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Temporal Dynamics of Co-circulating Lineages of Porcine Reproductive and Respiratory Syndrome Virus

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV), the etiological agent of PRRS, is one of the most important endemic viruses affecting the swine industry in the United States (Holtkamp et al., 2013) and globally (Stadejek et al., 2013; VanderWaal and Deen, 2018). The economic impact of the disease in the United States has been estimated at $664 million annually (Holtkamp et al., 2013). Clinical signs in affected farms vary by viral variant and according to the farm’s production stage (e.g., breeding or growing herd), herd management, immune status, and other factors (Goldberg et al., 2000). Premature farrowing can occur in 5–30% of sows in an affected farm, and up to 35% of piglets are stillborn during an outbreak (Christianson and Joo, 1994). Piglets may be born with low weight and can present with lethargy and anorexia, which can lead to a mortality of more than 70% among piglets (Pejsak et al., 1997). PRRSV-infected pigs are also susceptible to secondary infections leading to poor average daily gain and feed conversion, further increasing production loss (Solano et al., 1997; Xu et al., 2010). Up to 40% of United States breeding herds experience outbreaks annually (Tousignant et al., 2015a) and control of the disease in the United States, Europe, and globally is challenging due to high levels of antigenic variability and its rapidly expanding genetic diversity (Frossard et al., 2013; Brar et al., 2015; Guo et al., 2018; Smith et al., 2018).

Porcine reproductive and respiratory syndrome virus was first recognized almost simultaneously in Europe (Wensvoort et al., 1991) and North America (Collins et al., 1992) in the late 1980s and early 1990s, but genetic differences suggested a much earlier evolutionary divergence between the North American and European viral types. Thus, PRRSV is divided into two major phylogenetic clades, PRRSV Type 1 (more prevalent in Europe) and Type 2 (more prevalent in North America) (Shi et al., 2010a, b; Stadejek et al., 2013). Within each clade, high levels of genetic and antigenic diversity exist and cross-protection is only partial (Roberts, 2003; Kim et al., 2013; Correas et al., 2017). Genetic similarities between PRRSV isolates have been used as a tool to understand disease transmission and epidemiology (Kapur et al., 1996; Wesley et al., 1998), and several different strategies have been used for classifying isolates of PRRSV into epidemiologically meaningful groups. For PRRSV Type 2, the most commonly used classification system is based on restriction fragment length polymorphisms (RFLP) and sequencing, both of which are typically based on the open reading frame 5 (ORF5) portion of its genome (Kapur et al., 1996; Wesley et al., 1998). The ORF5 gene encodes for the major envelope protein (GP5), which plays a role in inducing virus neutralizing antibodies and cross-protection among PRRSV variants (Dea et al., 2000; Kim et al., 2013). RFLPs have been broadly adopted by the U.S. swine industry despite shortcomings, such as the fact that the genetic relationship between different RFLP types is unclear, the potential for two distantly related viruses to share the same RFLP type, and the instability of RFLP-typing when assessing isolates related to each other by as few as 10 animal passages (Cha et al., 2004). In 2010, a classification system based on the phylogenetic relatedness of the ORF5 portion of the virus’s genome was proposed (Shi et al., 2010a, b). This classification system aggregates isolates into phylogenetic lineages based on the ancestral relationships and genetic distance among isolates. Using this system, nine different lineages were described within PRRSV Type 2, each of which was estimated to have diverged between 1980 and 1992 (Shi et al., 2010b). Phylogeny-based classification of organisms is seen as the most powerful and robust instrument for distinguishing between variants of a viral population (Hungnes et al., 2000) and has been used in the study of other viral diseases (Liu et al., 2009). Phylogeny-based classification of PRRSV, rather than RFLP profiling, is expected to provide fewer ambiguities and more insight into the evolutionary relatedness amongst different variants. While the existence of PRRSV lineages is well established, the dynamics of their co-circulation within a given region has not been well documented.

Vaccination is often used as a tool to mitigate clinical impact and viral shedding (Holtkamp et al., 2011). Although specific practices vary across farms, gilts are typically vaccinated before entering the herd, and sometimes the sow herd is mass vaccinated during the year. Most commercial PRRSV vaccines currently sold in the United States are considered “modified live vaccines” (MLV), which means that the vaccine is an attenuated live virus. Vaccines against PRRSV show different degrees of protection against homologous and heterologous challenges (Cano et al., 2007; Díaz et al., 2012; Geldhof et al., 2012); the exact definition of what constitutes a homologous or heterologous challenge is often not clear, especially taking into consideration the genetic diversity existing within PRRSV Type 2 (Shi et al., 2010b). Five major PRRSV vaccines are commercialized in the United States, each developed using a different wild PRRSV isolate (lineages 1, 5, 7, and 8, with the lineage 5 vaccine being the most widely used historically).

Porcine reproductive and respiratory syndrome virus is known to possess a high mutation rate (Hanada et al., 2005; Brar et al., 2014). Genetic mutations for PRRSV are thought to result from RNA polymerase errors (Murtaugh et al., 2010) and from the lack of proofreading (Kappes and Faaberg, 2015). Coupled to that, genetic recombination events can contribute to PRRSV diversity (Forsberg et al., 2002). Thus, the emergence of new variants of PRRSV is expected to occur potentially through both mutation and recombination. Viral variants can quickly emerge in animals (Goldberg et al., 2003) even after inoculation with a single variant (Chang et al., 2002). Thus, the viral population within an animal can be referred to as a viral cloud or swarm (Lauring and Andino, 2010), which suggests that mutation has a considerable impact in virus diversification even on short time scales. In addition, it is assumed that the immune response removes genetic variants of the virus that it recognizes with high specificity, potentially creating selection pressure favoring antigenically divergent PRRSV variants (Murtaugh et al., 2010). Hypervariable portions of the viral genome may be subject to immune selective pressure (Chen et al., 2016); variation in proteins coded by those sites may play a role in evasion of host immune defenses (Ansari et al., 2006; Darwich et al., 2011). PRRSV vaccines are known to diminish the severity of clinical signs once an infection occurs, but not to prevent an infection from occurring (Lyoo, 2015). At the population scale, it can be expected that most animals have some level of immunity because of the high prevalence of natural infection and widespread use of vaccine. This creates the potential for immune-mediated selection to be a driver of PRRSV diversification and evolution (Murtaugh et al., 2010).

The identification of point mutations that are undergoing positive selective pressure is often interpreted as evidence of increased evolutionary fitness (Kryazhimskiy and Plotkin, 2008). One way to identify such sites is to evaluate dN/dS ratios, which measure the rate at which substitutions at non-synonymous sites (dN) occur relative to substitutions in synonymous sites (dS). Substitutions in synonymous sites are thought to be mostly neutral, but a higher occurrence of substitutions in non-synonymous sites can be interpreted as evidence of selective processes that favor changes in the protein sequence (Kosakovsky Pond and Frost, 2005). Positive selective pressure in sites that code for epitopes recognized by the host immune system are of special interest, because they suggest that the origin of such selective pressure, if present, could be driven by the host immune response.

The rapid evolution of PRRSV coupled with the periodic emergence of new and sometimes more virulent viral variants creates a need to continually update our knowledge on circulating PRRSV variants. Reports that show the waxing and waning of different viral types in the whole North America (Shi et al., 2010b) are helpful when understanding continent-wide status of PRRSV lineages. However, understanding viral dynamics on a regional scale could provide important insights into local evolutionary and ecological dynamics of PRRSV, including an improved understanding of how often new variants emerge or re-emerge within the region. Here, we describe the temporal dynamics of PRRSV occurrence in a swine-dense region of the United States, characterizing these patterns according to ORF5 genetic lineages and sub-lineages. We quantify the contemporary occurrence of each lineage, investigate the temporal dynamics and turnover of lineages, identify emerging sub-lineages, and examine evolutionary patterns for evidence of positive selective pressures.

Materials and Methods

Sequences available through the Morrison Swine Health Monitoring Project (MSHMP) were used for this analysis. Briefly, MSHMP is an ongoing voluntary producer-driven nation-wide monitoring program for endemic swine diseases that affect the U.S. swine industry. Based at the University of Minnesota (UMN), this program collects weekly reports on the infection status of sow farms from participating swine-producing companies, veterinary practices, and regional control programs, which serves to capture the occurrence of infectious diseases in the country (Tousignant et al., 2015a, b; Perez et al., 2016). Infection status data classifies farms into the following categories (Holtkamp et al., 2011): Status 1: positive-unstable, Status 2: positive-stable, either through use of live virus inoculation (2lvi) or use of vaccines (2vx); Status 3: provisional negative; and Status 4: negative. The main difference between positive-unstable (Status 1) and positive-stable (Status 2vx or 2lvi) is that unstable herds have an active clinical outbreak and are weaning PRRSV RT-PCR positive piglets. In contrast, PRRSV may be still present in positive-stable herds (through use of field virus inoculation or modified live vaccine) but clinical disease is controlled and piglets weaned from such farms are PRRSV-negative as a result of herd immunity, decreased shedding, and maternal antibodies (Holtkamp et al., 2011). MSHMP collects farm-level data from approximately 3.2 million sows, which represents approximately 50.5% of the United States breeding herd population (National Agricultural Statistics Service [NASS], Agricultural Statistics Board, and United States Deparment of Agriculture [USDA], 2018). Specific production systems (companies involved in pig production) participating in the project also share the ORF5 PRRSV sequences identified on their farms as part of routine veterinary management. For example, samples may be submitted by veterinary practitioners to determine if circulating PRRSV on the farm is the same or different from the vaccine virus or a previous variant present on the farm.

For this analysis, we analyzed 4,390 sequences reported between 2009 and 2017 from MSHMP participants located in a relatively isolated swine-dense region in the United States with an approximate area of 250 thousand square kilometers. Production systems operating in this region account for ∼12% of the United States sow population. Approximately 90% of farms within this region participate in MSHMP and in this project in particular. Sequences used in this study came mostly from sow (64.9% of sequences), nursery (16.8%) and finisher farms (14.7%), followed by boar stud farms (0.3%) and sequences without a description of their origin (3.3%). Sequences shared with us by project participants were sequenced according to standardized protocols adopted by laboratories at SDSU (Animal Disease Research and Diagnostic Laboratory et al., 2017), ISU (Zhang et al., 2017) and Eurofins Genomics. Of the ORF5 gene sequences used in this analysis, seven had fewer than 550 nucleotides. These were deemed incomplete and were excluded from further analysis. We also included 841 ORF5 gene sequences previously classified into nine different genetic lineages (Shi et al., 2010a, b) and added these to the collection of MSHMP sequences. These sequences, assembled from a database of sequences that spanned from 1989 to 2008, were used as guides to classify the MSHMP sequences into the previously described genetic lineages, and will be referred to here as “anchor” sequences. We also obtained the ORF5 gene sequences for five vaccines (Ingelvac PRRSV ATP – GenBank ID DQ988080.1, Ingelvac PRRSV MLV – GenBank ID AF066183.4 (both from Boehringer Ingelheim), Fostera PRRSV from Zoetis – GenBank ID KP300938.1, Prime Pac PRRSV RR from Merck – GenBank ID DQ779791.1, and Prevacent, from Elanco – GenBank ID KU131568.1). The Ingelvac PRRSV ATP and Fostera vaccines use isolates belonging to lineage 8, while Ingelvac PRRSV MLV uses a lineage 5 isolate, Prime Pac a lineage 7 isolate and Prevacent a lineage 1 isolate. We also obtained two PRRSV prototypes (Lelystad – GenBank ID NC_043487.1, and VR2332 – GenBank ID EF536003.1, which represent the prototypical European Type 1 and North American Type 2 viruses, respectively). The sequence dataset used here is available in Genbank under the accession numbers MN498289 – MN502669.

Sequences were aligned using the MUSCLE algorithm implemented in AliView (Larsson, 2014) using default settings. The alignment was then examined for the presence of recombinants using the Recombinant Detection Program version 4 (Martin et al., 2015), followed by removal of potential recombinants. In addition, duplicated sequences (with 100% nucleotide similarity) were identified and set aside for the allocation of sequences into lineages. The aligned and cleaned dataset was imported into Mega 7 (Kumar et al., 2016), where the genetic pairwise distance was measured as a percentage nucleotide difference. Using Stata 15 (StataCorp, 2017), each of the MSHMP sequences were assigned to the lineage that had the smallest genetic distance to an anchor. After sequences were classified into lineages, the duplicated sequences were allocated to their respective lineage group according to the sequence with 100% similarity that was kept in the lineage classification process. A flow-chart of these steps can be seen in Figure 1.

A maximum likelihood phylogenetic tree illustrating genetic relatedness of sequences was constructed based on 1,000 bootstraps, adopting the Tamura-Nei model for substitution of amino acids (Tamura and Nei, 1993; Kumar et al., 2016). ClusterPicker software was used to further stratify the most abundant lineage into sub-lineages (Ragonnet-Cronin et al., 2013), in a matter that seemed consistent with the tree main branches while still returning epidemiological meaningful sub-lineages. The phylogenetic tree was then colored according to the lineage classification and source of sequences (anchor versus MSHMP) using Microreact (Argimón et al., 2016). Traditional bootstrap support is estimated based on resampling and replication, which tends to yield low support particularly on deep branches and in large trees with hundreds or thousands of sequences (Lemoine et al., 2018). Branch support on the phylogenetic tree thus was evaluated using the bootstrap support by the transfer method (Lemoine et al., 2018). This method circumvents issues of traditional bootstrapping by assigning a gradual “transfer” index to each clade within the tree rather than a binary presence/absence index for the presence of a clade in each bootstrap (i.e., a clade is considered absent in the bootstrap replicate if the sequences found within the clade is different by even a single member). Temporal changes in the frequency of different lineages was tabulated by quarter of the year. Graphs representing the relative frequency of PRRSV lineages over time were constructed using Stata 15. The frequency with which each lineage occurred over different years was compared using trend analysis for proportions (using the ptrend command) in Stata 15 (StataCorp, 2017). For this test only, lineages with fewer than 10 sequences overall were grouped.

The ratio of synonymous to non-synonymous mutations (dN/dS) for all sites in the ORF5 gene region was calculated using the Single-Likelihood Ancestor Counting protocol (Kosakovsky Pond and Frost, 2005), implemented on the Datamonkey webserver (Pond and Frost, 2005). Because the analysis can only be performed on 500 sequences at a time, the analysis was repeated on ten random subsets of 500 sequences (after removal of 100% identical sequences). Sites were considered under positive selective pressure if the p-value associated with a higher rate of non-synonymous versus synonymous mutations was smaller than 0.05. The dN/dS (re-scaled for branch length) of all sites from different runs were averaged and the percentage of runs in which each codon was identified as under significant positive selection was calculated.

Lineage Classification

After removal of the seven inadequately sized and two recombinant sequences from the MSHMP data, the remaining 4,381 MSHMP sequences were classified in five different lineages. 70.9% (3,110 sequences) were classified as lineage 1, 10.0% (436) as lineage 5, 0.2% (9) as lineage 7, 2.2% (94) as lineage 8, and 9.2% (404) as lineage 9. A group of 7.5% (328) of the MSHMP sequences were genetically closer to the European Prototype (Lelystad) reference, and were thus classified as Type 1 PRRSV sequences. Lineage 1 was further separated into five sub-lineages (A to E). Out of the total 3,110 sequences in lineage 1, 48.7% (1515) were classified in lineage 1A, 13.9% (433) in lineage 1B, 37.2% (1157) in lineage 1C, 0.03% (1) in lineage 1D and 0.1% (4) in lineage 1E. The phylogenetic tree with all sequences used in the analysis can be seen on Figure 2. Using the Booster method (Lemoine et al., 2018), branch support on main branches (lineages and sub-lineages) was above 90%. The within- and between-lineage nucleotide pairwise genetic distance is shown in Table 1. In general, between lineage/sub-lineage distances are higher than within lineage variation. The distances between sub-lineages of lineage 1 seem to be smaller between them than between other lineages. Broad tree topology was similar when the tree was constructed using nucleotides or amino acids alignment (Supplementary Figure S2).

Temporal Dynamics of Lineage Occurrence

On average, the total number of sequences reported to MSHMP increased by 46 each year (Supplementary Table S1), and there was a clear seasonal pattern (Figure 3B). The first quarter of each year (January – March) was the one with highest number of sequences reported in all but 1 year. The relative frequency of each lineage changed through time (Figure 3A and Supplementary Table S1), and specific patterns are noteworthy. First, the absolute and relative occurrence of lineage 9 decreased over time from 68.4% (149 sequences) in 2009 to <1% (5 sequences) in the years 2014–2017. As lineage 9 occurrence declined, lineage 1 occurrence increased until it represented >60% of sequences reported in the period spanning 2011–2017. Within lineage 1, turnover in the dominant sub-lineages is apparent as the relative frequency of lineage 1C between 2009 and 2011 rose from 11.5% to 55.2%, then subsequently declined to approximately 10% of the sequences reported in years 2014–2017. Somewhat concurrently to the emergence of sub-lineage 1C, sub-lineage 1B increased from 1.8% to 27.4% in 2013, then subsequently declined to <2% of sequences reported in 2016 and 2017. Concomitant with the decrease in occurrence of lineage 1C and 1B was a sharp increase in the occurrence of lineage 1A. A single sequence of lineage 1A was observed in 2009, after which this sub-lineage was not detected in any subsequent years until 2014, at which point it was responsible for 37.3% of the sequences. By 2015, almost 75% of sequences belonged to this sub-lineage. Since then, the frequency in which this lineage has occurred decreased (68.4 and 57.3% of the sequences from 2016 and 2017, respectively).

To determine whether changes in sampling effort across time impacted general patterns observed here, we repeated the analysis five times, each time randomly sampling 50 ORF5 sequences per quarter. General patterns of lineage occurrence did not change, suggesting that patterns of lineage occurrence were not affected by sampling effort in each quarter (Supplementary Figure S1).

The visual patterns and turnover of lineages apparent in Figure 3A were shown to be statistically significant. The increase in the frequency of lineages 1A, 5, 9, and type 1 (p < 0.001) was significant, and changes in the grouped frequency of other lineages (a sum of lineages 1D, 1E, and 7, p = 0.0472) was also significant, but with a difficult interpretation since this is an aggregate of several uncommon lineages. Lineages 1B and 1C increased in frequency and then decreased (p < 0.001). Lineage 9 frequency decreased over time (p-value < 0.001), while lineage 8 occurrence remained unchanged (p-value = 0.958).

Evidence for Positive Selective Pressure

A total of 26 sites were identified as under positive selection in at least one Single-Likelihood Ancestor Counting run (Figure 4). Some sites were identified as under positive selection in all 10 runs, while others were only identified in some runs. Those identified in all runs (with the largest p-value across all runs), were sites 14 (p-value = 0.045), 30 (p-value = 0.012), 32 (p-value < 0.001), 33 (p-value < 0.001), 34 (p-value < 0.001), 35 (p-value < 0.001), 58 (p-value = 0.005), and 104 (p-value = 0.029). A list of all sites identified as under positive selection in at least one run can be found in the caption of Figure 4. Most of the sites positively selected were located in the first third of the PRRSV ORF5.

The infection status of farms part of MSHMP in the studied area over the study time span is shown in Figure 5. This data show two periods in which vaccine usage increased, the first one in mid-2012, and a second in approximately mid-2014. Not all farms that reported its status to MSHMP contributed to sequences to this analysis.

Future Research

Immune interaction between infections of differing PRRSV isolates remains poorly understood in swine. The vast adoption of control protocols that rely on imperfect immune response aimed mostly at reducing severity of upcoming infections (such as pre-exposure protocols with commercial vaccines or with lvi) suggests that a better understanding of the cross-immunity generated by infection with different isolates of the virus would be valuable to the industry as a whole. Prospective studies that obtain sera from sow farms under different pre-exposure regimens and follow the farms through time recording PRRSV occurrence would provide valuable information of potential cross-immunity in field conditions. Of interest also is the better understanding of how the spread different lineages/sub-lineages are related to epidemiological data, for example, animal movement data and farm proximity. This might allow for a better comprehension of drivers for PRRSV transmission while allowing for the evaluation of the effectiveness of practices aimed at reducing PRRSV risk (dead animals disposal, manure composting, filtering the air of farms, to name a few).

This study reflects data from a single United States region, which possibly does not reflect PRRSV diversity and temporal dynamics of the whole swine industry in the country (Shi et al., 2010b). That being said, the data presented here reflects a substantial portion of the U.S. swine industry in a region that is relatively spatially discontinuous from other swine producing regions in the United States. In addition, the general pattern of emergence and turnover of different lineages over time observed here describe an evolutionary phenomenon that is expected to also occur in other United States regions. A better understanding of the natural history of PRRSV can provide insights that can potentially aid in mitigating the impact of the emergence of new viral variants as well as serving as a basis for further work exploring the evolution of PRRSV and the effect this has on disease control, management and impact on the industry.

Conclusion

Here, we describe the occurrence of PRRSV over 9 years in a single United States region. We identified the emergence and turnover of different lineages and sub-lineages in the commercial pig population. Such rapid turnover in the dominant lineage through time suggests that temporal patterns of PRRSV occurrence are characterized by multi-strain dynamics, where different PRRSV variants potentially interact through immune-mediated competition or selection. However, cross-immunity between different PRRSV lineages elicited by natural or intentional infection is not fully understood, which hinders the effectiveness of disease control. More research is needed on drivers of evolution and emergence of new sub-lineages in order for the industry to be able to predict, prevent, and mitigate the impacts of PRRSV. Ongoing surveillance for PRRSV using molecular epidemiological methods is invaluable to characterize the evolution of the virus but also to identify recent and historical trends that help understanding the natural history of PRRSV in the United States.

Data Availability Statement

The sequence dataset used here is available in GenBank under the accessions numbers MN498289–MN502669.

Author Contributions

IP and KV analyzed, conceptualized, and designed the study. CC, JS, CV, and KV contributed to acquisition of the data. IP, CC, AR, JS, CV, DS, and KV interpreted the data. MM aided in early interpretation of data. All authors but MM were involved in drafting the manuscript and revising it critically for intellectual content and have given final approval of the version to be published.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.