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 floating 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).
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 neighbor-joining tree. Further phylogenetic analysis weremade between 33 Shandong strains and the 9 references sequences (Fig2.b). From theevolutionary tree, we can see that the outbreak case sequences and the sporadic case sequences are widely distributed. Similarly, strains isolated from different cities cross-distributed, not clustered by city. AB824116.1, K217, K403, k295, k322, k333 which have the totally same sequence formed a separate cluster. Outbreak strains K333, K323, K322 and K330, K352, K384, K385, K462, K514 which isolated from the same year 2015, are on different branches. there are two clusters generated by year. One formed by stains obtained in 2016 and the other 2019.
evolution rate and molecular clock analysis
The evolution rate and molecular clock phylogeny of 33 Shandong isolates were inferred using the Bayesian Markov chain Monte Carlo (MCMC) method in BEAST version 1.6.1. The time of the most recent common ancestor (tMRCA) with 95 % highest posterior density(HPD) was estimated for the global and Shandong clusters. The data were analyzed under the Hasegawa-KishinoYano (HKY) models with gamma distribution of among-site rate variation. The uncorrelated lognormal-distributed (UCLD) relaxed molecular clock, were implemented in our analysis. Both constant and exponential growth (EG) population size coalescent were used as tree prior. The MCMC chain was run for 30,000,000 steps and sampled every 1,000 steps. The first 3,000,000 steps of each run were discarded as burn-in. The molecular clock phylogenetic tree was established too. In the molecular clock phylogenetic tree (Fig3), Shandong isolatesdon’t segregated into a single cluster. Outbreaks and sporadic strains are dispersed inmolecular phylogenetic trees. Although different evolution rates were observed for some different branches, but most different branches have the similar evolution rate. The mean evolution rate of all 33 Shandong isolations with 9 reference isolations was 1.688× 10-3 substitutions per site per year. The mean evolution rate of 15 outbreak isolations and sporadic isolations was 4.73× 10-3 and 2.068× 10-3 substitutions per site per year separately.