Materials and Methods
A finishing pig farm (3400 heads) where five pathogens (ASFV, CSFV, PRV,
PRRSV and PCV2) had been detected was selected for the study. The farm
is located in Heilongjiang Province of China, and there is no
neighboring pig farm within 3 kilometers. The pig farm employs a fully
enclosed management model; personnel and materials entering and leaving
the area are disinfected, and the manure used to produce fertilizer was
treated appropriately. The internal layout of the pig farm is
reasonable; the buildings are at least 6 meters apart, ventilation is
good, disinfection is performed regularly, and necessary vaccines are
administered in a timely manner. Livestock are vaccinated with the
classical PRRSV modified-live virus (MLV) vaccine, classical swine fever
MLV vaccine, pseudorabies virus MLV vaccine, foot and mouth disease
(FMD) inactivated Vaccine and porcine circovirus 2 MLV vaccine. The pig
farm employs professional management and veterinary personnel.
Approximately 15-45 lung and lymph node samples from livestock on the
farm were submitted for laboratory testing approximately every 15 days
during the study period. During the breeding period, dead pigs were
dissected to collect samples, and the time of death and number of deaths
were recorded. The number of dead pigs was counted every day and summed
every 15 days. From September 2020 to January 2021, 283 clinical samples
were collected from this farm. Tissue sample disposal, RNA and DNA
extraction, cDNA preparation, RT-PCR analysis and genome sequencing were
conducted as previously described (Leng et al., 2014; Leng et al., 2012;
Zhang et al., 2015; Zhou et al., 2019). The primers used for virus
detection (AFSV, CFSV, PCV2, PRRSV and PRV) and complete genome
amplification have been reported previously (An et al., 2013; Cai et
al., 2012; Gao et al., 2017; Torres et al., 2017; Zhang et al., 2015;
Zhang et al., 2018).
All reference strains were downloaded from the NCBI, and the
corresponding sequences were compared and intercepted. Deduced amino
acid sequences were aligned by ClustalW with Lasergene software. All
sequences were aligned using MAFFT version 7 (Katoh and Standley, 2013)
with default parameters (https://mafft.cbrc.jp/alignment/software/) and
manually adjusted in MEGA6 (Tamura et al., 2013). We followed the same
rationale for the classification of sequences into lineages as
previously published (Shi et al., 2010). To identify evolutionary
relationships among strains on this farm, phylogenetic trees were
constructed in MEGA 6.0 by the neighbor-joining method with a bootstrap
value of 1,000 replicates and with the Kimura 2-parameter substitution
model. The trees were annotated and modified using Evolview (version
2.0) (https://www.evolgenius.info/evolview/#login) (He et al., 2016).
To analyze recombination events, similarity analysis was performed using
SimPlot software (v3.5), with a 200-bp window and a 20-bp step (Yu et
al., 2020).
The temporal signal in the phylogenetic datasets of NADC34-like PRRSV
was first investigated using TempEst to confirm the appropriateness of
the data for time-scaled phylogenetic tree reconstruction (Rambaut et
al., 2016). To estimate the evolution rate of the NADC34-like PRRSV
strain on this pig farm, a relaxed uncorrelated lognormal (UCLN)
molecular clock was used, with a flexible Bayesian SkyGrid plot (BSP)
demographic model and a general-time reversible model of nucleotide
substitution with gamma-distributed rate variation among sites (GTR+Γ),
allowing for partitions into codons in any of the three positions
(Barba-Montoya et al., 2020). The Markov chain Monte Carlo (MCMC)
algorithm was run for 200 million steps and sampled every 20,000 steps.
Convergence was assessed with effective sample size (ESS) values, and
ESS values over 200 were considered adequate. These analyses were
performed using BEAST (v1.10.4). Three independent runs were performed
in this study to prevent any local convergence. The BEAST results were
entered into Tracer to evaluate model convergence and consistency
between replicates.
GraphPad Prism 8.0 (San Diego, CA, USA) was used to perform the
statistical data analyses.