VDPV case investigation
The two-year-old boy was born on August 19, 2014. He received four scheduled doses of IPV (at 4, 5, 6, and 18 months) and completed routine polio immunization. His last vaccination date was March 14, 2016. On February 14, 2017, the patient experienced weakness and claudication of the right lower extremity. On February 16, his condition deteriorated with fever (37.5°C–38.5°C), diarrhea, and difficulty in walking. The patient was sent to a hospital in Zhumadian City and diagnosed with AFP. Nutritional myocardial and comprehensive rehabilitation treatments were administered after the diagnosis of viral myositis upon admission. The patient gradually recovered with regained strength in the right lower extremity and no residual paresis. On March 12, the fully-recovered patient was discharged from the hospital with a discharge diagnosis of viral myositis.
Viruses were isolated from the first two stool samples collected during hospitalization (February 23 and 24, 2017) and were identified as poliovirus serotype 3 Sabin-like using rRT-PCR; however, sequencing of the VP1 region of the virus revealed nine nucleotide changes consistent with VDPV3 classification. After the patient was discharged from the hospital, type 3 VDPV was detected again in the third stool sample collected during follow-up (April 5, 2017), with 10/900 (1.11%) nucleotide substitutions in the VP1 region compared with the type 3 Sabin strain. These three viral strains (CHN21006-1, CHN21006-2, and CHN21006-3) isolated at three time points shared eight nucleotide substitution sites. In the stool samples collected from the fourth to the seventh time periods (April 23, May 3, May 10, and May 19, 2017), the viral isolation results were negative, and no virus was detected. Therefore, it is considered that the excretion of poliovirus has been terminated.
Humoral immunity function was measured using blood samples from the patient collected on April 21, 2017. The results showed that the serum neutralizing antibody for Sabin type 1 was 1:16 and Sabin 3 was 1:512 (1: 4 is considered positive), while antibodies against hepatitis B, hepatitis C, and HIV were all negative. Based on all the results, the patient was diagnosed with VDPV classified as VDPV.
Investigation for AFP in close contacts and the environment
To investigate the scope of the virus epidemic, after identification of the VDPV case, investigations among 19 relatives and 20 close contacts who had contact with the patient were performed by testing stool samples for poliovirus. The vaccination certificates of the individuals who had contact with the patient were examined. Furthermore, sewage samples from household wastewater and nearby river water were also collected. All samples were negative for polioviruses.
Retrospective analysis of AFP cases and assessment of vaccination coverage
AFP cases were actively searched and crosschecked using the records of all county- and prefecture-level hospitals in Zhumadian city, Henan province. Medical records for both inpatients and outpatients were also reviewed in 23 county-level hospitals in Zhumadian. No additional AFP cases were found during active case searches in hospitals, and no additional AFP cases were observed during house-to-house searches in the villages and neighboring areas.
Through a vaccination information management system for children, the inoculation clinic A and adjacent inoculation clinic B were investigated. The vaccination rate of the third dose of polio vaccine for children under 1 year of age was 80.51%, while the rate of the fourth dose was only 68.93% in vaccination clinic A. The vaccination rates of the second and third doses of polio vaccine were 80.24% and 74.47% in vaccination clinic B respectively. The rate of complete polio vaccination in children under 5 years of age was above 97% in vaccination clinic B.
To elucidate the divergence and evolution of type 3 Henan aVDPV, a maximum-likelihood tree of sequence relationships in the entire P1 capsid region for Henan type 3 VDPVs and a set of divergent type 3 VDPVs was constructed (Figure 1). Representative VDPVs include Russian cVDPVs (GenBank accession numbers: MT645947–MT645951), Iranian iVDPVs (GenBank accession numbers: EU684056–EU684057), Belarusian aVDPV (GenBank accession numbers: FJ460226–FJ460227), and Indian aVDPV (GenBank accession number: KR259358).
The phylogenetic tree, which was based on the P1-coding region, revealed that all three Henan type 3 VDPVs could be grouped into a single cluster with divergence pathways different from those of Sabin 3, and they were distinct from the genetic clusters of other representative type 3 VDPVs (Figure 1).
Reversions of key neurovirulence determination sites
Based on the full-length genome sequencing analysis, CHN21006-1, CHN21006-2, and CHN21006-3 contained 41, 43, and 60 nucleotide substitutions, respectively, compared to Sabin3/Sabin1 strain. Most of the nucleotide substitutions in the coding region of the genome were synonymous, resulting in 9, 9, and 12 amino acid substitutions, respectively (Supplementary Table 1).
Notably, the nucleotide U at position 472 in the 5′ noncoding region was mutated to C. Moreover, the nucleotide C at position 2493 in the VP1 coding region was mutated to U, causing an amino acid substitution in VP1–6: Thr to Ile. Both mutations are neurovirulent reversion mutations usually observed in VDPV-associated paralytic poliomyelitis cases (24). However, the third nucleotide substitution identified as a key determinant of the attenuated phenotype of Sabin 3 (a U-to-C reversion at nucleotide 2034 in the VP3 coding region, which caused an amino acid substitution in VP3–91: Phe to Ser) did not revert.
Recombination features of Henan type 3 VDPVs
A similarity plot and bootscanning analysis of the complete genomes of the three Henan type 3 VDPVs indicated that they were Sabin 3/Sabin 1 recombinants (Figure 2). The upstream sequences (i.e., 5′-UTR, P1/capsid, and 5′ part of the P2/noncapsid sequences) up to nucleotide position 4899 were derived from the Sabin 3 strain (28, 30, and 38 nucleotide substitutions, respectively), and sequences downstream of nucleotide position 4899 (i.e., 3′ part of the P2/noncapsid sequences, P3/noncapsid sequences, and 3′-UTR) were derived from the Sabin 1 strain (13, 13, and 22 nucleotide substitutions, respectively) (Figure 2).
Antigenic divergence of Henan type 3 VDPVs
The amino acid sequences within or near the predicted neutralizing antigenic (NAg) sites were aligned with those of the Henan type 3 VDPV strains, Sabin 3 strain (GenBank accession No. AY184221), and type 3 wild poliovirus prototype strain P3/Leon/37 (GenBank accession No. K01392) (Figure 3). The Henan type 3 VDPVs had two shared amino acid replacement in the NAg sites, one is in NAg2 (VP2–164: Asn to Asp, and ), and the other is in NAg3b (VP3–77: Asp to Asn). Strain CHN21006-3 also had another amino acid replacement in NAg3b (VP2–172: Glu to Lys) (Figure 3).
Supplementary immunization activities (SIAs)
To prevent possible circulating VDPV, SIAs were conducted throughout the county. House-to-house SIAs were conducted in urban regions, covering 54,916 children under 7 years of age. The staff at all levels were trained to ensure the high quality of SIAs, including planning the SIA, overseeing the obligation and responsibility of staff, providing technical guidelines, and monitoring adverse events of OPV. It is worth mentioning that the bivalent OPV (bOPV, containing Sabin virus types 1 and 3) was used in SIAs, instead of the trivalent OPV (tOPV).