Introduction

Varicella-zoster virus (VZV) belongs to the Herpesviridae family, subfamily Herpesviridae alpha, and human herpesvirus type 3 (HHV-3)1. VZV possesses a genome of approximately 125 kb, consisting of 71 open reading frames (ORFs) and five repeat regions (R1–R5)2,3. It is an enveloped virus containing nine known glycoproteins in nine ORFs, namely gK (ORF5), gN (ORF9A), gC (ORF14), gB (ORF31), gH (ORF37), gM (ORF50), gL (ORF60), gI (ORF67) and gE (ORF68)4,5. These glycoproteins fulfill a variety of roles including viral entry, efflux, cell spreading, attachment, replication and envelopment6,7,8,9,10,11,12,13,14,15,16,17,18. It causes infections with two main clinical conditions: varicella and herpes zoster. Primary infection usually manifests as varicella and develops a latent phase in sensory ganglia.VZV reactivation can lead to herpes zoster, which is usually accompanied by neuralgia and postherpetic neuralgia (PHN)19,20. Although the clinical manifestations are typical, there is still a report showing that no VZV-induced rash manifestations were found in fatal cases21, suggesting that new variants may exist. With the wide application of real-time PCR detection technology and the advancement of whole genome sequencing technology, the monitoring of VZV has become more convenient.

The World Health Organization (WHO) recommended countries to establish a comprehensive disease surveillance system in its 2014 Position Paper22. In China, we have been monitoring the incidence of varicella since 2005, and molecular biology surveillance of VZV was started in Jilin Province in 2010.

Jilin Province lies in the northeast corner of China. Jilin Province lies in the northwest of the country of North Korea. The current total population of Jilin Province is 24 million.

In this study, we monitored the incidence of varicella in Jilin Province in the past nearly 20 years, and at the same time, we obtained vesicular fluid samples from the diseased population in the past 15 years to identify the genotype and analyze the nucleotide site variation.

This enables it to provide reference data for vaccine development, clinical diagnosis and treatment at the molecular level. It also contributes to the establishment of a national surveillance program for varicella and herpes zoster, including incorporating live attenuated varicella vaccine into the national routine immunization program and promoting the use of live attenuated varicella vaccine in the elderly.

Methods

Varicella epidemiological data

Varicella epidemiologic data on the number of reported varicella cases and annual incidence rates in Jilin Province were extracted directly from the National Notifiable Disease Reporting System (NNDRS) of the China Center for Disease Control and Prevention (China CDC) from 2005 to March 2024.

Specimen collection

Since VZV is the causative agent of both varicella and herpes zoster, clinicians collected vesicular fluid specimens from suspected varicella or herpes zoster cases from China-Japan Union Hospital of Jilin University, Changchun Infectious Disease Hospital, the Affiliated Hospital to Changchun University of Chinese Medicine and the Second Hospital of Jilin University from fall to winter every year from 2010 to March 2024. These specimens were stored at – 80 °C. In total, 92 specimens were collected, with 38 successfully sequenced (Table 1).

Table 1 Number of specimens from 2010 to March 2024.
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VZV qualitative detection

DNA extraction was performed using the qEx-DNA/RNA kit (Xi’an Tianlong Technology Co., Ltd., China) according to the manufacturer’s instructions. VZV Real-time fluorescence quantitative polymerase chain reaction (Real-time quantitative PCR)kit (Beijing Kinghawk Pharmaceutical Co.,Ltd.) was used for detecting all collected specimens according to the manufacturer’s instructions. Specimens with CT values less than 25 were selected for sequencing.

Reference sequences selection

The reference sequences used in this study were obtained from GenBank (https://www.ncbi.nlm.nih.gov/nuccore) under accession numbers NC001348 (Dumas), MF898328 (Baike), AB097933 (pOka), KJ767491 (YC01), DQ479957 (03–57), and DQ479957 (03–57). DQ479957 (03–500), DQ452050 (M2DR), DQ457052 (CA123), KF811485 (Pasadena, USA), JN704710 (457/2008), JN704709 (1483/2005) which were of Clade 1, 2, 2, 2, 3, 4, 5, 6, 9 and VIII, respectively23.

VZV genotype analysis

Polymerase chain reaction (PCR) was used to amplify specific fragments of 447 bp19 in ORF22 region and 347 bp in ORF38 region24. The PCR products were purified and the nucleotide sequences were determined by Sangon Biotech (Shanghai) Co. Ltd. Five SNPs (37902, 38019, 38055, 38081, and 38177) in ORF22 and one SNP (69424) in ORF38 were genotyped according to the international universal genotyping scheme25. typed. the PCR amplification kit was performed using (GoTaq® Green Master Mix).

Determination sequence of the nine glycoproteins of VZV

Nine regions of glycoprotein ORFs were amplified by polymerase chain reaction (PCR) using GoTaq® Green Master Mix (Promega, USA) and primers (see Table 2 for primer sequences). The PCR products were purified and the nucleotide sequences were determined by Sangon Biotech (Shanghai) Co. Ltd. and edited by Sangon Biotech (Shanghai) to obtain the full sequence of each glycoprotein.

Table 2 Primers for 9 glycoproteins.
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Bioinformatic analysis

Multiple sequences were aligned using MEGA 6 software version 6. Bioedit 7.0 software was utilized to identify nucleotide mutation sites after alignment.

Ethics statement

This study received approval from the Ethics Review Committee of China-Japan Union Hospital of Jilin University during its initial session (2020081904) and the Ethics Review Committee of the Jilin Province Center for Disease Control and Prevention (JLCDC20230002).

Results

Varicella epidemic profile

From 2005 to 2007, the reported incidence of varicella consistently remained below 20 per 100,000. Reports indicate one fatality each in both 2006 and 2007. From 2008 to 2012, the reported incidence rate rose above 20 per 100,000, peaking at 30.5 per 100,000 in 2009, with two deaths reported in 2008. From 2013 to 2016, the reported incidence fell below 20 per 100,000, with no reported deaths. From 2017 to 2019, the incidence rate exceeded 20 per 100,000, reaching a peak of 33.89 per 100,000 in 2019. Between 2020 and 2023, the reported incidence rate fluctuated between below and above 20 per 100,000. Two fatalities were reported in 2020 and 2021, respectively (Fig. 1).

Fig. 1
figure 1

The reported cases and incidence rate of varicella in Jilin Province, China during 2005-Mar 2024. The red number means death cases.

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VZV genotyping clade

After real-time quantitative PCR detection, a total of 38 positive samples were identified, with SNP loci 37902, 38019, 38055, 38081, 38177 and 69,424 being G, G, C, C, A, and G, respectively, which are the same as Clade 2.

Variation of 9 glycoprotein nucleotide sites, amino acid sites and potential N-glycation sites

In this study, the site variation analysis utilized Dumas’ nucleotide site (NC001348) as a reference, designating the starting position of each ORF for the nine glycoproteins as nucleotide site 1. Potential N-glycosylation sites were predicted based on the amino acid sequence Asn-X-Ser/Thr (excluding Asn-Pro-Ser/Thr). As shown in Table 3, only five specimens from Jilin Province exhibited synonymous mutations at the 59,355 locus (T59355C), representing potential N-glycosylation sites (aa781-783: NAF) in gB. No mutations at the 59,355 nucleotide locus have been reported in other regions.

Table 3 The predict potential N-glycation sites of 9 glycoprotein.
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All specimens exhibited identical nucleotide variants in the following glycoproteins: gB (A217C, A294C, and G390T), gH (G573T, C806T, and G2099A), gM (C1276T), gL (C162T, 454ATC458 insertion), gI (G 546 A), gE (C119T), and gC (A289G, T1226A, and T1254C). The variants A217C and G390T in gB, G573T in gH, C162T in gL, and A289G in gC were associated with pOka, YC01, Baike, and Pasadena. USA. The variants C1267T in gM, G2099A in gH, C119T in gE were observed in 9 other reference strains: pOka, YC01, Baike, 03-500, M2DR, CA123, Pasadena. USA, 457-2008, and 1483-2005.

In 38 samples, nucleotide mutation sites occurred once in gK (C170T, A298G), gN (C66T, C195T), gH (T2238C), gM (C1290T), gI (T239C, A460C, C776T), gE (T23C, G1869A), gC (G283T, C666T, C1273A, C1629T, A1632C). Nucleotide mutation sites were observed three times in gK G202T, gB (C627T, T2349C), gM (G112A), gC (T320A), while gK (T554G) and gI (A92G) were observed five times.

A total of 21 samples exhibited varying degrees of nucleotide site variation, among which 8 specimens (VZV12-5, VZV14-4, VZV14-6, VZV14-10, VZV16-5, VZV22-3, VZV23-1, and VZV23-2) presented a mutation site. Six specimens (VZV10-14, VZV14-12, VZV17-6, VZV19-3,VZV21-4,VZV22-2) displayed two mutation sites; four specimens (VZV10-16, VZV11-3, VZV12-1, and VZV23 -1) had three mutation sites. Two samples (VZV13 -3 and VZV14 -2) contained four mutation sites; one sample (VZV20 -4) possessed five mutation sites; and one sample (VZV21 -1) exhibited six mutation sites. All identified mutation sites are detailed in Tables 4, 5, 6 and 7.

Table 4 List of VZV nucleotide mutation of gK, gN and gB.
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Table 5 List of VZV nucleotide mutation of gH and gM.
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Table 6 List of VZV nucleotide mutation of gL, gI and gE.
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Table 7 List of VZV nucleotide mutation of gC.
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In gC, there is a repetitive region named R2, which consists of a 42-bp tandem sequence, the tandem sequences had 5 types which were named A, B, C, D and E, respectively and the 32-bp residue at the end of tandem sequence were named a, b, c, d and e, respectively (Table 8). The repeats of all specimens were 5–12. The length and repeats were shown in Table 9 for all specimens.

Table 8 List of the sequence of nucleotide and amino acid in R2. “_” means final 32-bp sequence was named a, b, c, d and e.
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Table 9 Length of gC and repeat structure of gC in R2.
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Discussion

This study presents critical reference data regarding the incidence of varicella and multiple mutation sites across nine glycoproteins of VZV, which are essential for the future assessment of the safety, efficacy, and immunogenicity of current vaccines. Such information is vital for advancing VZV vaccine development, enhancing clinical diagnosis and treatment protocols, as well as establishing national surveillance programs for varicella and herpes zoster.

Data on varicella incidence reveal a pattern of alternating high and low rates every 4–5 years. However, the COVID-19 pandemic appears to have disrupted this regular trend after 2019, resulting in a marked annual fluctuation between high and low incidence rates. Moreover, deaths were documented in the years 2006, 2007, and 2008, as well as in 2020 and 2021, underscoring the surveillance system’s sensitivity. In China, clinicians are responsible for the initial diagnosis of varicella cases and the entry of case information so that data can be obtained from NNDRS for epidemiologic analysis. The introduction of the disease and vaccination recommendations by healthcare professionals, as well as home isolation measures during the COVID-19 outbreak, may be the main reason for the decrease in incidence. Therefore, it is recommended that clinicians proactively collect cyst fluid samples to provide basic data for studying the pathogenesis of breakthrough cases and molecular level prevention and control of VZV26,27,28,29.

The prevalent VZV in Jilin Province from 2010 to 2024 has been Clade 2, with no other branches seen, which may be related to the lack of major population movements in Jilin Province in the last 100 years.

VZV is an enveloped virus, with nine known glycoproteins (gK, gN, gC, gB, gH, gM, gL, gI, and gE) that are instrumental in the virus’s entry, egress, cell-to-cell transmission, attachment, replication, and enveloping processes. In this study, we sequenced VZV samples from 38 patients suffering from varicella or herpes zoster, and examined the variations in nucleotide sites across these nine glycoproteins.

gK, which plays an important role in viral replication, is hydrophobic and has multiple transmembrane structural domains6. Four sites exhibited synonymous mutations in gK from seven specimens, consistent with previous report indicating gK stability. gN is essential for the maturation of gM and promotes viral proliferation, syncytium formation, and cell-to-cell transmission when it forms a complex with gM. gN inactivation decreases viral titer and affects syncytium formation7. Mutations at two nucleotide sites were found in the gN of only one of the 38 samples, but they were synonymous and did not cause amino acid changes, suggesting that the gN in Jilin VZV viruses was relatively stable from 2010 to March 2024.

gB is responsible for the initial infection of virus particles into cells and plays an important role in virus adsorption and penetration. It is the second most abundant glycoprotein in VZV. Research has shown that the gB of strain S2 in the Singapore subgroup is mainly dominated by mutations at positions 294 and 390, while there is no mutation in the Bangkok subgroup. All samples in this study share these three common nucleotide variation sites (A217C, A294C, and G390T), which are identical to the reference strains pOka, YC01, Baike, and Pasadena. USA, similar to the Singapore subgroup 2 strain, therefore this variant does not have a clear geographical distribution attribution. Meanwhile, as all samples in this study have these three identical variants, continuous monitoring is still needed to determine whether it is a strain specific expression of the Jilin strain.

gH is the third most abundant glycoprotein in VZV, which induces the production of neutralizing antibodies in the organism and also mediates virus transmission between cells10,11,12. C806T and G2099A appeared in all specimens from Jilin Province, which is different from the results of a previous study in China31. C806T mutation site did not appear in one specimen from Guangdong Province, which is located on the southern coast of China, in 2009. Regardless of whether this result is related to geographic location, there is a need to expand the sample size for continuous monitoring, and it is recommended that more provinces participate in VZV studies, share sequences, and work together to monitor whether there is a consistent trend of variation at these mutant sites.

gM plays a role in virus transmission between cells13,14,15. We also detected some gM variants in this study. First, the same C1267T site variant as the other nine reference strains was found in all samples. This is in contrast to the absence of the 1267 site variant previously found in some specimens from four other provinces in China32. Therefore, this is the first time that the C1267T site variant has been found in China. Secondly, the same mutation G112A, at site 112, occurred in three of the surveillance samples, and the time span of these three samples was eight years, and the same mutation at this site also appeared in the specimen from Shanghai in 200733. It is suggested that this mutation is not a de novo mutation and has a certain distribution in China.

VZV gL is a molecular chaperone of gH. gH: gL complex is required for gH maturation and membrane expression10, and the complex promotes cell membrane fusion, as well as the formation of a large number of multinuclei in transfected cells12,34. There is evidence that membrane fusion is induced by a multiprotein core complex composed of gB and gH/gL complexes11. In the present study, we also detected variants concerning gL. Firstly, a site variant of A76G was also detected, which also appeared in samples from Beijing in 2009, but has not been reported in China since then32. Secondly, an extra AGT codon was found in all the samples, which was the same as that in the reference strains Oka, YC01, M2DR, Pasadena.USA and 1483/2005, and further studies are needed to determine whether this variant affects the function of the proteins differently or even causes antigenic variability. It is noteworthy that the C162T site variant in gL, the A217C, A294C and G390T site variants in gB and the G573T site variant in gH are consistent with the site variants in the reference strains pOka, YC01, Baike and Pasadena.USA, which suggests that there is functional unity among gL, gB and gH.

gI plays a role in the maturation and distribution of gE to the cell surface17. It has been reported that the ORF of gI appears to be very stable and no gI mutations have been detected in vaccine strains4. The vaccine strain Baike selected in this study showed a degenerate base A800R (A/G) at position 800, which may result in an N→D amino acid mutation at the corresponding amino acid site. In addition, the presence of a mutation at the A92G site in gI was identified in five samples, which spanned the period from 2010 to 2023, and it is noteworthy that A92G resulted in an amino acid change aaQ31R, but the effect of this alteration on its function is still unclear. Third, an alteration in C776T (aaS259F) was seen in one sample in 2014 but not in other years and has not been reported, so it may be a transient occurrence,. Fourth, analysis of gI also revealed variants at the T239C and A460C, with T239C leading to the V→A amino acid mutation. Moreover, these 2 variants appeared in the COVID-19 post-pandemic, so whether these 2 new site variants are related to COVID-19 infection has not been similarly reported. In conclusion, the appearance of these missense mutation sites suggests that not only vaccine strains but also wild strains should be better monitored for VZV gI stability.

gE is the most abundant glycoprotein in VZV and plays an essential role in viral replication, assembly and mediation of VZV cell-to-cell transmission17. It stimulates the production of neutralizing antibodies and cell-mediated immune responses, with major antigenic epitopes located in the 1–161 amino acid fragment. Among them, regions 11–30, 71–90, 91–110 and 106–125 elicited the strongest cellular immune responses35,36. In the present study, the C119T (aaT40I) mutation was present in all reference strains and specimens from Jilin province, except for the Dumas strain, which is consistent with previous reports26,37,38,39,40. Unlike the missense mutation in aaD150N41, none of the synonymous mutations in T660C appeared42. Neither the T23C (aaV8A) nor G1869A mutations that appeared were found within the epitope. This preliminary indicates that the VZV gE epitope of the samples in this study is highly conserved. But T23C and G1869A appeared in 2021 and 2020, respectively, and the appearance of these two variants may be related to COVID-19.

The gC is thought to be critical for VZV replication in the skin8. It contains the R2 region that is essential for virus attachment43. Previously reported R2 structures include ABABAAx (Dumas, NC01348), AAABBBBx (MSP, AY548170), and ABBBBAAx (BC, AY548171)41. five nucleotide sequence combinations of the R2 region yield four amino acid sequence combinations A, B, C, D, E (Table 4). The combination of E was found only in the selected reference strain Baike due to the presence of concatenated base C1273Y (C/T) in the gC of Baike, which suggests that Baike should be subjected to continuous monitoring of nucleotide site variation and evaluation of immunization efficacy. In addition, this study identified 5–12 repetitive sequences in the R2 region, with cases of 5 repetitions observed in 2014, 2022, and 2023. It is noteworthy that during these years, measles outbreaks occurred in Jilin Province in 2014 and COVID-19 outbreaks occurred in 2022 and early 2023. Whether the number of sequence repeats in the R2 region was suppressed due to epidemics of other viruses needs to be further investigated. One case of VZV infection with a repeat number of 12 was observed in January 2024 in a pregnant woman who had severe symptoms of cutaneous herpes and was not receiving medication. These changes described above may reflect the impact of concurrent infectious disease epidemics and human interventions on VZV replication in the skin, and clinicians are advised to consider the epidemiologic context when evaluating VZV cases.

In addition, our study of the gC region revealed the presence of three nucleotide mutation sites outside of the R2 region, namely A289G, T1226A, and T1254C. The common feature of these three nucleotide mutation sites is that they are present in samples of the reference strains pOka, YC01, Baike, and Pasadena.USA and in all of the Jilin province, which at least suggests that these three mutated nucleotide sites are not unique to the Northeast Asian geographic region, but are perhaps specific to Jilin Province, which will require continuous monitoring in the future to support this conclusion. In addition to the three nucleotide mutation sites mentioned above, two nucleotide variants of CA123, C666T (aaA→T); T1629C (aaT→A), were found in two specimens from Jilin Province, respectively, in this study. Previously, VZV with both Clade1 and Clade3 characteristics was reported in Changchun City, Jilin Province39, and CA123 was Clade 5 on genetic branching, but the specimens from Jilin Province in the present study were Clade 2, which suggested that, in addition to serial testing of VZV from the perspective of molecular biology, emphasis should be placed on molecular epidemiological surveillance.

The VZV genome has an estimated mutation rate of 10^-6–10^-7/site/year and is highly conserved44. In this study, the researchers found that four mutated nucleotide sites appeared only once before the COVID-19 epidemic, while 12 mutated nucleotide sites were found for the first time after the COVID-19 epidemic. Given that nucleotide mutations in COVID-19 continue and its spread has not ceased, the effect of COVID-19 on VZV nucleotide mutations remains to be confirmed. Healthcare professionals are encouraged to test patients for COVID-19 infection when collecting blister fluid specimens.

The mechanism of attenuation of the live attenuated varicella-zoster vaccine remains incompletely understood. In a previous study, the varicella vaccine (Oka-Merck) was approximately 85% effective in healthy children, with less than 5% developing a mild vesicular rash45. Another study reported that the incidence of breakthrough cases (rash more than 42 days after vaccination) increased as the duration of vaccination increased46. Unfortunately, no vaccine-associated cases or breakthrough cases were monitored in this study. Therefore, clinicians should strengthen the inquiry and registration of vaccination information when seeing patients, whether they are varicella patients or herpes zoster patients, and collect specimens in a timely manner in order to further study the attenuation mechanism of VZV envelope glycoprotein at the molecular level.

In summary, this study reviewed the epidemiology of varicella in Jilin Province from 2005 to March 2024, and analyzed nucleotide site mutations in 9 known VZV envelope glycoproteins that occurred in the province between 2010 and March 2024. In light of China’s measles prevention and control strategy47, it is advocated for expediting or facilitating the establishment of a national strategy to prevent and control VZV transmission, mobilizing the entire society to extend its benefits to more people.