Evolving epidemiology of poliovirus serotype 2 following withdrawal of the serotype 2 oral poliovirus vaccine.

Although there have been no cases of serotype 2 wild poliovirus for more than 20 years, transmission of serotype 2 vaccine-derived poliovirus (VDPV2) and associated paralytic cases in several continents represent a threat to eradication. The withdrawal of the serotype 2 component of oral poliovirus vaccine (OPV2) was implemented in April 2016 to stop VDPV2 emergence and secure eradication of all serotype 2 poliovirus. Globally, children born after this date have limited immunity to prevent transmission. Using a statistical model, we estimated the emergence date and source of VDPV2s detected between May 2016 and November 2019. Outbreak response campaigns with monovalent OPV2 are the only available method to induce immunity to prevent transmission. Yet our analysis shows that using monovalent OPV2 is generating more paralytic VDPV2 outbreaks with the potential for establishing endemic transmission. A novel OPV2, for which two candidates are currently in clinical trials, is urgently required, together with a contingency strategy if this vaccine does not materialize or perform as anticipated.

Updated modelling of the prevalence of immunodeficiency-associated long-term vaccine-derived poliovirus (iVDPV) excreters.

Conditions and evidence continue to evolve related to the prediction of the prevalence of immunodeficiency-associated long-term vaccine-derived poliovirus (iVDPV) excreters, which affect assumptions related to forecasting risks and evaluating potential risk management options. Multiple recent reviews provided information about individual iVDPV excreters, but inconsistencies among the reviews raise some challenges. This analysis revisits the available evidence related to iVDPV excreters and provides updated model estimates that can support future risk management decisions. The results suggest that the prevalence of iVDPV excreters remains highly uncertain and variable, but generally confirms the importance of managing the risks associated with iVDPV excreters throughout the polio endgame in the context of successful cessation of all oral poliovirus vaccine use.

ICTV Virus Taxonomy Profile: Picornaviridae.

The family Picornaviridae comprises small non-enveloped viruses with RNA genomes of 6.7 to 10.1 kb, and contains >30 genera and >75 species. Most of the known picornaviruses infect mammals and birds, but some have also been detected in reptiles, amphibians and fish. Many picornaviruses are important human and veterinary pathogens and may cause diseases of the central nervous system, heart, liver, skin, gastrointestinal tract or upper respiratory tract. Most picornaviruses are transmitted by the faecal-oral or respiratory routes. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Picornaviridae, which is available at www.ictv.global/report/picornaviridae.

Zinc supplementation fails to increase the immunogenicity of oral poliovirus vaccine: a randomized controlled trial.

BACKGROUND: Polio eradication remains a challenge in Pakistan and the causes for the failure to eradicate poliomyelitis are complex. Undernutrition and micronutrient deficiencies, especially zinc deficiency, are major public health problems in Pakistan and could potentially affect the response to enteric vaccines, including oral poliovirus vaccine (OPV). OBJECTIVE: To assess the impact of zinc supplementation among infants on immune response to oral poliovirus vaccine (OPV). METHODS: A double-blind, randomized placebo-controlled trial was conducted in newborns (aged 0-14 days). Subjects were assigned to either receive 10mg of zinc or placebo supplementation daily for 18 weeks. Both groups received OPV doses at birth, at 6 weeks, 10 weeks and 14 weeks. Data was collected on prior immunization status, diarrheal episodes, breastfeeding practices and anthropometric measurements at recruitment and at 6 and 18 weeks. Blood samples were similarly collected to determine the antibody response to OPV and for micronutrient analysis. Logistic regression was used to determine the relationship between seroconversion and zinc status. RESULTS: Overall, 404 subjects were recruited. At recruitment, seropositivity was already high for poliovirus (PV) serotype 1 (zinc: 91.1%; control: 90.5%) and PV2 (90.0%; 92.7%), with lower estimates for PV3 (70.0%; 64.8%). By week 18, the proportion of subjects with measured zinc levels in the normal range (i.e. ≥60 µg/dL) was significantly greater in the intervention group compared to the control group (71.9%; 27.4%; p<0.001). No significant difference in seroconversion was demonstrated between the groups for PV1, PV2, or PV3. CONCLUSIONS: There was no effect of zinc supplementation on OPV immunogenicity. These conclusions were confirmed when restricting the analysis to those with measured higher zinc levels.

Surveillance during an era of rapidly changing poliovirus epidemiology in India: the role of one vs. two stool specimens in poliovirus detection, 2000-2010.

Since 2004, efforts to improve poliovirus detection have significantly increased the volume of specimen testing from acute flaccid paralysis (AFP) patients in India. One option to decrease collection and testing burden would be collecting only a single stool specimen instead of two. We investigated stool specimen sensitivity for poliovirus detection in India to estimate the contribution of the second specimen. We reviewed poliovirus isolation data for 303984 children aged <15 years with AFP during 2000-2010. Using maximum-likelihood estimation, we determined specimen sensitivity of each stool specimen, combined sensitivity of both specimens, and sensitivity added by the second specimen. Of 5184 AFP patients with poliovirus isolates, 382 (7.4%) were identified only by the second specimen. Sensitivity was 91.4% for the first specimen and 84.5% for the second specimen; the second specimen added 7.3% sensitivity, giving a combined sensitivity of 98.7%. Combined sensitivity declined, and added sensitivity increased, as the time from paralysis onset to stool collection increased (P = 0.032). The sensitivity added by the second specimen is important to detect the last chains of poliovirus transmission and to achieve certification of polio eradication. For sensitive surveillance, two stool specimens should continue to be collected from each AFP patient in India.

High degree of genetic diversity of non-polio enteroviruses identified in Georgia by environmental and clinical surveillance, 2002-2005.

Enterovirus surveillance data are useful for establishing temporal and geographical patterns of circulation and for virus characterization to determine phylogenetic relationships between strains. Almost no information is available on circulating enteroviruses in Georgia and the surrounding region. To describe enterovirus circulation in Georgia, determine relationships with previously characterized strains and assess the role of environmental and clinical enterovirus surveillance, this study analysed a total of 112 non-polio enterovirus isolates identified during 2002-2005 from sewage and human stool samples. Viruses were isolated in cell culture using standard methods and typed by partial sequencing of the VP1 gene. A total of 20 different non-polio enterovirus serotypes were identified over the 4-year period. The most commonly detected enteroviruses included echovirus (E) 6 (21 isolates; 18.8 %), E20, E3 and E7 (11 isolates each; 9.8 %), E11, coxsackievirus (CV) B4 and CVB5 (seven isolates each; 6.3 %), and E13, E19 and E30 (six isolates each; 5.4 %). Phylogenetic analysis showed that many serotypes were represented by more than one genetic lineage. The present study showed a very high degree of enterovirus diversity in Georgia and demonstrated the added value of environmental enterovirus surveillance, particularly in settings with limited clinical surveillance. Several serotypes would not have been detected without having both clinical and environmental surveillance in place. Several serotypes detected in Georgia were among those rarely reported in the USA and Europe (e.g. E3, E20 and E19). As the emergence of new genetic lineages of enterovirus in a particular area is often associated with large-scale outbreaks, continued monitoring of enterovirus strains by both environmental and clinical surveillance and genetic characterization should be encouraged.

Failure to detect enterovirus in the spinal cord of ALS patients using a sensitive RT-PCR method.

OBJECTIVE: To assess the association of enteroviruses (EV) with ALS by applying a sensitive seminested reverse transcription (RT) PCR protocol to the detection of enteroviral RNA in a blinded set of archived tissues from ALS and control cases. METHODS: The specimen set consisted of 24 frozen spinal cord samples from ALS cases, 17 frozen spinal cord samples from negative control (non-ALS) cases, and 5 frozen spinal cord positive control samples. The positive controls were two human spinal cord samples spiked with poliovirus (PV) and three spinal cords from PV-infected transgenic mice. A sensitive, EV-specific, seminested RT-PCR assay was used to detect EV genome in RNA extracted from the specimens and controls. RESULTS: The assay detected EV RNA in a 10(-5) dilution of infected mouse tissue. EV RNA was not detected in the ALS specimens or in specimens from control cases, despite the presence of amplifiable RNA as assessed by amplification with control primers, whereas all of the positive control specimens yielded the expected PV amplification product. CONCLUSION: The reported association between EV infection and ALS was not confirmed by testing this set of specimens with these sensitive methods.

An outbreak of acute haemorrhagic conjunctivitis in Melaka, Malaysia.

This paper reports a second outbreak of acute haemorrhagic conjunctivitis due to coxsackievirus A24 in peninsular Malaysia. Between June 2002 and early October 2003, 10,327 patients, comprising 3,261 children and 7,066 adults, were treated for acute conjunctivitis in 11 government health clinics in the Melaka Tengah district of the state of Melaka. The figure grossly underestimates the size of the outbreak; as no patients treated in private clinics in the same district were included. Institution and household surveillance showed that the commonest presenting clinical feature of the illness was eye-discharge (91.2%), followed by foreign body sensation (81.8%), pain (78.3%) and subconjunctival haemorrhage (74.4%). The mean duration of illness was 6.5 and five days for patients with and without subconjunctival haemorrhage respectively.

Persistence of vaccine-derived poliovirus following a mass vaccination campaign in Cuba: implications for stopping polio vaccination after global eradication.

BACKGROUND: With substantial progress made toward polio eradication, developing the appropriate strategy for discontinuing global oral poliovirus vaccine (OPV) after global eradication becomes increasingly important. At issue is the theoretical risk of independent circulation of potentially virulent OPV-derived strains. Because Cuba uses OPV only in mass campaigns, it represents an ideal site to assess vaccine-derived poliovirus persistence. METHODS: Infants born after the 1997 biannual mass campaigns were evaluated for past (neutralizing antibody) or current (virus excretion) evidence of vaccine-derived poliovirus exposure. We obtained sera and/or stool specimens from 861 infants; a second serum from 218 infants. RESULTS: All stool specimens were poliovirus negative. Of 762 infants, 113 (14.8%) had initially detectable poliovirus type 1 antibody, 193 (25.3%) type 2, and 94 (12.3%) type 3. A precipitous antibody decline occurred in initially positive sera. CONCLUSIONS: Our results suggest that in a country with high population immunity, vaccine-derived virus is unlikely to establish ongoing circulation.

Type-specific detection of echovirus 30 isolates using degenerate reverse transcriptase PCR primers.

Following an approach used to specifically identify polioviruses and enterovirus 71, we have developed reverse transcriptase (RT) PCR primers containing mixed-base residues or deoxyinosine at positions of codon degeneracy. These primers permit specific RT-PCR amplification of echovirus 30 (E30) sequences by targeting sites that encode conserved amino acid motifs within the major capsid protein, VP1. All 221 E30 strains tested, isolated in 16 countries over a 44-year period, yielded the predicted 158-bp PCR product. No specific products were obtained by PCR assays containing templates from any of the other 63 EV serotypes. Inosine-containing degenerate primers may be widely applicable to the identification of echovirus serotypes by PCR.

Pathologic studies of fatal cases in outbreak of hand, foot, and mouth disease, Taiwan.

In 1998, an outbreak of enterovirus 71-associated hand, foot, and mouth disease occurred in Taiwan. Pathologic studies of two fatal cases with similar clinical features revealed two different causative agents, emphasizing the need for postmortem examinations and modern pathologic techniques in an outbreak investigation.

Molecular evolution of a type 1 wild-vaccine poliovirus recombinant during widespread circulation in China.

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3'-terminal sequences of VP1 (115 nt) and the 5' half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3' half of 2A were more distantly related (<90% nucleotide sequence match) to those of other contemporary wild polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of approximately 3.7 x 10(-2) substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection.

Deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: clinical and pathological characteristics of the disease. For the Outbreak Study Group.

From April through June 1997, 29 previously healthy children aged <6 years (median, 1.5 years) in Sarawak, Malaysia, died of rapidly progressive cardiorespiratory failure during an outbreak of hand, foot, and mouth disease caused primarily by enterovirus 71 (EV71). The case children were hospitalized after a short illness (median duration, 2 days) that usually included fever (in 100% of case children), oral ulcers (66%), and extremity rashes (62%). The illness rapidly progressed to include seizures (28%), flaccid limb weakness (17%), or cardiopulmonary symptoms (of 24 children, 17 had chest radiographs showing pulmonary edema, and 24 had echocardiograms showing left ventricular dysfunction), resulting in cardiopulmonary arrest soon after hospitalization (median time, 9 h). Cardiac tissue from 10 patients showed normal myocardium, but central nervous system tissue from 5 patients showed inflammatory changes. Brain-stem specimens from 2 patients were available, and both specimens showed extensive neuronal degeneration, inflammation, and necrosis, suggesting that a central nervous system infection was responsible for the disease, with the cardiopulmonary dysfunction being neurogenic in origin. EV71 and possibly an adenovirus, other enteroviruses, or unknown cofactors are likely responsible for this rapidly fatal disease.

Trial of a supplemental dose of four poliovirus vaccines.

BACKGROUND: The immunogenicity of oral poliovirus vaccine (OPV), particularly the type 3 component, is lower in infants in most developing countries than in infants in industrialized countries. We conducted a multicenter trial in Oman to evaluate the response to a supplemental dose of four poliovirus vaccine formulations. METHODS: At nine months of age, infants were randomly assigned to receive inactivated-poliovirus vaccine (IPV), administered subcutaneously; trivalent OPV manufactured in the United States or in Europe; or monovalent type 3 OPV. Serum samples were collected at enrollment and 7 and 30 days later. All of the infants had previously received five doses of OPV. RESULTS: We enrolled 1025 infants; 785 (76.6 percent) met all the study requirements. At enrollment, 96.8 percent of the infants were seropositive for poliovirus type 1, 98.0 percent for type 2, and 88.0 percent for type 3. At 30 days there were no significant increases in type 3 seroprevalence or in the median antibody titer in the groups of infants who received OPV. Among the recipients of IPV, type 3 seroprevalence increased from 87.8 percent at enrollment to 97.1 percent at 30 days (P<0.001), and the median antibody titer increased from 1:228 to 1:1448 or higher (P<0.001). The rapid initial increase in the antibody titer suggests a secondary immune response. CONCLUSIONS: A supplemental dose of IPV has excellent immunogenicity and leads to increases in the titer of antibodies against type 3 poliovirus, whereas supplemental doses of the oral vaccines do not have these effects.

Serotype-specific identification of enterovirus 71 by PCR.

BACKGROUND: Enterovirus 71 and coxsackievirus A16 are closely related genetically and are causative agents of hand foot and mouth disease. Because enterovirus 71 is more often associated with severe neurological disease, there is a need to rapidly discriminate between enterovirus 71 and coxsackievirus A16 during hand, foot, and mouth disease outbreaks. OBJECTIVES: Our goal was to develop and evaluate a serotype-specific reverse transcription-polymerase chain reaction (RT/PCR)-based typing method for enterovirus 71. STUDY DESIGN: Two sets of PCR primers were designed to match conserved amino acid intervals of enterovirus 71. One diagnostic primer pair contains deoxyinosine at sites of 4-fold codon degeneracy. A second primer pair was designed for use in sequencing and molecular epidemiology studies. Primer pairs were tested on strains encountered in routine diagnostic samples. RESULTS: Using both sets of primers on a panel of 61 prototype enteroviral strains, both primer pairs gave strong positive signals for only enterovirus 71. These primers amplified all enterovirus 71 isolates tested and discriminated between enterovirus 71 and the most closely related enterovirus, coxsackievirus A16. CONCLUSIONS: Our RT-PCR assay can be used for specific identification of enterovirus 71 clinical isolates. Furthermore, the 484-bp product of one primer pair has proven useful in sequencing studies to identify distinct genotypes of enterovirus 71.

Comparison of classic and molecular approaches for the identification of untypeable enteroviruses.

Members of the family Picornaviridae are the most common viruses infecting humans, and species in several genera also infect a wide variety of other mammals. Picornaviruses have traditionally been classified by antigenic type, based on a serum neutralization assay. However, this method is time-consuming and labor-intensive, is sensitive to virus aggregation and antigenic variation, and requires a large number of antisera to identify all serotypes, even when antiserum pools are used. We developed generic reverse transcription (RT)-PCR primers that will amplify all human enterovirus serotypes, as well as many rhinoviruses and other picornaviruses, and used RT-PCR amplification of the VP1 gene and amplicon sequencing to identify enteroviruses that were refractory to typing by neutralization with pooled antisera. Enterovirus serotypes determined by sequencing were confirmed by neutralization with monospecific antisera. Of 55 isolates tested, 49 were of known enterovirus serotypes, two were rhinoviruses, and four were clearly picornaviruses but did not match any known picornavirus sequence. All four untyped picornaviruses were closely related to one another in sequence, suggesting that they are of the same serotype. RT-PCR, coupled with amplicon sequencing, is a simple and rapid method for the typing and classification of picornaviruses and may lead to the identification of many new picornavirus serotypes.

Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998.

Enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae), a common cause of hand, foot, and mouth disease (HFMD), may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity and rate of evolution of EV71, we have determined and analyzed complete VP1 sequences (891 nucleotides) for 113 EV71 strains isolated in the United States and five other countries from 1970 to 1998. Nucleotide sequence comparisons demonstrated three distinct EV71 genotypes, designated A, B, and C. The genetic variation within genotypes (12% or fewer nucleotide differences) was less than the variation between genotypes (16.5 to 19.7%). Strains of all three genotypes were at least 94% identical to one another in deduced amino acid sequence. The EV71 prototype strain, BrCr-CA-70, isolated in California in 1970, is the sole member of genotype A. Strains isolated in the United States and Australia during the period from 1972 to 1988, a 1994 Colombian isolate, and isolates from a large HFMD outbreak in Malaysia in 1997 are all members of genotype B. Although strains of genotype B continue to circulate in other parts of the world, none have been isolated in the United States since 1988. Genotype C contains strains isolated in 1985 or later in the United States, Canada, Australia, and the Republic of China. The annual rate of evolution within both the B and C genotypes was estimated to be approximately 1.35 x 10(-2) substitutions per nucleotide and is similar to the rate observed for poliovirus. The results indicate that EV71 is a genetically diverse, rapidly evolving virus. Its worldwide circulation and potential to cause severe disease underscore the need for additional surveillance and improved methods to identify EV71 in human disease.

Molecular epidemiology and genetic diversity of echovirus type 30 (E30): genotypes correlate with temporal dynamics of E30 isolation.

Echovirus type 30 (E30) (genus, Enterovirus; family, Picornaviridae) has caused large outbreaks of aseptic meningitis in many regions of the world in the last 40 years. U.S. enterovirus surveillance data for the period 1961 to 1998 indicated that the annual proportion of E30 isolations relative to total enterovirus isolations has fluctuated widely, from a low of 0% in 1966 to a high of 42% in 1998. Peaks of E30 isolations occurred in the years 1968 to 1969, 1981 to 1984, 1990 to 1993, and 1997 to 1998, coincident with large nationwide outbreaks of E30-associated aseptic meningitis. Analysis of the complete VP1 sequence (876 nucleotides) of 136 E30 strains isolated in geographically dispersed regions of the United States and nine other countries between 1956 and 1998 indicated that the currently circulating E30 strains are genetically distinct from those isolated 30 to 40 years ago. Phylogenetic reconstruction demonstrated the existence of at least four distinct genetic groups, three of which have not been isolated in North America since 1981. Two of the three groups disappeared during periods when E30 was isolated infrequently. All North American E30 strains isolated after 1988 were closely related to one another, and all post-1993 isolates were of the same lineage within this group. Surveillance data indicate that E30 causes large national outbreaks of 2- to 4-year durations, separated by periods of relative quiescence. Our results show that shifts in the overall genetic diversity of E30 and the predominant genetic type correlate temporally with the dynamics of E30 isolation. The sequence data also provide a basis for the application of molecular techniques for future epidemiologic investigations of E30 disease.

Specific detection of echoviruses 22 and 23 in cell culture supernatants by RT-PCR.

Reverse transcription-polymerase chain reaction (RT-PCR) methods are available for the rapid detection of enteroviruses in clinical specimens or virus isolates. Pan-enterovirus PCR primers, however, fail to amplify echovirus (E) type 22 or 23 because of their extreme sequence divergence from the other enteroviruses. We have developed an RT-PCR method to detect specifically E22 and E23 RNA directly in tissue culture supernatants without a viral RNA purification step. The E22/E23 primers successfully amplified 20 of 20 clinical isolates of E22 and 4 of 4 E23 isolates representing viruses isolated in 15 states over a 19-year period, as well as E22 and E23 prototype strains isolated in the 1950s. The primers did not amplify any of the other 64 enterovirus prototype strains.