The phylodynamic of H1a genotype measles virus in Jiangsu province, China, 2005-2017


 Background:
The change on the pressures during viruses evolving will cause changes in phylodynamic. To know phylodynamic characteristic of measles virus in high vaccination coverage era, the phylodynamic characteristic was analyzed using nucleoprotein gene sequences of measles viruses isolated from Jiangsu province of China from 2005 to 2017.
Methods:
Nucleoprotein gene sequences of measles viruses were used to analyze gene distance and construct phylogenetic tree with Markov chain Monte Carlo algorithm. The mean gene distance within each group was computed with MEGA7.0 software. The phylodynamic of measles virus circulating in Jiangsu province was constructed using Bayesian skyline plot analyses.
Results:
Our results showed that the decline trend is observed in the gene distance of nucleoprotein gene and effective population size of measles virus with time. Two clusters of H1a genotype shows multiple origins and the extinction of variants with time.
Conclusion:
These findings highlight that phylodynamic of measles viruses is a helpful tool to assess the effectiveness of epidemic control.


Background
The incidence and morbidity of measles have decreased dramatically due to use of effective measles vaccine [1,2]. To reach the goal of eliminating measles by the end of 2020 in the world, at least 95% measles immunization coverage at national level and minimum 95% coverage in all supplemental immunization activities have been implemented in most of countries since 2010 before or later [3][4][5]. As a result, the incidence of measles in these countries decreased rapidly and has been sustaining at historic low level, even measles was veri ed that is has been eliminated in some countries [6].
Before the vaccine era, the immune pressure measles virus facing in vivo was strong cross-immunity elicited by primary infection of other measles virus variants [7,8]. The prevalence features of measles virus in this era is generally biennial, explosive in classic epidemiology, and that many strains coexist in molecular epidemiology [8,9]. In vaccine era of high measles immunization coverage, measles virus is under overwhelming immune pressure. Consequently, the phylodynamic of measles virus changed. For example, molecular epidemiology of measles virus in USA showed the disappearance of partial genotypes ever circulating before measles was eliminated [9]. Therefore, to monitor phylodynamic of measles virus can provide a mean to help document the interruption of measles transmission and assess the effectiveness of measles control [10][11][12].
Jiangsu province, located in the east of China, is a developed region with high quality of public health service. The coverage of measles immunization has reached to 95% in 2005. Furthermore, two rounds of supplemental immunization activities were implemented before 2011. The incidence of measles has been sustaining at the historic low level since 2010 except 2015( Figure 1). The molecular epidemiology surveillance of measles virus was introduced in 2005 and H1a is the unique genotype before October, 2017. Only three D8 genotype cases were identi ed in 2017 and 2018, respectively [13]. The surveillance of antibody level among health population showed a higher herd immunity [14]. Measles virus encounters more immune pressure than before, and the corresponding characteristic may present in phylogenetic of measles virus in Jiangsu province. In this study, we reported the phylodynamic features of measles virus H1a genotypes isolated from 2005 to 2017 in Jiangsu province, China.

Collection of specimens and isolation of virus
Throat swabs were collected from patients with serologically con rmed cases of measles (IgM positive).
Specimens were kept in 3mL Copan Universal Transport Media and stored at 4℃, then transported into Jiangsu Provincial Center for Disease Control and Prevention to isolate measles virus. The specimens were inoculated onto Vero-Slam cells, and the cells were observed for cytopathic effect. Inoculated cells were blind-passaged up to three times before being discarded. Cells were harvested when the cytopathic effect was maximal.
The ampli cation and sequence of Measles virus N gene RNA was extracted from infected Vero-slam cells using QIAamp Viral RNA Mini Kit (Qiagen, GmbH, Hilden, City name, German). The N gene(634bp) was ampli ed using One step RT-PCR kit (Invitrogen) with primers MeV216(5'-TGGAGCTA TGCCATGGG AGT-3') and MeV214(5'-TAACAATGATGGAGGGTAGG-3') according to as described previously [15]. Puri ed PCR products were sequenced bidirectionally using the ABI 3730 DNA analyzer in Shanghai Sangon Biotech Company Limited. Sequences were edited manually using BioEdit software, and imported into MEGA7.0 software to conduct alignment using muscle program.

Calculation of mean gene distance of N gene within various groups by year
The N gene sequences of measles virus were grouped by year. The mean gene distance within each group was computed with MEGA7.0 software. Variance estimation method is bootstrap method, and number of bootstrap replications is set as 500.

Model selection and phylogenetic analysis
The best-t model for nucleotide substitution was tested in jModelTest v3.7 using Akaike information criterion (AIC). It was GTR+I+G (general time reversible model (GTR); proportion of invariable sites(I); gamma distribution(G). Then TMRCA was estimated using a coalescent-based Baysian method with GTR+I+G model in BEAST v1.8.4 software. The sequences were partitioned into 3-codon positions. The convergence of parameters was analyzed using Tracer v1.7.1. The effective sample size of each parameter was ensured more than 200. The maximum clade credibility tree was generated using TreeAnnotator v1.8.4. The dynamics of measles virus epidemic over time in Jiangsu province was estimated using Bayesian skyline plots, a non-parametric smoothing method for approximating past population dynamics.

Results
The Based on the sequence analysis of H and N genes, not only genotype of measles virus was identi ed[16-18], but also the source of wild types measles viruses associated with outbreaks was inferred [9,[19][20][21]. For example, the new genotype of measles emerged in a given region was associated with the imported virus by travelers between various countries [22,23]. Moreover, for a given genotype, the molecular diversity was observed between measles viruses with different geographic origins. Jennifer et al con rmed that resurgence of measles virus in 1994 was more closely related to wild type viruses previously circulating in Europe, Africa, and Japan than the indigenous virus circulating in USA [9]. The indigenous virus was interrupted after the 1989-1922 epidemic in USA. It suggests that to monitor molecular feature of measles virus can provide more helpful information on for classic epidemiology as well as our nding herein.
Measles virus has the ability of higher mutation and replication rates. This variation is modulated by two processes: the host's immune response to infection and the bottleneck at transmission. The former is elicited by primary infection in pre-vaccine era while by vaccine immunization in vaccine era. The latter includes incubation time, infectious periods, seasonality, population density, and so on. For measles virus, overwhelming immune pressure limits the chance of an adaptive response in vivo. It will lead the appearance of different phylodynamic in various vaccine immunization era. In pre-vaccine era, it shows that many strains coexist in phylogenetics [7]. Nowadays, more than 95% vaccine coverage has reached in many regions. Subsequently, immune pressure become the predominant drivers, and it will make partial epidemic strains die out, and the number of extinct strains will increase with the time of persistent high immune pressure. As described in this study, the cluster 1 disappeared in 2016 and only a small part of strains in cluster 2 survived in 2017.

Conclusion
In this study we describe the phylodynamic characteristic of measles virus in region with at least 95% measles immunization coverage. Our nding highlights the importance of molecular epidemiology of measles virus on epidemic surveillance.

Funding
This project was supported partly This work was supported by Six Talent Peaks Project in Jiangsu Province (WSN-017) and Jiangsu Province "333" project (LGY2016021). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and material
The datasets used in this study are available from the corresponding author on reasonable request.

Consent for publication
Not applicable

Competing interests
We declare no competing interests. Figure 1 The incidence of measles in Jiangsu province from 2005 to 2018