key: cord-0786289-cnwqvgy2
authors: Gorse, Geoffrey J.; Patel, Gira B.; Fan, Xiaofeng
title: Interpatient mutational spectrum of human coronavirus‐OC43 revealed by illumina sequencing
date: 2017-03-14
journal: J Med Virol
DOI: 10.1002/jmv.24780
sha: dcc160e2b4d3b8b9bc87dc48157b29d818d211da
doc_id: 786289
cord_uid: cnwqvgy2

Human coronaviruses (HCoV) are RNA viruses that cause respiratory tract infections with viral replication of limited duration. The host and viral population heterogeneity could influence clinical phenotypes. Employing long RT‐PCR with Illumina sequencing, we quantified the gene mutation load at 0.5% mutation frequency for the 4529 bp‐domain spanning the Spike gene (4086 bp) of HCoV‐OC43 in four upper respiratory clinical specimens obtained during acute illness. There were a total of 121 mutations for all four HCoV samples with the average number of mutations at 30.3 ± 10.2, which is significantly higher than that expected from the Illumina sequencing error rate. There were two mutation peaks, one at the 5′ end and the other near position 1 550 in the S1 subunit. Two coronavirus samples were genotype B and two were genotype D, clustering with HCoV‐OC43 strain AY391777 in neighbor‐joining tree phylogenetic analysis. Nonsynonymous mutations were 76.1 ± 14% of mutation load. Although lower than other RNA viruses such as hepatitis C virus, HCoV‐OC43 did exhibit quasi‐species. The rate of nonsynonymous mutations was higher in the HCoV‐OC43 isolates than in hepatitis C (HCV) virus genotype 1a isolates analyzed for comparison in this study. These characteristics of HCoV‐OC43 may affect viral replication dynamics, receptor binding, antigenicity, evolution, transmission, and clinical illness.

2, which is significantly higher than that expected from the Illumina sequencing error rate. There were two mutation peaks, one at the 5′ end and the other near position 1 550 in the S1 subunit. Two coronavirus samples were genotype B and two were genotype D, clustering with HCoV-OC43 strain AY391777 in neighbor-joining tree phylogenetic analysis. Nonsynonymous mutations were 76.1 ± 14% of mutation load. Although lower than other RNA viruses such as hepatitis C virus, HCoV-OC43 did exhibit quasi-species.

The rate of nonsynonymous mutations was higher in the HCoV-OC43 isolates than in hepatitis C (HCV) virus genotype 1a isolates analyzed for comparison in this study. These characteristics of HCoV-OC43 may affect viral replication dynamics, receptor binding, antigenicity, evolution, transmission, and clinical illness. CoV and Middle East respiratory syndrome (MERS) CoV. 3 HCoV-OC43 is prevalent among humans and genotype D has been prominent in recent years. [3] [4] [5] Little is known about how HCoV-OC43 genotypes persist in human populations, but continuous adaptation by viral antigenic genes in the Spike protein through genetic drift may be necessary. The Spike protein is the major antigenic protein and is under selection pressure by the host immune response; it is important for host range and tissue tropism. It is cleaved into S1 and S2 subunits for receptor binding and membrane fusion. The N-terminal domain of the S1 subunit is responsible for sugar receptor binding and the S2 subunit is responsible for fusion of viral and host membranes. 6 The S1 subunit is more divergent in sequence and the S2 subunit is more conserved. [1] [2] [3] 7 Human coronaviruses cause the common cold and influenza-like illnesses, but can be associated with more severe illnesses such as pneumonia, exacerbations of asthma and chronic obstructive pulmonary disease, croup, and bronchiolitis. In patients with chronic obstructive pulmonary disease studied during the 1998-1999 influenza season, 13.5% of illnesses were associated with HCoV-229E and HCoV-OC43 infection, while in another study between 2009 and 2013, 19% of acute respiratory illnesses in patients with cardiopulmonary diseases and 21.5% in healthy young adults were associated with HCoV. [8] [9] [10] [11] Coronavirus-associated illness was less severe than influenza but was associated with multiple respiratory and systemic symptoms, and hospitalization. 10 HCoV-229E and HCoV-OC43 infection rates of 2.8-26% in healthy young and elderly adults, high-risk adults, and hospitalized patients were reported during the winters of 1999-2003 and they contributed to medical disease burden. 12 Little is known about the degree of heterogeneity of HCoV-OC43 viral quasi-species present in upper respiratory secretions. If present, this may help explain persistent incidence of HCoV-OC43 infections in human populations, if the mutational changes result in antigenic drift.

This might allow escape from host immunity and contribute to virus infectivity and pathogenicity.

In the current study, we combined RT-PCR and Illumina sequencing to measure the diversity of HCoV-OC43 Spike gene quasi-species through direct count of the Spike gene mutations, determination of percent nonsynonymous mutation rates and comparison of these rates to (HCV), which is in the genus Hepacivirus, family Flaviviridae. HCV is an RNA virus with a heterogeneous population of quasi-species in chronically infected patients. [13] [14] [15] 2 | MATERIALS AND METHODS

We studied nasal and oropharyngeal swab specimens that were obtained from each of four patients early during symptomatic acute respiratory illness and positive for HCoV-OC43 nucleic acids by multiplex RT-PCR. 11 Serum and nasal wash specimens were obtained at the time of acute illness and 3-4 weeks after illness onset. They were assayed by enzymelinked immunosorbent assay for serum IgG and nasal wash IgA antibodies to tissue culture-adapted HCoV-OC43 (American Type Culture Collection #VR-1558, GenBank: NC_005147.1) that was inactivated by psoralen compound and long-wavelength ultraviolet light, as described. 10, 11, 16 Severity of acute respiratory illness was measured by two scores: a self-reported visual analogue scale of overall illness severity, ranging from 1 (mildest) to 10 (most severe), and a severity of influenzalike symptoms and signs score that was the sum of 16 symptoms and signs that were graded on a scale of 0 (absent)-15 (most severe) with a maximum score of 240, as described. 10, 11, 17 Respiratory and systemic symptoms of the acute illness were recorded. The patients gave written informed consent and the study was approved by the Institutional Review Boards at the VA St. Louis Health Care System and Saint Louis University.

Two recombinant clones from a previous study, #1701 and #1709, each containing a 9022 bp HCV insert, were used to estimate potential errors associated with Illumina sequencing. 14,15 Also, 19 HCV genotype 1a samples from an earlier report 3 were available for re-analysis and comparison in the current study. 

We first estimated the error rate associated with Illumina sequencing using two recombinant HCV clones. In doing so, raw sequence reads in fastq format were first filtered in PRINSEQ (v 0.19.5) for quality control, including read length ≥70 bp, mean read quality score ≥25, low complexity with DUST score ≤7, ambiguous bases ≤1% and all duplicates. 18 Filtered reads were mapped onto HCV genotype 1a prototype strain H77 (GenBank accession number AY009606) using a gapped aligner Bowtie 2. 19 Mapped files were then converted into binary format (BAM), sorted and indexed in SAMtools 20 followed by local realignment and base quality recalibration in Genome Analysis Toolkit (GATK). 21 Next, by converting post-alignment BAM files into mpileup format in SAMtools, the consensus sequence for each clone was called in VarScan (v 2.2.3) with the settings of ≥1,000 x coverage, ≥25 base quality at a position to count a read and ≥50% mutation frequency. 22, 23 The entire pipeline was repeated using individual consensus sequences. Mutations were called at each position in VarScan under the setting of 0.5% frequency and base quality from 15 to 40, followed by manual check in the Integrative Genomics Viewer. 22 Using the value of base quality to define a mutation from above analysis, similar procedures were applied to four patient samples. The HCoV-OC43 strain (GenBank AY391777) was used as the reference at initial mapping. Over the entire coronavirus Spike gene, the mutation load, the total number of mutations at a given site, was counted through sliding windows, size = 300 bp, overlap = 100 bp. Finally, under the frame of fulllength HCoV Spike gene (4086 bp), the nature of each mutation, either synonymous or nonsynonymous, was determined using a custom script. 24 

The consensus full-length HCoV spike sequences from four patients and reference sequences retrieved from GenBank were used for GORSE ET AL.

| 1331 phylogenetic analysis. The tree was constructed using neighborjoining approach under nucleotide substitution model of maximum composite likelihood in MEGA program (version 5.2). 25 

Statistical analyses were done with either two-tailed, unpaired Students test or Chi-square. When applicable, data were expressed as mean value and standard deviation. P < 0.05 was considered statistically significant. Table 1) . The patients had underlying chronic cardiopulmonary diseases and diabetes mellitus. The two illnesses were associated with greater than a fourfold increase in nasal wash IgA antibody titers but only one with at least a fourfold increase in serum IgG antibody titer to HCoV-OC43, comparing acute illness to convalescent specimens collected 3-4 weeks after illness onset (Table 1) .

Samples from subjects two and 6, both genotype B, were collected about 2 years apart in January 2010 and March 2012 from a younger 

The raw data output indicated 70.1% of bases read had a quality score (Table 3) .

Using a sliding window analysis with window size = 300 bp and overlaps = 100 bp, there were two mutation peaks, one at the 5′ end and the other at about Spike gene position number 1550 (Fig. 1) . Using consensus sequences, a Neighbor-joining tree showed phylogenetically that two of the subjects' samples were genotype D and two were genotype B strains of HCoV-OC43 ( Fig. 2A and B) . The two genotype B samples both had high mutation frequencies at spike gene nucleotide positions 79 (36.92% and 45.12%) and 81 (7.96% and 

A previously reported study found that HCV genotype 1a patients experiencing relapse after antiviral treatment (n = 19) had a higher average total mutation load measured through 454 sequencing. 26 These samples were re-analyzed for the current study using a base quality score >30 rather than 25 in the earlier report. 26 The average mutation load in HCV patient isolates was significantly higher than in HCoV-OC43 patient isolates (296.2 ± 102.2 vs 30.3 ± 10.2, P = 7.7 × 10 −5 ). However, nonsynonymous mutations as a percentage of the total mutations were higher among the HCoV-OC43 isolates than among the 19 HCV genotype 1a patient isolates (76.7 ± 14% vs 26 ± 8%, P = 3.5 × 10 −9 ).

The alignment of Spike genes of consensus amino acid sequences show differences between the four clinical strains and the prototype AY391777 strain particularly at the N and C terminal ends of the S1 subunit, and at the S1 and S2 subunit cleavage site, (a.a. 762-766) with a smaller number of amino acid differences in the S2 subunit (Fig. 3) .

The high genetic mutation rates at sites 79 and 81 correspond to several amino acid changes compared to the prototype strain between amino acid positions 20 and 31. Our four clinical isolates were of the genotypes B and D which have been described as circulating in recent years rather than genotype A. 4 Alignment of spike gene consensus amino acid sequences compared to the AY391777 prototype strain sequence (genotype A) indicates the majority of changes were in the S1 subunit.

The proteolytic cleavage site of S1 and S2 subunits for the four clinical strains in our study had the RRSRR motif rather than the RRSRG motif of the prototype strain. This G to R substitution at amino acid 766 may result in increased cleavability and the cleavage process may play a part in fusion activity and viral infectivity. 4, 35 There were minimal amino acid changes at Spike gene nucleotide isolates, although all four had a Y154V substitution. This is in the lectin domain of the S1 subunit involved in attachment of the virus to the cellular receptor which is a derivative of neuraminic acid. 36 The TQDG sequence may have evolutionary and functional importance, is not present in the closely related bovine coronaviruses and not inserted in some strains of HCoV-OC43. 36 Four critical sugar-binding residues Y168, E188, W190, and H191 were present in our HCoV-OC43 isolates corresponding to Y162, E182, W184, and H185 in the bovine coronavirus sequence. 37 The receptor-binding of SARS-CoV in the S1 carboxy-terminal domain around the receptor binding motif involving amino acids 479 and 487 are areas where nonsynonymous substitutions and genetic diversity occurred for our HCoV-OC43 isolate sequences. These areas are important for binding affinity to human angiotensin-converting enzyme two for SARS-CoV, and host immune responses. 38 Our isolates had nonsynonymous substitutions at amino acid residues 259 and 260-264 in the N-terminal domain of S1, and, in particular, had nonsynonymous amino acid substitutions within the region between amino acids 471 and 550 at a putative receptor binding domain of HCoV-OC43 (a.a. positions 339-549). 4 In other HCoV strains, for instance HCoV-229E, antibody neutralization of the virus is dependent on the antigenic phenotype of the S1 subunit region. 39 Hence, this may also be a factor for HCoV-OC43 needing further study. Our patients all had acute upper respiratory and systemic signs and symptoms. The small number evaluated here precludes conclusions about pathogenicity and viral mutation rate patterns, but this approach opens up pathways to future study.

In conclusion, quasi-species were present in our HCoV-OC43 strains involving areas of the S1 subunit of the Spike gene that may affect evolution of the viral binding process and antigenicity, as well as 

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