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From March to May 2006, type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from one case patient with acute flaccid paralysis (AFP) and six unimmunized healthy contacts in isolated mountain villages in Guangxi, China. We conducted epidemiological investigations in the affected communities and nucleotide sequence analyses of the cVDPV isolates. The results of the investigations showed that the AFP patient, an unimmunized 10-year-old boy, and five laboratory-confirmed contacts lived in the same village; one contact lived in a neighboring village. Only ~27% of children 5 to 10 years of age in the affected villages had received three or more doses of the trivalent oral poliovirus vaccine (OPV). Nucleotide sequence analyses revealed that the cVDPV isolates differed from the Sabin 1 (S1) isolate at 1.4 to 2.2% of VP1 nucleotide positions and shared 12 nucleotide substitutions within VP1. All isolates were S1/S2/S1/S3 recombinants sharing common recombination junctions. Key determinants of attenuation were replaced. Phylogenetic analysis suggested that the cVDPV circulated locally for ~12 months following the initiating OPV dose. No VDPVs were found after mass OPV immunizations, conducted from May to June 2006, that targeted all children <12 years of age. Our findings reinforce the point that VDPVs can emerge and spread in isolated communities with immunity gaps. Maintenance of sensitive AFP and poliovirus surveillance is essential to permit early detection and a rapid response to VDPV circulation.
Circulation of indigenous wild poliovirus (WPV) ceased in China in 1994 (23, 38, 44). High levels of population immunity have been maintained throughout most of the country, and vigorous immunization responses to the detection of poliovirus circulation have subsequently protected against widespread poliovirus transmission. The WPVs (WPV type 1 [WPV1] and WPV3), introduced into communities bordering Myanmar in 1995 and 1996, were associated with only four paralytic poliomyelitis (polio) cases (44), and WPV1 imported into Qinghai, China, from northern India was associated with only one case in 1999 (45) Keys to the success in China are (i) a strong routine immunization program with trivalent oral poliovirus vaccine (tOPV) supplemented by synchronized mass campaigns in the form of national immunization days (NIDs) and subnational immunization days (SNIDs) (38), (ii) sensitive surveillance for cases of acute flaccid paralysis (AFP) (47), and (iii) rapid, detailed characterization of poliovirus isolates (23, 47). The eradication of polio in China (40), whose population constitutes 23% of the world population, provided strong impetus to the World Health Organization's (WHO's) Global Polio Eradication Initiative, whose efforts have reduced the polio incidence by >99% since 1988, such that only four countries have never stopped WPV circulation (43).
Successful eradication of WPV, however, does not entirely eliminate risks of paralytic poliomyelitis. The primary weapon in polio eradication, OPV, is genetically unstable and can revert to increased neurovirulence during replication in the human intestine (19, 27, 35). Consequently, in all countries where OPV is used, there is a very low background rate of vaccine-associated paralytic poliomyelitis (VAPP) among OPV recipients and their close contacts (35). Exposure to OPV of persons with primary immunodeficiencies is associated with an ~3,000-fold higher VAPP risk (36) and the further risk of prolonged infection with immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) (19). An additional risk is the emergence and spread of circulating VDPVs (cVDPVs) in areas with low rates of OPV coverage (19). cVDPV outbreaks have occurred and been controlled in Egypt (46), Hispaniola (Haiti and the Dominican Republic) (18), the Philippines (34), Madagascar (32), Indonesia (13), Cambodia (10), Myanmar (10), and China (23). Within the past year, cVDPV outbreaks have occurred in Nigeria (type 2, 2005 to 2010), the Democratic Republic of Congo (type 2, 2008 to 2010), Ethiopia (type 2, 2008 to 2009; type 3, 2009 to 2010), Somalia (type 2, 2008 to 2009), and India (type 2, 2009 to 2010) (9) (for updates, see http://www.polioeradication.org/content/general/cvdpv_count.pdf). VDPVs are operationally defined as having >1% nucleotide sequence divergence from their parental Sabin strains in the VP1 capsid region (9, 19). In addition to the well-defined cVDPV and iVDPV categories, VDPV isolates are assigned to a third category, ambiguous VDPVs (aVDPVs), when there is no evidence of community circulation or immunodeficiency (9).
Since 1997, an average of one to two new VDPV isolates has been identified each year in China (23). The first cVDPV outbreak in China (three cases, four contacts), associated with type 1 virus, occurred in Guizhou Province in 2004 (23). The first reported iVDPVs (types 2 and 3) were isolated in Anhui Province in 2005 from a patient with X-linked agammaglobulinemia (10). In 2007, four aVDPVs were isolated from four patients and one was isolated from one healthy child in 2009.
From March to May 2006, type 1 VDPV was isolated from one AFP case and six healthy contacts within two neighboring mountain villages in the Guangxi Zhuang Autonomous Region of southern China. An important local risk factor for VDPV emergence was the low rate of OPV coverage among children 5 to 10 years of age. Although these isolates were originally classified as aVDPVs according to WHO criteria because only a single AFP case attributable to VDPV was found (8, 10), the sequence relationships among these isolates were consistent with cVDPV circulation for ~12 months after the initiating OPV dose. No VDPVs were detected in other villages with similar immunization status, suggesting that the cVDPV transmission was highly localized. In this report, we describe the epidemiological and virological investigations of the Guangxi cVDPV infections and discuss the factors that may have favored its emergence and localized transmission.
Following the current WHO virological classification standard (9), a VDPV case is identified by isolation of VDPV from the patient with AFP. The immunization history, the clinical symptoms, and the presence of residual paralysis for the AFP patient were investigated. The immunization histories for six healthy VDPV-infected children were also investigated. The serum IgG, IgM, and IgA levels of the patient and six healthy VDPV-infected children from the community were determined by standard methods (11).
To evaluate immunization coverage, a convenience survey of 469 children aged 1 to 10 years was conducted in Dahua and Duan Counties in China (Fig. (Fig.1).1). To determine the extent of the VDPV infection, single stool specimens were collected from 13 to 18 May 2006 from 64 healthy children ≤13 years of age in Hongwei and neighboring villages. Concurrently, an active search for AFP cases was conducted in Dahua and Duan by a review of hospital records in four county-level hospitals, by interviews of health care workers in five township-level hospitals, and by house-to-house visits in affected areas to detect any previously unreported AFP cases. An active AFP case search was subsequently extended to four neighboring counties. Two rounds of supplementary immunization campaigns with tOPV were conducted from 26 to 30 May and from 26 to 30 June 2006, and thereafter, coverage rates were estimated by convenience survey. Stool specimens were collected from 55 healthy children in Dahua from 15 June to 22 September to detect any residual VDPV infections.
Polioviruses were isolated by culture in RD and L20B cells (42). Poliovirus isolates were further characterized by neutralization with hyperimmune sera, PCR-restriction fragment length polymorphism (PCR-RFLP) analysis (3), and antigenic analysis by an enzyme-linked immunosorbent assay (ELISA) using highly specific cross-absorbed antisera (37, 42). The complete genomes of seven VDPV isolates were sequenced as described previously (25). The sequence relationships in the third-codon position (3CP) of the complete open reading frame (ORF) (nucleotides [nt] 743 to 7369) among the 7 VDPV isolates and the ancestral S1/S2/S1/S3 recombinant were summarized in a phylogenetic tree constructed by Bayesian Markov chain Monte Carlo analysis using the BEAST program (version 1.4) (12). The tree was rooted to the ORF of a simulated sequence of a nonmutated S1/S2/S1/S3 recombinant homologue to the observed VDPV recombinants. The time of the initiating OPV dose and divergence of different VDPV branches was estimated from the rate of 3CP substitutions into the ORF. The ratio of nonsynonymous to synonymous substitutions (Ka/Ks ratio) within the neutralizing antigenic (NAg) sites (as defined in Fig. Fig.3)3) were determined using the Pamilo-Bianchi-Li (PBL) method (22, 31) implemented in the MEGA3 software package (21).
Complete genomic sequences of the type 1 VDPVs described here were deposited in the GenBank database under accession numbers FJ859058 to FJ859064.
The Guangxi Zhuang Autonomous Region is a mountainous region in southern China (Fig. (Fig.1)1) with a subtropical climate, a total population of 46.6 million, and an average population density of 150 persons/km2. Dahua County (population, 410,000), home to the case patient, is located in Hechi City and has a per capita annual income of $247 (2005 estimate). Dahua County is surrounded on three sides by mountains, and the only entrance is through neighboring Duan County (Fig. (Fig.11).
The routine immunization schedule in China is four doses of tOPV given at ages 2, 3, and 4 months and 4 years. The nationwide rates of routine coverage with three or more doses of tOPV were reported to be ~95% over the past 10 years (48). Since 1991, Guangxi has conducted two annual rounds of supplementary tOPV campaigns (SNIDs) in the winter and spring targeting all children <4 years of age. The AFP surveillance system, established in Guangxi in 1991, reported non-polio-associated AFP rates of 2.2/100,000 children <15 years old in 2004 and 2.3 in 2005 (target rate, 2/100,000 [41, 43]). The last case associated with WPV (type 1) in Guangxi was reported in June 1992.
The case patient was an unimmunized 10-year-old boy (Table (Table1)1) who had lived in Hongwei, Dahua County, since 2001 (Fig. (Fig.1).1). Hongwei is a remote mountain village of 2,374 persons living in 604 households (2006 estimate) and had an average birth rate of 34 (1.4%) per year from 2000 to 2004. On 8 March 2006, the boy developed fever, headache, and cough. On 10 March he felt weakness of his right leg; 1 day later the weakness progressed to his left leg, while sensory functions remained intact. On 15 March, he was hospitalized in Duan County Hospital and was reported as an AFP case. He was discharged on 23 March with grade 2 muscle power in both legs; bilateral residual paralysis was present 60 days after onset. He had no travel history before the onset of paralysis. Type 1 VDPV was isolated from stool specimens collected on 18 and 19 March 2006.
Among 64 healthy children aged <1 to 13 years sampled from 13 to 18 May 2006 in Duhua and Duan Counties, type 1 VDPV was isolated from the stools of six (five in Hongwei and one in closely neighboring Yongping Village [in Duan County, with 1,948 persons living in 530 households and an average birth rate of 16 per year from 2000 to 2004]) (Fig. (Fig.1;1; Table Table1).1). The six laboratory-confirmed VDPV contacts were all female with a median age of 8.2 years (range, 4 to 13 years). They frequently played together with the case patient because of their close living locations. None of them had received a dose of OPV. The status of immunization against other vaccine-preventable diseases for the case patient and the six community contacts was unknown. However, serological tests showed that the case patient was positive for measles virus antibody and negative for tetanus antibody and hepatitis B virus surface antibody (HBsAb); and one, four, and five of the six contacts were negative for measles virus antibody, tetanus antibody, and HBsAb, respectively. The serum concentrations of IgG, IgM, and IgA of the case patient and the six community contacts were found to be within normal limits (data not shown), and there were no other indications of primary immunodeficiency in these children.
An immunization coverage survey of 469 children aged 1 to 10 years of age in Dahua and Duan Counties found that only 61.2% had received three or more doses of OPV. More critically, the rate of OPV coverage for children aged 1 to 5 years was 98.7%, but the rate of coverage for children aged 5 to 10 years was only 27.2%. Therefore, the most susceptible population for poliovirus infection was children >5 years old, consistent with the ages of six of the seven children found to be infected with VDPV (Table (Table11).
Review of 53,053 records from 2004 to 2006 in four county-level hospitals of Dahua and Duan found only the eight previously reported AFP cases. Review of the eight AFP cases by a clinical expert panel confirmed that only the case described in this report was compatible with polio. No suspected AFP case was found during the inquiry of five township-level hospital workers and house-to-house visits in Dahua and Duan Counties. In addition, active AFP case searches performed in respective county- and township-level hospitals of four neighboring counties found no previously unreported case. Retrospective analysis of AFP surveillance data in 2005 showed that the non-polio-associated AFP rates per 100,000 population aged <15 years were 4.4 in Dahua and 3.1 in Duan.
Two rounds of supplementary tOPV immunization campaigns targeting all children <12 years of age were conducted in Dahua, Duan, and four neighboring counties from 26 to 30 May and from 26 to 30 June 2006. Approximately 400,000 children were immunized in the two rounds, with the coverage rate estimated to be >96%. No VDPVs have been detected in Guangxi since June 2006 either from AFP cases or from 55 stool specimens collected from healthy children from 15 June to 22 September 2006.
Three of the seven type 1 VDPV isolates showed evidence of genetic drift by PCR-RFLP analysis, and all seven isolates were found to have double-reactive (reacting with both Sabin 1-specific and type 1 wild virus-specific cross-absorbed rabbit antisera) antigenic properties by ELISA (Table (Table1).1). The Guangxi isolates differed from the Sabin 1 isolate at 1.4 to 2.2% of the nucleotide positions in the VP1 region (906 nt), indicating that they were VDPVs (Table (Table1).1). The VDPVs shared 12 nt substitutions in VP1, suggesting the existence of a recent common ancestor and consistent with community circulation.
All cVDPV isolates were S1/S2/S1/S3 recombinants sharing common recombination junctions, confirming the existence of a common recombinant ancestral virus. Crossover sites mapped to the extreme 3′ end of the VP1 region (S1/S2; nt 3380 to 3381), near the 5′ end of the 2C region (S2/S1; nt 4267 to 4271), and near the 3′ end of the 3Dpol region (S1/S3; nt 5842 to 5868).
A Bayesian Markov chain Monte Carlo phylogenetic tree was constructed from the sequences at the 3CP of the complete ORF (6,627 nt) of the seven cVDPV isolates and a simulated nonmutated S1/S2/S1/S3 recombinant root sequence. The 3CP sequences were compared because they closely approximate synonymous sites (17), thereby largely screening out selected changes. The topologies of the trees of each recombinant interval were similar to the topology of the complete ORF (data not shown), suggesting that the recombination occurred soon after the initiating OPV dose, probably in the original tOPV recipient. The complete ORF tree diverged into two main branches from a common ancestral infection estimated to have occurred in about mid-December 2005 (95% highest posterior density [HPD], 19 September 2005 to 20 February 2006), ~3 months before the appearance of the AFP case (Fig. (Fig.2).2). The rate of fixation of 3CP substitutions into the ORF was estimated from the data set to be 3.4 × 10−2 3CP substitutions/site/year (overall rate, 1.2 × 10−2 total substitutions/site/year), similar to previous estimates for the poliovirus capsid region (17). Under the assumption of a strict molecular clock, we estimated that the initiating OPV dose was given in May 2005, ~10 months before the appearance of the Hongwei case (Fig. (Fig.2).2). The HPD intervals for the date on the initiating OPV dose, calculated from the variance in the evolution rate, were wide (11 October 2004 to 27 December 2005) because the cVDPV-containing specimens were taken over only a 2-month time interval.
Key determinants of the attenuated (G480 → A; VP465, S → A; VP1106, T → A) and temperature-sensitive (VP465, S → A; 3D73, H → F; G7441 → A) phenotypes of Sabin 1 (6) had reverted or had been exchanged out in the VDPV genomes (Table (Table2).2). In addition, substitutions at several other sites (G26 → A; U355 → C; VP360, K → T; VP190, I → M, L; VP1106, T → A; 2A134, T → S, A; 2B95, T → I; 3D53, N → D; 3D250, E → K; 3D362, I → T; C7410 → U) were shared among the cVDPVs from Guangxi, Hispaniola, and the Philippines outbreaks (Table (Table2).2). Most of the substitutions restored the consensus residues for type 1 poliovirus (capsid region) or for species C human enteroviruses (noncapsid and nontranslated regions) (7, 18, 34), consistent with the view that the Sabin 1 sequences are subject to negative selection during replication in humans and phenotypic revertants can emerge either by backmutation or recombination (19, 27).
All cVDPV isolates were found to have double-reactive antigenic properties in the ELISA using specific cross-absorbed antisera (Table (Table1).1). The amino acid sequences within or near the predicted NAg sites (5, 26, 30, 39) of the Guangxi cVDPVs were aligned with those of Sabin 1, its parental Mahoney strain, and representative type 1 cVDPVs from the outbreaks in Hispaniola and the Philippines (Fig. (Fig.3).3). Among the 4 or 5 amino acid replacements in the NAg sites of the Guangxi cVDPVs, 3 were shared with the Mahoney strain and the other 2 were shared with cVDPV isolates. One replacement (VP360, K → T) is associated with the loss of Sabin 1-like properties in antigenic assays (5). Another replacement (VP199, K → E, T) eliminated a trypsin cleavage site in NAg-1, characteristic of Sabin 1 (15). Most of the antigenic evolution of the Guangxi cVDPVs had stabilized by the time of the common ancestral infection, ~7 months after the initiating OPV dose. The Ka/Ks ratio within the NAg sites was 0.59 for the Guangxi cVDPVs, similar to the ratios for the type 1 cVDPVs from Hispaniola and the Philippines (0.47 to 0.52) and lower than the ratios for type 1 iVDPV isolates from immunodeficient patients in Taiwan (1.07 to 3.04) (1) and the United States (1.20 to 1.60) (J. Jorba, unpublished results).
The limited, highly localized cVDPV outbreak in 2006 in the neighboring villages of Hongwei and Yongping highlights the risks of cVDPV emergence and spread in small, isolated populations with critical immunity gaps. In this outbreak, the key risk group (n ≈ 250) was children aged 5 to 10 years, among whom the rates of OPV coverage were <30%. Younger children had high rates of OPV coverage, and older persons had acquired immunity from the intensive mass immunization campaigns of the 1990s and some had acquired immunity from natural WPV infection before 1993 (23, 38). If the potential paralytic attack rate of the cVDPV was similar to that of WPV1 (~1 paralytic case per 200 infections in nonimmune children) (28, 33) and most of the susceptible children in the two communities were exposed, then the expected paralytic case count would be ~1.
We cannot be certain that the cVDPV actually emerged from an OPV dose given in Hongwei or Yongping because the cVDPV appears to have been in circulation for ~10 months before appearance of the index AFP case. However, the inaccessibility of these communities makes local emergence likely. The lack of paved roads restricts the mobility of the local population and limits outside contact, and the rugged mountain paths were a barrier both to the delivery of quality routine immunization services and to virus dissemination. The absence of polio cases elsewhere in Guangxi, where the province-wide coverage rates for three doses of OPV is ~95% (48), is consistent with local emergence.
The observation that all isolates were S1/S2/S1/S3 recombinants sharing common recombination junctions is also consistent with limited VDPV circulation. Although vaccine/vaccine recombinants of Sabin 1 are relatively infrequent (16, 20), it is likely that the recombination occurred in the original tOPV recipient. Spread of the vaccine progeny was sufficiently limited that no subsequent recombination with various species C human enteroviruses (HEVs-C) was observed. This is in distinct contrast to the serial recombination with HEVs-C that is typically found during circulation of most cVDPVs (18, 19, 32, 34, 46) and WPVs (24). The likelihood of recombination with HEV-C increases with the frequency of poliovirus and HEV-C infections. Although the HEV-C carriage rate in Hongwei and Yongping in 2005 to 2006 is unknown, the VDPV was likely circulating throughout the 2005 enterovirus season, and close contact among infected school-age children and poor conditions of sanitation and hygiene may have facilitated virus transmission. Thus, vaccine/nonvaccine recombinants probably would have emerged had there been widespread VDPV circulation. The absence of HEV-C recombinants from among the Guangxi cVDPVs and the nonrecombinant type 1 cVDPVs from the 2004 Guizhou outbreak (23) demonstrates that recombination with HEVs-C is not required for cVDPV emergence and spread in the early phases of outbreaks.
Phylogenetic analysis has provided insights into the minimum period of cVDPV circulation in small, isolated communities. In a previous study, circulation of a type 1 VDPV in a rural undervaccinated community in Minnesota (population, ~161) was estimated to have been at least 3 to 4 months, whereas it was ~12 months in the villages of Hongwei and Yongping (combined populations, ~4,300). In Minnesota, the estimate of the circulation time may have been reduced by the small sample size (n = 5), whereas in Guangxi, circulation was likely halted by the strong immunization response.
It is of interest that the recent small VDPV outbreaks in China and Minnesota were associated with type 1. In contrast, the large and prolonged VDPV outbreaks in Egypt (46) and Nigeria (9) were associated with type 2. One possible explanation is that the substantially higher paralytic attack rate for poliovirus type 1 than for type 2 (28, 33) favors earlier detection of AFP cases associated with type 1 infection, especially in small populations. Moreover, emergence of type 2 cVDPVs probably requires wider gaps in tOPV coverage because of the greater tendency of type 2 vaccine virus to spread to unvaccinated contacts (2, 4, 14). For unknown reasons, outbreaks of type 3 cVDPV infections appear to be rare (9, 19).
Because of its large population, high population densities, numerous communities with poor sanitation, and large areas with subtropical and tropical climates, China is potentially at high risk of cVDPV emergence. China has mitigated those risks with high rates of routine OPV coverage nationwide (estimated in 2004 to be 93.8% for three or more doses of OPV among children aged 1 to 3 years ), sensitive AFP surveillance, comprehensive and definitive laboratory testing, and a rapid immunization response following detection of VDPVs. Since 1997, of the 18 different VDPVs isolated from AFP patients in China (>130,000 specimens have been screened), only 3 have been associated with outbreaks (23). Both the Guangxi and the Guizhou outbreaks were detected before circulation was widespread, were associated with very few AFP cases, and were halted by large effective immunization responses launched within 2 to 3 months of appearance of the first AFP case. Immediately following the detection of VDPV from the March AFP case in Guangxi, intensive screening for VDPVs was initiated by provincial and national-level polio laboratories.
Our findings reinforce the point that geographic isolation and limited outside contact do not protect against polio and that cVDPVs can emerge in any location where OPV is used at low rates of coverage (1, 13, 23, 32). Therefore, it is critical that high rates of polio vaccine coverage and sensitive AFP and poliovirus surveillance be maintained in all countries to mitigate the risks of poliovirus transmission.
This study was supported by National Key Technology R&D Program of China (project no. 2008BAI56B00), National Key Science and Technology Projects of China (project no. 2008ZX10004-008), and grant I8/181/978 from the World Health Organization.
We all report no conflict of interest.
Published ahead of print on 14 July 2010.