# Identification and Genomic Characterization of Anelloviruses in Patients with Chronic Lymphocytic Leukemia

Authors: Tingwang Jiang; Qiang Liu; Fenying Lu; Teng He; Xiaoli Xiang; Zhicheng Zhang; Ying Sun
Journal: Journal of Clinical Pharmacy and Therapeutics
 (2023)

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## Abstract

Purpose. Metagenomics has revealed that, in addition to the digestive tract, certain viruses are also commonly found in human blood. In order to explore and monitor potential novel viruses, three serum samples of patients with chronic lymphocytic leukemia were collected at the No. 2 People’s Hospital of Changshu City, China. Materials and Methods. We sequenced the virome of serum samples from three patients with chronic lymphocytic leukemia using an unbiased viral metagenomic approach and subsequently performed maximum likelihood phylogenetic analysis using MrBayes v3.2. In addition, pairwise sequence comparison was produced with ORF1 amino acid sequences of anelloviruses within Bayesian consensus tree. Results. Partial genomes of eight different anelloviruses containing the complete ORF1 gene have been identified. BLASTp results showed that the amino acid sequence identity of these viruses with the best match in GenBank was between 56.22% and 95.43%. Phylogenetic analysis based on ORF1 indicated that seven sequences belong to the genus Alphatorquevirus and one sequence belongs to the genus Gammatorquevirus. Conclusions. This virological investigation has increased our understanding of the diversity of anelloviruses in human serum, but further study is needed to verify its potential correlation with disease.


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## Body

## 1. Introduction

Anelloviruses are nonenveloped, circular, single-stranded DNA (ssDNA) viruses with a genome size of approximately 3.8 kb, which include the Torque teno mini virus (TTMV), Torque teno midi virus (TTMDV), Torque teno virus (TTV), and small anellovirus (SAV) [1]. According to the classification principles of the International Committee for the Taxonomy of Viruses (ICTV), the family Anelloviridae is mainly divided into 14 genera, including Alphatorquevirus, Betatorquevirus, Deltatorquevirus, Epsilontorquevirus, Etatorquevirus, Gammatorquevirus, Iotatorquevirus, Kappatorquevirus, Lambdatorquevirus, Mutorquevirus, Thetatorquevirus, Zetatorquevirus, and Gyrovirus. Anelloviruses are ubiquitous in the population, even in healthy individuals. In some countries, the infection rate of anellovirus is as high as 100%, which is prevalent among about 10% of blood donors in the United Kingdom and the United States. However, in most cases, anellovirus infection is considered asymptomatic [2, 3]. Anellovirus was first found in serum of a Japanese patient with posttransfusion hepatitis through a representative difference analysis (RDA) in 1997 [4]; the 500 nucleotide (nt) sequence (N22) was first characterized and further expanded to approximately 3700 nt (TA278 clone). At the end of 1998, two studies showed that there was an extra-GC-rich region of about 120 nt, which led to the discovery of the circular feature of the anellovirus genome (–3800 nt) [5].Although the anellovirus was initially suspected to be transmitted only through blood transfusion, its detection in various biological samples (plasma, saliva, and feces) revealed that the virus has multiple modes of transmission, especially the transmission of saliva droplets. Infection with these viruses often leads to life-long viremia, and their possible association with the disease is still under investigation. Studies have suggested that higher than usual viral loads may be related to severe idiopathic inflammatory myopathy [6], cancer [7], lupus [8], and leukemia [9, 10]. In animals, the anellovirus has been found in chimpanzees [11], chickens [12], pigs [13], cattle [14], sheep [15], and dogs [16]. In addition, vertical transmission has also been found in pigs [17].In this research, we investigated the viral composition in serum samples from patients with chronic lymphocytic leukemia. Several anelloviruses were identified, and their genetic relationship with currently known viruses was further studied. This study further broadened our understanding of the diversity of anelloviruses.


## 2. Materials and Methods

### 2.1. Sample Collection and Preparation

In March 2021, serum samples of three patients with chronic lymphocytic leukemia were collected at the No. 2 People’s Hospital in Changshu City, China. All patients have signed an informed consent form. Among them, two are male patients, aged 69 and 74, respectively, and another is an 80-year-old female patient. All samples were transported over dry ice. Prior to viral metagenomic analysis, all collected samples were diluted with Dulbecco’s phosphate-cushioned saline (DPBS) and vortexed for 5 min vigorously, and then incubated at 4°C for 30 min. After centrifugation (10 min, 15,000 g), the supernatants were harvested in 1.5 ml centrifuge tubes and stored at −80°C until examined [18].


### 2.2. Viral Metagenomic Analysis

A total of 500μL of supernatant was pipetted from the three samples (166.60 μL from each) and transferred into a new 1.5 mL centrifuge tube. The sample was centrifuged at 12,000 g for 5 min at 4°C and then filtered through a 0.45 μm filter (Merck Millipore, MA, USA) to remove cellular material and bacterial cell-sized particles [18]. Free DNA and RNA in the filtrates were then removed using DNase (Turbo DNase from Ambion, Thermo Fisher, Waltham, MA, USA; Baseline-ZERO from Epicentre, Charlotte, USA; and Benzonase from Novagen, Darmstadt, Germany) and RNase (Promega, Madison, WI, USA) to digest unprotected nucleic acid at 37°C for 60 min [19]. Total viral nucleic acids were then extracted using a QIAamp MinElute Virus Spin Kit (Qiagen, HQ, Germany) according to the manufacturer’s protocol. The total viral cDNAs were obtained by reverse transcription using SuperScript III reverse transcriptase (Invitrogen, CA, USA) and random hexamer primer (100 pmol), followed by DNA synthesis using Klenow fragment polymerase (New England Biolabs, MA, USA). The next-generation sequencing library was constructed using the Nextera XT DNA Sample Preparation Kit (Illumina, CA, USA), and the 250 bases paired ends with dual barcoding were then sequenced using the MiSeq Illumina platform. The sequenced raw reads were debarcoded using vendor software from Illumina for bioinformatics analysis. The data were processed using an in-house developed pipeline analysis running on a 32-node Linux cluster. Reads were considered duplicates if base positions 5–55 were identical and only one random copy was retained. Clonal reads were removed, and low sequencing quality tails were trimmed using Phred, and the quality threshold was set to a Phred score of 20 [18]. Adaptors were trimmed using VecScreen with the default parameters. The cleaned reads were de novo assembled using the ENSEMBLE assembler [20]. The resulting contigs and singlets were then aligned against a customized viral proteome database using BLASTx, which was compiled using the NCBI virus reference proteome (ftp://ftp.ncbi.nih.gov/refseq/release/viral/) to which was added viral proteins sequences from the NCBI nr fasta file (based on annotation taxonomy in Virus Kingdom) with an E value cutoff of <10–5 [18]. Contigs over the E value cutoff were further searched against viral protein families in vFam database [21] using HMMER3 to detect somewhat similar sequences with virus [22].


### 2.3. Acquisition and Annotation of Virus Genome

To assemble the virus genome, the contigs were assembled with Geneious Prime version 2022.0.1. Those assembled contigs with sequence length >2000 bp and the nonstructural protein or the structural protein were subjected to full characterization. Putative viral open reading frames (ORFs) were predicted by Geneious Prime with built-in parameters (minimum size: 300; genetic code: standard; start codons: ATG) [23]. The annotations of viral ORFs were performed based on the Conserved Domain Database.


### 2.4. Phylogenetic Analysis

To infer phylogenetic relationships, nucleotide and protein sequences of reference strains from different groups of corresponding viruses were downloaded from the GenBank database. Nucleotide and protein sequences were aligned using CLC Genomics Workbench 10.0 and further optimized using MUSCLE in MEGA v7.0 (24) and MAFFT v7.3.1 employing the E-INS-I algorithm [24]. All positions containing over 50% gaps were temporarily removed from alignments before phylogeny construction. Bayesian inference trees were then constructed by MrBayes v3.2 [25]. The Markov chain was run for a maximum of 1 million generations, sampling every 50 generations, and the first 25% of Markov chain Monte Carlo (MCMC) samples were discarded as burn-in [18]. All the Bayesian inference trees were confirmed using the maximum likelihood method by Mega v7.0 trees and were also constructed to confirm using software [26].


### 2.5. Quality Control

Standard precautions were used for all procedures to reduce the possibility of cross-sample contamination and nucleic acid degradation. Aerosol-battier pipette tips were used to reduce the possibility of sample cross contamination, and all the materials (including microcentrifuge tubes and pipet tips) used for nucleic acid extraction were RNase and DNase free. Nucleic acid samples were dissolved in DEPC-treated water, and RNase inhibitors were added. Sterile ddH2O (Sagon, Shanghai, China) was set as a blank control. During sequencing on the Illumina MiSeq platform, the blank control readings were subtracted from all the sample readings.


## 2.1. Sample Collection and Preparation

In March 2021, serum samples of three patients with chronic lymphocytic leukemia were collected at the No. 2 People’s Hospital in Changshu City, China. All patients have signed an informed consent form. Among them, two are male patients, aged 69 and 74, respectively, and another is an 80-year-old female patient. All samples were transported over dry ice. Prior to viral metagenomic analysis, all collected samples were diluted with Dulbecco’s phosphate-cushioned saline (DPBS) and vortexed for 5 min vigorously, and then incubated at 4°C for 30 min. After centrifugation (10 min, 15,000 g), the supernatants were harvested in 1.5 ml centrifuge tubes and stored at −80°C until examined [18].


## 2.2. Viral Metagenomic Analysis

A total of 500μL of supernatant was pipetted from the three samples (166.60 μL from each) and transferred into a new 1.5 mL centrifuge tube. The sample was centrifuged at 12,000 g for 5 min at 4°C and then filtered through a 0.45 μm filter (Merck Millipore, MA, USA) to remove cellular material and bacterial cell-sized particles [18]. Free DNA and RNA in the filtrates were then removed using DNase (Turbo DNase from Ambion, Thermo Fisher, Waltham, MA, USA; Baseline-ZERO from Epicentre, Charlotte, USA; and Benzonase from Novagen, Darmstadt, Germany) and RNase (Promega, Madison, WI, USA) to digest unprotected nucleic acid at 37°C for 60 min [19]. Total viral nucleic acids were then extracted using a QIAamp MinElute Virus Spin Kit (Qiagen, HQ, Germany) according to the manufacturer’s protocol. The total viral cDNAs were obtained by reverse transcription using SuperScript III reverse transcriptase (Invitrogen, CA, USA) and random hexamer primer (100 pmol), followed by DNA synthesis using Klenow fragment polymerase (New England Biolabs, MA, USA). The next-generation sequencing library was constructed using the Nextera XT DNA Sample Preparation Kit (Illumina, CA, USA), and the 250 bases paired ends with dual barcoding were then sequenced using the MiSeq Illumina platform. The sequenced raw reads were debarcoded using vendor software from Illumina for bioinformatics analysis. The data were processed using an in-house developed pipeline analysis running on a 32-node Linux cluster. Reads were considered duplicates if base positions 5–55 were identical and only one random copy was retained. Clonal reads were removed, and low sequencing quality tails were trimmed using Phred, and the quality threshold was set to a Phred score of 20 [18]. Adaptors were trimmed using VecScreen with the default parameters. The cleaned reads were de novo assembled using the ENSEMBLE assembler [20]. The resulting contigs and singlets were then aligned against a customized viral proteome database using BLASTx, which was compiled using the NCBI virus reference proteome (ftp://ftp.ncbi.nih.gov/refseq/release/viral/) to which was added viral proteins sequences from the NCBI nr fasta file (based on annotation taxonomy in Virus Kingdom) with an E value cutoff of <10–5 [18]. Contigs over the E value cutoff were further searched against viral protein families in vFam database [21] using HMMER3 to detect somewhat similar sequences with virus [22].


## 2.3. Acquisition and Annotation of Virus Genome

To assemble the virus genome, the contigs were assembled with Geneious Prime version 2022.0.1. Those assembled contigs with sequence length >2000 bp and the nonstructural protein or the structural protein were subjected to full characterization. Putative viral open reading frames (ORFs) were predicted by Geneious Prime with built-in parameters (minimum size: 300; genetic code: standard; start codons: ATG) [23]. The annotations of viral ORFs were performed based on the Conserved Domain Database.


## 2.4. Phylogenetic Analysis

To infer phylogenetic relationships, nucleotide and protein sequences of reference strains from different groups of corresponding viruses were downloaded from the GenBank database. Nucleotide and protein sequences were aligned using CLC Genomics Workbench 10.0 and further optimized using MUSCLE in MEGA v7.0 (24) and MAFFT v7.3.1 employing the E-INS-I algorithm [24]. All positions containing over 50% gaps were temporarily removed from alignments before phylogeny construction. Bayesian inference trees were then constructed by MrBayes v3.2 [25]. The Markov chain was run for a maximum of 1 million generations, sampling every 50 generations, and the first 25% of Markov chain Monte Carlo (MCMC) samples were discarded as burn-in [18]. All the Bayesian inference trees were confirmed using the maximum likelihood method by Mega v7.0 trees and were also constructed to confirm using software [26].


## 2.5. Quality Control

Standard precautions were used for all procedures to reduce the possibility of cross-sample contamination and nucleic acid degradation. Aerosol-battier pipette tips were used to reduce the possibility of sample cross contamination, and all the materials (including microcentrifuge tubes and pipet tips) used for nucleic acid extraction were RNase and DNase free. Nucleic acid samples were dissolved in DEPC-treated water, and RNase inhibitors were added. Sterile ddH2O (Sagon, Shanghai, China) was set as a blank control. During sequencing on the Illumina MiSeq platform, the blank control readings were subtracted from all the sample readings.


## 3. Results

### 3.1. Viral Metagenomic Overview

The library generated a total of 97,814 raw sequence reads on the Illumina MiSeq platform. The BLASTx results containing sequencing data from the library generated by DIAMOND (data format) were imported into Megan6 software for generation of RMA-6 format files, which were further used for classification and visualization. After bioinformatics analysis, a total of 6,419 sequences reads were hit with virus (TableS1), accounting for 6.56% of the total raw reads. About 13 viral families were identified; the most abundant virus family was Anelloviridae (79.83% of the total virus reads), followed by Siphoviridae (6.04%), Myoviridae (5.55%), Mimiviridae (3.19%), Podoviridae (1.17%), and Phycodnaviridae (1.06%) (Figure 1(a) and Table S1). In addition, there are 1,766 reads that have little match with known viruses (E value >10–5) and maybe the potential novel viruses. In total, 472 contigs were obtained by assembling; among them, the longest contig belonged to the family of Anelloviridae (Figure 1(b)).Figure 1
Virome detected in serum samples and the length distribution of assembled contigs. (a) The percentage of reads in different viral families. (b) The length of assembled contigs.
(a)(b)


### 3.2. Identification of Novel Viruses of Anelloviridae

In this research, eight different partial anellovirus sequences (temporarily named HumanAV1-8) were obtained using the assemble sequences program in Geneious Prime version 2022.0.1 and were confirmed that those anelloviruses originated from the clinical samples by PCR; partial genome sizes of these eight anelloviruses were 3,547, 2,949, 3,373, 3,812, 2,740, 3,697, 3,678, and 3,113 bp, with GC contents of 49.8, 51.0, 49.9, 52.4, 39.9, 50.4, 50.3, and 51.6%, respectively, and their amino acid sequence identity with the best match in GenBank ranged from 56.22% to 95.43%. The genomes of all these eight anelloviruses have a large ORF1, which encodes the putative Rep protein.Phylogenetic analysis combined with BLASTp based on the ORF1 amino acid sequence indicated that HumanAV1 shared the highest 90.34% amino acid identity with unclassified Anelloviridae (QUD39751), and the best matches of HumanAV2 and HumanAV8 were Torque teno virus 24 (YP_009505729), sharing 72.29% and 72.75% amino acid sequence identity, respectively. HumanAV3 and HumanAV7 shared the highest amino acid identity of 90.63% and 90.50% with the SEN virus (AAS18428) in GenBank, respectively. The ORF1 of HumanAV4 had the highest amino acid identity (88.51%) with Torque teno virus (AAK01942), and the identity between HumanAV5 and its best matching strain Torque teno midi virus 6 (YP_009505762) was 56.22%. While, HumanAV6 shared the highest amino acid identity (95.43%) with Torque teno midi virus 6 (YP_009505762). Except for HumanAV5, the remaining seven viruses were clustered with the genusAlphatorquevirus, and HumanAV5 was close to the genus Gammatorquevirus (Figure 2).Figure 2
Phylogenetic analysis of the anelloviruses identified in serum. Bayesian inference tree based on amino acid sequences of ORF1 of viruses from Anelloviridae. Viruses detected in this study are marked with a red star. Each scale bar represents 0.5 amino acid substitutions per site.Distance matrix analysis of the 48 amino acid sequences of ORF1 showed that pairwise identity ranging from 26% to 100%, where HumanAV1-8 shared 26–99% identity with other viruses, respectively. Among them, HumanAV5 has the lowest identity with all other matched sequences, while HumanAV3 and HumanAV7 were the opposite (Figure3).Figure 3
Pairwise sequence comparison produced with ORF1 amino acid sequences within the Bayesian consensus tree. The anelloviruses identified in this study are highlighted. The percent identities were shown by color saturation, ranging from low (blue) to high (red).


## 3.1. Viral Metagenomic Overview

The library generated a total of 97,814 raw sequence reads on the Illumina MiSeq platform. The BLASTx results containing sequencing data from the library generated by DIAMOND (data format) were imported into Megan6 software for generation of RMA-6 format files, which were further used for classification and visualization. After bioinformatics analysis, a total of 6,419 sequences reads were hit with virus (TableS1), accounting for 6.56% of the total raw reads. About 13 viral families were identified; the most abundant virus family was Anelloviridae (79.83% of the total virus reads), followed by Siphoviridae (6.04%), Myoviridae (5.55%), Mimiviridae (3.19%), Podoviridae (1.17%), and Phycodnaviridae (1.06%) (Figure 1(a) and Table S1). In addition, there are 1,766 reads that have little match with known viruses (E value >10–5) and maybe the potential novel viruses. In total, 472 contigs were obtained by assembling; among them, the longest contig belonged to the family of Anelloviridae (Figure 1(b)).Figure 1
Virome detected in serum samples and the length distribution of assembled contigs. (a) The percentage of reads in different viral families. (b) The length of assembled contigs.
(a)(b)


## 3.2. Identification of Novel Viruses of Anelloviridae

In this research, eight different partial anellovirus sequences (temporarily named HumanAV1-8) were obtained using the assemble sequences program in Geneious Prime version 2022.0.1 and were confirmed that those anelloviruses originated from the clinical samples by PCR; partial genome sizes of these eight anelloviruses were 3,547, 2,949, 3,373, 3,812, 2,740, 3,697, 3,678, and 3,113 bp, with GC contents of 49.8, 51.0, 49.9, 52.4, 39.9, 50.4, 50.3, and 51.6%, respectively, and their amino acid sequence identity with the best match in GenBank ranged from 56.22% to 95.43%. The genomes of all these eight anelloviruses have a large ORF1, which encodes the putative Rep protein.Phylogenetic analysis combined with BLASTp based on the ORF1 amino acid sequence indicated that HumanAV1 shared the highest 90.34% amino acid identity with unclassified Anelloviridae (QUD39751), and the best matches of HumanAV2 and HumanAV8 were Torque teno virus 24 (YP_009505729), sharing 72.29% and 72.75% amino acid sequence identity, respectively. HumanAV3 and HumanAV7 shared the highest amino acid identity of 90.63% and 90.50% with the SEN virus (AAS18428) in GenBank, respectively. The ORF1 of HumanAV4 had the highest amino acid identity (88.51%) with Torque teno virus (AAK01942), and the identity between HumanAV5 and its best matching strain Torque teno midi virus 6 (YP_009505762) was 56.22%. While, HumanAV6 shared the highest amino acid identity (95.43%) with Torque teno midi virus 6 (YP_009505762). Except for HumanAV5, the remaining seven viruses were clustered with the genusAlphatorquevirus, and HumanAV5 was close to the genus Gammatorquevirus (Figure 2).Figure 2
Phylogenetic analysis of the anelloviruses identified in serum. Bayesian inference tree based on amino acid sequences of ORF1 of viruses from Anelloviridae. Viruses detected in this study are marked with a red star. Each scale bar represents 0.5 amino acid substitutions per site.Distance matrix analysis of the 48 amino acid sequences of ORF1 showed that pairwise identity ranging from 26% to 100%, where HumanAV1-8 shared 26–99% identity with other viruses, respectively. Among them, HumanAV5 has the lowest identity with all other matched sequences, while HumanAV3 and HumanAV7 were the opposite (Figure3).Figure 3
Pairwise sequence comparison produced with ORF1 amino acid sequences within the Bayesian consensus tree. The anelloviruses identified in this study are highlighted. The percent identities were shown by color saturation, ranging from low (blue) to high (red).


## 4. Discussion

In recent years, great progress in viral metagenomics has allowed researchers to detect viruses on the human skin and in vivo [27]. Genomic studies on human samples have revealed dozens of unknown viruses in our intestines, lungs, skin, and blood [28]. Some of these newly identified viruses may be related to the emergence and development of diseases, but it is also possible that in most cases, some of these viruses are harmless in most humans. Understanding how these newly discovered viruses affect humans will allow us to determine whether their infections can be prevented and treated.Anelloviruses are widely distributed, and any individual carrying the anellovirus may become a potential source of infection. It has been reported that anellovirus can be detected in healthy people, and the detection rate of anellovirus in serum or blood of thalassemia patients, long-term hemodialysis patients, and hemophilia patients treated with clotting factors will be higher than that of normal people [29, 30]. Anellovirus was first found in serum of patients with posttransfusion hepatitis and has the ability to grow on hepatocytes in vivo or in vitro. Some studies have found that anellovirus infection may be related to fulminant hepatitis, acute recurrent hepatitis, and hemophilia liver failure [31], and it was also found that the histopathological grade of patients coinfected with hepatitis C virus (HCV) and anellovirus was higher than that of patients infected with HCV alone [32]. However, another series of studies found that there was no difference in disease severity between these two types of patients [33].


## 5. Conclusion

In this study, we analyzed three serum samples from patients with chronic lymphocytic leukemia collected at the No. 2 People’s Hospital of Changshu City, China, using viral metagenomics approach, and eight anelloviruses belonging to the genusAlphatorquevirus or genus Gammatorquevirus with the complete region of ORF1 were identified, which expanded our understanding of the diversity of DNA viruses in a limited population to a certain extent. This is the first time that the anelloviruses have been detected in the blood samples of patients with chronic lymphocytic leukemia. This study provides data support for the correlation between anelloviruses and chronic lymphocytic leukemia. But limited by the number of samples, a definite conclusion cannot draw that anelloviruses are associated with chronic lymphocytic leukemia. Further dynamic monitoring of these viruses is necessary in the future.



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*Source: 4125745-2023-02-09.xml*