Varicella-Zoster Virus—Genetics,

Molecular Evolution and Recombination

Daniel P. Depledge and Judith Breuer

Contents

1 The Genetics of Varicella-Zoster Virus .............................................................................. 2

1.1 Structure of the VZV Genome................................................................................... 2
1.2 Structural Variation in the VZV Genome.................................................................. 3
1.3 The Coding Potential of the VZV Genome............................................................... 5
1.4 The Kinetics of Gene Expression and Genome Replication..................................... 7
2 The Molecular Evolution of Varicella-Zoster Virus........................................................... 8
2.1 The Evolution of VZV Genetic Screening ................................................................ 8
2.2 Establishment of a Unified Virus and Clade Nomenclature ..................................... 13
2.3 The Role of Homologous Recombination in Shaping VZV Evolution.................... 13
2.4 Genome Diversity and the Global Phylogeography of VZV.................................... 14
2.5 Evolution of VZV Clades .......................................................................................... 16
References .................................................................................................................................. 17

Abstract This chapter first details the structure, organization and coding content of
the VZV genome to provide a foundation on which the molecular evolution of the
virus can be projected. We subsequently describe the evolution of molecular pro-
filing approaches from restriction fragment length polymorphisms to single
nucleotide polymorphism profiling to modern day high-throughput sequencing
approaches. We describe how the application of these methodologies led to our
current model of VZV phylogeograpy including the number and structure of geo-
graphic clades and the role of recombination in reshaping these.

D. P. Depledge (&)
Institute of Virology, Hannover Medical School (MHH), Hannover, Germany
e-mail: depledge.daniel@mh-hannover.de
Department of Microbiology, NYU School of Medicine, New York, USA
J. Breuer
Department of Infection & Immunology, University College London, London, UK

Current Topics in Microbiology and Immunology (2023) 438: 1–23

https://doi.org/10.1007/82_2021_238
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021
Published Online: 11 August 2021 1
2 D. P. Depledge and J. Breuer

1 The Genetics of Varicella-Zoster Virus

1.1 Structure of the VZV Genome

Varicella-zoster virus (VZV) was first isolated in 1953 by Thomas Weller (Weller
et al. 1958) who showed that the virus could infect a wide variety of human cell
types and that strains of virus recovered from patients with varicella or herpes zoster
could not be distinguished by their cultural attributes. At the same time, Dr.
Robert E. Hope-Simpson, a general practitioner, began a programme of study in
which he carefully examined *3500 patients, including 192 Hz cases, who visited
his practice over a 16-year period. This, in combination with careful reading of the
available anatomical and epidemiological literature regarding herpes zoster led to
his famous hypothesis that: ‘Following the primary infection (chickenpox), virus
becomes latent in the sensory ganglia, where it can be reactivated from time to time
(herpes zoster)’ (Hope-Simpson 1965). Eighteen years later, this hypothesis was
proven by Donald J. Gilden’s crucial discovery of VZV DNA in latently infected
human ganglia (Gilden et al. 1983).
The enveloped VZV virion comprises a nucleocapsid and tegument surrounded
by a host-derived lipid bilayer embedded with viral glycoproteins (Padilla et al.
2003; Storlie et al. 2008). These proteinaceous particles are pleomorphic to
spherical in shape, *150–200 nm in diameter. Upon entry to the host cell, tegu-
ment proteins are released into the cytoplasm to inhibit antiviral responses and
initiate transcription of viral genes [reviewed in Virgin et al. (2009)]. The nucle-
ocapsid contains itself docks with the nuclear membrane and delivers the
double-stranded DNA (dsDNA) viral genome into the nucleus.
The isolation and examination of the double-stranded DNA genome through
restriction fragment length maps of VZV laid the foundations for producing the first
VZV genome sequence. Restriction endonucleases are naturally occurring enzymes
of bacteria and archaea which cleave foreign DNA at specific nucleotide sequences
(e.g. EcoRI recognizes and cleaves (|) the sequence G|AATTC), evolving as host
defences against viruses. Restriction enzymes were first used to analyse VZV DNA
in 1977 (Oakes et al. 1977). Following digestion of DNA with a restriction enzyme,
the fragments are separated on denaturing acrylamide gels and the sizes of each
fragment estimated by comparison with DNA of known molecular weight and size
(https://www.ncbi.nlm.nih.gov/probe/docs/techrflp/). The resulting fingerprint
reflects the location of the sequence recognized by the restriction enzyme in the
genome and is altered by genetic variation.
In 1981, Dumas and colleagues used the restriction enzymes Xba1, Bgl1 and
Pst1 in combination with Southern blotting and fragment hybridization to describe
the organization of the VZV genome (Dumas et al. 1981). These findings were
confirmed by Stephen E. Straus and colleagues in 1981 who used 11 restriction
enzymes for their analysis and additionally confirmed that the results were con-
sistent with the genome existing in a circular state (Straus et al. 1981). These
findings were later confirmed by additional studies making use of restriction
Varicella-Zoster Virus—Genetics, Molecular Evolution … 3

enzymes which also reported that the VZV genome exists in at least two isomeric
configurations (Straus et al. 1982; Ecker and Hyman 1982).
The first full genome sequence for VZV was obtained using the M13-
dideoxynucleotide sequencing approach and published by Davison and Scott
(1986). They showed that VZV genomes are approximately 125 kilo base pairs
(kbp) in size with a mean G + C content of 46% (Fig. 1a). In common with other
human-infective herpesviruses, the genome comprises two unique segments, termed
the unique long (UL) and unique short (US), that are flanked by inverted terminal
repeat (TR) and internal repeat (IR) structures with high G + C content (68% for
the TRL/IRL and 59% for the IRS/TRS) (Fig. 1a-b) (Oakes et al. 1977; Dumas et al.
1980, 1981; Straus et al. 1981, 1982; Davison and Scott 1986; Ludwig et al. 1972;
Richards et al. 1979; Zweerink et al. 1981; Martin et al. 1982). The very short
(*88 bp) TRL and IRL sequences flank the UL region, while the long (7.3 kbp) IRS
and TRS sequences flank the US region. While the composite structure of the VZV
genome allows for inversion of the US region (Fig. 1c) (Dumas et al. 1981),
inversions of the UL region have only been reported in small numbers (*5%) of
virions. These genomes are not thought to be functional due to the disruption of
sequence required for viral DNA cleavage (Kinchington et al. 1985; Kaufer et al.
2010).

1.2 Structural Variation in the VZV Genome

A key observation provided by the generation of restriction fragment length

polymorphism maps for VZV was that the variation in fragment lengths could be
mapped to specific regions of the genome. This led to the identification and
characterization of six tandem direct reiterations (termed R1, R2, R3, R4, and R5) of
short repeat sequences, one of which (R4) is found in both the IRS and TRS
(Fig. 1b). With the exception of R5, all reiteration regions were reported to be
GC-rich (Ruyechan et al. 1985).
First, Casey and colleagues showed that the R4 region comprises variable
numbers of GC-rich 27 bp repeat units (Casey et al. 1985). The same group
characterized the R2 repeat region in ORF14 which codes for glycoprotein C and
showed it to comprise 42 bp GC-rich repeat units, which again varied in number
between different strains (Kinchington et al. 1986). The first VZV genome sequence
revealed that the R1 and R3 repeat sequences are present in ORFs 11 and 22, while
R5 which lies in the non-coding regions between ORFs 60 and 61 was charac-
terized by Hondo in 1988 (Hondo and Yogo 1988). In 1988, Kinoshita and col-
leagues cloned and sequenced fragments containing R1 and demonstrated that the
combinations of 18 and 15 bp sequences which made up the repeat element differed
between unrelated isolates (Kinoshita et al. 1988). Some length variation also
occurred following tissue culture passage, suggesting instability. Interestingly, R1
length variation was found between viruses isolated from a vesicle and the ganglion
in the same patient although several analyses by different groups of two or more
4 D. P. Depledge and J. Breuer

Fig. 1 Organization of the VZV genome. a The 125 kb VZV genome has an average GC content
of 47% with local increases in the reiterative and terminal repeat regions. b Schematic
representation of the VZV genome structure comprising the terminal repeat long (TRL), unique
long (UL), internal repeat long (IRL), internal repeat short (IRS), unique short (US), terminal repeat
short (TRS), reiterative repeat regions (R1–R5) and the origin of replication (OriS). c The VZV
genome is arranged in two equally prevalent isomeric configurations that differ only in the
orientation of the US. d The recently updated VZV transcriptome (Braspenning et al. 2020)
includes 71 transcripts encoding canonical ORFs with their UTRs defined (grey), 39 alternative
isoforms of existing RNAs (orange), 7 fusion RNAs (dark blue) and 17 putative polyadenylated
ncRNAs (red). Wide and thin boxes indicate canonical CDS domains, while thin boxes indicate
UTRs, respectively. Absence of CDS regions indicates that the respective VZV RNA has an
uncertain coding potential. Reiterative repeat regions R1, R2, R3, and R5, as well as both copies of
the R4 and OriS repeats shown as purple (R1–R5) and yellow boxes (OriS) embedded in the
genome track

viruses from different vesicles in the same patient have without exception found the
viruses to be identical (Kinoshita et al. 1988; Shibuta et al. 1974; Pichini et al. 1983;
Takayama et al. 1988). The instability of R1 mirrored findings for R3 (Hayakawa
et al. 1986). Here, Hayakawa and colleagues found that the Hpa1 fragment K,
which contains R3, showed length variation on passage, while fragments F and G
Varicella-Zoster Virus—Genetics, Molecular Evolution … 5

remained stable up to high passage. The Hpa1 K fragment also varied among
patients who had shared a room and infected one another (Hayakawa et al. 1986).
A definitive analysis, however, was obtained by the repeated passage and genome
sequencing of one virus which revealed that R1 OriS and R4 elongated by 1–2
repeat units after 72 passages, R2 and R5 remained stable and R3 was completely
altered in its structure (Grose et al. 2004).
The length variation of these repeat regions led several groups to explore their
use for genotyping. Initial evaluations showed mis-duplication for R2 resulting in
PCR ladders, while the R4 was unstable on low passage (Takada et al. 1995). R5
was stable and had three alleles, comprising different combinations of the 88 and
22 bp units. Further studies confirmed the difficulties of amplifying R2, R3 and R4,
and the stability, albeit uninformative nature of R5 (Hawrami et al. 1997; Yoshida
and Tamura 1999; Yoshida et al. 2003; Sauerbrei and Wutzler 2007). R1
sequencing revealed a large number of variants with certain sequences predomi-
nating both among UK and Japanese strains (Hawrami et al. 1997; Yoshida et al.
1999). Multiple R1 alleles were found in one patient who died from HZ (Kinoshita
et al. 1988) and in the ocular fluid from patients with acute retinal necrosis (Abe
et al. 2000). This may reflect slippage due to increased viral replication since all
these patients were immunosuppressed. Importantly, the recent analysis of wild
type VZV genomes collected as part of the Shingles Prevention Study also showed
expansion and contraction of the reiteration regions both within and between dis-
tinct viral populations and strains (Jensen et al. 2020; Harbecke et al. 2020).
The only other report of structural variation in VZV concerns the lab strain
VZV32 in which a 2158 bp deletion was identified within ORF12 (Cohrs et al.
2017). Notably, this deletion would also ablate the transcription and synthesis of
ORF12-ORF13 fusion proteins (Braspenning et al. 2020). To date, there are no
other reports of such large-scale structural variations (i.e. deletions, insertions, or
translocations of sequences > 1000 bp) in wild type VZV genomes.

1.3 The Coding Potential of the VZV Genome

The original annotation (Davison and Scott 1986) of the VZV genome reported 70
genes, later termed ORFs. Three of these genes were duplicate, present in both the
IRS and TRS segments that flank the US region. Only a single gene, encoding
ORF42, was predicted to contain a spliced intron. A further two ORFs, ORF9A and
ORF33.5 were reported in 1997 (Ross et al. 1997; Preston et al. 1997). Both of
these ORFs are encoded by RNAs that originate from transcription start sites within
existing ORFs (ORF9 and ORF33, respectively) and terminate at the same cleavage
and polyadenylation sites as the RNAs encoding ORF9 and ORF33. This poly-
cistronic arrangement of RNAs within a gene cluster is a common feature of
herpesviruses (Depledge et al. 2019; Stern-Ginossar et al. 2012; O’Grady et al.
6 D. P. Depledge and J. Breuer

2016; O’Grady et al. 2019; Arias et al. 2014; Bencun et al. 2018; Whisnant et al.
2020) with many additional putative ORFs identified in the latest VZV reannotation
(Braspenning et al. 2020). In 2000, a second spliced VZV RNA was identified,
initially termed as ORF S/L and later renamed as ORF0 (Kemble et al. 2000).
Unusually, the spliced intronic region lies entirely within the 3ʹ UTR of the
ORF0-encoding transcript although a mutation at the primary stop codon in some of
the vaccine-strains results in a C’ terminal extended protein product (Kemble et al.
2000). Further evidence of spliced VZV RNAs was obtained in 2010 when
Tomohiko Sadaoka and colleagues reported an abundance of internally spliced
RNAs originating in the ORF50 (glycoprotein M) locus (Sadaoka et al. 2010). In
2017, the discovery of the VZV latency-associated transcript (VLT), located anti-
sense to ORF61, provided the first multiply spliced VZV RNA (Depledge et al.
2018a). The subsequent development and application of Oxford Nanopore
Sequencing allowed, for the first time, a comprehensive reannotation of the VZV
transcriptome (Braspenning et al. 2020) that demonstrated the existence of addi-
tional spliced transcript isoforms, including several candidates that encode ‘fusion’
proteins (Fig. 1d). Transcripts encoding ‘fusion’ proteins contain in-frame splice
junctions that join coding sequence domains from otherwise distinct proteins,
resulting in a fusion and potential new function for the resulting protein. These
should not be confused with proteins capable of fusion with membranes. The
coding capacity of the non-canonical transcript isoforms reported in this study is
varied (Braspenning et al. 2020). 17 are predicted to be ncRNAs based on the
absence of coding sequences (CDS) longer than 30 amino acid. The remainder
predominantly encodes canonical proteins either in their native form or with short N
′ or C′ terminal extensions. Western blotting and mass-spectrometry approaches
cannot resolve these differences so alternative approaches such as ribosome pro-
filing are still required. Several fusion transcripts were also reported, and the
encoded fusion proteins confirmed by Western blotting approaches where anti-
bodies were available. The most notable of these is the VLT-63 fusion protein that
has been implicated as a key transactivator during reactivation from latency
(Ouwendijk et al. 2020).
Despite these improvements to the depth and breadth of the VZV transcriptome
annotation, it remains likely that the full transcriptional potential of VZV has yet to
be revealed and there remains a need for studies that examine the full diversity of
viral proteins synthesised during infection, as well as a detailed analysis of the
putative non-coding RNAs, including microRNAs (miRNAs) and other short
non-coding RNAs. While the former has been partially tackled through mass-
spectrometry analyses, sensitivity remains an issue. The study of miRNAs and
small non-coding RNAs is also still an area of active study with contrasting results
(Depledge et al. 2018a; Markus et al. 2017; Umbach et al. 2009).
Varicella-Zoster Virus—Genetics, Molecular Evolution … 7

1.4 The Kinetics of Gene Expression and Genome

Replication

In common with other herpesviruses, VZV transcripts are designated as

Immediate-Early (IE), Early (E) or Late (L), based upon their expression profile
during lytic infection. While the kinetic classification of most VZV transcripts was
initially derived from their homology to HSV-1 counterparts, a small number were
experimentally validated (Lenac Roviš et al. 2013; Reichelt et al. 2009). Recently, a
comprehensive survey of the VZV transcriptome using long read sequencing pro-
vided the first complete annotation and kinetic classification of 136 polyadenylated
RNAs produced during lytic VZV infections (Braspenning et al. 2020). This
analysis described six distinct categories of transcription, mirroring what has pre-
viously been reported for human cytomegalovirus, a related betaherpesvirus
(Weekes et al. 2014). The six kinetic categories identified for VZV transcription are
IE, True-Early, Early-Late, Leaky-Late, True-Late and Transactivated/True-Late.
IE transcripts are transcribed immediately following entry of the viral genome
into the host cell nucleus and predominantly encode transcriptional regulators
required for transactivation of E and L genes. The eight VZV IE transcripts were
defined by expression in the presence of cyclohexamide (CHX), a protein synthesis
inhibitor. These include RNA00-2 (pORF0), RNA04-1 (pORF4/IE4), RNA15-2
(putative ncRNA), RNA43-2 (putative ncRNA), RNA61-1 (pORF61/IE61),
RNA61-2 (Nʹ terminal truncated pORF61) and RNA63a/b-1(pORF63/IE63). Three
of the encoded proteins, pORF4, pORF61 and pORF63, all of which have been
demonstrated to be transcriptional regulators (Wang et al. 2009; Michael et al.
1998; Kost et al. 1995) are also present as tegument proteins in the VZV virion
(Kinchington et al. 1995).
Fifty-four VZV transcripts are designated as Early and generally encode proteins
involved in DNA replication. Early transcripts, like IE transcripts, are expressed in
the presence of phosphonoacetic acid (PAA), an inhibitor of viral DNA replication
that prevents transcription of Late genes. 30 of the Early VZV transcripts are
subcategorized as True-Early, while 24 are subcategorized as Early-Late. This
distinction arises from whether relative transcript abundance is reduced (True-
Early) or maintained (Early-Late) following viral DNA replication (Braspenning
et al. 2020).
Similarly, the 69 Late transcripts can be categorized as either Leaky-Late
(n = 28), or True-Late (n = 41), dependent on whether low-level expression is
observed in the presence of PAA. Late transcripts encode structural proteins that are
crucial for virion formation and egress. The most intriguing set of transcripts belong
to the transactivator/True-Late (TA/TL) category. Here, RNA49-1, RNA58-1 and
RNA62-1 show limited expression during the IE phase (i.e. are expressed at
comparatively low levels in the presence of CHX), no expression during the E
phase and high expression during the Late phase. RNA62-1 encodes pORF62/IE62,
the dominant VZV transcriptional regulator (Perera et al. 1993; Moriuchi et al.
1994) and a major component of the VZV virion (Kinchington et al. 1992). IE62
8 D. P. Depledge and J. Breuer

transactivates all three kinetic classes of VZV genes in the absence of other viral
proteins including all IE genes, ORF4, ORF61, ORF62 and ORF63. By contrast,
IE4, IE61 and IE63 do not or minimally stimulate the ORF61 promoter (Wang et al.
2009; Michael et al. 1998; Kost et al. 1995; Ruyechan 2010). Host transcription
factors are also thought to contribute to viral gene expression (Ruyechan 2010),
while VZV replication has also been shown to proceed upon transfection of cell
with viral DNA (i.e. in the absence of virion proteins) (Cohen 2010).
Importantly, VZV transcriptomes vary little between cell types and viral strains,
including neurons (Braspenning et al. 2020; Baird et al. 2014; Markus et al. 2014;
Jones et al. 2014; Sadaoka et al. 2016), suggesting a prominent role for either
commonly expressed cellular transcription factors, or viral proteins in coordinating
VZV gene expression.
In contrast to lytic infections, transcription during VZV latency is highly
restricted. Here, studies of post-mortem human trigeminal ganglia, acquired within
4–9 h of death, have shown VZV transcription is limited to the latent VLT isoform
and, in some TG, the VLT-63 fusion transcript (Depledge et al. 2018a; Ouwendijk
et al. 2020). A similar observation was made in iPSC-derived sensory neurons;
although here, only the five-exon latent VLT isoform was detectable during latency
with the VLT-63 fusion transcript only detected following treatment with ani-
somycin, a JNK inhibitor (Ouwendijk et al. 2020). While the pattern of gene
expression during VZV reactivation from latency remains unknown, initial studies
have shown the VLT-63 fusion transcript encodes a pVLT-pORF63 fusion that
retains a broad transactivation capability and that transactivation of latently infected
neurons with a vector expression pVLT-pORF63 results in viral reactivation
(Ouwendijk et al. 2020). Whether this reactivation takes the form of successive
waves of gene expression [i.e. the phase I, phase II phenomenon reported for the
related alphaherpesvirus HSV-1 (Kim et al. 2012)], remains unknown.

2 The Molecular Evolution of Varicella-Zoster Virus

2.1 The Evolution of VZV Genetic Screening

The ability to produce and compare maps of genetic variation is central to the
molecular epidemiology of viruses including VZV. Through the use of molecular
epidemiology approaches, we are able to derive information on the origins, trans-
mission, spread and evolution of viruses. This in turn provides insights into
within-host evolution, pathogenesis and viral genetic determinants of virulence. The
five predominant methods that have been used for VZV are: restriction fragment
length polymorphism (RFLP) analysis, length polymorphisms of reiteration regions
(LPRR, discussed in detail in Sect. 1.2 above), restriction site polymorphisms
(RSP), single nucleotide polymorphism (SNP) profiling and whole genome
sequencing (WGS).
Varicella-Zoster Virus—Genetics, Molecular Evolution … 9

Restriction fragment length polymorphism (RFLP) analysis

As described above, restriction endonucleases are naturally occurring enzymes of
bacteria and archea which cleave foreign DNA at signature nucleotide sequences.
These can be isolated and applied to digest DNA into fragments that are subse-
quently separated and sized on denaturing acrylamide gels. Initial studies using the
enzymes EcoR1 and Hind1 failed to distinguish between five isolates of
varicella-zoster (Oakes et al. 1977); however, a later study of seven VZV isolates,
two collected in Japan and five collected in the USA, was more successful
(Richards et al. 1979). In this study, cleavage patterns were generated using five
enzymes EcoR1, Hind1, Sma1, BamH1 and Ava1. Ava1 maps were identical for all
seven isolates, while between 0 and 3 restriction fragments were generated by the
other four enzymes. The authors did not find any obvious groupings of isolates, and
notably, there was not obvious distinction between the Japanese and US isolates
(Richards et al. 1979). The authors also observed that VZV was less variable than
the herpes simplex virus control and that no differences between low (10) and high
(36) passage viruses were obvious. The genomic stability of VZV and the appli-
cability of RFLP as an epidemiological tool were confirmed by Zweenik and col-
leagues. They analysed two strains KmcC and AW which were passaged up to 71
and 30 times, respectively, in the human fibroblast line WI-38 (Zweerink et al.
1981). Comparison of KMcC p6, 46 and 72 using six restriction enzymes showed
minor differences between virus harvested at pass 72, as compared with p6 and p42,
which were themselves identical. There was no difference between AWp6 and p30.
Much of the impetus for molecular genotyping of VZV came from the early
success by Takahashi and colleagues in developing a live-attenuated vaccine strain
(vOka), which was quickly shown to be safe and immunogenic in children
(Takahashi et al. 1974). Genotyping of parental and vaccine Oka strains revealed
several differences using the Hpa1 enzyme, an extra band in vOka when digested
by Kpn1 or Sma1, but no differences in Hind111, EcoR1 or Bgl1 digests (Ecker and
Hyman 1982). Not long afterwards, Martin and colleagues published unique dif-
ferences between BamH1, Bgl1 and Hpa1 digests of the vOka vaccine strain and six
wild type strains collected in the USA (Martin et al. 1982). Small differences
between the wild type strains were noted and led Straus to examine a larger set of
17 viruses taken from patients with varicella and zoster, including four high passage
viruses (Martin et al. 1982; Straus et al. 1983).
Using a combination of seven enzymes, Hind111, Xbl1, EcoR1, Bgl1, Pst1,
BamH1 and Sma1, Straus and colleagues noted that all 17 isolates differed from
each other, but that five were more similar and that the enzymes EcoR1, Hind111
and Sma1 appeared to be the most informative for distinguishing between strains
(Straus et al. 1983). Importantly, this paper showed for the first time that two strains
of VZV from members of the same family who contracted varicella from the same
source were identical by BamH1, EcoR1, Hind111 and Sma1 digests and different
from other viruses in the study. Also, viruses from three different vesicles in the
same person were identical to each other and different from viruses isolated from
other unrelated individuals. Straus and colleagues were also able to identify that
10 D. P. Depledge and J. Breuer

most differences between viruses arose from length variation of certain key frag-
ments. The value of the accumulating molecular epidemiological data at this time
was illustrated by a seminal paper in which Straus and colleagues used RFLP to
show that the virus which had caused primary varicella in a leukaemic child was
identical to the virus recovered from the lesions of herpes zoster occurring in the
same child three years later (Straus et al. 1984). This seminal finding provided proof
of the original concept outlined by Hope-Simpson, namely that herpes zoster is
caused by reactivation of virus which has persisted latently following primary
varicella infection (Hope-Simpson 1965).
Restriction site polymorphisms (RSP)
Two studies in the 1980s observed that digestion patterns of Bgl1 and Pst1 distin-
guished US wild type clinical strains rom the Oka vaccine and vaccine related strains
which caused clinical symptoms (Martin et al. 1982; Gelb et al. 1987). Following on
from this in 1989, Adams and colleagues identified a Bgl1 site in a BamH1 fragment,
D, which was stably present in the vaccine, parental Oka and three wild type
Japanese strains, but absent in wild type American strains (Adams et al. 1989).
Unlike other informative sites, none of the cleaved products of BamH1 fragment D
showed length variation in different viruses. Moreover, a 1940 bp Hind111 sub-
fragment of the BamH1 D fragment could not be cleaved further by other enzymes.
Together this suggested that the Bgl1 site, which distinguished the the vOka strain
and wild type isolates collected in Japan from wild type isolates collected in the US
strains, was present as a single nucleotide polymorphism within a stable region.
Exploiting this, LaRussa and colleagues developed a PCR to detect this Bgl1 site,
which is located in ORF54 for use as an easy method to distinguish vaccine and wild
type rashes. Finding that the Bgl1 site was also positive in 3 of 20 clinical varicella
and herpes zoster strains isolated in the USA, LaRussa and colleagues (LaRussa
et al. 1992), using data from the restriction maps, identified a Pst1 site in ORF38
which had previously been found to be positive in all strains isolated in the USA
tested and negative in the vaccine. Another SNP which distinguished between iso-
lates from Japan and Europe was identified as causing the substitution of a histidine
to a proline at nucleotide 10 in the ORF10 protein (Kinchington and Turse 1995).
This abrogates binding of an antibody directed against the N terminus of the ORF 10
peptide and could theoretically provide a serological test to differentiate between
isolates typically found in Japan and those typically found in Europe.
Single nucleotide polymorphism (SNP) profiling
Following publication of the Dumas genome sequence and the advent of PCR,
detection of single nucleotide polymorphisms (SNPs) became the most widely used
method for genotyping of viruses as well as other organisms (Faga et al. 2001;
Barrett-Muir et al. 2001; Loparev et al. 2004). The development SNP-typing tools
for molecular epidemiological analysis of VZV were pioneered simultaneously by
two US groups and one in the UK. SNPs are sites that contain single base pair
variations (Collins et al. 1997, 1998). These sites generally are considered biallelic
due to the limited number of transversions that have been found (Brookes 1999).
Varicella-Zoster Virus—Genetics, Molecular Evolution … 11

Each allele must occur in more than 1% of the population to be considered an SNP
(Kruglyak 1997; Wang et al. 1998). Individuals who share many of the same SNPs
are likely to have arisen from a common ancestor and can therefore be grouped by
inheritance (Collins et al. 1997). SNP alleles occurring at 10% or more frequency
are useful in allelic discrimination.
In 2001, a UK-based group published data on SNPS occurring in 10 UK isolates of
VZV, seven cases of varicella and three zoster (Barrett-Muir et al. 2001). Here, 50
primer pairs, each amplifying a 500 bp fragment and spaced out across the genome at
3000 base pair intervals, were designed. Denaturation and reannealing of PCR prod-
ucts produced homoduplexes and heteroduplexes which could be separated on dena-
turing gels and used to identify fragments for which heteroduplex mobility was shifted
compared with that of the homoduplex. Fifteen fragments within the 10 genomes
showed heteroduplex mobility shift, and Sanger sequencing revealed 29 polymor-
phisms. Using a total of eleven SNPs, three distinct groups of viruses were identified as
circulating in the UK. Further analysis of the SNP patterns enabled the selection of a
subset of SNPs, located in ORFs 1, 21, 50 and 54 that could satisfactorily discriminate
between the three groups of viruses, as well as the attenuated vaccine strain vOka.
These findings were confirmed and extended following a survey of over 75 VZV
strains isolated from multiple continents including Africa (Zambia, Guinea-Bissau),
Asia (India, Bangladesh, Singapore, Japan) and North America (Barrett-Muir et al.
2003; Quinlivan et al. 2002). Phylogenetic analysis of these strains led to the
assignment of four geographically distinct clades termed C, J, B and A. Concurrently, a
US-based group was utilizing a similar approach—this one targeting five glycoproteins
as well as ORF62. Here, a total of 61 SNPs were identified, 21 across the glycoproteins
and 40 within the ORF62 coding sequence (Faga et al. 2001). This led to four distinct
phylogenetic groups, A, B, C and D, being defined (Wagenaar et al. 2003). At the
same time, a second US-group, based at the centres for Disease Control and Prevention
(CDC) identified a 447 bp fragment of ORF22 that was sufficiently polymorphic to
segregate three phylogenetic clades which were termed European (E), Mosaic (M) and
Japanese (J) (Loparev et al. 2004). Here, however, the use of two distinct clade
classification schemes (ABCD vs. EMJ) caused confusion for several years prior to the
emergence of whole genome sequencing as a viable alternative and the corresponding
establishment of a unified nomenclature (Sect. 2.2 below).
Whole genome sequencing (WGS)
Prior to 2006, only seven full length VZV genome sequences were available:
Dumas, MSP, BC, pOka and the Biken, Merck and GSK Oka vaccines.

Note that the usage of the terms strain, isolate and variant is not subject to
strict definitions, and as such, these are often use interchangeably in the case of
VZV and other herpesviruses. Recently, stricter definitions have been sug-
gested but are still not universally applied. One pertinent example was supplied
by Prof. Vincent Racaniello of Columbia University (https://www.virology.ws/
2021/02/25/understanding-virus-isolates-variants-strains-and-more/).
12 D. P. Depledge and J. Breuer

Isolate is used for a virus that was isolated from an infected host and
propagated in culture. Functionally, an isolate is not distinct from a strain
other than that strains may possess experimentally proven differences in
properties of infection, spread and reproduction.
A variant is an isolate whose genome sequence differs from that of a
reference virus. No inference is made about whether the change in genome
sequence causes any change in the phenotype of the virus.
A strain is a variant that possesses unique and stable phenotypic char-
acteristics. Such characteristics can only be ascertained by the results of
experiments done in the laboratory, in cells in culture and in animals, coupled
with observations made in infected humans.
In the context of VZV, no significant differences in phenotype have been
reported between genetically distinct viruses except for those observed in the
live-attenuated vOka vaccine virus (Gomi et al. 2002), it is South Korean
counterpart (Jeon et al. 2016), and an attenuated virus derived through
extensive passaging in cell culture (Peters et al. 2006).

The advent of short-read sequencing technologies released by Illumina, 454 and

ABI revolutionised the ability of researchers to capture, assemble and publish
whole viral genome sequences with comparative ease. In the context of VZV, this
led to the release of a further thirteen strains in 2006 and the construction of the first
VZV phylogenies (Peters et al. 2006; Norberg et al. 2006a; Tyler et al. 2007).
While the reiterations could not be resolved using short-reads, these were obtained
by Sanger sequencing for some of the new genomes. Importantly, many of these
new strains were isolated in geographically diverse regions of North America, and
one was isolated in Morocco.
Despite the advances in whole genome sequencing technologies, the ability to
sequence viruses directly from clinical DNA extracts was effectively impossible due
to the vast amounts of host DNA present. Initial approaches, thus, relied on growing
clinically isolated viruses in culture for a few passages before sequencing. While
this made sequencing a genome possible, the relative expense precluded this
approach from becoming commonplace. Moreover, concerns remained over whe-
ther viral diversity was being lost and new mutations being introduced as a result of
culturing.
In 2010, Depledge and colleagues developed a novel adaptation of the
SureSelect target enrichment approach and demonstrated an ability to successfully
capture, sequence and assemble viral genomes from clinical samples (Depledge
et al. 2011). Further refinement of this approach (Depledge et al. 2014a) led to an
explosion in full length VZV genome sequences sampled from geographically
distinct regions including Guinea-Bissau (Depledge et al. 2014b), the USA,
Singapore, Sweden and Germany (Jensen et al. 2020; Harbecke et al. 2020;
Norberg et al. 2015). At the time of writing, there are now over 150 complete VZV
genomes available on Genbank (https://www.ncbi.nlm.nih.gov/genbank/).
Varicella-Zoster Virus—Genetics, Molecular Evolution … 13

2.2 Establishment of a Unified Virus and Clade

Nomenclature

While the emergence of VZV phylogenies were a welcome development, each uti-
lized its own system for naming clades and grouping viruses. This confusing situation
was, however, rapidly resolved during a meeting to discuss a common nomenclature,
organized in London in July 2008 (Breuer et al. 2010). Four principles were agreed
upon: the nomenclature should (1) be distinct from the three established nomencla-
tures, (2) reflect the clade structure, (3) reflect the order in which full genome
sequences became available and (4) describe the provenance of the strain. To that
end, the proposed nomenclature for VZV was established, and a prototypic viruses
for each of the (initial) five clades were chosen. A putative sixth clade was later
confirmed (Norberg et al. 2015). Furthermore, a set of 28 SNPs across 15 ORFs were
identified as being the minimum complement of SNPs required to identify a putative
clade. Finally, a scheme for naming newly sequenced viruses was proposed although
its usage by the wider VZV community has been minimal to date.

2.3 The Role of Homologous Recombination in Shaping

VZV Evolution

In a viral context, genetic recombination may occur when at least two viral genomes
co-infect the same host cell and exchange genetic segments (see Fig. 1 from Norberg
et al. (2015) (100) for a comprehensive explanation of this process). The structure of
the crossover site defines the mode of recombination (Austermann-Busch and Becher
2012; Scheel et al. 2013). Homologous recombination (HR) occurs in the same site in
both parental strands. By contrast, illegitimate recombination (IR) (Lai 1992), some-
times referred to as non-homologous recombination, occurs at different sites of the
genetic fragments involved (Galli et al. 2010). For a broader review of the modes and
mechanisms of viral recombination, we direct the reader to Pérez-Losada et al. (2015).
A number of studies have now shown HR is a major force in shaping the evolution of
herpesviruses (Norberg et al. 2006a; Norberg 2010; Bowden et al. 2004, 2006). These
include studies focussed on HSV-1 (Norberg et al. 2004, 2006b, 2011), HSV-2
(Norberg et al. 2007), HCMV (Lassalle et al. 2016) and the varicellovirus genus (Kolb
et al. 2017). HR has previously demonstrated for HSV-1 and HSV-2 with recombi-
nation rates shown to be higher in the latter (Norberg 2010; Norberg et al. 2007).
For VZV, coinfection with multiple genotypes has been shown to occur both
in vitro and in vivo (Quinlivan et al. 2009; Depledge et al. 2018b; Dohner et al.
1988), and reinfection with VZV can result in the reestablishment of latency with
later reactivation to cause herpes zoster (Quinlivan et al. 2002). Several published
studies provided evidence that at least some of the VZV clades likely emerged
through inter-clade recombination (Peters et al. 2006; Norberg et al. 2006a; McGeoch
2009), while recent studies also provided evidence of intraclade recombination
14 D. P. Depledge and J. Breuer

(Norberg et al. 2011; Zell et al. 2012). These studies also speculated that the rate of
detected recombinants may increase as more genomes are sequenced. In 2015, an
analysis of 115 full length VZV genome sequences demonstrated that VZV recom-
bination is frequent both within and between clades. While this has raised occasional
concern about the potential for recombination events between wild type virus and the
live-attenuated vOka strain, no such recombinant has ever been reported, despite
extensive and ongoing surveillance (Simberkoff et al. 2010).

2.4 Genome Diversity and the Global Phylogeography

of VZV

Using 249 VZV genome sequences downloaded from Genbank in July 2021, a new
phylogenetic network was generated and curated (Fig. 2). Collectively, these
genome sequences represented seven clades and at least three inter-clade recom-
binants. A description of the viruses present in each clade, and their notable
characteristics, follows:

Fig. 2 VZV network phylogeny. Phylogenetic network showing distribution of VZV clades.
A phylogenetic network was constructed using SplitsTree4 (Huson and Bryant 2006) and
represents the alignment of 249 VZV genomes downloaded from Genbank in July 2021. Repeat
elements within the genomes (R1–R5, OriS and the terminal repeat region) were masked prior to
network construction. VZV genome sequences falling outside the major classed are classed as
recombinant (R) and are marked in red
Varicella-Zoster Virus—Genetics, Molecular Evolution … 15

Clade 1
The first sequenced VZV isolate, strain Dumas, serves as the prototypic Clade 1
genome sequence (Davison and Scott 1986). Clade 1 sequences are typically
derived from Europe and USA and are the most numerous (Jensen et al. 2020;
Norberg et al. 2015; Zell et al. 2012).
Clade 1 also includes two wild type VZV strains containing gE mutations which
confer an accelerated cell spread phenotype (Grose et al. 2004).
Clade 2
VZV isolates from this clade are dominant in Japan and South Korea. However, the
majority of sequenced VZV genomes assigned to clade 2 are derived from isolated
collected in the USA, UK and Germany. The prototypic genome sequence for
Clade 2 is derived from strain pOka, which also serves as the parent to the
live-attenuated vOka strain (Gomi et al. 2002). Interestingly, while VZV isolates
derived from rashes caused by vOka cluster tightly with strain pOka, VZV clade 2
genome sequences isolated in Europe, and the USA are placed on a distinct
subnode from strain pOka. Thus, there remains a need to generate more full genome
sequences from circulating wild type virus in Japan and South Korea in order to
assess whether individual SNPs may segregate clade 2 isolates derived from dif-
ferent geographic regions.
Clade 3
One of the two European/US clades (along with clade 1), clade 3 has been
extensively sampled across several studies (Norberg et al. 2015; Zell et al. 2012).
The four prototypic strains, 11, 22, 03–500 and HJO, were first published in 2006
with the majority of recent additions derived from VZV genomes isolated in Europe
(Peters et al. 2006; Norberg et al. 2006a, 2015; Zell et al. 2012).
Clade 4
Initially comprising just two prototypic sequences, strain 8 (Tyler et al. 2007) and
strain M2DR (Loparev et al. 2007), clade 4 shares SNPs with both clade 1 and clade
2 and may represent an ancestral recombination event. The addition of more full
length VZV genome sequences from both the USA and Europe suggest this clade is
geographically widespread (Jensen et al. 2020).
Clade 5
Dominant in Africa, Malaysia and Indonesia. The prototypic clade 5 sequence is
strain CA123, isolated in California and first reported in 2007 (Loparev et al. 2007)
although this remains controversial due to its distal placement in the tree relative to
VZV clade 5 isolates collected from Guinea-Bissau (Depledge et al. 2014b) and
Singapore (Norberg et al. 2015). Currently over 35 full length genome sequences
cluster within the clade 5 node with an intra-node bifurcation separating isolates
collected in Africa versus those collected in Europe and the USA. No single SNP
directs this segregation. Clade 5 viruses possess a unique R1 region structure, not
16 D. P. Depledge and J. Breuer

seen in other clades, although recent reports of a few clade 3 viruses with this
structure, potentially representing recombinant viruses, require further confirmation
(Jensen et al. 2020).
Clade 6
First defined in 2015, clade 6 currently comprises three full length genomes. The
prototypic member, strain var160, was isolated from a seven year old male in
Mexico (Garcés-Ayala et al. 2015). A second clade 6 sequence was isolated in
France in 2013 (Norberg et al. 2015). Recent analyses suggest this to be a
recombinant between clades 1, 2, 3 and 5 (Pontremoli et al. 2020).
Clade 9
First defined in 2020, the prototypic strain 457/2008 was isolated from a five-year
old female in Germany (Zell et al. 2012). Two additional sequences make up the
(putative) clade, strains 12–135 and 13–169, but are placed on a somewhat distal
node. Mostly likely, clade 9 represents a recombination event between clades 1, 2, 3
and 5 that is distinct from that which gave rise to clade 6 (Pontremoli et al. 2020).
Inter-clade recombinants
A total of six putative inter-clade recombinants have been reported (Norberg et al.
2015; Zell et al. 2012). Three of these (1483/2005, Cli/UK/CSF/2909/2011, and
Cli/UK/CSF/3009/2011) are orphan sequences that do not cluster with others.
Moreover, the two UK samples have since been shown to be derived from mixed
populations (Depledge et al. 2018b). The remaining three (isolates 12–212, 13–103,
13–353) cluster together and form a deep subnode with clade 3 sequences (Jensen
et al. 2020), Whether these represent a new clade or simply a cluster of recombi-
nants remains uncertain until such time as deeper sampling of clade 3 genomes is
performed.

2.5 Evolution of VZV Clades

Analysis of highly conserved parts of the herpesvirus genomes supports the

hypothesis that speciation of VZV (and other herpesviruses) has occurred in tandem
with their cognate hosts, (McGeoch et al. 2006). The mutation rates derived from
these highly conservative analyses, some tenfold less than other herpesviruses, date
the most recent common ancestor (MRCA) for the VZV clades as around
100,000 years ago, a time consistent with the early migrations of humans from
Africa (Wagenaar et al. 2003; Zell et al. 2012). More recent analyses, however,
[reviewed in Breuer (2020)] including phylogeographical modelling (Pontremoli
et al. 2020) suggest the most recent common ancestor for VZV clades to be some
10,000 years ago, co-incidental with substantial human migrations in Eurasia and
Africa (Reich 2018). These analyses place the likeliest origin of currently circu-
lating VZV clades as European. More work to sequence VZV from around the
Varicella-Zoster Virus—Genetics, Molecular Evolution … 17

world in particular from undersampled regions such as South America, the Middle
East, Sub-Saharan Africa and the Western Pacific region and from ancient genomes
would further confirm or refute these findings.

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