Keywords
genome sequencing, Respirovirus, phylogenetic analysis
Introduction
The family Paramyxoviridae consists of viruses that are known to
affect a wide range of species including humans, pigs, cattle, poultry
and companion animals. Porcine parainfluenzavirus type 1, PPIV-1
(species Porcine respirovirus 1 ) was first detected in rectal and
nasopharyngeal swabs obtained from pigs in a slaughterhouse in Hong Kong
(Lau et al., 2013). Upon full genome sequence analysis, the authors
proposed a novel paramyxovirus. PPIV-1 is a member of theRespirovirus genus within the Paramyxoviridae family. It
has a negative sense, single-stranded RNA genome of approximately 15
kilobases in length consisting of six genes (3′‐N‐P‐M‐F‐HN‐L‐5′). These
genes encode for major proteins: nucleocapsid, phosphoprotein, matrix,
fusion, hemagglutinin-neuraminidase and large proteins, respectively,
and two accessory proteins that are associated with the phosphoprotein
(Henrickson, 2003; Lau et al., 2013; Palinski et al., 2016; Park et al.,
2017, 2019). The paramyxoviral hemagglutinin-neuraminidase (HN) protein
is responsible for the attachment of the virus to the target cells,
whereas the fusion (F) protein directly mediates the fusion of the
membranes (Morrison, 2003).
Full genome sequence analyses have revealed that the Human
parainfluenzavirus 1 and the Sendai virus of mice and other rodents are
the most similar viruses to PPIV-1 genetically (Palinski et al., 2016).
Challenge trials in the United States revealed that inoculation of
conventional and CD/CD piglets showed no mortality and minimal morbidity
despite significant viral replication (Welch et al., 2018). In a recent
study, the same research group found a significant reduction in viral
genome copies in BALF, tracheal swab and turbinate in PPIV-challenged
animals, that were previously vaccinated with an RNA particle vaccine
(Welch et al., 2020).
- Materials and Methods,
- Sample collection and viral nucleic acid extraction
The presence of PPIV-1 has only been reported in Hong Kong and the
United States so far, and the aim of our work was to screen large scale
pig herd in Hungary for the presence of the virus. We have analyzed
15–15 (3–3 piglets from five litters) nasal swab samples obtained from
three-week-old piglets originating from 22 farms altogether. The study
was conducted in compliance with the provisions of Directive 2010/63/EU,
Hungarian Act XXVIII/1998 and the Hungarian Ministerial Decree No.
40/2013. (II.14.).
The nasal swabs were vortexed in PBS, and RNA was isolated in a QIAcube
automatic instrument using QIAmp cador Pathogen Mini Kit (Qiagen,
Hilden, Germany) according to the manufacturer’s recommendations.
Detection and phylogenetic analyses of PPIV-1 in clinical
samples
Endpoint PCR for the first round detection of respiroviruses was
performed by amplifying a 555 bp fragment of the L gene using degenerate
primers (5′-GACTCATCTACTAACGGNTAYGARA-3′ and
5′-CACAAACATCTTGCTACTWATDATNGT-3′) described in Lau et al. (2013).
Real-time quantitative RT-PCR to detect the N gene of PPIV-1 and
subsequent melting point analysis was performed in a Rotor-Gene Q
instrument (Qiagen) using QuantiNova SYBR Green RT-PCR Kit (Qiagen),
with specific primers (5′-TACAATATATGTGGGTGATCCTTACT-3′ and
5′-GCCTGAATCTTCATGATCTTCTAAA-3′) as described previously in Lau et al.
2013. Positive control RNA obtained from a PPIV-1 isolate was kindly
provided by Phillip C. Gauger (Department of Veterinary Diagnostic and
Production Animal Medicine, College of Veterinary Medicine, Iowa State
University). The L gene product used for diagnostic PCR and a partial F
gene segment was sequenced by the use of primers and conditions
described in Park et al., 2019.
Briefly, PCR products were subjected to gel electrophoresis and
amplicons with suitable length were cut out of the gel and purified by
the Qiagen Gel Extraction Kit (Qiagen). Bidirectional Sanger sequencing
reaction was performed with BigDye™ Terminator v3.1 Cycle Sequencing Kit
(Thermo Fisher Scientific, Ljubljana, Slovenia) with the corresponding
primers, and the capillary electrophoresis was carried out by a
commercial provider (Hungarian Natural History Museum).
The obtained sequences were proofread, assembled using E-INS-i method of
the online software mafft version 7 (Katoh & Toh, 2008) and aligned
against available PPIV-1 genomes, and reference respirovirus sequences
downloaded from the GenBank. Maximum Likelihood (ML) analyses were
conducted using MEGA version X (Kumar, Stecher, Li, Knyaz, & Tamura,
2018) .
Results and Discussion
The initial respirovirus screening of the 22 herds revealed the presence
of positive samples in the case of one herd. In order to gather more
information on the within-herd infection dynamics we performed
cross-sectional nasal swab sampling directly from that herd: 20–20
samples were obtained from 2, 4, 6 and 8-week-old piglets. The samples
were analyzed by real-time quantitative RT-PCR. Results of the
second-round PCR are shown in Figure 1. Out of 20 samples obtained from
the 4-week-old age group, we found 13 positives, 8 in the 6-week-old
group and 5 in the 8-week-old group. Only one positive sample was found
among the 2-week-old animals. The highest viral burden was observed
among the 4-week-old animals, where the mean Cq value was 28,39 (±5,09),
but the differences between the groups were not statistically
significant (Figure 1).
Melting curve analysis has been conducted after every real-time run and
surprisingly there was a 2.5 °C difference between the melting point of
the positive control (76 °C) and the positive samples (73.5 °C),
suggesting multiple nucleotide differences between the two amplicons.
After sequencing three positive samples (GenBank accession numbers:
MT765271–3) with low Cq values, initial BLAST analysis of the partial L
and F gene sequences confirmed the presence of PPIV-1 in our samples.
The sequences obtained were almost identical to each other with less
than 5 nucleotide differences among each other. Maximum Likelihood trees
constructed with a 1636 nucleotide long part of the F gene and the 555
nucleotide long part of the L gene revealed almost identical topology
(Figure 2). Our sequences grouped together with other PPIV-1 sequences
supported by relative high bootstrap values. Pairwise similarities were
calculated with every available PPIV-1 sequence and the highest values
were found in the case of two sequences originating from Hong Kong
submitted by Lau et al. (2013): 95.6% (acc. no.: JX857410) and 92.2%
(acc. no.: JX857409). All other PPIV-1 sequences, including the recently
submitted ones from the USA, were less than 92% similar.
Interestingly, the partial F and L sequences obtained from our samples
showed the highest similarity values to the very first PPIV-1 genomes
submitted from Hong Kong in 2013, whereas the overall genetic difference
compared to the recently described North American isolates was around
10%. The genetic difference was already visible during the real-time
PCR investigations where the melting curve analyses revealed 2.5 °C
difference between our samples and the positive control originating from
the USA. The pairwise alignment of the amplicons’ sequences revealed 6
mismatches 5 of which resulted in smaller GC content of our samples,
explaining the decreased melting point.
To the authors’ knowledge, this is the first report of porcine
parainfluenzavirus 1 outside Hong Kong and the United States of America.
Out of the 22 herds screened we only found positive animals in one herd,
where the second-round sampling revealed significant virus circulation
among the young animals. Both the number of positive nasal swabs and the
viral copy numbers were the highest among the 4-week-old animals. Elder
animals had fewer positive samples and lower viral amounts.
These results are in harmony with previous data where 37.2% of all the
PPIV-1positive cases found at the Veterinary Diagnostic Laboratory of
the Iowa State University originated from nursery units (Gauger et al.,
2018). On the contrary, however, among the 22 screened, we only found a
single herd infected with the virus whereas it seems to be widespread in
the USA (Gauger et al., 2018; Palinski et al., 2016). We could not,
however, successfully identify the source of infection in our case, as
the farm had no prior contact to pigs of personnel coming from either
USA or Hong Kong, China.