Epistemic_Statements  

CORD-19:40812d7b28ab3c043610f6e224b3f62806af6eb6 JSONTXT 11 Projects

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Id Subject Object Predicate Lexical cue
T1 526-688 Epistemic_statement denotes However, the antigenic variations among different PEDV strains are less explored, although they may contribute to the failure of PEDV vaccines in Asian countries.
T2 689-911 Epistemic_statement denotes In addition, the evolution of PEDV results in variants with distinct genetic features and virulence differences; thus PEDV can serve as a model to explore the molecular mechanisms of coronavirus evolution and pathogenesis.
T3 1971-2128 Epistemic_statement denotes Contaminated feed and feed ingredients may serve as vehicles for PEDV transmission to naïve pigs (Dee et al., 2014 (Dee et al., , 2016 Pasick et al., 2014) .
T4 3478-3549 Epistemic_statement denotes Therefore, the development of updated PEDV vaccines is urgently needed.
T5 3647-3874 Epistemic_statement denotes Throughout this review, the terms "classical" and "emerging" PEDV strains correspond to the PEDV prototype CV777-like strains that have appeared since the 1970s and those PEDV strains detected globally after 2010, respectively.
T6 4202-4295 Epistemic_statement denotes This nomenclature is proposed based on sequence analysis as explained in detail in Section 2.
T7 5410-5812 Epistemic_statement denotes However, not until 1984 were the causative agents, PEDV H, CH and J strains, especially the J strain, isolated in primary fetal pig intestinal cell cultures and confirmed to be PEDV by immunofluorescence assay (IFA), two-way cross virus neutralization (VN), and in vivo pig cross-protection assays comparing tissue culture-adapted (TC) PEDV and TGEV, and their respective antisera (Xuan et al., 1984) .
T8 5813-5937 Epistemic_statement denotes However, the sequence information for the early PEDV strains (H, CH, and J) is unavailable (personal communication with Drs.
T9 7014-7288 Epistemic_statement denotes In Japan, PEDV outbreaks were reported from the early 1980s and temporarily disappeared in 2006, probably due to the use of an attenuated PEDV 83P-5 (or P-5V) strain vaccine (Nisseiken Co. Ltd., Japan) since 1997 Sato et al., 2011; Song et al., 2015b; Song and Park, 2012) .
T10 7411-7495 Epistemic_statement denotes Since 2011, the attenuated PEDV DR13 vaccine also has been used in the Philippines .
T11 7589-7828 Epistemic_statement denotes We will refer to these historic PEDV strains as classical strains because such viruses are still detected in the field, such as CHN/SD-M/2012 and THA/EAS1/2014, to differentiate them from the emerging strains detected worldwide after 2010.
T12 9620-9695 Epistemic_statement denotes However, no complete genomic sequence data are available for these strains.
T13 9824-9983 Epistemic_statement denotes However, except for the Ukraine (Dastjerdi et al., 2015) , no highly virulent PEDV infections were reported in the European, African and Australian continents.
T14 10704-10855 Epistemic_statement denotes Although the amino acid sequences of the S proteins of S INDEL strains are closer to the classical strains than to the highly virulent US strains (Fig.
T15 11682-11814 Epistemic_statement denotes Mutations, especially deletions and/or insertions in the S protein may change the pathogenicity and tissue tropism of coronaviruses.
T16 12779-12957 Epistemic_statement denotes Interestingly, a single US PEDV strain, PC177, with a 197 aa-deletion in a similar position (aa 34-230) of the S protein, was isolated from Vero cell culture (Oka et al., 2014) .
T17 14822-15329 Epistemic_statement denotes KJ645696; 1 nt different from Iowa106 strain at the genomic level) were probably the result of several recombination events: the ORF 1a and 1b region was most similar to that of the highly virulent strains CHN/AH2012 and CHN/ZMDZY/2011, respectively; the S1 and S2 region of the S protein was most similar to that of the classical strain CHN/CH/S/1986 and the highly virulent ZMDZY/2011 strain, respectively; and the 3 end (E, M, and, N genes) was dissimilar to that of either AH2012 or ZMZDY/2011 strains .
T18 15750-15936 Epistemic_statement denotes Not surprisingly, this strain was detected from a swine farm that is located in the same province as the CHN/ZMDZY/11 strain, and both CV777 and DR13 PEDV vaccines were used at the farm.
T19 15937-16160 Epistemic_statement denotes Although both PEDV and TGEV belong to the Alphacoronavirus genus and cause indistinguishable enteritis (Saif et al., 2012) , no recombinant virus between TGEV and PEDV had been found until recently (Boniotti et al., 2016) .
T20 16161-16351 Epistemic_statement denotes This recombinant porcine enteric coronavirus, Italy/213306/2009 strain, has potential parental strains TGEV H16 strain (major, backbone) and classical PEDV CV777 strain (minor, S gene) (Fig.
T21 16617-16760 Epistemic_statement denotes It is important to determine the virulence and tissue tropism of these chimeric viruses and to monitor how they evolve in the swine population.
T22 16806-17074 Epistemic_statement denotes Sequence analysis suggests the potential origin of emerging PEDV strains Phylogenic analysis of the full-length genomes and the genome organization showed that all PEDV strains are most closely related to bat alphacoronaviruses within the Alphacoronavirus genus ( Fig.
T23 17075-17272 Epistemic_statement denotes 1) Tang et al., 2006; Vlasova et al., 2014) , suggesting that PEDV strains probably originally evolved from bat alphacoronaviruses and were transmitted cross-species directly or indirectly to pigs.
T24 18758-18988 Epistemic_statement denotes Since PEDV has a relatively low mutation rate (6.2 × 10 −4 substitutions/site/year) similar to TGEV and other animal coronaviruses (Jarvis et al., 2015) , it seems likely that the PEDV strains have diverged due to host adaptation.
T25 18989-19099 Epistemic_statement denotes On the other hand, evidence supporting a different origin of the two distinct clusters of PEDV strains exists.
T26 19930-20008 Epistemic_statement denotes How such a unique feature was obtained by these PEDV strains is still unknown.
T27 20009-20123 Epistemic_statement denotes If the changes result in a replication advantage for PEDV strains, the genetic changes may evolve and be selected.
T28 20124-20261 Epistemic_statement denotes However, the process for acquiring the individual insertion and deletions in PEDV, or intermediate PEDV strains, has not been determined.
T29 20262-20386 Epistemic_statement denotes It is also possible that PEDV strains obtained this region from a BtCoV/512/2005-like coronavirus directly by recombination.
T30 20387-20747 Epistemic_statement denotes Although complete genomic sequences are lacking for many PEDV strains, information from the S protein may highlight when the emerging non-S INDEL strains evolved since the S genes are available for some samples collected before 2010, and the emerging non-S INDEL and classical groups can be clearly differentiated in an S protein tree (Sun et al., 2015b) (Fig.
T31 20753-20852 Epistemic_statement denotes It appears that the non-S INDEL strains, such as KOR/Chinju99/1999 strain, had emerged before 2010.
T32 21060-21161 Epistemic_statement denotes To date, when, where and how the original emerging non-S INDEL PEDV strains evolved is still unknown.
T33 21162-21348 Epistemic_statement denotes Recently, Khatri (2015) reported that the highly virulent PEDV grew in duck intestinal epithelial cells, raising the question of whether there are other reservoirs besides bats for PEDV.
T34 21349-21401 Epistemic_statement denotes All those questions need to be investigated further.
T35 21690-21979 Epistemic_statement denotes 2) , non-S INDEL PEDV strains from China are more closely related to each other than to the strains from the US and European countries, except for CHN/AH2012 that is closer to the US strains (with nt identity of 99.33-99.61%) than to the Chinese strains (with nt identity of 98.10-99.50%).
T36 22388-22571 Epistemic_statement denotes Therefore, those regions are probably not immediate targets in the recent evolution of emerging PEDV strains and there is a lack of enough information to generate evolution inference.
T37 22850-22994 Epistemic_statement denotes It is probably due to recombination among PEDV strains, which is reported as a major evolution pattern for coronaviruses (Makino et al., 1986) .
T38 23228-23540 Epistemic_statement denotes Recently, a novel method using sequence motifs, which was defined as "a highly conserved region at the same position in the genome that was exclusively shared between several of the PEDV strains", was developed to study the relationship among emerging PEDV strains detected in 2013-2014 (Yamamoto et al., 2016) .
T39 23746-23978 Epistemic_statement denotes The non-S INDEL strains showed an overall high similarity to the highly virulent CHN/AH2012 strain across the genome except for the S1 region where they showed the highest similarity to another highly virulent CHN/ZMDZY/2011 strain.
T40 24289-24495 Epistemic_statement denotes It is notable that the Ukraine, Japanese and Korean emerging non-S INDEL strains are within the NA non-S INDEL, but not the emerging Asian clade, suggesting global spread of the NA non-S INDEL strains (Fig.
T41 24782-24914 Epistemic_statement denotes Surprisingly, no S INDEL strains were reported from China where both the classical and the emerging PEDV strains are co-circulating.
T42 24915-25105 Epistemic_statement denotes The S INDEL strains could the United States; VNM, Vietnam)/strain name/year of sample collection (Genbank accession number) followed by passage (P) number for tissue culture-adapted strains.
T43 25327-25856 Epistemic_statement denotes (1) in general the S INDEL PEDV strains cause milder diarrhea in pigs compared to the highly virulent emerging strains, so clinical samples may not be collected for diagnosis; and (2) many diagnostic tests or surveillance studies did not differentiate S INDEL strains from classical (if the S1 region of the S gene is the target) or emerging non-S INDEL PEDV strains (if S2, ORF3, E, M or N region is the target), such as using real-time RT-PCR targeting the N gene or sequence analysis of the S1 region only (Oka et al., 2014) .
T44 25857-26167 Epistemic_statement denotes Interestingly, based on the genetic motif analysis (Yamamoto et al., 2016) In summary, based on genetic analysis, the global PEDV strains are divided into two major groups: the classical PEDV strains that first emerged in 1970s in Europe and the highly virulent PEDV strains that emerged in 2010 in China (Fig.
T45 26175-26378 Epistemic_statement denotes Many questions remain unanswered, such as whether the later strains evolved directly from the former strains, and when the non-S INDEL and S INDEL emerging strains were introduced into the US and Europe.
T46 26905-27036 Epistemic_statement denotes Because coronaviruses are RNA viruses and recombination occurs frequently, more PEDV variants are expected to emerge in the future.
T47 28420-28556 Epistemic_statement denotes However, exceptions were reported as described subsequently (Have et al., 1992; Lin et al., 2015b; Ma et al., 2016; Zhou et al., 1988) .
T48 29020-29246 Epistemic_statement denotes (1992) reported that sera collected from a putative coronavirus-infected mink cross-reacted with both PEDV and TGEV N proteins, supporting the idea that these viruses were distinct species, but partially related antigenically.
T49 30500-30554 Epistemic_statement denotes The reasons for these unexpected findings are unknown.
T50 30555-30728 Epistemic_statement denotes However, the antigenic cross-reactivity observed between PEDV and TGEV strains raised concerns about the specificity of PEDV whole virus or N protein-based serologic assays.
T51 31026-31172 Epistemic_statement denotes However, another PEDV N protein-based ELISA assay showed no cross-reactivity with TGEV (Purdue and Miller) and PRCV antisera (Okda et al., 2015) .
T52 31173-31368 Epistemic_statement denotes We speculate that convalescent TGEV Miller antisera collected from the field and displaying low cross-reactivity with PEDV N proteins might be interpreted as background or non-specific reactions.
T53 31369-31483 Epistemic_statement denotes This could be the reason why the antigenic cross-reaction between TGEV and PEDV strains was reported infrequently.
T54 31484-31663 Epistemic_statement denotes Truncation of the antigenic site in the N-terminal region could help exclude the possible serological cross-reactivity and enhance the sensitivity/specificity of the immunoassays.
T55 31664-31846 Epistemic_statement denotes Alternatively, PEDV membrane (M) and S proteins could serve as specific antigens for immunoassays (Fan et al., 2015; Gerber et al., 2014; Knuchel et al., 1992; Paudel et al., 2014) .
T56 32301-32537 Epistemic_statement denotes Because viral neutralizing epitopes are located on the S protein, the results of PEDV S protein-based immunoassays correlated well with viral neutralization (VN) titers (Paudel et al., 2014) , which is important for clinical protection.
T57 32723-32917 Epistemic_statement denotes However, different antigenicity among S epitopes of the relevant PEDV strains (Lin et al., 2015b; Wang et al., 2015) also raises concerns for the comprehensive detection of diverse PEDV strains.
T58 33833-34009 Epistemic_statement denotes However, a whole virus-based ELISA and Western blot showed two-way antigenic cross-reactivities between PEDV VBS2 strain and PDCoV Michigan/8977/2014 strain (Ma et al., 2016) .
T59 34010-34168 Epistemic_statement denotes Sequence analysis of the N proteins showed 4 motifs (47- are conserved between PEDV and PDCoV, probably leading to the cross-reactivities in the immunoassays.
T60 34169-34372 Epistemic_statement denotes Interestingly, no antigenic cross-reactivity between PEDV and PDCoV was detected in either virus-infected cells or intestinal tissues using CCIF or immunohistochemistry (IHC) staining (Ma et al., 2016) .
T61 34373-34616 Epistemic_statement denotes The discrepancy of different assays was suggested to result from different sensitivity among immune assays and/or the exposure of hidden epitopes on viral N proteins by protein denaturing steps in the ELISA and Western blot (Ma et al., 2016) .
T62 34783-34905 Epistemic_statement denotes However, the same panel of antibodies did not cross-react with PDCoV in our CCIF assays (Lin and Saif, unpublished data) .
T63 35065-35252 Epistemic_statement denotes If PDCoV is found to be widespread in swine and a factor in enteric disease, it is possible that PDCoV may be included in future multivalent vaccines against swine enteric viral diseases.
T64 35461-35574 Epistemic_statement denotes Thus, periodic updates of vaccines may be required to ensure sufficient efficacy against emerging virus variants.
T65 35807-36050 Epistemic_statement denotes However, more evidence based on field observations, viral gene sequence analysis and serologic assays suggested that the emerging highly virulent (non-S INDEL) PEDV strains differed antigenically in various degrees from classical PEDV strains.
T66 36557-36689 Epistemic_statement denotes To date, the use of classical PEDV strainbased inactivated and attenuated vaccines may still help to reduce the severity of disease.
T67 36690-36890 Epistemic_statement denotes However, many pig herds followed the routine vaccination program, but still experienced high mortality rates among newborn piglets caused by emerging highly virulent PEDV strains Song et al., 2015b) .
T68 37090-37330 Epistemic_statement denotes Initially, sequence comparisons of neutralizing epitopes on the S protein suggested that the antigenic variations between classical and emerging non-S INDEL (highly virulent) PEDV strains are the major reason for vaccine failure (Table 3) .
T69 38003-38271 Epistemic_statement denotes In addition, bioinformatics predicted that the S protein of highly virulent PEDV strains changed in primary/secondary structures, high-specificity N-glycosylation sites, potential phosphorylation sites, and palmitoylation sites (Chiou et al., 2015; Hao et al., 2014) .
T70 38272-38355 Epistemic_statement denotes These changes may affect viral antigenicity and change viral neutralizing activity.
T71 39719-39811 Epistemic_statement denotes However, inconsistences in cross-reactivity were evident using antisera from different pigs.
T72 40806-40916 Epistemic_statement denotes However, the homologous titers were two-fold higher than the heterologous titers in CCIF, ELISA and VN assays.
T73 40917-41288 Epistemic_statement denotes Overall, the above studies showed that pigs or mice immunized with classical attenuated PEDV vaccine strains usually displayed similar or lower serum antibody titers against the emerging highly virulent PEDV strains (2-16 fold differences), suggesting the classical PEDV vaccine strains may only provide partial cross-protection against the highly virulent PEDV strains .
T74 41289-41388 Epistemic_statement denotes However, in vivo cross-protection studies are needed to confirm the in vitro cross-reactivity data.
T75 41389-41617 Epistemic_statement denotes Although differences in antigenicity between classical and highly virulent PEDV clusters have been studied, the antigenic variations among PEDV strains within the same phylogenetic cluster are less studied in serological assays.
T76 41835-42083 Epistemic_statement denotes (2015) reported that the highly virulent PEDV strains from China had an increased number of substitutions within the S1 domain compared with the highly virulent American strains, probably due to the longer circulation time of PEDV in pigs in China.
T77 43284-43438 Epistemic_statement denotes However, three (JY5C, JY6C and JY7C) of these highly virulent PEDV strains had one additional mutation (L521H) in the neutralizing epitope COE (Table 3) .
T78 44086-44300 Epistemic_statement denotes The significance of these amino acid differences in VN epitopes among these highly virulent PEDV strains has not yet been examined by using MAbs in serological assays or by in vivo cross-protection studies in pigs.
T79 44301-44382 Epistemic_statement denotes However, it is likely that higher antigenic variations will appear in the future.
T80 44383-44675 Epistemic_statement denotes In addition, the amino acids in neutralizing epitopes of S INDEL strains were the same as that of the majority of NA highly virulent strains (Table 3 ), suggesting their cross-neutralization ability and confirmed by our previously reported in vitro and in vivo studies (Lin et al., 2015a,b) .
T81 45035-45103 Epistemic_statement denotes However, the homologous titers were higher than heterologous titers.
T82 45104-45275 Epistemic_statement denotes Alternatively, serum samples collected from pigs inoculated with S-INDEL PEDV showed similar homologous and heterologous titers by IFA and VN assays (Chen et al., 2016b) .
T83 45276-45489 Epistemic_statement denotes However, in vivo cross-protection also can be affected by additional factors, such as cellular immune responses and in suckling pigs by the lactating sow's milk production (Goede et al., 2015; Lin et al., 2015a) .
T84 45490-45621 Epistemic_statement denotes Also serum viral neutralizing antibody level may not reflect IgA viral neutralizing antibody level in milk (Scherba et al., 2016) .
T85 45622-45756 Epistemic_statement denotes Whether the circulating antibody plays a role in the protection of piglets against PEDV is still questionable (Poonsuk et al., 2016) .
T86 45757-45887 Epistemic_statement denotes Thus, in vivo cross-protection may not always be consistent with in vitro virus neutralizing antibody levels (Lin et al., 2015b) .
T87 45998-46178 Epistemic_statement denotes Continuous monitoring of genetic and antigenic changes in PEDV strains is critical to control epidemic outbreaks and for vaccine development (Chattha et al., 2015; Mumford, 2007) .
T88 46179-46451 Epistemic_statement denotes However, there are limitations in the comprehensive characterization of antigenic variations among PEDV strains, although cross-protection activity is a primary criterion for vaccine strain selection, and immunity is thought to be a major driving force of virus evolution.
T89 46452-46696 Epistemic_statement denotes First, unlike the viral genomic information accessible from Gen-Bank, it is difficult to collect large numbers of PEDV strains and produce the corresponding highly specific pig antisera for measuring cross-reactive neutralizing antibody titers.
T90 46697-46816 Epistemic_statement denotes Few virus strains are adapted to cell culture, and in the US it is difficult to import foreign isolates for comparison.
T91 46817-46926 Epistemic_statement denotes Antibody specificities can be influenced by the backgrounds of the experimental animals (Lin et al., 2015b) .
T92 46927-47207 Epistemic_statement denotes In addition to inferring the antigenicity by sequence data Sun et al., 2013) , expression of recombinant proteins in eukaryotic systems and generating antiserum and/or MAbs in laboratory animals could be alternative methods to explore the antigenic variations among PEDV strains .
T93 47208-47595 Epistemic_statement denotes However, whether the structure of recombinant proteins reflects the proteins in the intact virus (Makadiya et al., 2016) , or whether cross-reactive neutralizing antibody profiles defined using antisera from injected small laboratory animals are similar to those observed using post-infection sera obtained from their natural host in the field needs to be confirmed (Chia et al., 2014) .
T94 47596-47796 Epistemic_statement denotes Secondly, in the field a post-infection/convalescent antiserum contains polyclonal antibodies that may react with the conserved epitopes and may not reveal the antigenic variations among PEDV strains.
T95 47797-47962 Epistemic_statement denotes The antigenic variations among different TGEV strains could be distinguished by MAbs (Simkins et al., 1992) , but not by conventional antisera (Laude et al., 1986) .
T96 47963-48149 Epistemic_statement denotes Therefore, production of a comprehensive panel of PEDV neutralizing MAbs and epitope mapping studies will help to more precisely identify the regions contributing to antigenic variation.
T97 48407-48594 Epistemic_statement denotes When large numbers of viral strains and antiserum data were included, the results were difficult to combine and data below the sensitivity threshold of the assay could not be interpreted.
T98 48595-48661 Epistemic_statement denotes Moreover, results from different laboratories may be inconsistent.
T99 48936-49174 Epistemic_statement denotes Multiple datasets of the serological assays from different laboratories worldwide could be combined for constructing a comprehensive PEDV antigenic cartography by a computational framework approach (Cai et al., 2010; Smith et al., 2004) .
T100 50397-50650 Epistemic_statement denotes In the antigenic map, the S INDEL Iowa106 strain and the S 197aa-del PC177 strain were located between the highly virulent PEDV PC22A and the classical CV777 strain, showing a well correlation between the phylogenetic distance and antigenic differences.
T101 52211-52352 Epistemic_statement denotes However, not all of the pathological features of prototype PEDV CV777 infection were consistently observed for other PEDV strains (Table 4 ).
T102 52546-52808 Epistemic_statement denotes However, in these very young piglets, no PEDV antigens or nucleic acids were detected in epithelial cells lining the crypts, and positive-staining cells were lacking in the colon by IHC staining (Kim et al., 1999) and in situ hybridization (Kim and Chae, 2003) .
T103 52911-53143 Epistemic_statement denotes Therefore, the pathogenicity of TGEV/PEDV infection in pigs depended on the viral strains, the age of pig, how piglets were derived (CD) or if naturally farrowed, the colostrum/milk status (CD) and environment variations (Table 4 ).
T104 54882-54977 Epistemic_statement denotes Whether the regenerative capacity of the crypts is affected by PEDV infection is still unknown.
T105 56111-56289 Epistemic_statement denotes This data indicates that limited numbers of enterocytes were still available to sustain PEDV replication, but to lower levels during the prolonged infection (Lin et al., 2015a) .
T106 56290-56438 Epistemic_statement denotes Fifth, pigs of all ages can be infected by the highly virulent PEDV, but pigs develop age-dependent resistant to PEDV disease Madson et al., 2014) .
T107 56581-56728 Epistemic_statement denotes However, only mild diarrhea occurred in 2-and 4-week-old pigs and no clinical signs were observed in 8-and 12week old pigs (Shibata et al., 2000) .
T108 57177-57549 Epistemic_statement denotes However, the highly virulent PEDV strains caused severe growth retardation in weaned pigs and significant impairment in productivity, such as lower farrowing rates, lower total pigs and pigs born alive per litter and decreased nursing performance of sows (Goede and Morrison, 2016; Lin et al., 2016) , leading to the overall negative economic impacts on the pork industry.
T109 57808-58118 Epistemic_statement denotes In addition to antigenic variation, these disease characteristics also explained why current highly virulent PED outbreaks were so devastating in the major pig producing countries in Asia (Choi et al., 2014; Oka et al., 2014; Sun et al., 2012) and North America (Stevenson et al., 2013; Vlasova et al., 2014) .
T110 58124-58267 Epistemic_statement denotes INDEL PEDV strains showed lower virulence than the emerging non-S INDEL PEDV strains, but still can induce severe disease in some circumstances
T111 58268-58381 Epistemic_statement denotes The first US PEDV S INDEL strain, OH851, was isolated from conventional pigs without significant clinical signs .
T112 58542-58873 Epistemic_statement denotes In Japan, one-week-old, specific pathogen-free (SPF) piglets inoculated with S INDEL PEDV ZK-O strain had delayed onset of PEDV fecal RNA shedding, lower fecal diarrhea scores and fecal viral RNA titers compared with the highly virulent PEDV ZK-CHR stain, suggesting S INDEL strains may be less pathogenic (Yamamoto et al., 2015) .
T113 59021-59559 Epistemic_statement denotes However, the virulence of S INDEL PEDV Iowa106 was lower than that of the highly virulent PEDV strains as evident by: (1) a longer incubation time (delayed onset of clinical signs and the peak of viral RNA shedding); (2) a shorter duration of diarrhea; (3) relatively higher VH:CD ratios; (4) a lower percentage of PEDV positive enterocytes; (5) more limited intestinal regions of virus infection (crypt not involved); and (6) overall lower piglet mortality (18%) compared with the highly virulent PC21A strain (55%) (Lin et al., 2015a) .
T114 60084-60332 Epistemic_statement denotes However, individual and litter variations, as commonly observed in the field , were reported only in our piglet studies: the mortality of S INDEL Iowa106-inoculated piglets was 0% in three litters, but 75% in the fourth litter (Lin et al., 2015a) .
T115 60661-60914 Epistemic_statement denotes Therefore, although the consensus was that the S INDEL strains were of lower virulence (Lin et al., 2015a,b; Wang et al., 2014; Yamamoto et al., 2015) , they still induced severe disease in some circumstances, such as if the sow became infected and ill.
T116 60915-61158 Epistemic_statement denotes If sows had decreased appetite and milk production, the lactogenic immunity could be disrupted or decreased (Chattha et al., 2015) and their suckling piglets were likely to die (Lin et al., 2015a; Mesquita et al., 2015; Stadler et al., 2015) .
T117 61159-61316 Epistemic_statement denotes Also piglets with lower birth weight might compete less efficiently for milk and receive less nutrition and lower lactogenic immunity than their littermates.
T118 61317-61470 Epistemic_statement denotes The severity of clinical signs caused by S INDEL PEDV infection showed a negative correlation with the birth body weight of piglets (Lin et al., 2015a) .
T119 61471-61777 Epistemic_statement denotes Highly virulent TGEV strains, such as US Miller at low cell culture passage level 3 (passage 3), is associated with short incubation time, extensive spread of infection throughout the small intestine, infection in higher portions of small intestinal enterocytes and marked damage to the villous epithelium.
T120 63444-63569 Epistemic_statement denotes Unlike TGEV and PRCV, however, the results of our IHC staining did not suggest changes in tissue tropism of the PC177 strain.
T121 63800-63905 Epistemic_statement denotes Both findings suggest that the large deletion in the S1 NTD may be responsible for the reduced virulence.
T122 64173-64348 Epistemic_statement denotes However, the extreme C-terminus of the FL2013S gene has a unique 21 nt-deletion, leading to a 7 aa-deletion (FEKVHVQ) in comparison with other highly virulent PEDV sequences .
T123 64439-64674 Epistemic_statement denotes Whether these large deletions or early termination of S protein alone alter viral infectivity, pathogenicity and replication efficiency can be further examined by reverse genetics technology (Beall et al., 2016; Jengarn et al., 2015) .
T124 65019-65274 Epistemic_statement denotes Because PEDV causes up to 100% mortality in neonatal pigs (<7-day-old), the aim of vaccination for PEDV is to immunize the sows to induce adequate maternal immunity to passively protect neonatal piglets from PED (Chattha et al., 2015; Saif et al., 2012) .
T125 65275-65503 Epistemic_statement denotes This objective may be accomplished by the selection of the optimal viral strain, whose antigenicity is close to that of the epidemic strain, but of minimal pathogenicity in young piglets, as well as the optimal vaccine approach.
T126 65509-65905 Epistemic_statement denotes Concerns about the use of autogenous PEDV strains for feedback or for inactivated vaccines to induce protection in pigs Before effective PEDV vaccines became available in the US and many Asian countries, intentional infection of sows with an autogenous PEDV strain during gestation, feedback exposure was used to induce lactogenic immunity for piglets (Chattha et al., 2015; Song et al., 2015b) .
T127 66355-66467 Epistemic_statement denotes However, the safety of using this strategy as a disease control method was questionable (Chattha et al., 2015) .
T128 66659-66849 Epistemic_statement denotes To date, detailed efficacy studies of the PEDV inactivated (Zoetis, Florham Park, NJ) or alphavirus vectored (Harrisvaccines, Ames, IA) vaccines conditionally licensed in the US are lacking.
T129 66850-67029 Epistemic_statement denotes Whether the US PEDV S INDEL strains could be used as an effective live vaccine to cross-protect piglets from highly virulent PEDV strains has been investigated in several studies.
T130 67432-67590 Epistemic_statement denotes However, these in vitro immunological assays only examined the IgG antibodies in serum and may not reflect lactogenic antibodies in vivo challenge conditions.
T131 67591-67869 Epistemic_statement denotes Because PEDV infects and replicates in the intestinal mucosa, development of mucosal immunity that relies on the quality and quantity of antibodies present in the intestinal mucosa or colostrum/milk, is critical for optimal active protection (Chattha et al., 2015; Saif, 1999) .
T132 68334-68624 Epistemic_statement denotes However, the PEDV-challenged, passively immunized suckling piglets showed extreme litter variations with morbidity rates ranging from 0% to 100%, although their mothers were naturally pre-exposed to an S INDEL PEDV strains and re-boosted with the virulent PEDV strain (Goede et al., 2015) .
T133 69351-69541 Epistemic_statement denotes Attenuated PEDV generated via serial cell culture passages, as well as the naturally occurring mild S INDEL PEDV strains, may serve as live attenuated vaccine candidates Sato et al., 2011) .
T134 69542-69741 Epistemic_statement denotes High cell culture passage level of PEDV strains could facilitate the generation of viruses that replicate well in cell culture, but less efficiently in the natural host, leading to viral attenuation.
T135 69742-69903 Epistemic_statement denotes High titers of virus stocks that are free of other pathogens and can be generated and used to develop cost-effective inactivated and/or live attenuated vaccines.
T136 70901-70964 Epistemic_statement denotes However, none of the changes occurred in neutralizing epitopes.
T137 70965-71303 Epistemic_statement denotes Although detailed data regarding the serological cross-reactivity has not been reported, the S proteins of YN-P1 and YN-P144 had a high (99.13%) sequence identity.In the same study, PEDV YN-P144 was tested safe for pregnant sows and newborn piglets, and induced immunity in sows and their piglets against a highly virulent PEDV challenge.
T138 71565-71966 Epistemic_statement denotes However, concerns remain that the attenuated PEDV strains may recover their virulence via mutation in the natural hosts or generate new virulent strains by exchanging their genes with field strains by recombination (See Section 2 for examples of PEDV recombination) unless the viral genes responsible for recombination are defective, or suicide strains are engineered (limited replication competency).
T139 71967-72211 Epistemic_statement denotes The use of reverse genetics technology to generate PEDV infectious clones will allow targeted mutations in virulence genes and the engineering of genomic changes that limit PEDV recombination in vivo (Beall et al., 2016; Jengarn et al., 2015) .
T140 72212-72456 Epistemic_statement denotes Truncation of ORF3 protein observed in attenuated PEDV CV777 (Wang et al., 2012) and ORF3 gene mutations in attenuated DR13 (Song et al., 2003) suggested that ORF3 could be a region other than the S gene associated with viral virulence in vivo.
T141 72457-72582 Epistemic_statement denotes Previous studies indicated that PEDV ORF3 encodes an ion channel protein and regulates virus production (Wang et al., 2012) .
T142 72583-72759 Epistemic_statement denotes However, truncation of ORF3 also occurred in low-passaged (P15) YN strain suggesting that it may be related to cell culture adaption, and not necessarily to viral attenuation .
T143 73128-73305 Epistemic_statement denotes With the accumulation of knowledge regarding the molecular mechanisms of PEDV attenuation, reverse genetics will help to rationally design PEDV vaccine candidates in the future.
T144 73805-73942 Epistemic_statement denotes Partial cross-protection between emerging S-INDEL and non-S INDEL PEDV strains and between classical and emerging PEDV strains may exist.
T145 74148-74317 Epistemic_statement denotes Naturally occurring mild PEDV strains, TC PEDV strains, and the infectious clone-derived attenuated strains can be considered as live attenuated PEDV vaccine candidates.