Susceptibility of common family Anatidae bird species to clade 2.3.4.4e H5N6 high pathogenicity avian influenza virus: an experimental infection study

Background There were large outbreaks of high pathogenicity avian influenza (HPAI) caused by clade 2.3.4.4e H5N6 viruses in the winter of 2016–2017 in Japan, which caused large numbers of deaths among several endangered bird species including cranes, raptors, and birds in Family Anatidae. In this study, susceptibility of common Anatidae to a clade 2.3.4.4e H5N6 HPAI virus was assessed to evaluate their potential to be a source of infection for other birds. Eurasian wigeons (Mareca penelope), mallards (Anas platyrhynchos), and Northern pintails (Anas acuta) were intranasally inoculated with 106, 104, or 102 50% egg infectious dose (EID50) of clade 2.3.4.4e A/teal/Tottori/1/2016 (H5N6). Results All birds survived for 10 days without showing any clinical signs of infection. Most ducks inoculated with ≥ 104 EID50 of virus seroconverted within 10 days post-inoculation (dpi). Virus was mainly shed via the oral route for a maximum of 10 days, followed by cloacal route in late phase of infection. Virus remained in the pancreas of some ducks at 10 dpi. Viremia was observed in some ducks euthanized at 3 dpi, and ≤ 106.3 EID50 of virus was recovered from systemic tissues and swab samples including eyeballs and conjunctival swabs. Conclusions These results indicate that the subject duck species have a potential to be a source of infection of clade 2.3.4.4e HPAI virus to the environment and other birds sharing their habitats. Captive ducks should be reared under isolated or separated circumstances during the HPAI epidemic season to prevent infection and further viral dissemination. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-022-03222-7.

Background
To date, H5 subtype high pathogenicity avian influenza viruses (HPAIVs) have spread to domestic poultry and wild birds in over 70 countries in Asia, Europe, the Middle East, and Africa since late 1996 when the virus, recognized as the precursor of present circulating viruses (Goose/Guangdong [Gs/GD]-like viruses) was isolated from a goose in Guangdong Province, China [1]. Initial reports of high pathogenicity avian influenza (HPAI) were largely confined to poultry populations, and lethal Gs/GD-like virus infection was not detected in wild birds. In 2002, however, outbreaks of Gs/GD-like viruses were reported for the first time in waterfowl and other captive/ wild birds in two waterfowl parks in Hong Kong [2]. Subsequently, more than a thousand migratory birds on Lake Qinghai in China died from HPAIV infection in May and June 2005 [3]. Multiple HPAI outbreaks in wild birds have since been reported at numerous locations around Open Access *Correspondence: yamaguti@tottori-u.ac.jp Faculty of Agriculture, Avian Zoonosis Research Center, Tottori University, Tottori 680-8553, Japan the world. Phylogenetically, based on the hemagglutinin (HA) genes, Gs/GD-like viruses have been organized into 10 clades, numbered clades 0-9 [4]. Among them, clade 2.3.4.4 HPAIVs have been selected as major circulating strains worldwide since late 2013 [5]. The clade 2.3.4.4 HPAIVs had genetically evolved and grouped into four clusters, A-D [6]. Subsequently, these viruses have been reclassified into eight clusters (a-h) by WHO [7]. In this article, the WHO classification was used to describe the subclades of clade 2.3.4.4 HPAIVs, even when citing publications that used the previous classification scheme.
In the winter of 2016-2017, clade 2.3.4.4e (previously, Group C) H5N6 HPAIVs caused many outbreaks of HPAI in poultry and wild birds in the East Asian countries [8][9][10]. Japan experienced the largest HPAI outbreaks in wild birds. A total of 218 wild/captive birds, including endangered species, died as a result of infection or were euthanized as a preventive measure [11,12]. Successive outbreaks occurred in Izumi Plain in Kagoshima Prefecture, which is a major overwintering site for migratory birds [11]. Virus infection was confirmed in dead/debilitated hooded cranes (Grus monachal) and white-naped cranes (Grus vipio), which are classified as Vulnerable according to the International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species [13]. Reared cackling geese (Branta hutchinsii leucopareia, a Critically Endangered category in the IUCN Red List) in Higashiyama Zoo and Botanical Gardens in Aichi Prefecture also died of infection [12]. Viral infections were coincidentally observed in Family Anatidae bird species including Eurasian wigeons (Mareca penelope) and Northern pintails (Anas acuta) sharing their habitat with cranes in the Izumi Plain, and mallards (Anas platyrhynchos) and Eurasian wigeons co-housed with the cackling geese in Higashiyama Zoo. These incidents suggest that the risk of HPAIV infections in endangered bird species is correlated with the viral susceptibility of Anatidae.  [24]; Wang et al. [25] reported that some 2.3.4.4e HPAIVs demonstrated relatively higher pathogenicity in mallards, accompanied by the excessive expression of iNOS in the brain.
In the present study, we assessed susceptibilities of common Anatidae (Eurasian wigeon, mallard, and Northern pintail) to a clade 2.3.4.4e H5N6 HPAIV to assess their importance as a source of infection to environment and other birds, including threatened species, and to determine why clade 2.3.4.4e HPAIVs caused HPAI across East Asian countries. Migratory flyways of these ducks in East Asia were tracked using global positioning system technology in addition to classical bird banding studies [26][27][28][29], and revealed that they were probably involved in transboundary HPAIV dissemination. The results of this study contribute to the understanding of the 2016-2017 outbreaks, and may help to respond better to future HPAI outbreaks.

Results
Eight of 30 captured ducks (3 Eurasian wigeons, 4 mallards, and 1 Northern pintail) were seropositive for influenza A virus by competitive enzyme-linked immunosorbent assay (cELISA) (see Additional file 1). Among them, 2 wigeons and 1 mallard had low titers (2-4) of hemagglutination inhibition (HI) antibody against the challenge strain. Another mallard (M120) had an H7N7 subtype low pathogenicity avian influenza virus detected in a cloacal sample. Based on these results and the criteria as described in Materials and Method section, the ducks were grouped and applied for the subsequent infectious experiment (The experimental designs are described in Additional file 2).
Each of seven Eurasian wigeons, mallards, and Northern pintails was inoculated with 10 6-2 50% egg infectious dose (EID 50 ) of the clade 2.3.4.4e HPAIV A/teal/ Tottori/1/2016 (H5N6) (Tottori/1) and observed for 10 days (Table 1). All the ducks survived without showing any clinical signs during the observation period. The HI testing showed that all the ducks inoculated with 10 6 EID 50 of the virus (W104-W106, M114-M116, and P124-P126) seroconverted against the challenge virus by 10 days post inoculation (dpi). Among them, some ducks, including one cELISA-positive mallard (M115), shed viruses via oral route for a maximum of 10 days, peaked at 3-5 dpi. One each of wigeon and mallard, W106 and M116, respectively, also shed a relatively low titer of virus via the cloacal route. Virus was additionally recovered from a conjunctival swab of one mallard, M116, at 10 dpi, despite no virus shedding being detected in oral/cloacal samples at 7 dpi. Among the ducks inoculated with 10 4 EID 50 of the virus, one juvenile wigeon and one juvenile mallard, W108 and M117, respectively, seroconverted (128-256 HI titers) and shed virus via the oral and/or  18:127 cloacal routes. Viral shedding and antibody responses were not observed in an adult wigeon and an adult mallard (W107 and M118, respectively) which had a pre-HI antibody titer of 2, or two Northern pintails (P127 and P128). None of the ducks inoculated with 10 2 EID 50 of the virus shed any virus. Among them, one wigeon, W109, showed 4-fold change in its serum HI titer after the virus challenge. Notably, throughout the experiment, relatively low titers of viruses remained in the pancreas of several ducks at 10 dpi. No histopathological lesions or antigen-positive cells were observed in the tissues of ducks euthanized at 10 dpi (data not shown).
Viral tropism was assessed in the euthanized ducks at 3 dpi with 10 6 EID 50 of the virus ( Table 2). None of the ducks showed any clinical signs during the observation period. Viruses at titers of ≤ 10 6.3 EID 50 were recovered from all the samples (other than the wing shafts) of a wigeon and a pintail (W102 and P121, respectively) and viremia was confirmed. Systemic infection was also observed in one pintail (P123) with viruses disseminated to various tissues at titers of ≤ 10 6.5 EID 50 . Notably, the three ducks with systemic infection shed relatively higher titers of the virus (10 4.5-5.7 EID 50 ) via conjunctival route, accompanied by viral replication in their eyeballs. Three mallards (M111-M113) and one Northern pintail (P122) had relatively low titers of the virus in the limited tissues. No virus was isolated from two Eurasian wigeons (W101 and W103). None of the ducks had any obvious histopathological lesions at 3 dpi, other than the eyes of two pintails (P121 and P123), which showed mild nonsuppurative conjunctivitis which was composed of subconjuncitival inflammatory infiltrate and epithelial degeneration (Fig. 1A). Immunohistochemistry confirmed a small number of viral antigen-positive cells in some tissues of one wigeon (W102) and two pintails (P121 and P123), with the viruses at titers of ≥ 10 4 EID 50 /g ( Table 2 and Fig. 1B).

Discussion
In this study, three adult Anatidae captured in the winter of 2018-2019 (W107, W109, and M118) had low titers of serum HI antibody against the challenge virus before the infection experiments, indicating that they were infected with antigenically similar influenza viruses and subsequently survived in the field. Other reports also showed that H5 HA specific antibody was detected in serum and egg yolk of wild duck populations in Asian countries [30,31]. The HI seropositive Anatidae did not shed the virus after the challenge of the clade 2.3.4.4e H5N6 HPAIV. As observed in a previous study: mallards and Mandarin ducks with homologous immunity did not contribute to virus transmission [32,33]. Antibody raised against HPAIV infection in wild ducks is likely to be maintained as observed in a surviving mallard in a zoo [12]. These results indicate that wild duck are involved in transboundary dissemination of HPAIV. In island countries such as Japan, increases and decreases in the seroprevalence in migratory ducks is probably correlated with the occurence of HPAI every few years [34]. The results of this study support this hypothesis as no ducks captured in 2019-2020 had pre-HI antibody against the challenge virus, in contrast to those captured in 2018-2019 (See Additional file 1); HPAI outbreaks subsequently occurred in Japan in the winter of 2020-2021 [35]. The challenge virus was recovered from two cELISA-positive juvenile mallards, suggesting that heterologous antibody is less likely to inhibit propagation of HPAIV in Anatidae.
Most of the Anatidae inoculated with ≥ 10 4 EID 50 of Tottori/1 showed subclinical infection. Similar results have been reported in Mandarin ducks with clade 2.3.4.4e  [6,9,11,36,37]. Among them, the group C2 strains, including Tottori/1, were the most widely distributed strains in Japan in the winter of 2016-2017 [9]. The relatively low pathogenicity of the C2 strains probably contributes efficient virus transport by migratory Anatidae flocks. The ages of the duck subjects should also be considered to explain the differences in the pathogenesis of clade 2.3.4.4e H5N6 HPAIVs. Previous reports revealed that the past H5N1 HPAIVs showed higher pathogenicity in younger domestic ducks [38,39]. Juvenile Anatidae captured in this study were presumed to be several months old and to have been born during the summer in Northern nesting areas such as Siberia and Alaska, and then migrated to Japan for wintering. The pathogenicity of Tottori/1 in younger ducks warrants further study.
Virus shedding from the infected Anatidae was mainly observed via the oral route as in previous experimental infection studies of Anatidae (Eurasian wigeons and mallards) and clade 2.3.4.4b and 2.3.4.4c H5 HPAIVs [14,20]. Similarly, some ducks in this study orally shed the virus at titers of ≥ 10 4 EID 50 , in 1-5 dpi, suggesting that these duck species might be a source of infection for other wild bird species in field settings. Further, two Eurasian wigeons shed virus into the cloaca in late phase of infection. A similar observation was made in Mandarin ducks infected with a clade 2.3.4.4c H5N8 HPAIV [16]. One possibility is that the viruses orally shed into drinking water were retaken, and entered the digestive tracts under the experimental conditions. Alternatively, virus that replicated in the respiratory tract may have spread hematogenously.

Viral titers in blood and swabs (log EID 50 /mL)
Notably, several Anatidae, including two Northern pintails inoculated with 10 2 EID 50 of the virus, had low titers of virus in the pancreas at 10 dpi. Such tissue tropism was also observed in Baikal teals that died of natural infection caused by a clade 2.3.4.4c H5N8 HPAIV [40]. It remains unclear why the HPAIVs showed high tissue tropism for the pancreas in Anatidae. Pancreatic enzyme activity may contribute to cleavage activation of viral HA and isolation efficiency in experimental studies. In this study, virus detection in the pancreas and cloacal swabs was not correlated, indicating that the remaining viruses in the pancreas were not involved in virus shedding via the digestive tract.
Viremia and systemic infection were confirmed in two Anatidae. Their clinical outcomes after 3 dpi were unclear. Generally, such Anatidae are likely to be weakened and/or dead in field settings where birds have to feed by themselves, and they then become a source of infection by consumption by scavengers. Virus at titers of ≥ 10 4.5 EID 50 was also recovered from the eyeballs and conjunctival swabs of three ducks, and mild conjunctivitis were confirmed in the Northern pintails. Continuous virus detection in conjunctival swabs has been reported in domestic ducks inoculated with H5N1 HPAIVs [41], and antigen has been detected in ocular tissues [42]. Scavenging birds usually begin feeding on the eyes of carcasses, or attack live animals in certain instances, initially pecking out the eyes or feeding on open wounds [43]; HPAIVs in ocular tissues of ducks probably contribute to virus transmission.

Conclusion
This study showed that ≥ 10 4 EID 50 of the clade 2.3.4.4e HPAIV caused inapparent infection in Anatidae, sometimes accompanied by viral shedding. The number of examined Anatidae was limited and their profiles (sex, age, and seroprevalence) were not uniform. However, the results reflect the circumstances of HPAIV infection in Anatidae populations in field settings. Age, history of infection, and individual differences may affect the clinical outcome. Transboundary dissemination of the virus by migratory birds, including Anatidae, is still a concern. Recently, clade 2.3.4.4b HPAIVs have become predominant in East Asian countries [44], and antigenically different clade 2.3.4.4h HPAIVs, evolved from clade 2.3.4.4e HPAIVs, have also been detected in China and Vietnam [45]. Assessing the pathogenicity using wild Anatidae should continue to be used to estimate the risk of infection in poultry and endangered species. The results of this study contribute to the accumulation of knowledge about the susceptibility of migratory ducks to H5 HPAIVs.

Virus
Clade 2.3.4.4e HPAIV Tottori/1 was used for experimental infection of ducks. The strain was isolated from a fecal sample of teal in Tottori City located in the Midwestern region of Japan [46]. The virus was categorized into the C2 group of the clade 2.3.4.4e, which has the most popular gene constellation in the 2016-2017 outbreak in Japan [9]. The accession numbers of the gene sequences were LC199865-199872. Virus was propagated in 10-day-old chicken embryos (Aoki Breeder Farm, Tochigi, Japan) for 48 h at 35°C. After the incubation period, eggs were chilled at 4°C for 12 h. The allantoic fluid was harvested and stored as virus stock at −80°C.

Birds
Ten each of three Anatidae species (Eurasian wigeon, Mallard, and Northern pintail) were captured at Togo and Koyama Ponds in Tottori Prefecture in Japan in two consecutive winter seasons, January and December 2019 (see Additional file 1). Their ages (juvenile or adult) were identified by feather growth and molt. The pharyngolaryngeal, cloacal, and conjuntival swabs were collected and examined for influenza A virus antigen via rapid diagnostic kits (ESPLINE INFLUENZA A & B-N, Fujirebio Inc., Tokyo, Japan) and/or for virus isolation by egg inoculation. Blood was also collected, and subsequently checked for specific serum antibody against the challenge virus, Tottori/1, by HI testing [47]. These sera were also assayed for influenza A subtype viruses using a cELISA kit (IDEXX Influenza A Ab Test, IDEXX laboratories, ME, USA). The ducks were house at Tottori University for a maximum of 5 weeks. The ducks were grouped according to the following criteria: serum HI titers under the detection limit (< 2 HI) were preferentially applied to 10 6 EID 50 inoculation group as stated below; cELISApositive ducks were impartially distributed to each group as possible; their age (adult/juvenile) and sex were also taken into consideration for grouping to reduce biases.

Experimental design
Seven of each duck were intranasally inoculated with 200 μL of allantoic fluid containing the Tottori/1 at 10 6 , 10 4 , or 10 2 EID 50 , then observed for clinical signs at 24-h intervals for 10 days (see Additional file 2). Pharyngolaryngeal and cloacal swabs were collected at 1, 2, 3, 5, 7, and 10 dpi to assess viral shedding. The swabs were collected in 2 mL of nutrient broth medium (Nissui Pharmaceutical, Tokyo, Japan) with 10 mg of streptomycin sulfate (Meiji Seika Pharma, Tokyo, Japan) and 1 × 10 4 units of penicillin G (Meiji Seika Pharma). At the end of the 10-day period, the ducks were also checked for specific antibodies against the challenge virus in serum by HI testing. The surviving birds were euthanized by isoflurane (Fujifilm Wako Pure Chemical Corporation, Tokyo, Japan) inhalation at an overdose after collection of conjunctival swabs and blood at 10 dpi, and their tissues (brain, trachea, breast muscle, lung, liver, pancreas, spleen, heart, kidney, colon, eyeball, and wing shaft) were sampled for virus isolation and histopathological study, as described below. The remaining three of each duck species were intranasally inoculated with 200 μL of allantoic fluid containing the virus at 10 6 EID 50 , then euthanized at 3 dpi. The samples were collected in the same manner as above.
Portions of the tissue samples were homogenized using a Multi-Bead Shocker (Micro Smash ™ MS-100R, Tomy Seiko, Tokyo, Japan) at 3,000 rpm for 30 s to create a 10% (weight/volume) organ emulsion in nutrient broth medium with antibiotics. Samples serially tenfold diluted in phosphate buffered saline with streptomycin sulfate and penicillin G were inoculated into 10-day-old chicken embryos. Eggs were incubated at 35°C for 48 h. Hemagglutination testing [48] was then performed using allantoic fluid, and the EID 50 was calculated using the Reed and Müench method [49]. The sampled tissues were also subjected to histopathological analysis. Tissues fixed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Corporation) were processed according to routine methods, then embedded in paraffin wax. Sections were stained with haematoxylin and eosin for histopathological examination. Immunohistochemical staining was also performed using antigen retrieval solution, 0.05% citraconic anhydride, pH 7.4 (Immunosaver; Nissin EM, Tokyo, Japan), mouse antiinfluenza A virus matrix protein monoclonal antibody (clone GA2B; Serotec Ltd., Oxford, UK), and the Simple Stain MAX-PO (M) kit (Nichirei Bioscience Inc., Tokyo, Japan), in accordance with the manufacturers' instructions.


Lactiplantibacillus plantarum 0111 Protects Against Influenza Virus by Modulating Intestinal Microbial-Mediated Immune Responses

There are some limitations of traditional influenza vaccines concerning novel mutant strains. Therefore, it is particularly important to develop preventive means for antigen-unrelated types of influenza viruses. Recent studies have shown that probiotics can modulate the immune system and reduce the severity of viral infections. In this study, we investigated the potential of Lactiplantibacillus plantarum 0111 against influenza virus H9N2. Challenge experiments showed that L. plantarum 0111 pretreatments could effectively improve mice’s survival rate and weight loss and reduce the inflammatory cytokines IL-6 and TNF-α in the lungs and bronchoalveolar lavage fluid (BALF) along with the degree of lung and intestinal injury. FMT experiment demonstrates that the protective effect produced by L. plantarum 0111 is associated with gut microorganisms. In addition, 16S high-throughput sequencing of the mouse intestinal microbiota showed that L. plantarum 0111 remodeled the intestinal microbiota after H9N2 infection and maintained the gut microbiota balance. In a mouse model, the oral administration of L. plantarum 0111 increased IFN-β expression in the serum and BALF. At the same time, the transcript levels of IFN-β and related ISGs in the intestine and lungs of mice were also increased. In addition, the activation and polarization of T cells in mesenteric lymph nodes (MLNs) and the spleen were detected by flow cytometry, and the results showed that L. plantarum 0111 modulated cytokines in T cells and increased IgA expression in B cells in the MLNs and spleen. Thus, L. plantarum 0111 may improve gut microbiota-mediated immune responses and thus, resist infection by the influenza virus, and it could be used as an effective preventive measure against the influenza virus.

There are some limitations of traditional influenza vaccines concerning novel mutant strains. Therefore, it is particularly important to develop preventive means for antigen-unrelated types of influenza viruses. Recent studies have shown that probiotics can modulate the immune system and reduce the severity of viral infections. In this study, we investigated the potential of Lactiplantibacillus plantarum 0111 against influenza virus H9N2. Challenge experiments showed that L. plantarum 0111 pretreatments could effectively improve mice's survival rate and weight loss and reduce the inflammatory cytokines IL-6 and TNF-α in the lungs and bronchoalveolar lavage fluid (BALF) along with the degree of lung and intestinal injury. FMT experiment demonstrates that the protective effect produced by L. plantarum 0111 is associated with gut microorganisms. In addition, 16S high-throughput sequencing of the mouse intestinal microbiota showed that L. plantarum 0111 remodeled the intestinal microbiota after H9N2 infection and maintained the gut microbiota balance. In a mouse model, the oral administration of L. plantarum 0111 increased IFN-β expression in the serum and BALF. At the same time, the transcript levels of IFN-β and related ISGs in the intestine and lungs of mice were also increased. In addition, the activation and polarization of T cells in mesenteric lymph nodes (MLNs) and the spleen were detected by flow cytometry, and the results showed that L. plantarum 0111 modulated cytokines in T cells and increased IgA expression in B cells in the MLNs and spleen. Thus, L. plantarum 0111 may improve gut microbiota-mediated immune responses and thus, resist infection by the influenza virus, and it could be used as an effective preventive measure against the influenza virus.
Keywords: probiotics, influenza virus, IFN-β, T cells, gut microbiota INTRODUCTION According to disease severity, avian influenza viruses are divided into low and highly pathogenic viruses (LPAIs and HPAIs). Some LPAIs, such as the H9N2 subtype, can transmit between birds, mammals, and humans, which usually causes economic losses to the breeding industry and poses a serious threat to human health (Peiris et al., 1999;Zhang et al., 2013Zhang et al., , 2020. In 1998, human infection with the H9N2 subtype influenza virus was first discovered in southern China (Guo et al., 1999), followed by increasing case reports of H9N2 infection of humans (Song and Qin, 2020) and even cases of death due to infection with this virus (Peacock et al., 2019). Viruses directly infect humans while providing heavy ligands for some or even all of the internal genes of highly pathogenic influenza viruses, such as H7N9 (Pu et al., 2015), which pose a significant threat to public health. H9N2 can replicate in respiratory and gastrointestinal cells, and through pathological damage, immunosuppression causes damage to the host (Yao et al., 2014;Zhang et al., 2014;Li et al., 2016;Zhu et al., 2018). Vaccination against AIV is usually a practical preventive approach, but it is not enough to completely resist viral invasion because viral mutations occur quickly (Ujike et al., 2010). Therefore, it is essential to study and develop new therapeutic approaches to cope with emerging heterologous influenza viruses.
The World Health Organization defines probiotics as live microorganisms that confer a health benefit to the host when administered in adequate amounts. In addition, probiotics can effectively promote the health and growth of host animals by regulating the balance of microorganisms in the intestinal tract, enhancing the barrier function of intestinal epithelial cells, inhibiting the growth and adhesion of pathogenic microorganisms, and regulating immunomodulatory effects (Bubnov et al., 2015;Hu et al., 2017;Cao et al., 2020). Lactic acid bacteria are the most common probiotics and have been widely used and considered safe for humans and animals (Slattery et al., 2019). The metabolites they produce, such as organic acids and bacteriocins, have beneficial effects on the host (Yousefi et al., 2019). In addition, lactic acid bacteria stimulate and regulate the immune system of the intestinal mucosa, protecting in the event of pathogenic and viral infections of the organism (Surendran Nair et al., 2017). A previous study showed that oral administration of Lactobacillus pentosus increased IgA production, thereby protecting mice from lethal influenza virus infection (Kobayashi et al., 2011). At the same time, probiotics also have side effects. Probiotic consumption is associated with D-lactic academia and acidosis in adults and infants on probiotic-containing formula, and d-lactic acidosis is the culprit for brain fogginess (Papagaroufalis et al., 2014;Rao et al., 2018).
The gut microbiome functions include nutritional effects, metabolic effects, and immune stimulation (Sánchez et al., 2017). It has been shown that H9N2 avian influenza virus infection causes disturbance of the host gut microbiota . After the gut microbiome imbalance, it cannot effectively play the physiological role of a normal gut microbiome, damages the body's immune function, and reduces the body's clearance of influenza virus (Pang et al., 2018). At the same time, oral probiotics help establish a mutually beneficial symbiotic intestinal micro-ecological balance system (Hu et al., 2017). Therefore, it is important to explore probiotics to regulate intestinal flora dysregulation caused by the influenza virus.
Lactiplantibacillus plantarum 0111, a natural strain, was screened from the intestine of mice and had high immune activity and adherence. In the present study, the oral administration of L. plantarum 0111 improved survival and reduced body weight loss in infected mice. In addition, L. plantarum 0111 modulates the intestinal microbiota and the immune system and could be a candidate strain to prevent influenza virus infection.

Bacteria, Cells, Viruses, and Animals
Lactiplantibacillus plantarum 0111 was kindly provided by the Shandong Baolai Lilai Bioengineering Co., Ltd., NCBI accession number: MT907445, using MRS medium. After 16 h of anaerobic culture at 37°C, the bacteria were centrifuged at 4000g for 10 min at 4°C, washed three times with sterile phosphatebuffered saline (PBS, pH = 7.4), and then resuspended in PBS for subsequent application. Madin-Darby canine kidney (MDCK cell line) cells were preserved in this experiment and cultured with DMEM, FBS, 10,000 U/ml penicillin, and 10,000 μg/ml streptomycin at 37°C in 5% CO 2 . To ensure cell viability, the cells were recovered before use and passaged for more than three generations.
H9N2 was preserved by our laboratory (Shi et al., 2014). The median tissue culture infective dose (TCID 50 ) was used as the titer level for the influenza virus. SPF grade 6-week-old C57BL/6 female mice (n = 90, five in each cage) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. These mice were allowed free access to water and food in SPF Animal Farming Center. All animal experiments met the requirements of the 113 Animal Management and Ethics Committee of Jilin Agricultural University (JLAU20200704001).

Experimental Design and Sample Collection
Lactiplantibacillus plantarum 0111 (10 8 ) was gavaged in a volume of 200 μl for 7 consecutive days, and the control group was gavaged with PBS. Experiment groups were challenged with H9N2 (10 6.5 EID 50 ) on day 0. The mice were anesthetized by the intraperitoneal injection of sodium pentobarbital, and H9N2 was infected by nasal drops . All mice were monitored every day for 14 days, and their body weight and survival rate were recorded. Fecal samples are collected on day 7 and immediately stored at −80°C for total DNA extraction and sequencing. In another repeat experiment, after 7 days of gavage of L. plantarum 0111, serum and bronchoalveolar lavage fluid (BALF) were collected from mice to detect type I interferon, and the lungs and jejunum of mice were collected for qPCR Frontiers in Microbiology | www.frontiersin.org analysis. A single-cell suspension was prepared for flow cytometry analysis in the spleen, Peyer's patches, and MLNs . the lungs and jejunum of mice were collected on day 7 post-infection, fixed in 4% formaldehyde for histopathological analysis. Lung tissue and BALF were prepared to detect viral titers and inflammatory cytokines. BALF was inserted into the trachea using a 1 ml sterile syringe and fixed, while 800 μl of cold PBS was injected, repeatedly withdrawn 15 times, transferred into a 1.5 EP tube, and centrifuged at 2,000 rpm for 10 min; the supernatant was frozen at −80°C.

Antibiotic Treatment and FMT
To eliminate as much as possible the intestinal microbial background of mice, ampicillin (1 mg/ml), streptomycin (5 mg/ ml), vancomycin (0.25 mg/ml), and mycomycin (1 mg/ml; Solarbio) were added to the sterile drinking water of mice for 6 days, followed by FMT. Mouse feces from the PBS + H9N2 and 0111 + H9N2 groups were collected on the seventh day and processed as described previously (Yang et al., 2021). Briefly, fecal samples were placed in a sterile N 2 incubator, mixed thoroughly, and then plugged into a cell filter to collect bacteria and centrifuged at 12,000 rpm for 10 min. The samples were subsequently added to 10% glycerol, stored at −80°C. FMT before replacing the glycerol with PBS and stained with methylene blue staining to count the number of viable microorganisms. Mice were gavaged in 100 μl volumes (n = 5). One day later, all mice were intranasally attacked with H9N2 (10 6.5 EID 50 ), and their survival and weight loss were monitored 14 days after infection. FMT experiment was repeated twice independently.

Illumina Sequencing
A 16S rRNA sequencing library of the V3-V4 hypervariable region was constructed according to a previous study in our laboratory (Chen et al., 2020). See S Methods for specific methods. The primer sequences were as follows: forward primer 5′-GTACTCCTACGGGAGGCAGCA-3′; reverse primer 5′-GTGGACTACHVGGGTWTCTAAT-3′. This sequence has been logged into NCBI (SRP301018). Raw data sequencing first removed barcode and primer sequences, then spliced them to obtain raw tags. Raw tags were used to remove chimeras and short sequences to obtain clean tags. Sequences were subjected to a classification operation (cluster). The sequences were classified into many OTUs according to their similarity through the classification operation. Sequences were typically OTU partitioned at a 97% similarity level, followed by bioinformatics statistical analysis of the OTUs. Finally, α diversity, β diversity, and different species screened were analyzed according to OTU and a taxonomic rank.

Flow Cytometry Analysis
To determine the cellular immune response induced by L. plantarum 0111, we euthanized mice on day 0 and subsequently collected spleens, Peyer's patches, and MLNs and prepared single-cell suspensions. FACS analysis was performed using PerCP-Cy5.5-labeled anti-mouse CD3 antibody, PE-Cy7-labeled anti-CD4 antibody, APC-Cy7-labeled anti-CD8 PE-labeled anti-IFN-γ antibody, APC-labeled anti-IL-4 antibody, FITC-labeled anti-TNF antibody, APC-labeled anti-B220 antibody, and FITC-labeled anti-IgA antibody. All antibodies were purchased from Becton, Dickinson, and Company in the United States. Experiments were performed three times, and data compensation and analysis were performed using FlowJo v7.6.1. In parallel, IFN-α and IFN-β expression in serum and BALF at day 0 and IL-6 and TNF-α expression in the lung supernatant and BALF on day 7 were analyzed using the MU Anti-Virus Response Panel (13-plex, LEGENDplexTM, 740,622) according to the manufacturer's instructions, and data analysis was performed using LEGENDplex v8.0.

Virus Titer Detection
To determine the virus titer, an equal weight of lung tissue was homogenized in DMEM to obtain a serially diluted 10-fold suspension of tissue homogenate. Moreover, it was titrated in 96-well culture plates of MDCK cells. The Reed-Muench method was adopted to calculate the titer and was expressed as log 10 TCID 50 /g of lung tissue.

Quantitative PCR
According to the manufacturer's instructions, we extracted total RNA from the mouse intestine and lung using a Universal RNA Extraction Kit (Takara). Reverse transcription was performed using TransScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANSGEN China). Triple qPCRs were subsequently performed in an Applied Biosystems 7,500 (Life Technologies, United States of America) using PerfectStart™ Green qPCR SuperMix (TRANSGEN China). Thermal cycling conditions were 94°C for 30 s, followed by 40 cycles of 95°C for 5 s and then 60°C for 30 s. Data were collected using an Applied Biosystems 7,500 (Life Technologies, United States of America) and analyzed by the threshold cycle (ΔΔCT) method using 7,500 Software v2.3. The qPCR primers are designed to avoid contamination of the genome by cross-intron design, thereby inhibiting the amplification of genomic DNA. Primers are shown in Supplementary Table S1.2.

Histological Analysis
Mouse lung tissues and jejunum were fixed in 4% formaldehyde and subsequently embedded in paraffin. Tissues were dehydrated and stained in graded alcohols and then sectioned. At least two tissue sections (3 μm) from each sample were stained with hematoxylin-eosin (HE) and finally analyzed using an inverted fluorescence microscope (Leica Microsystems, Germany) in our laboratory.

Statistical Analysis
All data were expressed as the mean ± SEM. Statistical analysis was performed using one-way ANOVA and t-tests with GraphPad Prism 8.0 (GraphPad Software). The p values are indicated as follows: *p < 0.05; **p < 0.01; and ***p < 0.001.

The Protective Effect of Lactiplantibacillus plantarum 0111
We determined 108 CFU as the optimal dose (Supplementary Figures S1B,E). Subsequently, mice were challenged with the H9N2 subtype avian influenza virus and examined for health status over 14 days. After the viral challenge, mice usually died between day 5 and day 8 after infection ( Figure 1B). The 0111 + H9N2 group provided 40% more protection against H9N2 attack than the PBS + H9N2 group (p < 0.05). In terms of body weight, except for the control group, the bodyweight of mice in the other groups gradually decreased after being challenged, and by day 7, the 0111 + H9N2 group showed a trend of gradual weight regain and reached more than 80% by day 14 (Figure 1A). In contrast, the PBS + H9N2 group continued to decline until day 7. Therefore, L. plantarum 0111 pretreatment improved survival and weight loss in mice. Histological analysis of the lungs and jejunum of mice from each group showed that the H9N2 influenza virus caused damage to the lungs and the jejunum. Foamy exudates and damaged alveolar structures were observed in mice from the PBS + H9N2 group (Figures 1C,F). The alveolar structures were intact, and lung damage was not present in the 0111 + H9N2 group (Figures 1D,G). Meanwhile, the jejunal wall of mice in the PBS + H9N2 group (Figures 1I,L) was thickened, intestinal villi were shed, and inflammatory cells in the lamina propria were infiltrated compared with the PBS group (Figures 1K,N). However, no damage state was found in the 0111 + H9N2 group (Figures 1J,M). Thus, L. plantarum 0111 pretreatment could mitigate the lung and jejunum injury caused by H9N2.
Five days after infection with H9N2, the mice were euthanized to prepare lung homogenates, and the supernatant was inoculated into MDCK cells to determine the TCID 50 ( Figure 1O). After viral infection, mice in the 0111 + H9N2 group had approximately 1.6-fold lower virus than mock-treated mice (p < 0.05). In the meantime, inflammatory cytokines are produced excessively during influenza infection, and inflammatory cytokine storms can cause severe lung damage. Therefore, the measurement of IL-6 and TNF-α inflammatory cytokines in the lungs and BALF could help understand the mechanism of lung injury caused by H9N2. At 5 days post-infection, the levels of IL-6 detected in the lungs (p < 0.05) and BALF (p < 0.01) were significantly lower than those in the PBS + H9N2 group (Figures 1P,Q). In addition, the level of TNF-α in the BALF (p < 0.05) was also significantly lower than that in the PBS + H9N2 group, while no difference was produced in the lungs (Figures 1R,S). L. plantarum 0111 decreased the production of proinflammatory cytokines induced by H9N2 infection.

The Protection Provided by Lactiplantibacillus plantarum 0111 Is Associated With the Gut Microbiota
Lactiplantibacillus plantarum primarily colonizes the intestinal tract and exerts a probiotic effect. To verify whether the protective effect of L. plantarum 0111 is associated with gut microorganisms, we performed FMT on mice (Figure 2A). The result showed that mice treated with the 0111 + H9N2 group improved survival and weight loss, while mice treated with the PBS + H9N2 group showed the same susceptibility as the control group (Figures 2B,C), demonstrating the protective effect of L. plantarum 0111 through the gut microbiota. The α diversity was evaluated using the Chao1, observed species, and Shannon indices. The results showed that H9N2 infection significantly increased the number of actually observed OTUs compared with the PBS group (p < 0.05), while the number of OTUs was not disturbed in the oral 0111 group (Figure 3B). In addition, although the Chao1 and Shannon indices were not significantly different, there was an upward trend compared with the PBS group, whereas the 0111 group tended to be stable (Figures 3A,C). In the meantime, NMDS (nonmetric multidimensional scaling) was used to analyze the β diversity in samples from different groups. In striking agreement with the α diversity, H9N2 infection caused significant differences in biological populations compared with the PBS group, whereas no significant differences were observed in the 0111 group ( Figure 3D). Therefore, NMDS was adopted to analyze the degree of difference between different samples to assess β diversity. The results were calculated for statistical significance using the t-test (N = 5/ group). *P < 0.05. P, challenged H9N2 cells after pretreatment with PBS; M, challenged H9N2 cells after pretreatment with L. plantarum 0111. N, pretreatment with PBS.
oral administration of L. plantarum 0111 can effectively maintain the intestinal imbalance caused by H9N2 infection.

Lactiplantibacillus plantarum 0111 Remodels the Gut Microbiota After H9N2 Infection
To investigate the effect of L. plantarum 0111 on gut microbial composition in mice infected with H9N2, we analyzed bacteria at the phylum ( Figure 4A) and genus ( Figure 4B)

LEfSe Analysis of Landmark Commensal Species
To fully understand the effect of L. plantarum 0111 on the gut microbiota of H9N2-infected mice, we performed LEfSe (LDA effect size) analysis. The LEfSe branch diagram (Supplementary Figure S4) and LDA scores ( Figure 5A) were used to assess changes in gut microbes. LEfSe analysis showed that Faecalibaculum (Figure 5B) was significantly decreased in group P compared with group N, while it tended to rise in group M. In comparison, Akkermansia ( Figure 5C) and Alistipes ( Figure 5D) were significantly increased in group P. Still, it correspondingly decreased in group M. At the same time, Lachnospiraceae-NK4A136-group was significantly increased in group P compared with group N (Figure 5E), and showed normal levels in group M. Therefore, L. plantarum 0111 can maintain intestinal homeostasis after H9N2 infection by interacting with commensal microorganisms.

Lactiplantibacillus plantarum 0111 Increases the Expression of IFN-β and ISGs
It has been shown that specific microorganisms derived from the gut can resist viral infections by increasing the expression of type I interferons (Yang et al., 2021). Type I interferons play a crucial role in the infection phase and are important in assessing antiviral capacity. The expression levels of IFN-α and IFN-β in serum and BALF were measured in mice after 7 days of continuous oral administration of L. plantarum 0111.
The results showed that the oral administration of L. plantarum 0111 could enhance the expression levels of IFN-β in serum as well as BALF (p < 0.05; Figures 6C,D). Notably, the expression levels of IFN-α were not significantly different in either the serum or BALF (Figures 6A,B). Therefore, pretreatment with L. plantarum 0111 enhanced the induction of IFN-β but not IFN-α.
The ability of the host to suppress viral infection largely depends on the effectiveness of the initial antiviral innate immune response, which leads to the upregulation of IFNs, followed by ISGs. PKR, MX1, OaS1, and ISG15 are the main antiviral proteins produced by IFN stimulation (Lenschow et al., 2007;Schoggins, 2019). Next, we tested type I interferon and ISG expression after L. plantarum 0111 pretreatments. We measured the relative mRNA levels of IFN-α, IFN-β, and the four ISGs in the intestine and lungs of mice by Q-PCR. The results of examining the intestine showed that the transcript levels of MX1 (p < 0.05) and ISG15 (p < 0.01) were increased by approximately 1.6-fold compared with the control group. At the same time, the transcript level of IFN-β was also elevated (p < 0.05; Figure 6F). Notably, the transcript levels of IFN-α, PKR, and OaS1 were not significantly elevated. The results of detecting the lungs showed that the expression of IFN-β was increased by 1.8-fold compared with the control group, while the expression level of ISG15 was also significantly increased in the intestine (p < 0.01). Unlike the intestine, the expression level of OaS1 was significantly increased in the lungs compared with the control group (p < 0.001). At the same time, the expression of IFN-α, PKR, and MX1 was not elevated in the lungs (Figure 6E).

Lactiplantibacillus plantarum 0111 Regulates T and B Cell Responses
The activation and polarization of T cells in MLNs and spleen were detected by flow cytometry (Supplementary Figure S2). The 0111 group significantly increased the percentages of CD3 + CD4 + IFN-γ + T cells (Figure 7D; p < 0.01) and CD3 + CD8 + IFN-γ + T cells (Figure 7F; p < 0.05) in MLNs compared with the PBS group, although a similar trend was not observed in the spleen (Figures 7E,G). On the other hand, the 0111 group had an increased percentage of CD3 + CD4 + IL-4 + T cells in both the MLNs (p < 0.05) and spleen (p < 0.01; Figures 7H,I).
In addition, the 0111 group significantly increased the percentages of CD3 + CD4 + TNF-α + T cells (Figure 7K; p < 0.01) and CD3 + CD8 + TNF-α + T cells (Figure 7M; p < 0.01) in the spleen compared with the PBS group, while no difference was observed in the MLNs (Figures 7J,L). The numbers of B220 + IgA + B cells in Peyer's patches were examined. The number of B220 + IgA + B cells (p < 0.01) was significantly increased (Figures 7A-C), demonstrating that 0111 had a regulatory effect on cytokines within T cells and increased the expression of IgA within B cells in the MLN and spleen.

CORRELATION ANALYSIS
Pearson correlation results are shown in Figure 8A. At the expression level of type I interferon, Ruminococcus-1, Lachnospiraceae-NK4A136-group, and Aerococcus in group P were significantly positively correlated with serum IFN-β expression negatively correlated with serum IFN-α expression, while Turicibacter also showed a negative correlation with serum IFN-β. Ruminiclostridium-6, Ruminococcus-1, and Eubacteriumxylanophilum-group were significantly negatively correlated with IFN-α expression in BALF. Akkermansia was negatively correlated with IFN-β in serum and BALF in group M. In contrast, Bifidobacterium was positively correlated with IFN-β in serum and BALF. Faecalibaculum and Lachnoclostridium in group P were positively correlated with intestinal IFN-β transcript levels at the transcriptional level, while Alistipes was negatively correlated with them. Notably, there was a significant positive correlation between Turicibacter and IFN-β transcript levels in the lungs.
In group M, Bacteroides and Odoribacter were positively correlated with intestinal IFN-α transcript levels, while Ruminococcaceae UCG-014 was significantly negatively correlated with intestinal and lung IFN-α transcript levels. In addition, correlation networks were plotted for Actinobacteria, Verrucomicrobia, Firmicytes, and Bacteroidetes to show the relationships among some important members belonging to the four dominant phyla (Figures 8B-D).

DISCUSSION
Here, we demonstrate that oral administration of L. plantarum 0111 can prevent and protect against H9N2 influenza virus infection in mice. We also reveal that oral administration of L. plantarum 0111 can maintain the balance of intestinal microbes and modulate the immune response to mitigate the aggression caused by the influenza virus. Viral infection heavily relies on host transfer RNA (tRNA) for viral RNA decoding, which is a major determinant of the success of viral infection (Ou et al., 2020). Factors controlling tRNA use in the intestine remain unexplored, but microbiome and virome can influence this, and thus by inference, L. plantarum could as well (Zhang et al., 2021). There is increasing evidence that probiotics provide immune protection by regulating multiple aspects of the innate and adaptive immune systems (Giorgetti et al., 2015). The potential antiviral mechanisms of probiotics in the enteropulmonary axis and mucosal immune system may play a role in the prevention and supportive treatment of SARS-CoV-2 infection (Sundararaman et al., 2020). Therefore, probiotics may be involved in activating immune responses and actively participate in the protection of respiratory tract infections. In the present study, mice orally administered L. plantarum showed excellent protection against lethal influenza virus infection, as indicated by reduced weight loss and viral load in the lungs and improved survival. Thus, L. plantarum 0111, a new Lactobacillus strain, may have anti-influenza virus activity.
The main role of commensal bacteria is to maintain normal homeostasis by regulating the host's immunity, development, and physiology (Yitbarek et al., 2018). Microorganism-associated molecular patterns (MAMPs) of bacteria, including lipopolysaccharide, flagellin, peptidoglycan, formyl peptides and unique nucleic acid structures, as well as gut microbe-derived SCFAs, tryptophan metabolites, and polyamines, can modulate the gut immune system (Rooks and Garrett, 2016). H9N2-type AIV infection can cause dysbacteriosis in the intestine . Probiotic supplementation enables probiotics to dominate the gut, maintaining gut health (Sanders et al., 2019). A mathematical analysis predicts that the diversity of microbial communities in the gut may influence the stability of the intestinal system (Coyte et al., 2015). The 16S sequencing results showed that H9N2 infection altered the gut microbial structure in this study. Lachnoclostridium,  Odoribacter, and Lachnospiraceae-NK4A136-group were only present in the H9N2-infected group and not in the supplemented 0111 group. Faecalibaculum is beneficial for intestinal health and has been shown to have antitumor properties Zagato et al., 2020). Supplementation with L. plantarum 0111 restored the dramatic decreases in the abundances of Faecalibaculum and Lactobacillus caused by H9N2 infection, thus maintaining the balance of intestinal microorganisms. It has been shown that Alistipes is a pathogenic factor in colorectal cancer, and deregulation of Alistipes abundance affects the intestinal microenvironment (Parker et al., 2020). In this study, H9N2 infection resulted in an elevation of Alistipes abundance and was inversely correlated with the transcript level of intestinal IFN-β. Notably, supplementation with L. plantarum 0111 significantly reduced Alistipes abundance. A commensal bacterium of the mucus layer of Akkermansia can regulate metabolism, immune responses, and gut health protection (Zhai et al., 2019). Akkermansia is the most important propionic acid producer and an important modifier of immunity, both in humans and mice (Al-Lahham et al., 2010;Su et al., 2021). Akkermansia-related protein enrichment regulates the intestinal barrier by activating TLR2 and TLR4 to induce the production of specific cytokines (Ottman et al., 2017). Thus, L. plantarum 0111 may act synergistically with other probiotics to activate the immune system. The pathogenesis of viral infection is associated with an excessive inflammatory response (de Jong et al., 2006;Wang et al., 2010). It has been reported that during influenza virus infection, a large number of inflammatory cells infiltrate the lungs and produce excessive inflammatory cytokines, forming a cytokine storm resulting in severe lung injury (Kobasa et al., 2007;Cillóniz et al., 2009). A significant reduction in IL-6 production in the lungs was found after administering Bifidobacterium bifidum to mice infected with lethal influenza A (H1N1; Iwabuchi et al., 2011). Type I interferons play an important role in antagonizing viral replication, monitoring the immune system, and inhibiting cytopathic effects as a powerful tool for the host to protect against infection by foreign pathogens (Fuertes et al., 2013;Rönnblom, 2016). After invading the body, the influenza virus activates the host's innate immune response and induces the production of type I interferon, which then binds to its receptors IFNAR1 and IFNAR2 and activates downstream signaling pathways, thereby activating the The results were calculated for statistical significance using the t-test. *P < 0.05; **P < 0.01.
transcription and expression of downstream ISGs (Pestka, 2007). In addition to canonical ISG transcriptional regulation mechanisms, non-canonical ISG transcriptional regulation plays an important role . For example, bacteria can affect the transcription of ISGs by altering epigenetic regulators of transcription (Alphonse et al., 2021). The expression products of ISGs can directly promote the antiviral effect of the body and further induce type I interferon, thereby indirectly enhancing the antiviral effect of the body (Schoggins, 2014). Many ISGs, OAS1, Mx1, PKR, and ISG15, are common ISGs (Schoggins, 2019). Previous studies have shown that probiotics can reduce the viral load and thus exert a protective effect (Goto et al., 2013;Park et al., 2013). Lactococcus lactis JCM5805 and Lactobacillus gasseri SBT2055 increased the expression of antiviral factors in the lung, which is similar to our findings (Nakayama et al., 2014;Jounai et al., 2015). In the present study, the transcription levels of IFN-β, OAS1, Mx1, and ISG15 in the lungs and intestines of mice pretreated with L. plantarum 0111 were correspondingly increased, and the lung viral load was significantly reduced after viral infection. Therefore, the probiotic regulatory pathway may depend on the natural immune system and is closely linked to type I interferons.
In diseased individuals, the ability of commensal gut bacteria to modulate the host immune system has been demonstrated (Perez-Lopez et al., 2016). Commensal bacteria also modulate regulatory T cells by recognizing the bacteria themselves or their metabolites/products by immune cells and greatly affect mucosal immunity (Hall et al., 2008;Goto et al., 2016). Probiotic supplementation of healthy neonates significantly enhanced IFN-γ expression, indicating that probiotics play an important The P values were adjusted according to the Bonferroni and Hochberg procedures to determine the false discovery rate. Asterisks indicate a significant correlation between bacterial groups and type I interferon (*P < 0.05, **P < 0.01, ***P < 0.001). P, challenged H9N2 cells after pretreatment with PBS; M, challenged H9N2 cells after pretreatment with L. plantarum 0111; N, pretreatment with PBS.
role in improving Th1 immune responses. In addition, bifidobacteria were shown to have powerful potential to modulate humoral and cellular immune responses and induce a balanced Th1/Th2 immune response against influenza infection (Mahooti et al., 2019). Our study determined the differentiation direction of Th cells into Th1 and Th2 subtypes by the percentage of IFN-γ and IL-4 produced by CD4 + T cells. The results showed that the expression of IFN-γ and IL-4 in TH cells was increased to different extents after treatment with L. plantarum 0111, indicating their potential regulatory effects on humoral and cellular immunity. IFN-γ can induce virus-infected cells to express viral antigens, increase the ability of the immune system to recognize and kill infected cells and resist viral replication (Maggi et al., 1992). NK cells are early antiviral effector cells that can directly kill infected cells. IL-12 can promote the proliferation of NK and Th1 cells, and the proliferation of Th1 cells may also affect the proliferation of NK cells to resist viral infection. In addition, IFN-γ produced by Th1 cells could further activate NK cells, suggesting the possibility of NK cells exerting antiviral effects (Komastu et al., 1998). In the meantime, it has been shown that probiotics elevate IgA expression in B cells within gut-associated lymph nodes and intestinal secretory IgA (Kikuchi et al., 2014). The results of this experiment are consistent with these results, suggesting that IgA is essential for intestinal protection at the initial stage of influenza virus infection. In summary, L. plantarum 0111 protects mice from damage caused by H9N2, and this protection may be a function of the innate and acquired immune response mediated by the gut microbiota. Therefore, L. plantarum 0111 could be a candidate strain to prevent the influenza virus.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found at: NCBI Sequence Read Archive; SRP301018.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Management and Ethics Committee of Jilin Agricultural University (JLAU20200704001).


Rapid restructurization of conformationally-distinct alpha-synuclein amyloid fibrils at an elevated temperature

Protein aggregation in the form of amyloid fibrils is linked with the onset and progression of more than 30 amyloidoses, including multiple neurodegenerative disorders, such as Alzheimer’s or Parkinson’s disease. Despite countless studies and years of research, the process of such aggregate formation is still not fully understood. One peculiar aspect of amyloids is that they appear to be capable of undergoing structural rearrangements even after the fibrils have already formed. Such a phenomenon was reported to occur in the case of alpha-synuclein and amyloid beta aggregates after a long period of incubation. In this work, we examine whether incubation at an elevated temperature can induce the restructurization of four different conformation alpha-synuclein amyloid fibrils. We show that this structural alteration occurs in a relatively brief time period, when the aggregates are incubated at 60 °C. Additionally, it appears that during this process multiple conformationally-distinct alpha-synuclein fibrils all shift towards an identical secondary structure.

INTRODUCTION
Protein aggregation into highly-structured amyloid fibrils is linked with the onset and progression of over 30 amyloidoses, including neurodegenerative Alzheimer's or Parkinson's diseases Chiti & Dobson, 2017). Currently, the process of fibril formation is still not completely understood (Lee & Terentjev, 2017;Chaudhuri et al., 2019), which, in turn, has resulted in very few approved drugs or treatment modalities (Mehta et al., 2017;Doig et al., 2017;Maurer et al., 2018;Cummings et al., 2020). It is projected that the already high number of patients afflicted with these disorders will continue to rise in the upcoming years (Brookmeyer, Gray & Kawas, 1998;Arthur et al., 2016). In order to prevent this and discover potential cures, a better understanding of both the formation pathways (Brännström et al., 2018), as well as the resulting aggregates (Fändrich et al., 2018) is necessary.
Protein misfolding and association into fibrillar structures proceeds through multiple stages. The first step in amyloid aggregation is primary nucleation-a process during which native protein molecules misfold and assemble into a stable, β-sheet-rich nucleus (Buell et al., 2014;Meisl et al., 2014). Once a stable nucleus is formed, it can then incorporate other, homologous protein molecules into its structure and elongate into a protofibrillar aggregate. The process of elongation occurs at the ends of the fibrillar structure, which act as both a catalyst and template for the conversion of native protein/ peptide molecules (Milto, Botyriute & Smirnovas, 2013;Gurry & Stultz, 2014;Rodriguez et al., 2018). When amyloid fibrils reach a critical length, they can experience fragmentation-a process during which the aggregate breaks into two shorter fibrils, each with its own aggregation-catalyzing ends. The incidence of fragmentation depends on both the structural stability of the aggregate, as well as the environmental conditions (Nicoud et al., 2015). Another process, which occurs once amyloid fibrils form, is surface-mediated nucleation (also referred to as secondary nucleation) (Foderà et al., 2008;Gaspar et al., 2017;Törnquist et al., 2018). During this step, the surface of aggregates acts as a catalyst for the formation of new amyloid nuclei. Unlike elongation at fibril ends, this process does not template the aggregate's structure onto the nuclei, but only increase their probability of formation (Sneideris, Milto & Smirnovas, 2015).
It is usually considered that once the aggregation reaction uses up all or most of the available monomers, then the amyloid formation process is complete, with only certain other events occurring, such as fibril lateral association (Fujiwara, Matsumoto & Yonezawa, 2003) or large cluster formation (Manno et al., 2006). However, this may not be the case. It was shown that during a long incubation time, alpha-synuclein (Sidhu et al., 2017) and amyloid beta fibrils (Ma et al., 2013) experienced structural alterations. It was hypothesized that this type of aggregate "maturation" may also be required for fibrils to obtain a higher level of infectivity (Yamaguchi et al., 2005). If this restructurization occurs for other amyloids as well, then it may also have an impact on in vitro experiments, making incubation time (Morel et al., 2010) a crucial factor. Even if the exact same reaction solution and protocol is used, the aggregates may undergo structural rearrangement after their formation (Mahdavimehr, Katebi & Meratan, 2018). If the assay employs a set timeframe of fibrillization, then uninhibited aggregation would yield fibers sooner than in the inhibitor reaction solution and they would incubate for a relatively longer time. This could result in aggregates with seemingly different structural aspects (Pellarin et al., 2010) and skew the experimental data, as well as conclusions of the study.
In this work, we generated alpha-synuclein fibrils under previously reported experimental conditions, which yield multiple structurally-distinct aggregates from an identical protein solution (Toleikis et al., 2021). The resulting four distinct fibril solutions were then incubated at 60 C and their structural alterations were observed using CD, infrared and UV/Vis spectroscopy, atomic force microscopy, as well as ThT-binding assays (Hoyer et al., 2002;Ziaunys, Sakalauskas & Smirnovas, 2020). We show that all four different types of fibrils rapidly converge into aggregates with similar secondary structures and that this change occurs in the matter of hours.

Initial fibril preparation
Alpha-synuclein (a-syn) was purified as described previously (Šneideris et al., 2015), lyophilized, and stored at −20 C prior to use. Further fibril preparation procedures were done under similar conditions as described previously (Toleikis et al., 2021). The a-syn powder was dissolved in a phosphate-buffered saline (PBS, pH 7.4) and filtered through a 0.22 µm syringe filter, after which the protein concentration was determined using a Shimadzu UV-1800 spectrophotometer (ε 280 = 5,960 M −1 cm −1 ). The protein solution was then combined with a 10 mM thioflavin-T (ThT) solution and PBS to result in a final protein concentration of 70 and 100 µM ThT. The solution was then distributed to 96-well half-area non-binding plates (cat. No 3881, Fisher Scientific, Hampton, NH, USA) (each well contained 100 µL of the solution and one 3 mm diameter glass-bead (cat. No 104015, Merck, Kenilworth, NJ, USA) and sealed with Nunc sealing-tape. The 96-well plates were incubated at 37 C in a ClarioStar Plus plate reader, under constant 600 RPM agitation for 48 h, with measurements taken every 5 min (excitation wavelength was 440 nm, emission -480 nm). For aggregation monitoring under 60 C, all sample preparation and incubation procedures, apart from the set temperature, were identical. After aggregation, the plates were cooled down to room temperature prior to further examination. Aggregation lag time and apparent rate constants were determined as described previously (Ziaunys et al., 2021a).

Excitation-emission matrices (EEM)
The procedure of EEM acquisition was based on a previous study on alpha-synuclein aggregation (Ziaunys et al., 2021a). In short, 96-well plates containing the fibril samples were placed in a ClarioStar Plus platereader and incubated at 25 C for 5 min. Bound-ThT excitation-emission matrices (EEM) were obtained by first scanning the sample fluorescence emission intensity at a fixed wavelength (485 nm), using a range of excitation wavelengths (430-460 nm). Afterwards, the intensity was scanned under a range of emission wavelengths (470-500 nm), using a fixed excitation wavelength (445 nm). The data was combined into an EEM by using ClarioStar MARS 3D spectrum function. EEM "centers of mass" were calculated as described previously (Ziaunys & Smirnovas, 2019). In short, the center of mass was calculated for the top 10% intensity values from each EEM. All the maximum position values were then plotted in a single graph for comparison. Since alpha-synuclein and other amyloid fibril ThT-binding characteristics can be related to their structure/morphology (Sidhu et al., 2018;Ziaunys et al., 2021a;Ziaunys et al., 2021b), eight out of the 96 samples with distinct EEM maximum positions were selected for further replication and analysis. This selection procedure was done in order to improve the likelihood of obtaining different types of alpha-synuclein fibrils, without the necessity to analyze the entirety of the large number of samples.

Fibril reseeding
In order to generate a higher quantity of aggregates, the eight selected samples (100 µL) were each combined with 400 µL of the initial reaction solution, containing 70 µM a-syn monomers. The resulting solutions were placed in 1.5 mL test-tubes (each containing two 3 mm glass beads) and incubated at 37 C with constants 600 RPM agitation for 24 h in a Digital Heating Shaking Dry bath (Fisher Scientific, Hampton, NH, USA). The resulting aggregate solutions were then combined with 2 mL of the initial reaction solution, divided into 1.5 mL test-tubes (500 µL each) and incubated identically for another 24 h. The resulting 2.5 mL solutions were combined with 2.5 mL of initial reaction solutions and incubated as described previously, resulting in 5 mL volume a-syn aggregate solutions, which were then cooled down to room temperature.
Each fibril solution was then distributed to 1.5 mL test-tubes (1 mL each) and centrifuged at 10,000×g for 15 min, after which the supernatant (900 µL) was removed and replaced with an identical volume of the initial buffer solution, which did not contain a-syn monomers. These centrifugation and fibril pellet resuspension steps were repeated three times, in order to remove non-aggregated protein monomers. This was done in case the aggregation reactions at 37 C did not use up all available monomers (the resulting samples may also contain a fraction of monomers due to an equilibrium between native and aggregated proteins). Afterwards the samples were combined to a final volume of 5 mL. All further experimental procedures were performed using only these replicated and resuspended samples.

Fibril incubation
Each 5 mL fibril sample was vigorously agitated for 10 s and then distributed to sets of three 1.5 mL test-tubes (1.5 mL solution volume, each test-tube contained two 3 mm glass beads). The first set was kept at room temperature, while the other two sets were incubated at 60 C under constant 600 RPM agitation for either 24 or 48 h. During the incubation procedures, aliquots of each sample were taken for further analysis by Fourier-transform infrared (FTIR) and circular dichroism (CD) spectroscopies, atomic force microscopy (AFM) and UV/Vis spectroscopy.
To measure the changes in bound-ThT EEM positions, the selected samples were placed into 96-well half-area non-binding plates (100 µL volume, one 3 mm glass bead in each well) and incubated in a ClarioStar Plus plate reader at 60 C under constant 600 RPM agitation. EEMs were scanned every hour. Both the scanning and analysis procedure was identical to the one described in the previous method section.

Fourier-transform infrared (FTIR) spectroscopy
For FTIR measurements, 500 µL of each aggregate sample was centrifuged at 10,000 × g for 15 min, after which the supernatant was removed and replaced with 500 µL D 2 O (containing 400 mM NaCl in order to improve fibril sedimentation (Mikalauskaite et al., 2020)). The centrifugation and resuspension procedure was repeated four times. After the final step, the fibril pellet was resuspended into 50 µL D 2 O and mixed vigorously for 10 s. FTIR spectra were acquired as described previously (Mikalauskaite, Ziaunys & Smirnovas, 2022), which were then baseline corrected and normalized to the same band area in a range between 1,700 and 1,595 cm −1 . All data processing was done using GRAMS software.

Circular dichroism (CD) spectroscopy
For CD spectroscopy measurements, aliquots of each fibril sample (50 µL) were taken every hour for the first 10 incubation hours, then after 24 and 48 h. The samples were cooled down to 25 C and placed in a 0.1 mm pathlength cuvette. The CD spectra were measured between 200 and 250 nm, using a Jasco J-815 spectropolarimeter. For each sample, three spectra were recorded and averaged, after which a PBS solution spectrum was subtracted from each sample spectrum. The data was then analyzed using BeStSel Protein Circular Dichroism (Micsonai et al., 2015) spectra analysis software, in order to determine the content of secondary structures.

Atomic force microscopy (AFM)
AFM sample preparations and measurements were performed as described previously (Ziaunys et al., 2021a). Before sample deposition, freshly cleaved mica surface was modified with (3-aminopropyl) triethoxysilane (APTES). 0.5% (% v/v) APTES solution (30 µL) was spread on the surface of the mica, incubated at room temperature for 5 min, gently washed with 2 mL of H 2 O and dried using airflow. 30 µL aliquots of each sample were placed on APTES-modified mica and left to adsorb for 60 s. The mica were then gently washed with 3 mL of H 2 O and dried using airflow. AFM images were acquired using a Dimension Icon (Bruker) atomic force microscope and analyzed using Gwyddion 2.5.5 software as described previously (Ziaunys et al., 2021a).

UV/Vis spectroscopy
Each fibril sample (500 µL) was supplemented with 5 µL 10 mM ThT and vigorously agitated for 10 s. The samples were then placed in a 3 mm pathlength cuvette and sample absorbance spectra were scanned in the 200-600 nm range using a Shimadzu UV-1800 spectrophotometer (three repeats were scanned for each sample and averaged). After this, 200 µL of each sample was centrifuged at 10,000×g for 15 min and 100 µL of the supernatant was carefully removed. The supernatant absorbance spectra were scanned as described previously. In order to determine the absorbance spectra of bound-ThT molecules, the supernatant spectra were subtracted from the fibril-ThT spectra and baseline corrected between 300 and 550 nm using Origin 2018 software baseline subtraction function with three anchor points at each side of the peak.

RESULTS
In order to test the effect that an elevated temperature has on alpha-synuclein (a-syn) fibril structure, different conformation aggregates were first generated. Since a-syn is capable of spontaneously forming structurally distinct fibrils under identical experimental conditions (Toleikis et al., 2021;Ziaunys et al., 2021a) at random, a large number of samples (n = 96) was aggregated for an initial assessment. Potential conformationally-distinct fibrils were then identified by scanning their bound-ThT excitation-emission matrices (EEM) and selecting eight samples from the set, based on their unique EEM positions (Fig. 1A) (Ziaunys et al., 2021a). This was done in order to increase the likelihood of obtaining different structure samples, without the necessity to analyze the entire set. The selected samples were then replicated as described in the Materials and Methods section in order to generate a higher quantity of aggregates. To determine which of the samples contained significantly different secondary structures, an aliquot from each was scanned using Fourier-transform infrared spectroscopy (FTIR).
Five of the samples contained aggregates with similar secondary structures (Figs. 1B, 1C), with the FTIR spectrum main maximum at 1,627 cm −1 and a shoulder at 1,636 cm −1 (same position minima in the second derivatives, which are positions associated with hydrogen bonds in the beta-sheet structure (Barth, 2007)). They also had a minimum in the second derivative at 1,666 cm −1 , which is related to turn/loop motifs. Due to their similarities, these five samples were regarded as Type 1 fibrils. The VI sample shared some similarities to Type 1 aggregates in the turn/loop motif position (1,666 cm −1 ), however, the main maximum was shifted to 1,624 cm −1 , suggesting a different type of hydrogen bonding in the beta-sheet structure. Since this sample FTIR spectra was different from all other seven and had a main maximum position between other two group spectra positions, it was regarded as consisting of Type 2 fibrils. The other two remaining samples (VII and VIII) shared a similar main maximum position at 1,623-1,624 cm −1 , with significant variation in the turn/loop motif regions (minima at 1,662 and 1,673 cm −1 for Type 3, 1,668 cm −1 for Type 4). These four replicated fibril types were then used in all further experimental procedures.
Before incubating the selected fibrils at an elevated temperature, the aggregates were first centrifuged and resuspended into their initial buffer solution, in case they contained a significant concentration of non-aggregated protein. This was done to avoid any possible additional aggregation occurring during the incubation procedure. The resuspended fibrils were then incubated at 60 C with constant agitation for 24 or 48 h as described in the Materials and Methods section. After this step, a portion of each sample was used to scan their FTIR spectra.
In the case of Type 1 fibrils (Figs. 2A, 2E) after 24 h of incubation, there was a shift of the FTIR spectrum main maximum position towards 1,625-1,626 cm −1 and a reduction of the shoulder at 1,636 cm −1 with minimal changes in the region associated with turn/loop motifs. This suggests that incubation at an elevated temperature resulted in the reduction of weaker hydrogen bonds and the formation of stronger ones. Further incubation at an elevated temperature did not yield any notable changes. An opposite effect was observed for the Type 3 and Type 4 samples (Figs. 2C, 2D, 2G, 2H). Here, the main maximum positions shifted towards a larger wavenumber (from 1,623-1,624 to 1,625-1,626 cm −1 ). Surprisingly, the resulting FTIR spectra were almost completely identical to the one which formed when Type 1 fibrils were incubated (Figs. 2A, 2E). The Type 2 sample spectrum (Figs. 2B, 2F) experienced only very minor changes during incubation, which was to be expected, as it had a similar spectrum to the incubated fibril spectra, as seen for Type 1, 3 and 4 fibrils. Taken together, this indicates that the a-syn fibril polymorphism, which existed when aggregation occurred at 37 C, was completely negated when the fibrils were incubated at an elevated temperature. In order to verify if this newly-formed fibril type is the result of restructurization or simply a temperature-induced association/clumping, each 48 h incubation sample was reseeded at 37 C as described in the Materials and Methods section and the resulting aggregate FTIR spectra were scanned. Interestingly, all four of the a-syn samples retained the same identical spectra as were obtained after incubation at 60 C. This suggests that the newly-formed fibril type is capable of templating its structure under lower temperature conditions as well and does not revert back to its original state. Comparing all four FTIR spectra before and after incubation shows how they all become practically identical and overlap with each other (Figs. 2I, 2J).
Considering that an elevated temperature resulted in the formation of aggregates with an identical secondary structure, it was interesting to compare them to fibrils which originally formed at 60 C. Based on the kinetic data, alpha-synuclein aggregation proceeded significantly quicker when subjected to a higher temperature. The process average lag time was reduced three times (Fig. 3A) and the apparent rate constant of fibril elongation increased two times (Fig. 3B). The formed aggregate FTIR spectra were almost completely identical to the incubated fibril spectra, with the same main maximum position and overlapping spectra in the turn/loop motif region. This indicates that an elevated temperature leads to the same specific alpha-synuclein conformation, regardless if the aggregates were prepared at 37 C or 60 C.
Since incubation led to changes in fibril secondary structure, it was important to also analyze any possible morphological differences. Comparing the AFM images of all three fibril Types (Figs. 4A-4D, with their 48 h incubation counterparts (Figs. 4E-4H)), it can be observed that both the initial and incubated samples contained long fibers with some of them having periodicity patterns. Based on a visual inspection, it may appear that the incubated samples contained a larger number of fibrils with periodicity patterns, however, such a conclusion is difficult to make based on AFM images alone. Comparing the heights of fibrils (Fig. 4I) revealed that in all three cases, the average values were slightly higher for incubated samples (1-2 nm difference). This could be the result of the aforementioned morphological changes or fibril lateral association, however, the small difference in average values, coupled with no differences in fibril width (Fig. 4J) led to an assumption that incubation had no substantial effect on these parameters. Interestingly, the distances between periodic repeats of fibrils also did not change upon incubation (Fig. 4K), which means that the alterations in secondary structure did not have an influence on this morphological parameter. Additionally, all four cases (control and Figure 4 Atomic force microscopy (AFM) images of different fibril types before and after incubation. AFM images of Type 1 (A, E), Type 2 (B, F), Type 3 (C, G) and Type 4 (D, H) fibrils, as well as height (I), width (J) and periodicity (K) distribution before and after incubation respectively. All images are of identical 5 × 5 µm scale. Fibril height, width and periodicity were determined as described in the Materials and Methods section. Distribution box plots (n = 50) indicate the interquartile range and error bars are for one standard deviation.
Full-size  DOI: 10.7717/peerj.14137/ fig-4 incubated), did not have a homogenous distribution, as both non-periodic and periodic fibrils could be detected in all samples. This suggests that the restructurization may not affect certain subgroups of aggregates, which may require a significantly longer time to change, as shown by Sidhu et al. (2017).
Since there was a slight increase in fibril height, there existed a possibility that parts of the unstructured protein region became incorporated into the aggregate core region. In order to examine this possibility, CD spectra of all four fibril types were scanned after each hour of incubation for 10 h, as were the 24 and 48 h samples. When comparing the initial and end-point samples, for Type 1 (Fig. 5A) and Type 3 fibrils (Fig. 5C) there was a Figure 5 Alpha-synuclein fibril secondary structure element distribution during incubation at 60 C. The secondary structures for Type 1 (A), Type 2 (B), Type 3 (C) and Type 4 (D) fibrils were determined by scanning each sample's CD spectra after different periods of incubation and fitting the data using BeStSel Protein circular dichroism spectra analysis software. The normalized root mean square deviation (NRMSD) of each sample's secondary structure element distribution is displayed under their respective distribution graphs.
Full-size  DOI: 10.7717/peerj.14137/ fig-5 slight increase in the fraction of beta-sheets and a reduction in either turn or unstructured regions. For the other two fibril types (Figs. 5B, 5D), there were no notable deviations observed. In all cases, however, the change was rather marginal and, due to the relatively large normalized root mean square deviation values (NRMSD) (Micsonai et al., 2015), could not be considered as statistically significant.
Examining the absorbance spectra of fibril-bound ThT (Figs. 6A-6D) revealed that there were no sizeable variations in either the maximum absorbance value position or the maximum absorbance itself. This indicates that the structural rearrangements did not cause significant changes to the amount of bound ThT molecules. Interestingly, the same cannot be said about the sample optical density. There was a clear disparity between the Type 2 fibril sample and the rest (Fig. 6E), where the Type 2 sample had an OD 600 of 0.18, while for Type 1, 3 and 4 it was 0.10-0.12. After 24 h of incubation, there was only a minimal increase in OD 600 for the Type 2 sample, while the other three aggregate samples experienced a substantial OD 600 increase to 0.16-0.17. Additional incubation caused a further convergence of the OD 600 values. Considering that the fibrils were resuspended into the initial reaction solution, which did not contain a-syn monomers, additional aggregation can be ruled out as a cause of such a significant increase in optical density. This leaves the possibility that aggregate structural rearrangement caused an increase in the sample light scattering properties. This is further supported by the fact that the Type 2 sample experienced the least significant change in OD 600 , similarly to it having the least notable FTIR spectrum shift. In order to examine whether there was also a shift in bound-ThT EEM maximum positions, the four different fibril types were incubated as described in the Materials and Methods section at 60 C and their EEM spectra were scanned every hour. The initial scans were performed after the samples reached 60 C (after 10 min of incubation). During the first 4 h, the EEM positions shifted significantly (Fig. 6F), suggesting a change in the ThT-binding properties of the aggregates (as a note, the 60 C EEM positions do not correlate to the Fig. 1 positions due to the elevated sample temperature, which reduces the dye's fluorescence quantum yield and may influence the mode of binding). After 5-6 h of incubation, all four fibril type bound-ThT EEM positions converged to a similar position, which falls in line with the FTIR results. Comparing this information to the minimal change observed in ThT absorbance spectra, it appears that the structural alterations mainly affect the dye's mode of binding, rather than the quantity of surface-associated molecules. These results suggest that most major changes to the fibril structures occur during the initial 4 h of incubation.

DISCUSSION
Taking into consideration all the results presented in this work, it appears that different conformation a-syn fibrils undergo rapid restructurization at an elevated temperature. While physiological temperatures were also reported to cause aggregate maturation, the process spanned over several days or even months (Ma et al., 2013;Sidhu et al., 2017). In this case, based on changes in FTIR spectra and sample optical density, significant alterations to the secondary structure occurred in less than 24 h. If we use ThT fluorescence as an indicator of distinct structure aggregates, then the ThT excitation-emission matrix assay displays that most of the significant changes happened during the initial 4 h of incubation. If this is the case, then it is possible that a-syn fibril maturation is a condition-dependent process, which is greatly escalated at higher temperatures.
Another interesting aspect was the apparent convergence of multiple distinct conformation fibrils into one type of dominant aggregate with the increase of the solution's temperature. The four fibril types with distinct FTIR spectra all rapidly changed into one specific conformation, which was also capable of replicating its structure at the initial 37 C temperature, similarly to the initial four aggregate types. The structural alterations also do not appear to be caused by a significant increase in the fibril beta-sheet content (as seen during CD analysis), but rather due to a restructurization of existing secondary structure elements. This hints at a possible meta-stability of the fibrils generated at 37 C and that their existence is only facilitated by a high energy barrier needed to cross into the higher stability structure. Such a hypothesis is supported by the FTIR spectra similarity of incubated fibrils and aggregates prepared at 60 C (Fig. 3C). However, while this may be the case for pre-existing fibril conversion, the barrier does not appear to prevent the initial formation of such aggregates (Type 2 fibrils). In the AFM images, we can observe each initial sample containing fibrils with periodicity patterns. Such fibers seem to be the dominant type of structure after incubation and their presence in each sample suggests that they can readily form at 37 C as well. Despite this appearance, it is difficult to quantitatively verify such an observation due to the nature of AFM imaging.
While this, in itself, is an interesting phenomenon, such a rapid aggregate restructurization may have a negative impact both during in vitro drug screenings and may even have certain physiological implications. If ThT fluorescence intensity or sample optical density are used as a means of identifying changes in aggregate concentration, then such restructurization events may provide false-negative or false-positive results, as seen in the OD 600 (Fig. 6E) and ThT (Fig. 6F) assays. If experimental procedures involve the characterization of fibril structural parameters, then this type of rapid change to secondary structure may also lead to skewed experimental results and conclusions. Finally, if elevated temperatures can increase the incidence of the restructurization events, then this may be one of the many possible factors that influence the onset of neurodegenerative disorders, by stabilizing a certain fibril conformation.

CONCLUSIONS
Overall, it is apparent that an elevated temperature can not only significantly enhance the rate of alpha-synuclein fibril restructurization/maturation, but also result in the stabilization of a certain aggregate structure. This process also leads to changes in bound-ThT signal intensity and optical density, as well as alterations in fibril secondary structure, which should be taken into account during studies of both alpha-synuclein and other amyloid proteins.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
The authors received no funding for this work.