Comparative Study on Molecular Epidemiology of Measles H1a Outbreak and Sporadic Cases in Shandong Province, 2013-2019

Background: Measles caused by measles virus (MeV) is a highly contagious viral disease which has also been associated with complications including pneumonia, myocarditis, encephalitis, and subacute sclerosing panencephalitis. The current study collected33 strains from 2013 to 2019 in 13 cities of Shandong province and separate them into 2 group, outbreak cases and sporadic cases. Comparative of genetic characterization between 15 outbreak strains and 18 sporadic strains was made using nucleotide sequencing of the C-terminal region of the N protein gene(N-450). Results: The results showed that all 33 stains belonged to genotype H1a. The outbreak strains and sporadic strains distribute crossly in phylogenetic tree. Sequences alignment revealed some interesting G and A transversion, which change the amino acids, on sites 1317, 1422, 1543. The nucleotide sequence and amino acid homologies of 15 Shandong outbreak isolates were 98%–100% (0–10 nucleotide variation) and 97.7%–100%, for sporadic isolations, they are 97.3%–100% and 96.6%–100% respectively. The mean evolution rate of 15 outbreak isolations and 18 sporadic isolations was 4.73× 10 -3 and 2.068× 10 -3 substitutions per site per year separately, which is higher than the study made before 2002. Conclusions: This report compared epidemic and genetic characteristics of outbreak strains and sporadic strains, and raise evolutionary study of sporadic cases may be helpful for discovery of the possibility of outbreak, especially in the stage of measles elimination. a cluster. However outbreak strains K333, K323, K322 and K330, K352, K384, K385, K462, K514 which isolated from the same year 2015, are on different branches. These above suggest the complexity of measles transmission in Shandong: there are both single-chain transmission across year and multi-chain transmission in the same year in Shandong province. The comparative of outbreak and sporadic strains in evolution rate was made by the Bayesian Markov chain Monte Carlo (MCMC) method in BEAST, and the result shows the mean evolution rate of outbreak isolations and sporadic isolations was 4.73× 10 -3 and 2.068× 10 -3 substitutions per site per year. Although the difference of evolution rate between outbreak and sporadic strains is small, but it is higher than previous study( Rima et al. (1997) estimated the rate of mutation for MeV in the eld to be 5 ×10 -4 substitutions per base per year[18] and a similar estimate of 4 ×10 -4 was made by Jenkins et al. (2002)[20]). Whether measles evolved faster recent years than previous years, and whether outbreaks always evolved a bit faster than sporadic cases. All of these questions require more cases and further studies to answer in our next step study. Measles virus; N-450: carboxy-terminal 450 nucleotides of signaling lymphocyte-activation molecule; CPE:cytopathic effect; Analyses; Carlo; of most recent HKY: UCLD: uncorrelated lognormal-distributed;


Introduction
Measles caused by measles virus is a highly contagious viral disease which has also been associated with complications including pneumonia, myocarditis, encephalitis, and subacute sclerosing panencephalitis. It is one of the leading causes of death among young children worldwide, with a fatality rate as high as 15% in children [1]. Even though a safe and cost-effective vaccine is available, there were 110,000 measles deaths globally in 2017, mostly among children under age 5 years [2]. Measles virus is an enveloped virus with a nonsegmented, negative-sense RNA genome that is 15,894 nucleotides in length. It belongs to the genus Morbillivirus in the Paramyxovirinae sub-family. MeV have 24 genotypes recognized to date (A, B1-B3, C1-C2, D1-D11, E, F, G1-G3 and H1-H2) [3,4] and there are four predominant measles genotypes currently circulating worldwide: D8, B3, H1 and D4 [5]. MeV is genotyped using carboxy-terminal 450 nucleotides of N-region (N-450), the most variable sequence in the MeV genome, or the entire protein-coding region of the H gene [6].
The WHO Measles and Rubella laboratory Network (LabNet) has been established tomonitor progress toward mortality reduction and elimination of measles. Besides serologic testing, another important function of the network is to support the genetic characterization of currently circulating measles viruses. Shandong measles laboratory is amember of the LabNet. The traditional epidemiology characterization had changed in recent years, that is, the season distribution was delayed and the age distribution waschanged; there was great difference among different provinces on the incidence of measles [7]. Outbreak was not the main form of measles in china, and many cases of measles are sporadic. Genetic information is a key factor for improving our understanding of virus transmission pathways [8]. The genetic analysis of the N-450 is a useful tool for establishing whether connections exist between concurrent measles cases [9]. So our study made an effort to seak whether if there is any genetic correlation between the sporadic cases and outbreaks by comparing N-450 region.

Sample collection and virus isolation
We enrolled 15 measles outbreak cases and 18 measles sporadic cases from Shandong measles surveillance system from 2013 to 2019. All the enrolled cases are de nited according to the diagnostic criteria of measles. Throat swabs samples were collected from individuals identi ed as measles after obtaining informed consent from patients or their parents.
Clinical specimens were inoculated on Vero/SLAM (signaling lymphocyte-activation molecule; also known as CDw150) cells [10] and the cells were observed for cytopathiceffect (CPE). Inoculated cells were blind-passaged up to three times before being discarded. Cells were harvested when the CPE was maximal. sequence ampli cation and sequencing All isolations were sequenced and genotyped using the standard protocols. Brie y, viral RNA was extracted from 140 μl Virus culture medium according to the manufacturer's instructions (Omega, USA). RNA was reconstituted in 60 μl nuclease-free waterand stored at -80°C until tested. Measles virus N gene was ampli ed using primers; MeV-F: 5'-GGGAGGCTTGAACTTTGG-3'; MeV-R: 5'-TCCGTGTCTGAGCCTTGT-3'.
One-step RT-PCR was performed using Takara PrimeScript RT Master Mix. The PCR program was followed by 98°C for 10 s and 30 cycles of 98°C 1 s, using the Omega Gel Extraction kit. Sequences of the PCR products were obtained using BigDye terminator chemistry version 3.0 according to the manufacturer's protocol for both sense and antisense strands on an automated ABI PRISM 3100 DNA Sequencer (PerkinElmer, Beijing, China).

Sequence analysis
Sequences were analyzed using Sequencer (Gene CodesCorporation, Ann Arbor, MI, USA) and aligned using version 7.0 of BioEdit (www.mbio.ncsu.edu/BioEdit/BioEdit.html). Phylogenetic analyses were performed and trees were generated using the Molecular Evolutionary Genetics Analyses (MEGA 7.0) software based on N-450 coding region. Phylogenetic trees were constructed by comparison with the reference strai-ns de ned by the WHO previously reported or references committed among 2013-2016 using the neighbor-joining method. The reliability of the groupings was estimated using bootstrap resampling of 1000 replicates. Genebank number of reference sequences use were: LC002659.1, AB824116.1, AB915260.1, KF533141.1, KT031805.1, MH375617.1, MN602401, MK628225,AB915260.1. The evolution rate and molecular clock phylogeny of global measles isolates were inferred using the Bayesian Markov chain Monte Carlo (MCMC) method in BEAST version 1.6.1.

Epidemiological information
Out of the 15 outbreak cases, 10 cases were males and 5 were females. In terms of age, 8 cases were children under 15 years old and 8 adults. For 18 sporadic cases selected in this study, 9 were males and 9 were females; 6 were children under 15 years old and 12 were adults. Except 2 cases were oating population from other provinces, 31 cases involved in this study were resident population. All cases which clinical symptoms informations were available had fever and rash, most of them accompanied with cough, less with Carta Symptoms, Conjunctivitis or Kirschsprung, and rarely with lymph node enlargement or joint pain, only 4 cases accompanied with serious complications (table 1). No matter outbreak cases or sporadic cases, most cases had less than two doses of vaccine or not been vaccinated at all (table 1). In this study, we tried to make sure there are both outbreak strains and sporadic strains in the same isolation time (table.1). The outbreak and sporadic strains we selected from 13cities which have certain consistency in geographical distribution. Separation areas and quantities are shown below ( Fig.1).

Sequences alignment
All genetic changes in the Shandong isolates in our study were base substitutions, and no deletion, insertions, or frame-shift mutations, but some interesting mutations were found. Sequences named AB824116.1 which is a reference sequence obtained from Genebank and K217, K314, K403 which were isolated from sporadic cases, together with k295, k322, k333 obtained from outbreaks generated G and A transversions on sites 1317, 1422, 1543(the position were aligned by the measles reference sequence) at the same time comparing to other sequences in our study. All these transversions induced amino changes. Then we aligned these sequences and found all the sequencesseparated in different years, 2013-2015, are totally the same. There are also unique mutation on site 1417 of k540 and 1581 of k314, which all induced amino change. k540 and k314 are all the sporadic case sequence. The nucleotide sequence and amino acid identities of 15 Shandong outbreak isolates were 98%-100% (0-10 nucleotide variation) and 97.7%-100%, for sporadic isolations, they are 97.3%-100% and 96.6%-100% respectively. We separated 33 Shandong isolations into two groups,sporadic group and outbreak group. The distances between the 2 group was 0.008.
Genotype and phylogenetic analysis PCR products of the 33 viral isolates in the COOH-terminus of the nucleoprotein gene were available and then sequenced. All 33 viral isolations together with 9 reference sequences obtained from Genebank were genotyped as H1a (Fig2.a), using WHO reference strains sequences by neighborjoining tree. Further phylogenetic analysis weremade between 33 Shandong strains and the 9 references sequences (Fig2.b)

Discussion
China is now in the phase of accelerated measles control and Shandong province wasalso in the phase of measles control. Although from 2016, measles incidence remainslow until 2021, and most of measles are sporadic cases, but before 2020 there are still several outbreaks occurred in Shandong. There are many factors correlated with occurrence of measles outbreak, such as accumulation of susceptible children, seasonal factors, density of population and so on [12][13][14][15][16]. This study made some effort to explored the factors causing measles outbreaks from another perspective: We included 33 isolates from outbreak or sporadic cases from 13 cities of Shandong provinces from 2013 to 2019; And then we compare the outbreak sequences to the sporadic sequences of N-450 to study whether if measles outbreak is related to genetic differences.
As reported previously, measles virus has high genetic stability and there is very little variation in the N sequences of viruses isolated from the same chain of transmission [17]. Moreover, very little genetic changes revealed from the same genotype that were collected several years apart [18,19].
Similarly, In our study, no matter outbreak sequences or sporadic sequences isolated from different cities or different years have high identities of nucleotide and protein. And the distances between the two group areonly 0.008. These results demonstrate measles strains epidemic in Shandong keep high stability between the outbreak and sporadic strains. However there were some interesting mutations found in our study. Sequences named AB824116.1 which is a reference sequence obtained from Genebank and K217, K314, K403 which were isolated from sporadic cases, together with k295, k322, k333 obtained from outbreaks generated G and A transversions on three sites (site 1317, 1422, 1543) at the same time.  [20]). Whether measles evolved faster recent years than previous years, and whether outbreaks always evolved a bit faster than sporadic cases. All of these questions require more cases and further studies to answer in our next step study.
Annual measles outbreaks with high vaccination rate also continues to occur. Although these outbreaks typically involve importation and mostly affect unvaccinated individuals, there are surprisingly high numbers of vaccine failure among one-and two-dose recipients of measles vaccine who were infected (approximately 2 -12% for children immunized at/around one year of age . Similarly with previous study, in our study, all cases no matter outbreak or sporadic had less than two dose measles vaccines.
Although in our comparative study, there was no genetic difference were found between the outbreak strains and the sporadic strains, but we found that both the study of the outbreak cases and the evolutionary study of the sporadic cases are important. Evolutionary study of the sporadic cases may have a certain value for the discovery of the outbreak hazard in the surveillance of measles, especially in the stage of measles elimination. There may be accumulation of sporadic in the early stages of each outbreak.

Declarations
Newly generated N-450 sequences of Shandong measles virus isolates have been submitted to GenBank, under the following accession numbers: OL807629-OL807661. All sequences data that support the ndings of this study have been deposited in GeneBank.
Authors' contributions SW, CW, LZ and AX conceived the study and drafted the paper, XL, PX, YL and QX gathered and analyzed the data, and MC, ZT participated in RNA extraction. SW and CW are co-rst authors. All authors read and approved the nal manuscript.

Competing interests
The authors declare that they have no competing interests.  Tables   Table. 1 Symptoms and measles vaccination of measles cases together with the isolation time Figure 2 Evolutionary relationships of taxa The evolutionary history was inferred using the Neighbor-Joining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer thephylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The differences in the composition bias among sequences were considered in evolutionary comparisons. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair.
There were a total of 450 positions in the nal dataset. Evolutionary analyses were conducted in MEGA7. Sequences labeled by light blue circles are shandong strains in Fig2.a; Sequences labeled by red circles are outbreak strains and green triangles are sporadic strains from shandong; Two clusters generated by year were colored by green and deep blue. MCMC tree of the N-450 sequences of 33 Shandong isolates with 9 reference isolation visualized in FigTree. The outbreak sequences were colored red and the sporadic ones were green. Andthe branches were colored by rate, from blue to red.