Concomitant Transmission of Dengue, Chikungunya, and Zika Viruses in Brazil: Clinical and Epidemiological Findings From Surveillance for Acute Febrile Illness.

BACKGROUND: Since their emergence in the Americas, chikungunya (CHIKV) and Zika (ZIKV) viruses co-circulate with dengue virus (DENV), hampering clinical diagnosis. We investigated clinical and epidemiological characteristics of arboviral infections during the introduction and spread of CHIKV and ZIKV through northeastern Brazil. METHODS: Surveillance for arboviral diseases among febrile patients was performed at an emergency health unit of Salvador, Brazil, between September 2014 and July 2016. We interviewed patients to collect data on symptoms, reviewed medical records to obtain the presumptive diagnoses, and performed molecular and serological testing to confirm DENV, CHIKV, ZIKV, or nonspecific flavivirus (FLAV) diagnosis. RESULTS: Of 948 participants, 247 (26.1%) had an acute infection, of which 224 (23.6%) were single infections (DENV, 32 [3.4%]; CHIKV, 159 [16.7%]; ZIKV, 13 [1.4%]; and FLAV, 20 [2.1%]) and 23 (2.4%) coinfections (DENV/CHIKV, 13 [1.4%]; CHIKV/FLAV, 9 [0.9%]; and DENV/ZIKV, 1 [0.1%]). An additional 133 (14.0%) patients had serological evidence for a recent arboviral infection. Patients with ZIKV presented with rash and pruritus (69.2% each) more frequently than those with DENV (37.5% and 31.2%, respectively) and CHIKV (22.9% and 14.7%, respectively) (P < .001 for both comparisons). Conversely, arthralgia was more common in CHIKV (94.9%) and FLAV/CHIKV (100.0%) than in DENV (59.4%) and ZIKV (53.8%) (P < .001). A correct presumptive clinical diagnosis was made for 9%-23% of the confirmed patients. CONCLUSIONS: Arboviral infections are frequent causes of febrile illness. Coinfections are not rare events during periods of intense, concomitant arboviral transmission. Given the challenge to clinically distinguish these infections, there is an urgent need for rapid, point-of-care, multiplex diagnostics.

Diagnostic performance of commercial IgM and IgG enzyme-linked immunoassays (ELISAs) for diagnosis of Zika virus infection.

BACKGROUND: Serologic detection of Zika virus (ZIKV) infections is challenging because of antigenic similarities among flaviviruses. OBJECTIVE: To evaluate the sensitivity and specificity of commercial ZIKV IgM and IgG enzyme-linked immunoassay (ELISA) kits. METHODS: We used sera from febrile patients with RT-PCR-confirmed ZIKV infection to determine sensitivity and sera from RT-PCR-confirmed dengue cases and blood donors, both of which were collected before ZIKV epidemics in Brazil (2009-2011 and 2013, respectively) to determine specificity. RESULTS: The ZIKV IgM-ELISA positivity among RT-PCR ZIKV confirmed cases was 0.0% (0/14) and 12.5% (1/8) for acute- and convalescent-phase sera, respectively, while its specificity was 100.0% (58/58) and 98.3% (58/59) for acute- and convalescent-phase sera of dengue patients, and 100.0% (23/23) for blood donors. The ZIKV IgG-ELISA sensitivity was 100.0% (6/6) on convalescent-phase sera from RT-PCR confirmed ZIKV patients, while its specificity was 27.3% (15/55) on convalescent-phase sera from dengue patients and 45.0% (9/20) on blood donors' sera. The ZIKV IgG-ELISA specificity among dengue confirmed cases was much greater among patients with primary dengue (92.3%; 12/13), compared to secondary dengue (7.1%; 3/42). CONCLUSIONS: In a setting of endemic dengue transmission, the ZIKV IgM-ELISA had high specificity, but poor sensitivity. In contrast, the ZIKV IgG-ELISA showed low specificity, particularly for patients previously exposed to dengue infections. This suggests that this ZIKV IgM-ELISA is not useful in confirming a diagnosis of ZIKV infection in suspected patients, whereas the IgG-ELISA is more suitable for ZIKV diagnosis among travelers, who reside in areas free of flavivirus transmission, rather than for serosurveys in dengue-endemic areas.

Congenital brain abnormalities during a Zika virus epidemic in Salvador, Brazil, April 2015 to July 2016.

BackgroundNorth-eastern Brazil was the region most affected by the outbreak of congenital Zika syndrome that followed the 2015 Zika virus (ZIKV) epidemics, with thousands of suspected microcephaly cases reported to the health authorities, mostly between late 2015 and early 2016. Aim: To describe clinical and epidemiological aspects of the outbreak of congenital brain abnormalities (CBAs) and to evaluate the accuracy of different head circumference screening criteria in predicting CBAs.MethodBetween April 2015 and July 2016, the Centers for Information and Epidemiologic Surveillance of Salvador, Brazil investigated the reported cases suspected of microcephaly and, based on intracranial imaging studies, confirmed or excluded a diagnosis of CBA. Sensitivity, specificity and positive and negative predictive values of different head circumference screening criteria in predicting CBAs were calculated.ResultsOf the 365 investigated cases, 166 (45.5%) had confirmed CBAs. The most common findings were intracranial calcifications and ventriculomegaly in 143 (86.1%) and 111 (66.9%) of the 166 CBA cases, respectively. Prevalence of CBAs peaked in December 2015 (2.24 cases/100 live births). Cases of CBAs were significantly more likely to have been born preterm and to mothers who had clinical manifestations of arboviral infection during pregnancy. None of the head circumference screening criteria performed optimally in predicting CBAs.ConclusionThis study highlights the magnitude of neurological consequences of the ZIKV epidemic and the limitations of head circumference in accurately identifying children with CBA. Gestational symptoms compatible with ZIKV infection should be combined with imaging studies for efficient detection of suspect CBAs during ZIKV epidemics.

Variation in Aedes aegypti Mosquito Competence for Zika Virus Transmission.

To test whether Zika virus has adapted for more efficient transmission by Aedes aegypti mosquitoes, leading to recent urban outbreaks, we fed mosquitoes from Brazil, the Dominican Republic, and the United States artificial blood meals containing 1 of 3 Zika virus strains (Senegal, Cambodia, Mexico) and monitored infection, dissemination, and virus in saliva. Contrary to our hypothesis, Cambodia and Mexica strains were less infectious than the Senegal strain. Only mosquitoes from the Dominican Republic transmitted the Cambodia and Mexica strains. However, blood meals from viremic mice were more infectious than artificial blood meals of comparable doses; the Cambodia strain was not transmitted by mosquitoes from Brazil after artificial blood meals, whereas 61% transmission occurred after a murine blood meal (saliva titers up to 4 log 10 infectious units/collection). Although regional origins of vector populations and virus strain influence transmission efficiency, Ae. aegypti mosquitoes appear to be competent vectors of Zika virus in several regions of the Americas.

Time Lags between Exanthematous Illness Attributed to Zika Virus, Guillain-Barré Syndrome, and Microcephaly, Salvador, Brazil.

Zika virus infection emerged as a public health emergency after increasing evidence for its association with neurologic disorders and congenital malformations. In Salvador, Brazil, outbreaks of acute exanthematous illness (AEI) attributed to Zika virus, Guillain-Barré syndrome (GBS), and microcephaly occurred in 2015. We investigated temporal correlations and time lags between these outbreaks to identify a common link between them by using epidemic curves and time series cross-correlations. Number of GBS cases peaked after a lag of 5-9 weeks from the AEI peak. Number of suspected cases of microcephaly peaked after a lag of 30-33 weeks from the AEI peak, which corresponded to time of potential infections of pregnant mothers during the first trimester. These findings support the association of GBS and microcephaly with Zika virus infection and provide evidence for a temporal relationship between timing of arboviral infection of pregnant women during the first trimester and birth outcome.

Influenza-like illness in an urban community of Salvador, Brazil: incidence, seasonality and risk factors.

BACKGROUND: Our understanding of the epidemiology of influenza is limited in tropical regions, which in turn has hampered identifying optimal region-specific policy to diminish disease burden. Influenza-like illness (ILI) is a clinical diagnosis that can be used as a surrogate for influenza. This study aimed to define the incidence and seasonality of ILI and to assess its association with climatic variables and school calendar in an urban community in the tropical region of Salvador, Brazil. METHODS: Between 2009 and 2013, we conducted enhanced community-based surveillance for acute febrile illnesses (AFI) among patients ≥ 5 years of age in a slum community emergency unit in Salvador, Brazil. ILI was defined as a measured temperature of ≥ 37.8 °C or reported fever in a patient with cough or sore throat for ≤ 7 days, and negative test results for dengue and leptospirosis. Seasonality was analyzed with a harmonic regression model. Negative binomial regression models were used to correlate ILI incidence with rainfall, temperature, relative humidity and the number of days per month that schools were in session while controlling for seasonality. RESULTS: There were 2,651 (45.6% of 5,817 AFI patients) ILI cases with a mean annual incidence of 60 cases/1,000 population (95% CI 58-62). Risk of ILI was highest among 5-9 year olds with an annual incidence of 105 cases/1,000 population in 2009. ILI had a clear seasonal pattern with peaks between the 35-40th week of the year. ILI peaks were higher and earlier in 5-9 year olds compared with > 19 year olds. No association was seen between ILI and precipitation, relative humidity or temperature. There was a significant association between the incidence of ILI in children 5-9 years of age and number of scheduled school days per month. CONCLUSIONS: We identified a significant burden of ILI with distinct seasonality in the Brazilian tropics and highest rates among young school-age children. Seasonal peaks of ILI in children 5-9 years of age were positively associated with the number of school days, indicating that children may play a role in the timing of seasonal influenza transmission.

Unveiling invisible farm-to-farm PRRSV-2 transmission links and routes through transmission tree and network analysis.

The United States (U.S.) swine industry has struggled to control porcine reproductive and respiratory syndrome (PRRS) for decades, yet the causative virus, PRRSV-2, continues to circulate and rapidly diverges into new variants. In the swine industry, the farm is typically the epidemiological unit for monitoring, prevention, and control; breaking transmission among farms is a critical step in containing disease spread. Despite this, our understanding of farm transmission still is inadequate, precluding the development of tailored control strategies. Therefore, our objective was to infer farm-to-farm transmission links, estimate farm-level transmissibility as defined by reproduction numbers (R), and identify associated risk factors for transmission using PRRSV-2 open reading frame 5 (ORF5) gene sequences, animal movement records, and other data from farms in a swine-dense region of the U.S. from 2014 to 2017. Timed phylogenetic and transmission tree analyses were performed on three sets of sequences (n = 206) from 144 farms that represented the three largest genetic variants of the virus in the study area. The length of inferred pig-to-pig infection chains that corresponded to pairs of farms connected via direct animal movement was used as a threshold value for identifying other feasible transmission links between farms; these links were then transformed into farm-to-farm transmission networks and calculated farm-level R-values. The median farm-level R was one (IQR = 1-2), whereas the R value of 28% of farms was more than one. Exponential random graph models were then used to evaluate the influence of farm attributes and/or farm relationships on the occurrence of farm-to-farm transmission links. These models showed that, even though most transmission events cannot be directly explained by animal movement, movement was strongly associated with transmission. This study demonstrates how integrative techniques may improve disease traceability in a data-rich era by providing a clearer picture of regional disease transmission.

Mapping the Dynamics of Contemporary PRRSV-2 Evolution and Its Emergence and Spreading Hotspots in the U.S. Using Phylogeography.

The repeated emergence of new genetic variants of PRRSV-2, the virus that causes porcine reproductive and respiratory syndrome (PRRS), reflects its rapid evolution and the failure of previous control efforts. Understanding spatiotemporal heterogeneity in variant emergence and spread is critical for future outbreak prevention. Here, we investigate how the pace of evolution varies across time and space, identify the origins of sub-lineage emergence, and map the patterns of the inter-regional spread of PRRSV-2 Lineage 1 (L1)-the current dominant lineage in the U.S. We performed comparative phylogeographic analyses on subsets of 19,395 viral ORF5 sequences collected across the U.S. and Canada between 1991 and 2021. The discrete trait analysis of multiple spatiotemporally stratified sampled sets (n = 500 each) was used to infer the ancestral geographic region and dispersion of each sub-lineage. The robustness of the results was compared to that of other modeling methods and subsampling strategies. Generally, the spatial spread and population dynamics varied across sub-lineages, time, and space. The Upper Midwest was a main spreading hotspot for multiple sub-lineages, e.g., L1C and L1F, though one of the most recent emergence events (L1A(2)) spread outwards from the east. An understanding of historical patterns of emergence and spread can be used to strategize disease control and the containment of emerging variants.

Refining PRRSV-2 genetic classification based on global ORF5 sequences and investigation of their geographic distributions and temporal changes.

IMPORTANCE: In this study, comprehensive analysis of 82,237 global porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) open reading frame 5 sequences spanning from 1989 to 2021 refined PRRSV-2 genetic classification system, which defines 11 lineages and 21 sublineages and provides flexibility for growth if additional lineages, sublineages, or more granular classifications are needed in the future. Geographic distribution and temporal changes of PRRSV-2 were investigated in detail. This is a thorough study describing the molecular epidemiology of global PRRSV-2. In addition, the reference sequences based on the refined genetic classification system are made available to the public for future epidemiological and diagnostic applications worldwide. The data from this study will facilitate global standardization and application of PRRSV-2 genetic classification.

Porcine Reproductive and Respiratory Syndrome (PRRSV2) Viral Diversity within a Farrow-to-Wean Farm Cohort Study.

Describing PRRSV whole-genome viral diversity data over time within the host and within-farm is crucial for a better understanding of viral evolution and its implications. A cohort study was conducted at one naïve farrow-to-wean farm reporting a PRRSV outbreak. All piglets 3-5 days of age (DOA) born to mass-exposed sows through live virus inoculation with the recently introduced wild-type virus two weeks prior were sampled and followed up at 17-19 DOA. Samples from 127 piglets were individually tested for PRRSV by RT-PCR and 100 sequences were generated using Oxford Nanopore Technologies chemistry. Female piglets had significantly higher median Ct values than males (15.5 vs. 13.7, Kruskal-Wallis p < 0.001) at 3-5 DOA. A 52.8% mortality between sampling points was found, and the odds of dying by 17-19 DOA decreased with every one unit increase in Ct values at 3-5 DOA (OR = 0.76, 95% CI 0.61-0.94, p = 0.01). Although the within-pig percent nucleotide identity was overall high (99.7%) between 3-5 DOA and 17-19 DOA samples, ORFs 4 and 5a showed much lower identities (97.26% and 98.53%, respectively). When looking solely at ORF5, 62% of the sequences were identical to the 3-5 DOA consensus. Ten and eight regions showed increased nucleotide and amino acid genetic diversity, respectively, all found throughout ORFs 2a/2b, 4, 5a/5, 6, and 7.

Dynamic network connectivity influences the spread of a sub-lineage of porcine reproductive and respiratory syndrome virus.

Swine production in the United States is characterized by dynamic farm contacts through animal movements; such movements shape the risk of disease occurrence on farms. Pig movements have been linked to the spread of a virulent porcine reproductive and respiratory syndrome virus (PRRSV), RFLP type 1-7-4, herein denoted as phylogenetic sub-lineage 1A [L1A]. This study aimed to quantify the contribution of pig movements to the risk of L1A occurrence on farms in the United States. Farms were defined as L1A-positive in a given 6-month period if at least one L1A sequence was recovered from the farm. Temporal network autocorrelation modelling was performed using data on animal movements and 1,761 PRRSV ORF5 sequences linked to 494 farms from a dense pig production area in the United States between 2014 and 2017. A farm's current and past exposure to L1A and other PRRSV variants was assessed through its primary and secondary contacts in the animal movement network. Primary and secondary contacts with an L1A-positive farm increased the likelihood of L1A occurrence on a farm by 19% (p = .04) and 23% (p = .03), respectively. While the risk posed by primary contacts with PRRS-positive farms is unsurprising, the observation that secondary contacts also increase the likelihood of infection is novel. Risk of L1A occurrence on a farm also increased by 3.0% (p = .01) for every additional outgoing shipment, possibly due to biosecurity breaches during loading and transporting pigs from the farm. Finally, use of vaccines or field virus inoculation on sow farms one year prior reduced the risk of L1A occurrence in downstream farms by 36% (p = .04), suggesting that control measures that reduce viral circulation and enhance immunological protection in sow farms have a carry-over effect on L1A occurrence in downstream farms. Therefore, coordinated disease management interventions between farms connected via animal movements may be more effective than individual farm-based interventions.

Measuring How Recombination Re-shapes the Evolutionary History of PRRSV-2: A Genome-Based Phylodynamic Analysis of the Emergence of a Novel PRRSV-2 Variant.

While the widespread and endemic circulation of porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) causes persistent economic losses to the U.S. swine industry, unusual increases of severe cases associated with the emergence of new genetic variants are a major source of concern for pork producers. Between 2020 and 2021, such an event occurred across pig production sites in the Midwestern U.S. The emerging viral clade is referred to as the novel sub-lineage 1C (L1C) 1-4-4 variant. This genetic classification is based on the open reading frame 5 (ORF5) gene. However, although whole genome sequence (WGS) suggested that this variant represented the emergence of a new strain, the true evolutionary history of this variant remains unclear. To better elucidate the variant's evolutionary history, we conducted a recombination detection analysis, time-scaled phylogenetic estimation, and discrete trait analysis on a set of L1C-1-4-4 WGSs (n = 19) alongside other publicly published WGSs (n = 232) collected over a 26-year period (1995-2021). Results from various methodologies consistently suggest that the novel L1C variant was a descendant of a recombinant ancestor characterized by recombination at the ORF1a gene between two segments that would be otherwise classified as L1C and L1A in the ORF5 gene. Based on analysis of different WGS fragments, the L1C-1-4-4 variant descended from an ancestor that existed around late 2018 to early 2019, with relatively high substitution rates in the proximal ORF1a as well as ORF5 regions. Two viruses from 2018 were found to be the closest relatives to the 2020-21 outbreak strain but had different recombination profiles, suggesting that these viruses were not direct ancestors. We also assessed the overall frequency of putative recombination amongst ORF5 and other parts of the genome and found that recombination events which leave detectable numbers of descendants are not common. However, the rapid spread and high virulence of the L1C-1-4-4 recombinant variant demonstrates that inter-sub-lineage recombination occasionally found amongst the U.S. PRRSV-2 might be an evolutionary mechanisms that contributed to this emergence. More generally, recombination amongst PRRSV-2 accelerates genetic change and increases the chance of the emergence of high fitness variants.

Potential Novel N-Glycosylation Patterns Associated with the Emergence of New Genetic Variants of PRRSV-2 in the U.S.

Glycosylation of proteins is a post-translational process where oligosaccharides are attached to proteins, potentially altering their folding, epitope availability, and immune recognition. In Porcine reproductive and respiratory syndrome virus-type 2 (PRRSV-2), positive selection pressure acts on amino acid sites potentially associated with immune escape through glycan shielding. Here, we describe the patterns of potential N-glycosylation sites over time and across different phylogenetic lineages of PRRSV-2 to better understand how these may contribute to patterns of coexistence and emergence of different lineages. We screened 19,179 PRRSV GP5 sequences (2004−2021) in silico for potential N-glycosylated sites. The emergence of novel combinations of N-glycosylated sites coincided with past PRRSV epidemics in the U.S. For lineage L1A, glycosylation at residues 32, 33, 44, 51, and 57 first appeared in 2012, but represented >62% of all L1A sequences by 2015, coinciding with the emergence of the L1A 1-7-4 strain that increased in prevalence from 8 to 86% of all L1A sequences from 2012 to 2015. The L1C 1-4-4 strain that emerged in 2020 also had a distinct N-glycosylation pattern (residues 32, 33, 44, and 51). From 2020 to 2021, this pattern was responsible for 44−47% of the L1C sequences, contrasting to <5% in years prior. Our findings support the hypothesis that antigenic evolution contributes to the sequential dominance of different PRRSV strains and that N-glycosylation patterns may partially account for antigenic differences amongst strains. Further studies on glycosylation and its effect on PRRSV GP5 folding are needed to further understand how glycosylation patterns shape PRRSV occurrence.

Ecological and evolutionary dynamics of multi-strain RNA viruses.

Potential interactions among co-circulating viral strains in host populations are often overlooked in the study of virus transmission. However, these interactions probably shape transmission dynamics by influencing host immune responses or altering the relative fitness among co-circulating strains. In this Review, we describe multi-strain dynamics from ecological and evolutionary perspectives, outline scales in which multi-strain dynamics occur and summarize important immunological, phylogenetic and mathematical modelling approaches used to quantify interactions among strains. We also discuss how host-pathogen interactions influence the co-circulation of pathogens. Finally, we highlight outstanding questions and knowledge gaps in the current theory and study of ecological and evolutionary dynamics of multi-strain viruses.

Temporal stability of swine movement networks in the U.S.

As a consequence of multi-site pig production practiced in North America, frequent and widespread animal movements create extensive networks of interaction between farms. Social network analysis (SNA) has been used to understand disease transmission risks within these complex and dynamic production ecosystems and is particularly relevant for designing risk-based surveillance and control strategies targeting highly connected farms. However, inferences from SNA and the effectiveness of targeted strategies may be influenced by temporal changes in network structure. Since farm movements represent a temporally dynamic network, it is also unclear how many months of data are required to gain an accurate picture of an individual farm's connectivity pattern and the overall network structure. The extent to which shipments between two specific farms are repeated (i.e., "loyalty" of farm contacts) can influence the rate at which the structure of a network changes over time, which may influence disease dynamics. In this study, we aimed to describe temporal stability and loyalty patterns of pig movement networks in the U.S. swine industry. We analyzed a total of 282,807 animal movements among 2724 farms belonging to two production systems between 2014 and 2017. Loyalty trends were largely driven by contacts between sow farms and nurseries and between nurseries and finisher farms; mean loyalty (percent of contacts that were repeated at least once within a 52-week interval) of farm contacts was 51-60 % for farm contacts involving weaned pigs, and 12-22% for contacts involving feeder pigs. A cyclic pattern was observed for both weaned and feeder pig movements, with episodes of increased loyalty observed at intervals of 8 and 17-20 weeks, respectively. Network stability was achieved when six months of data were aggregated, and only small shifts in node-level and global network metrics were observed when adding more data. This stability is relevant for designing targeted surveillance programs for disease management, given that movements summarized over too short a period may lead to stochastic swings in network metrics. A temporal resolution of six months would be reliable for the identification of potential super-spreaders in a network for targeted intervention and disease control. The temporal stability observed in these networks suggests that identifying highly connected farms in retrospective network data (up to 24 months) is reliable for future planning, albeit with reduced effectiveness.

Integrating animal movements with phylogeography to model the spread of PRRSV in the USA.

Viral sequence data coupled with phylodynamic models have become instrumental in investigating the outbreaks of human and animal diseases, and the incorporation of the hypothesized drivers of pathogen spread can enhance the interpretation from phylodynamic inference. Integrating animal movement data with phylodynamics allows us to quantify the extent to which the spatial diffusion of a pathogen is influenced by animal movements and contrast the relative importance of different types of movements in shaping pathogen distribution. We combine animal movement, spatial, and environmental data in a Bayesian phylodynamic framework to explain the spatial diffusion and evolutionary trends of a rapidly spreading sub-lineage (denoted L1A) of porcine reproductive and respiratory syndrome virus (PRRSV) Type 2 from 2014 to 2017. PRRSV is the most important endemic pathogen affecting pigs in the USA, and this particular virulent sub-lineage emerged in 2014 and continues to be the dominant lineage in the US swine industry to date. Data included 984 open reading frame 5 (ORF5) PRRSV L1A sequences obtained from two production systems in a swine-dense production region (∼85,000 mi2) in the USA between 2014 and 2017. The study area was divided into sectors for which model covariates were summarized, and animal movement data between each sector were summarized by age class (wean: 3-4 weeks; feeder: 8-25 weeks; breeding: ≥21 weeks). We implemented a discrete-space phylogeographic generalized linear model using Bayesian evolutionary analysis by sampling trees (BEAST) to infer factors associated with variability in between-sector diffusion rates of PRRSV L1A. We found that between-sector spread was enhanced by the movement of feeder pigs, spatial adjacency of sectors, and farm density in the destination sector. The PRRSV L1A strain was introduced in the study area in early 2013, and genetic diversity and effective population size peaked in 2015 before fluctuating seasonally (peaking during the summer months). Our study underscores the importance of animal movements and shows, for the first time, that the movement of feeder pigs (8-25 weeks old) shaped the spatial patterns of PRRSV spread much more strongly than the movements of other age classes of pigs. The inclusion of movement data into phylodynamic models as done in this analysis may enhance our ability to identify crucial pathways of disease spread that can be targeted to mitigate the spatial spread of infectious human and animal pathogens.

Phylogenetic Structure and Sequential Dominance of Sub-Lineages of PRRSV Type-2 Lineage 1 in the United States.

The genetic diversity and frequent emergence of novel genetic variants of porcine reproductive and respiratory syndrome virus type-2 (PRRSV) hinders control efforts, yet drivers of macro-evolutionary patterns of PRRSV remain poorly documented. Utilizing a comprehensive database of >20,000 orf5 sequences, our objective was to classify variants according to the phylogenetic structure of PRRSV co-circulating in the U.S., quantify evolutionary dynamics of sub-lineage emergence, and describe potential antigenic differences among sub-lineages. We subdivided the most prevalent lineage (Lineage 1, accounting for approximately 60% of available sequences) into eight sub-lineages. Bayesian coalescent SkyGrid models were used to estimate each sub-lineage's effective population size over time. We show that a new sub-lineage emerged every 1 to 4 years and that the time between emergence and peak population size was 4.5 years on average (range: 2-8 years). A pattern of sequential dominance of different sub-lineages was identified, with a new dominant sub-lineage replacing its predecessor approximately every 3 years. Consensus amino acid sequences for each sub-lineage differed in key GP5 sites related to host immunity, suggesting that sub-lineage turnover may be linked to immune-mediated competition. This has important implications for understanding drivers of genetic diversity and emergence of new PRRSV variants in the U.S.

Emergence of a New Lineage 1C Variant of Porcine Reproductive and Respiratory Syndrome Virus 2 in the United States.

We report an ongoing regional outbreak of an emerging porcine reproductive and respiratory syndrome virus (PRRSV2) variant within Lineage 1C affecting 154 breeding and grow-finishing sites in the Midwestern U.S. Transmission seemed to have occurred in two waves, with the first peak of weekly cases occurring between October and December 2020 and the second starting in April 2021. Most of cases occurred within a 120 km radius. Both orf5 and whole genome sequencing results suggest that this represents the emergence of a new variant within Lineage 1C distinct from what has been previously circulating. A case-control study was conducted with 50 cases (sites affected with the newly emerged variant) and 58 controls (sites affected with other PRRSV variants) between October and December 2020. Sites that had a market vehicle that was not exclusive to the production system had 0.04 times the odds of being a case than a control. A spatial cluster (81.42 km radius) with 1.68 times higher the number of cases than controls was found. The average finishing mortality within the first 4 weeks after detection was higher amongst cases (4.50%) than controls (0.01%). The transmission of a highly similar virus between different farms carrying on trough spring rises concerns for the next high transmission season of PRRS.

Phylogenetically Distinct Near-Complete Genome Sequences of Porcine Reproductive and Respiratory Syndrome Virus Type 2 Variants from Four Distinct Disease Outbreaks at U.S. Swine Farms over the Past 6 Years.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to mutate, causing disruptive PRRS outbreaks in farms that lead to reproductive failure and respiratory disease-associated mortality. We present four new PRRSV type 2 variants in the United States belonging to four distinct orf5 sublineages within lineage 1.