UK circulating strains of human parainfluenza 3: an amplicon based next generation sequencing method and phylogenetic analysis

Abstract

Background: Human parainfluenza viruses type 3 (HPIV3) are a prominent cause of respiratory infection with a significant impact in both pediatric and transplant patient cohorts. Currently there is a paucity of whole genome sequence data that would allow for detailed epidemiological and phylogenetic analysis of circulating strains in the UK. Although it is known that HPIV3 peaks annually in the UK, to date there are no whole genome sequences of HPIV3 UK strains available. Methods: Clinical strains were obtained from HPIV3 positive respiratory patient samples collected between 2011 and 2015. These were then amplified using an amplicon based method, sequenced on the Illumina platform and assembled using a new robust bioinformatics pipeline. Phylogenetic analysis was carried out in the context of other epidemiological studies and whole genome sequence data currently available with stringent exclusion of significantly culture-adapted strains of HPIV3. Results: In the current paper we have presented twenty full genome sequences of UK circulating strains of HPIV3 and a detailed phylogenetic analysis thereof. We have analysed the variability along the HPIV3 genome and identified a short hypervariable region in the non-coding segment between the M (matrix) and F (fusion) genes. The epidemiological classifications obtained by using this region and whole genome data were then compared and found to be identical. Conclusions: The majority of HPIV3 strains were observed at different geographical locations and with a wide temporal spread, reflecting the global distribution of HPIV3. Consistent with previous data, a particular subcluster or strain was not identified as specific to the UK, suggesting that a number of genetically diverse strains circulate at any one time. A small hypervariable region in the HPIV3 genome was identified and it was shown that, in the absence of full genome data, this region could be used for epidemiological surveillance of HPIV3.

Keywords: circulating strains; epidemiology; human parainfluenza 3; phylogenetics.

Figure 1.

Primers used for amplicon generation for full genome sequencing ( a) and the position of the amplicons along the human parainfluenza viruses type 3 (HPIV3) genome. 12 primer sets ( a) were designed and used to generate overlapping amplicons covering the entire HPIV3 genome, as shown in ( b).

Figure 2.

Total samples tested positive for human parainfluenza viruses type 3 (HPIV3) by Public Health England (PHE) diagnostic laboratory during 2011–2017 ( a) and provenance of sequenced clinical strains ( b). Total number of samples that have tested positive for HPIV3 in PHE diagnostic laboratory of a major teaching hospital each month are shown in ( a) for the period 2011–2017. The data has been extracted from the local hospital database and is separated by age. The provenance of sequenced clinical strains collected between 2011 and 2015 is shown in ( b). All samples originated from the upper airway and 12/20 samples were from hospital A. PMH = past medical history; NPA = Nasopharyngeal aspirate; URT = upper respiratory tract; VUD = volunteer unrelated donor (transplant); ALL = acute lymphocytic leukaemia.

Figure 3.

Depth of coverage achieved for laboratory strain ( A), strain 153 ( B) and FastQC statistics for both sequences ( C). Consistent coverage of above 1000 was achieved over the full length of the genome of both sequences excluding the very 5′ and 3′ prime ends. The length of the final sequence was 15409 base pairs, as the forward primer (26 bases) of the first amplicon, and the reverse primer of the last amplicon (27 bases) were removed in the pipeline.

Figure 4.. Molecular Phylogenetic analysis of human parainfluenza viruses type 3 (HPIV3) full length genome by Maximum Likelihood method.

The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible + I + G model and 1000 bootstrap repetitions. The tree with the highest log likelihood (-42087.04) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 56 nucleotide sequences. Evolutionary analyses were conducted in MEGA7. Clusters, subclusters and strains were identified using Automatic Barcode Gap Discovery and genetic distances of 0.043 (cluster); 0.02 (subcluster) and 0.015 (strain) were identified. All strains (and cluster 2) are colored for ease of visualization and tracking.

Figure 5.. Relative site by site evolutionary rate of the human parainfluenza viruses type 3 (HPIV3) genome.

Mean (relative) evolutionary rate are shown for each site next to the site number with a window of 200. These rates are scaled such that the average evolutonary rate across all sites is 1. This means that sites showing a rate < 1 are evolving slower than average, and those with a rate > 1 are evolving faster than average. These relative rate were estimated under the General Time Reversible model (+G+I). The analysis involved 56 nucleotide sequences. The position along the HPIV3 genome is shown on the x axis with the hypervariable region identified between positions 4703 to 5160. Evolutionary analyses were conducted in MEGA7.

Figure 6.. Molecular Phylogenetic analysis of human parainfluenza viruses type 3 (HPIV3) hypervariable region by Bayesian Phylogenetics using BEAST.

The evolutionary history was inferred by using Bayesian Phylogenetics based on the TRN +I model using BEAST v1.8.4. The tree with the highest log likelihood (-2020.47) using path sampling and stepping stone analysis is shown. A strict clock and a constant coalescent prior were used. The MCMC length was 10,000,000. Convergence was assessed with Tracer and the maximum clade credibility tree was generated with Tree Annotator. Dates of strain emergence (in years) are shown in the figure legend. Automatic Barcode Gap Discovery was used to analyse genetic distances and 0.1 (cluster) and 0.04 (subcluster) were defined. Subclusters A-E are shown next to their respective branches. All strains and cluster and subcluster labels are colored identically to the phylogenetic analysis using full genome ( Figure 4) to demonstrate near identical clustering patterns.

Figure 7.. Molecular Phylogenetic analysis of human parainfluenza viruses type 3 (HPIV3) HN coding region by Maximum Likelihood method.

The evolutionary history was inferred by using the Maximum Likelihood method based on the TRN +G model and 1000 bootstrap repetitions. The tree with the highest log likelihood (-4490.43) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 56 nucleotide sequences. Evolutionary analyses were conducted in MEGA7. Clusters, subclusters and strains were identified using Automatic Barcode Gap Discovery and genetic distances of 0.043 (cluster); 0.02 (subcluster) and 0.01 (strain) were identified. All strains (and cluster 2) are colored consistent with Figure 4 for ease of visualization and tracking.