and you need to speak to that in alignments and phylogenetic trees to delineate the 2016 vs, 2017 outbreak.

12. In line with #10, the supplementary phylogenetic trees are suboptimal and provide minimal information as to the H1av diversity circulating in the country and how they related to the rest of Europe. You do not provide any real background information of the H1av subtype in the introduction and discussion. You mention your sequences on line 152 but what do they correspond to?

13. Line 53, source #17, I did not find any data in this source that clarified that this vaccine provides protection to the Denmark subtype. While this is inferred from the H1avN1 component in the vaccine provides protection against your H1av do you have a proper source for this subtype?

14. Line 58- another unwanted effect reported by others in the swine influenza literature is vaccine associated enhanced respiratory disease (VAERD), please consider citing.

15. Line 24 abstract- state what the subtype is and provide a reference strain (see #10).

16. Figure 3 would be better resprented if a supplementary table showed the HI titers for each of the 20 pigs.

17. Figure 2 needs more descriptive labels to reflect before and after vaccination.

18. Figure 3, as already mentioned, many of your sows are sero-postive to flu, this data indicated that vaccination really didn’t do much. Also, did you sequence the P5-U4, HB4, and VB4 viruses before doing the HI? Were they grown in MDCK cells? Please include in materials and methods.

19. Fig. 3 is limiting as we have no idea about the antigenic cross-reactivity of sera to these H1av viruses. Any naiive serum as a control? Any positive swine serum you could obtain specific to the vaccine component from another group or previous study? it appears that all pigs have immunity to the 2016 strain (1st round- P5-U4), approximately half have immunity to the 2017 strain (HB-4) this highly impacts how the piglets might respond with these maternal antibodies.

Reviewer #2: In this study, the authors describe the dynamics and variation of swine Influenza viruses (swIAV)in one swine herd in Denmark before and after vaccination and how maternally derived antibodies alter the viral diversity and immune response. The authors were able to sample three age groups of pigs, piglets, weanlings and sows when the herd was infected with an outbreak strain that replaced the enzootic strain and then after mass administration of a commercially available swine influenza vaccine. Viruses were isolated from nasal swabs from these animals; viral genomes were sequenced using Sanger and Illumina sequencing and compared to the circulating viruses. The authors discover that immunization did positively select for some key epitopes in the HA antigen in the progeny viruses that likely helped these viruses adapt. Testing HA specific antibody titers in these animals pre- and post-immunization revealed that animals responded to both the pre-vaccination enzootic strain as well as the novel outbreak strain; they also observed prolonged viral shedding in the piglets post vaccination sows and ascribe it to the presence of maternal derived antibodies.

The manuscript is well written and most data analyses are well thought out. Sequence data are submitted to NCBI and will be available upon publication.

The major critiques of the manuscript are as follows

1. Sample positivity is defined by qPCR Ct values <36. This is dependent on the amount of RNA utilized for the one-step qRT-PCR they employ in this manuscript. There is no mention of the amount of RNA from each sample that was used for the qRT-PCR assay. Please clarify that samples that were considered flu negative were not due to lack of sufficient RNA in the reaction. Please clearly state the amounts of RNA used for qRT-PCR.

2. Please show the multiple alignments of the original enzootic H1N1, HB4 and VB4 strain HAs as supplemental material.

3. Line 337. please clarify what two-seven weeks (is it twenty seven, is it two through seven).

4. In Table2, please provide range of the Ct values instead of the mean. This would help understand the spread of the data across each time point as well as compare run efficiencies.

5. What Ct threshholds were utilized in the multiplexing assays used (line 309)?

6. Please speculate on why a majority of the amino acid changes in HA were located in the positions 91-307 between screening test and 1st sampling isolates. Do these changes improve viral fitness?

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Reviewer #1: No

Reviewer #2: No

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18 Oct 2019

Dear Editor and reviewers,

Thank you a lot for your comprehensive review and very useful comments that indeed will improve the paper. We have tried to address and if needed, to incorporate all of your comments as described in details below:

Editor:

The notion that the 2017-outbreak virus ‘evolved’ from the 2016 virus and why this is the case based on the number of differences in the HA1 amino acid sequence. Describe other evidence for this.

- The 2017 virus did not evolve from the 2016 and I have now corrected the section which describes the differences between the 2016 strain and the 2017 strain, so it now does not contain the word “mutation”, which could indicate that the 2017 evolved from the 2016 strain (line 382). Furthermore, this has also been emphasized in the discussion, where it is now mentioned that the 2017-outbreak virus likely were introduced from an external source (line 568-570).

As the vaccination appears to provide limited protection, as all the pigs are already seropositive please clarify the pre- vs. post-vaccination results (1st vs. 2nd sampling).

- The impact of the pre-existing immunity is now discussed (line 529-537). However, it is also underlined that in the majority of herds choosing to implement swIAV vaccination, there will be pre-existing immunity toward swIAV, which can impact the vaccine efficacy, which also highlight the importance of sharing the results of this study.

In your Methods, please state the volume of the blood samples collected for use in your study.

- This has now been stated in line 127.

Please amend your current ethics statement to confirm that your named ethics committee specifically approved this study.

- The statement has now been edited and refers to the Danish legislation, which does not require a specific license for the samplings performed in this field study.

We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing.

- All sequences have been uploaded in NCBI Genbank and will be available upon publication. This statement and the accession number are now both included in the manuscript and in the revised cover letter, and the reference strains highlighted in the manuscript are presented with their specific accession number.

We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

- As the data in the two cases are not core parts of the study the two phrases has been deleted from the manuscript.

Reviewer #1:

General Comments:

1. The purpose of describing the screening process is unclear. Line 239, what is the purpose of the 30 screening samples and what is the result? You mention the screening process in the study design on lines 98-100, but is this simply to obtain a 2016 virus for comparison? Please clarify.

- The purpose for the screening samples has now been emphasized in line 256-259.

2. It appears that all sows were sero-positive by ELISA (Fig. 2A). It appears all sows are sero-positive to the 2016 strain (Fig. 3, left) while half are to the 2017 strain and half are not (Fig, 3 left). How does vaccinating an already flu sero-positive make a difference? It seems vaccination provides no benefit.

- Thanks for pointing this out. We have added a paragraph in the discussion section where this is discussed (line 529-537). Moreover, it was not the same sows included in sampling round 1, which were later vaccinated and included in sampling round 2. This has now been underlined in the study design (line 121-122).

3. As shown in Figure 1B, do you know which sow gave birth to each ear-tagged piglet? This is imperative in deciphering maternal antibody impacts. Showing the piglet data based on the mother sow serology data (Fig 3), and not simply barn location is important.

- Yes, as mentioned in the materials and methods the sows were sampled and identified before birth, and all the piglets were ear-tagged at birth. However, this has been further emphasized in line 113 and in the Figure text of Figure 2 (line 282). Moreover, an additional supplementary table (S1 table) has now been included in the manuscript, presenting individual sow and piglet data.

4. Figure 2 in connection with Table 1, is awfully hard to decipher, and just showing %positive does not do the data justice. Could they have been exposed to the virus a week or two earlier and were now recovered and that is why they were not detected in nasal swabs at week 1? These and other questions are not answered, we are left guessing with a simple %summary of the data. Because of this it makes the data minimally impactful, these pigs have different immune-exposure histories, it is not appropriate to lump the data like this.

- Figure 2 is meant to give an overview on the different infection patterns observed during the first and second sampling round, as clear differences in infection time is observed. However, the pigs were not sampled every week, so of course we could have missed some infections. However, it is still clear from the data that fewer pigs were infected at week 1, and as pigs shed IAV for approx. 1 week and there is an incubation period before they start shedding, it is very unlikely that the piglets has already recovered from an swIAV infection at one week of age. However, to allow the reader to access the data on individual pigs, another supplementary table (S1 table) has been added where the details of each litter and sow can be found in regards to both infection status, ELISA antibodies and HI-titers.

5. Table 3- coughing index, this is rarely shown in the swine influenza literature as a measure of infection as pigs can be asymptomatic to an active infection depending on the strain. Some pigs don’t cough at all during infection. Since you only have 1 of 73 pigs actively shedding virus after week one in the first sampling and you found no real differences this does not justify a stand-alone table. This table can be summarized in one sentence instead that “while there was nasal discharge indicative of infection (Table 1), there were no discernable differences in coughing.” Alternatively this data should be shown in bar graph form for one to even interpret.

- You are right that in some herds/batches the swIAV infection may be subclinical, but we have previously shown that the coughing index in some cases are significantly related to infection with swIAV in pigs (Ryt-Hansen et al. Vet Res (2019)). Even though no difference in coughing index was seen during the first sampling round, there was an association between an increased coughing index during the second sampling, and the table thereby highlight that no clinical effect of the vaccination was observed. The caption now specifies that a p value was not calculated for week 3 during the 1st sampling round, as only one litter was swIAV positive.

6. This ties into #1-2. For instance, you show in figure 2A that all the pigs in the first round have ELISA antibodies. How did they become sero-positive and to what extent? From the 2016 or 2017 outbreak? This isn’t really answered until Fig. 3. Also we have no idea if maternal antibodies for the piglets are derived from an infection or vaccination. It appears that vaccination was minimally effective since all your pigs were sero-positive to begin with so this make interpreting and vaccination data in 1st sampling round vs. 2nd sampling round difficult.

- As also emphasized in the answer to comment #4 a new section in the discussion has now been added to emphasize that the effects seen after vaccination both can be due to antibodies stimulated naturally in the sow or as a response to the vaccination.

7. The impression is given (lines 340-348) that the outbreak virus H1 in 2017 somehow evolved from the circulating strain a few months earlier in 2016 because you use the word “mutation”. This is not known, as you very well could have had two co-circulating viruses on the farm and then through reassortment they share the other 7 segments besides the HA. Also in Table 5 data I find it very difficult to believe that immune pressure lead by vaccination lead to this many changes in the HA. Please provide evidence that this many HA1 can change due to vaccination pressure in this short of a time frame. A more plausible explanation is you have 2 co-circulating strains on the farm and the vaccine preferentially prevented on type while the other was allowed to persist. While I am not an evolutionary biologist I very rarely see dN/dS ratios presented in flu studies in which viruses are circulating in less than a year’s duration, these types of analysis are done to look at evolution over decades.

- The word “mutation” has now been changed to “differences”, so that the reader does not interpret that the 2017 outbreak strain evolved from the 2016 strain. The 2016 and the 2017 strain were not detected at the same time point and there is no evidence that the two strains circulated together for a longer time period, but rather that the outbreak strain replaced the 2016 endemic strain. Furthermore, it has been emphasized in the discussion that the 2017 was most likely introduced into the herd from an external source (line 568-570)

- In the discussion it is clearly stated that we do not know if the evolution is driven by immunity stimulated by the vaccine or by natural immunity towards the herd strain, and that further studies are needed to investigate this. However, the evolutionary analysis clearly showed an increased selection pressure directed towards antigenic important sites in sampling round 2 after start of vaccination.

- The dN/dS analysis has mostly been used to look at evolution over a longer time period. However the analysis can also be used over a shorter time frame, as long as you don’t compare them to dN/dS ratios calculated over a longer time frame. In this study the time frame is the same in sampling 1 and sampling 2, and we therefore think it is scientific sound to compare them.

8. You state the amino acid numbering scheme is based on the first methionine. In the flu literature H1 numbering occurs after the polypeptide sequence is cleaved, are you using this numbering scheme?

- International publications on H1 IAVs have both used numbering from the 1st methionine and the polypeptide sequence “DTIC”. I have now only included the DTIC numbering.

9. The data would be highly strengthened if we were shown an amino acid alignment of the 2016 strain, the 2017 strain and the viruses sequences with difference in amino acids in the H1, if there are duplicate sequences they can be lumped, however Table 5 does not suffice for depicting the evolution. There isn’t even the known amino acid changes shown in “codon” in table 5.

- Another figure (Fig 3) containing an alignment of the vaccine strain H1av component, the enzootic screening H1av, the H1av of the 1st sampling and the H1av of the 2nd sampling has been added in the manuscript. This alignment also contains the antigenic sites. Moreover, the amino acid changes have been included in Table 5 and the specific antigenic sites are noted.

10. The strains used in the vaccine are known, please state them and speak to them and if the amino acid sequence is known, maybe even include it in the alignment (suggested in #8).

- The vaccine strains are now stated in the introduction (line 53-54) and the avian HA protein from one of the vaccine strains has also been included in the alignment with accession number (Fig 3). Furthermore, a section in the discussion has been added discussing the vaccine stains of the vaccine used in the study (line 590-595).

11. All relevant viral segments for at least one represenative strain circulating on the farm in 2016 and in 2017 needs to be deposited into a proper publicly accessible database (NCBI) and referenced in the manuscript (A/Sw/xxx/2016 for example), this doesn’t need to be done with every isolate but virologists need a reference and you need to speak to that in alignments and phylogenetic trees to delineate the 2016 vs, 2017 outbreak.

- In the results section, the accession number of all sequences obtained during the study is given. In addition, I now refer to three representative strains of the study both with the sequence ID A/Sw/xxx/2016/2017 and accession number.

12. In line with #10, the supplementary phylogenetic trees are suboptimal and provide minimal information as to the H1av diversity circulating in the country and how they related to the rest of Europe. You do not provide any real background information of the H1av subtype in the introduction and discussion. You mention your sequences on line 152 but what do they correspond to?

- It is not the meaning to show the H1av diversity within Denmark, but rather to illustrate the genetic diversity of the sequences obtained at the 1st and 2nd sampling round. Therefore the S1 and S2 figures are only cited in relation to the evolutionary analysis. The trees are molecular clock trees with a timeline below, which indicates how fast the viral strains evolved, therefore no other reference strains can be included in the trees. In the caption of the phylogenetic tree now include a statement that the names of the sequences observed in the tree are similar to the ID that the sequences are given by NCBI Genbank. A short description and an additional reference on the H1av-like viruses in Denmark have been included in the introduction.

13. Line 53, source #17, I did not find any data in this source that clarified that this vaccine provides protection to the Denmark subtype. While this is inferred from the H1avN1 component in the vaccine provides protection against your H1av do you have a proper source for this subtype?

- We acknowledge that this description was a bit misleading. There is no proper evidence that the vaccine covers all the Danish H1 variants, but the trivalent vaccine includes both one strain with a H1 gene and one strain with a N3 that belong to the linkage as the H1N2 virus. Therefore this vaccine is extensively used in Denmark to provide protection against the Danish H1N2 strain. However, we can discuss if the variations between the vaccine strains and the circulating strain have an impact on vaccine efficiency, but this should be tested in another setup. The sentence has now been revised and extended (line 53-57)

14. Line 58- another unwanted effect reported by others in the swine influenza literature is vaccine associated enhanced respiratory disease (VAERD), please consider citing.

- VAERD has now been mentioned and cited in the introduction.

15. Line 24 abstract - state what the subtype is and provide a reference strain (see #10).

- This has now been added.

16. Figure 3 would be better resprented if a supplementary table showed the HI titers for each of the 20 pigs.

- This has now been implemented in S1 table.

17. Figure 2 needs more descriptive labels to reflect before and after vaccination.

- More descriptive labels have now been added to Figure 2.

18. Figure 3, as already mentioned, many of your sows are sero-postive to flu, this data indicated that vaccination really didn’t do much. Also, did you sequence the P5-U4, HB4, and VB4 viruses before doing the HI? Were they grown in MDCK cells? Please include in materials and methods.

- As mentioned above the sero-positive sows at the time of vaccination is now discussed in line 529-537.

- The viruses were grown and sequenced before doing HI-test, and this has now been emphasized in line 225-226.

19. Fig. 3 is limiting as we have no idea about the antigenic cross-reactivity of sera to these H1av viruses. Any naiive serum as a control? Any positive swine serum you could obtain specific to the vaccine component from another group or previous study? it appears that all pigs have immunity to the 2016 strain (1st round- P5-U4), approximately half have immunity to the 2017 strain (HB-4) this highly impacts how the piglets might respond with these maternal antibodies.

- As stated in the materials and methods section, hyperimmune sera raised against the full vaccine and the H1av component of the vaccine were used as controls for the HI-test. Moreover, an H3N2 strain was used as control to ensure that the sows of the 2nd sampling indeed had been vaccinated. However, no naïve serum control was used. There is always a risk of cross-reactivity when performing HI-test. However, the sera obtained from the 1st and 2nd sampling round, were tested against the same strains, so if there was cross-reactivity it would be the case for all included samples, and therefore we can still observed differences between the two samplings.

Reviewer #2:

The major critiques of the manuscript are as follows:

1. Sample positivity is defined by qPCR Ct values <36. This is dependent on the amount of RNA utilized for the one-step qRT-PCR they employ in this manuscript. There is no mention of the amount of RNA from each sample that was used for the qRT-PCR assay. Please clarify that samples that were considered flu negative were not due to lack of sufficient RNA in the reaction. Please clearly state the amounts of RNA used for qRT-PCR.

- We did not measure the amount of RNA following extraction, and this has been emphasized in the material and methods (line 144-152) and is also mentioned in the discussion (line 510-512).

2. Please show the multiple alignments of the original enzootic H1N1, HB4 and VB4 strain HAs as supplemental material.

- An alignment has now been included in the manuscripts as Fig 3.

3. Line 337. please clarify what two-seven weeks (is it twenty seven, is it two through seven).

- This has not been corrected (374-375).

4. In Table2, please provide range of the Ct values instead of the mean. This would help understand the spread of the data across each time point as well as compare run efficiencies.

- The range of Ct values at each sampling time has now been added.

5. What Ct threshholds were utilized in the multiplexing assays used (line 309)?

- The threshold has now been stated in the materials and methods (line 151-152).

6. Please speculate on why a majority of the amino acid changes in HA were located in the positions 91-307 between screening test and 1st sampling isolates. Do these changes improve viral fitness?

- This has now been emphasized in the discussion (line 578-580).

Submitted filename: Response to reviewers.docx

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24 Oct 2019

Acute Influenza A virus outbreak in an enzootic infected sow herd: Impact on viral dynamics, genetic and antigenic variability and effect of maternally derived antibodies and vaccination

PONE-D-19-25024R1

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Reviewers' comments:

1 Nov 2019

PONE-D-19-25024R1

Acute Influenza A virus outbreak in an enzootic infected sow herd: Impact on viral dynamics, genetic and antigenic variability and effect of maternally derived antibodies and vaccination

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