Monitoring viral genomic sequences in transfusion‐transmitted viruses

Monitoring genomic sequences of blood‐borne viruses infecting blood donors enables blood operators to undertake molecular epidemiology, confirm transfusion transmission and assess and characterize molecular and serological screening assays. The purpose of the study was to determine how blood operators globally value viral diversity surveillance and to assess its impact.


INTRODUCTION
Rapid advances and continuous improvements in serology assays and nucleic acid amplification testing (NAT) have reduced the risk of transfusion-transmitted infectious diseases (TTIDs). In addition, the introduction of molecular screening has stimulated the development and use of increasingly sophisticated molecular methods to confirm initial screening results. Concomitantly, the development of molecular biology has led to an unprecedented increase in the knowledge of the genetics of TTIDs including viruses, and, in particular, their diversity.
Differences in the nucleic acid sequences of a viral genome are driven by genetic variability leading to phenotypic changes. This is the result of various drivers, such as the natural history of viral infection, high replication rates and host-pathogen interactions [1]. This can occur through a range of mechanisms including the high intrinsic error rate of the viral reverse transcriptases or the RNA-dependent RNA polymerases, reassortment or template switching [2]. RNA viruses tend to display greater amounts of genetic diversity than DNA viruses, while diversity is higher in single-stranded compared with doublestranded viruses [2,3]. Viruses with a smaller genome also tend to exhibit more genetic diversity than those with larger genomes [2,4].
There is wide variability in the genetic diversity of blood-borne viruses, such as the single-stranded RNA viruses, hepatitis C virus (HCV) [5] and human immunodeficiency virus (HIV) [6], and the partially double-stranded DNA virus, hepatitis B virus (HBV) [7,8]. Based on genetic diversity, viruses can be separated into genotypes and eventually sub-genotypes. HIV-1 is broken down into four groups (main [M], outlier [O], non-M [N] and P), with group M subdivided into nine subtypes [9], while HCV is classified into at least eight genotypes, each with several subtypes (over 80 in total) [10,11], and HBV into nine genotypes with subtypes identified in all genotypes except E and G [12]. Inter-genotype genetic recombination, leading to new viral circulating recombinant forms (CRFs), has also been documented for HIV and HBV [13,14].
Different methodological approaches are used to characterize and monitor genomic sequences in viruses infecting blood donors and eventually recipients. First, direct amplification of viral DNA or RNA by nested (RT)-PCR methods is usually needed to generate viral amplicons in sufficient quantity and length to obtain informative viral sequences. It might be necessary to increase the amount of viral nucleic acid template in the amplification reaction by increasing the volume of plasma in the nucleic acid extraction procedure or by concentrating viral particles in the sample prior to extraction [15][16][17].
Whole or partial genome sequencing is then performed by standard Sanger or next-generation sequencing (NGS) methods. Sanger sequencing is based on the random incorporation of dideoxynucleotides. NGS methods, however, allow for higher throughput, automated sequencing. Non-sequencing approaches, such as strainspecific nucleic acid tests or proteomics can also be used [18,19].
Viral genetic characterization can be performed by blood transfusion centres either in-house, using technologies adapted to local resources and infrastructure, through regional or national reference centres, or, via international collaborative networks.
The analysis of viral genomic sequences in blood donors can have both direct and indirect impacts on transfusion safety. It may shed light on nucleic acid and amino acid variations that can reduce the performance of NAT or serological detection by altering primers/probes hybridization or antigenicity [20][21][22]. Genetic polymorphism may also affect the natural history of infection and negatively impact viraemia and antigenemia, which may challenge the analytical sensitivity of detection assays [23]. Identification of the molecular features responsible for detection failure has proven essential to improve not only molecular and serological blood screening tests but also both qualitative and quantitative viral diagnosis. Monitoring viral genomic sequences can also allow investigation of cross-reactive samples leading to false-positive results, as shown in donors vaccinated against the Japanese encephalitis virus and tested falsely reactive for West Nile virus (WNV) RNA [24]. Characterization of TTIDs is essential for assessing the transmissibility of (re)emerging viruses, and the effectiveness of new screening strategies on the residual risk to blood safety. However, in the absence of a recipient pre-transfusion sample free of viral markers, a high genetic similarity of the viral strains found in both donor and recipient is required to definitively differentiate between transfusion transmission, reactivation of persistent viral infection and iatrogenic infection, especially in highly endemic areas [25]. In addition, molecular epidemiology is essential for monitoring the constant genetic evolution of viruses that may result in changes in genotype geographical distribution and the emergence of inter-genotype CRFs and viral variants with potential differences in their replicative and infectious properties, pathogeny and sensitivity to antiviral treatments or vaccines [26][27][28]. Examining diversity in specific genes can provide insight into single or multiple nucleotide polymorphisms associated with phenotypic changes. For example, a single amino acid change to the WNV NY99 genotype resulted in the development of the WN02 genotype, which had a shorter extrinsic incubation period leading to rapid spread across the United States eventually displacing the original NY99 strain [26,29].
Monitoring the prevalence of viral variants in blood donors appears to be important to assess the risk of transfusion transmission and future epidemiological changes in order to continuously evaluate and improve the performance of screening tests to ensure blood safety. Blood operators are in a unique position to enable investigations for variants of transfusion-transmitted viruses.
However, the place of this type of molecular investigation in the blood transfusion field is still a matter of debate. An interactive session focused on monitoring transfusion-transmitted virus diversity was developed by the Virology subgroup of the International Society of Blood Transfusion TTID Working Party (ISBT TTID WP) during the 31st regional congress of the ISBT-ISBT in Focus!-in June 2021. Following on from widespread interest during and after this session, the present study was conceived to understand how blood operators globally value monitoring for viral diversity and assess the potential impact of monitoring for viral diversity in the transfusion and blood operator fields. Response data were analysed, and basic graphs were generated using the GraphPad Prism v9.4.1 software.

RESULTS
Thirty-two questionnaires were received that contained sufficient data to be analysed. Twenty-nine respondents provided information about the World Health Organization (WHO) income classification of their country, with 13 (45%), 10 (35%) and 6 (21%) classified as high, middle, and low income, respectively. Of all respondents, 11 (34%) identified themselves as blood service/operator, 7 (22%) as transfusion medicine laboratory, 9 (28%) as both transfusion medicine and blood service/operator, 4 (13%) as other (including virology research laboratory, reference laboratory, industry and medical practitioner) and 1 (3%) did not provide the information. Among the 28 respondents whose activities are related to blood donation qualification, preparation and distribution of blood products as well as post-donation expertise, 46%, 14% and 29% were from national, regional and local organizations, respectively (Table 1). One (4%) was from a regional (blood screening)/national (reference laboratory) hybrid structure, and no information was provided for two (7%). National organizations were more frequent in high-income countries (80%) compared to middle-(33%) and low-income countries (50%). There was no difference between middle-and low-income countries with local organizations accounting for 44%-50% (data not shown). Four respondents had activities not linked to blood donations.
Excluding four respondents identified as virology research laboratory, industry, medical practitioner or without identification. b Hybrid regional (blood screening)/national (reference laboratory) structure. c Not available (information not provided by the respondent).
T A B L E 2 Estimating the potential impact of viral diversity monitoring on blood service/operator policy and public health activities. Blood service/operator policy Public health activities Participants noted a range of benefits from monitoring viral diversity, including identification and monitoring of viral variants that may compromise the performance of molecular and serological screening tests, surveillance of circulating viral strains evading therapeutic and immunoprophylactic treatments, investigation of TTID, monitoring national and/or international molecular epidemiology, and improving knowledge of the natural history of infections ( Figure 1). A similar importance was attributed to these different benefits, with the exception of the last. However, just under half of the participants (47%, n = 15) reported that they currently monitor viral diversity, with 40% (6/15) indicating that this monitoring was conducted in a national/ regional reference centre, followed by 40% (6/15) in-house at the local level. One participant reported the viral diversity to be monitored both in-house and in a national/regional reference centre, and the remaining two in a consortium of blood services and a public F I G U R E 1 Perceived benefits of monitoring viral diversity. Participants ranked benefits from 1 (not relevant) to 5 (high importance). Columns represent means, with error bars showing standard deviations. TTID, transfusion-transmitted infectious disease.
F I G U R E 2 Number (a) and types of viruses (b) reported by 15 participants who monitor viral diversity.
health institute. Of those 15 responders, monitoring 4 or more viruses was the most common approach (54%), followed by monitoring for a single virus (20%) (Figure 2a). HBV genetic diversity was monitored by the majority of participants (80%), followed by HCV and HIV (73%), then HEV (47%) (Figure 2b). Sanger sequencing and NGS were the methods used by 60% (9/15) and 47% (7/15) of respondents, respectively, including two respondents reporting the use of both methods ( Figure 3). One participant reported using non-sequencing molecular methods (e.g., genotype-specific nucleic acid tests). NGS was used exclusively in national/regional reference centres or public health institutes, whereas Sanger sequencing was mainly used in-house locally. Although the use of NGS was limited to high-and middleincome countries, there was no significant difference overall compared with the use of Sanger sequencing that was also used in three low-income settings (data not shown). Of 14 respondents interested in implementing viral diversity monitoring, 9 (65%), 2 (14%), 2 (14%) and 1 (7%) preferred NGS, Sanger sequencing, non-sequencing molecular methods and serotyping, respectively ( Figure 3).
Overall, the main limitations to implementing viral diversity monitoring were reported to be financial, followed by inadequate infrastructures, and lack of political support ( Figure 4) favouring the use of regional or national reference centres (Table 3).
Although developing an international collaborative network was the next most frequent response (24%), no responder classified as low by the WHO national income classification favoured this option. The development of suitable in-house testing based on local resources was also on the radar of 17% of participants.

DISCUSSION
Blood safety is directly challenged by the continuous, and mostly unpredictable, emergence of new viruses (e.g., SARS-CoV-2) and variants of well-characterized viruses that are not limited to those viruses known to be highly variable (e.g., parvovirus B19). Indeed, viral variants have been repeatedly identified in blood donors in recent years [28,[30][31][32]. Therefore, monitoring viral genomic sequences appears clearly an important initiative for blood operators as agreed by 97% of participants to the present survey. This figure may be biased by the number of participants in the present survey (n = 32) who may represent primarily individuals already actively involved in viral genetic surveillance and may constitute a limitation of the study. However, only slightly less than half of the respondents (47%) indicated that they were currently monitoring viral genomic sequences. An imbalance between participants from countries with different resource levels could also introduce a bias. Although the majority (45%) of respondents were from countries classified as high income, countries classified as middle (35%) and low (21%) income were also represented.
The reasons for the limited monitoring of viral genomic sequences appear to be primarily the lack of financial resources, irrespective of income classification. Limited financial resources result in a lack of adequate infrastructure, and limited access to advanced technologies, but possibly also the lack of qualified staff. Viral genetic monitoring and characterization require a sufficient number of qualified, trained and competent staff. It might be particularly challenging for blood operators to establish training programmes and develop valorization measures to retain experienced staff [32]. Expertise in viral genetics was also reported as a key limiting factor, suggesting that efforts should be made to more actively involve clinicians and researchers with expertise in molecular virology, epidemiology and infectious diseases in the blood transfusion field. Ultimately, the development of methods for monitoring viral genomic sequences will depend heavily on political will to allocate the necessary resources, as participants indicated.
Different methods exist for monitoring viral genomic sequences.
Genotype-specific (RT)-PCR amplification is a relatively inexpensive approach that does not require very advanced technology. However, the level of information provided remains limited. Sanger sequencing of PCR amplicons of full-length or partial viral genomes directly or after cloning remains a method of choice to characterize viral diversity. NGS is becoming an attractive alternative for 47% of participants.
Compared to Sanger sequencing, NGS can identify a greater diversity of variants and provide the information to enable the broader comparison of genetic relationships in a population of viruses [33]. However, NGS requires costly sophisticated equipment, not only for sequencing itself but also for sample preparation and expensive maintenance. The development of portable third-generation sequencing, based on nanopore technology, is a promising alternative, which applies long-read single-molecule sequencing directly to amplified whole viral genomes, allowing also for the direct identification of recombination events within and between virus species [34]. This technology has been rapidly implemented successfully in complicated clinical situations to provide rapid viral genome consensus sequencing from field-collected samples in resource-limited settings [35]. However, genomic data analysis is complex and may require additional bioinformatics resources and staff training, regardless of the NGS methodology used [36]. Despite these limitations, NGS-based approaches may offer a greater level of data enrichment that may allow for more substantial analyses [18]. Ultimately, the choice of methodological approach will depend on the type of infrastructure available.
Various operational models have been proposed for monitoring viral genomic sequences, ranging from in-house to national and regional reference centres. The majority of participants favoured the use of regional or national reference centres, which present the advantage of re-grouping resources and expertise, and potentially allow for the implementation of the most advanced technologies.
However, this option requires initially expensive infrastructure, adequate centralized management and a strong political will to provide financial resources over the long term. Small local facilities may be T A B L E 3 Best-fit model of viral diversity monitoring based on replies from 28 participants, with blood transfusion activities, a stratified according to WHO national income classification.

WHO national income classification
Models Low (n = 6) Medium (n = 10) High (n = 12) Total (n = 28)  [37]. The role of blood operators as members of the one-health community became evident with the SARS-CoV-2 pandemic, which saw blood operators undertake large seroprevalence studies to inform public health policy [38]. Moreover, blood donor viral sequences have informed various public health initiatives [39,40]. In front of the globalization of human and animal population travel, human activities with increased risk of zoonotic infections, and the environmental changes leading to the geographical expansion of viral vectors, blood operators and the transfusion medicine community should be prepared to actively engage in surveillance of emerging viral infections as a major actor in public health and in order to ensure optimal blood safety.
In conclusion, monitoring for genetic diversity of blood-borne viruses appears important given the many applications for blood safety and global public health. It is important for enhanced collaboration between blood operators and public health authorities to enable the greatest benefits to be afforded. While national and regional reference centres may provide the most suitable place for such monitoring, international collaborations should not be overlooked.
Overcoming financial hurdles will be important. This review of practices inevitably leads to questions regarding the next steps that blood operators and the transfusion medicine community can play in the genetic characterization of blood-borne viruses. There are three core options including (1) conceptualization of specific multi-regional studies to characterize blood-borne viruses identified in blood donors; (2) development of networks to support the implementation of viral molecular characterization, especially in limited-resourced settings; and (3) creation of grant-writing teams to identify and compete for research grants focused on this theme.