Antiserum Pools Research Articles

Streptococcus pneumoniae serotype epidemiology among PCV-10 vaccinated and unvaccinated children at Gertrude's Children's Hospital, Nairobi County: a cross-sectional study.

Background:Streptococcus pneumoniae (SP n) serotype replacement and emergence of multidrug resistant SP n has exacerbated the need for continuous regional serotype surveillance. We investigated SP n serotypes circulating among children ≤5 years in Nairobi County. Methods:Streptococcus pneumoniae stocks stored at -70°C in brain heart infusion medium were thawed at room temperature for 30 minutes. In total, 10 µl of the stored SP n cells were suspended in 50 µl PBS and gently vortexed. About 10 µl of the suspended cells were added on to a glass slide and mixed with 10 µl pooled antisera. The glass slide was swirled gently while observing for any reaction. The process was repeated with individual groups under various antisera pools. Those serotypes that did not belong to any pool were typed directly until a positive agglutination reaction was observed. The cells/PBS/serotype-specific antisera mixture on the glass slide were covered with a coverslip and observed under a phase contrast microscope at ×100 objective lens with oil emulsion. Results: Out of the 206 subjects sampled, 20.39% (n=42) were found to be carriers of SP n. About 52% (n=22) of the SP n carriers had received the recommended dose of PCV-10, while 48% (n=20) of the carriers had not. Almost all (n=41; 19.90% of subjects) isolates contained non-vaccine type SP n serotypes, while n=1 of the serotypes (in 0.49% of subjects) were untypeable. Serotypes 28F, 6A, 11A, 3 and 7C were prevalent in both vaccinated and unvaccinated children, whereas serotypes 23A, 17F, 35F, 48, 13 and 35B, and 23B, 20, 19B, 21, untypeable, 15B and 39 were found among unvaccinated and vaccinated groups, respectively. Conclusions: All SP n serotypes isolated from the subjects sampled were non PCV-10 vaccine type. Therefore Kenyan children receiving PCV-10 vaccine are not protected.

Vaccination against urinary tract infections caused by <i>E. coli</i>

Uropathogenic Escherichia coli (UPEC) causes uncomplicated urinary tract infection (UTI) depicts a prevalent and potentially uncompromising infectious disease. In this analysis, we explained the functions of an immunoproteomics concept to vaccine development that has been successfully employed to recognize vaccine targets in other pathogenic bacteria. Pyelonephritis strains E. coli CFT073 are used for outer membrane isolation mimics urinary tract environment in which iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells are included. During experiments of UTI, the antigens that induce the humoral immune response is to identified, two-dimensional gel electrophoresis are employed for the isolation of outer membrane protein and probed using pooled antisera from 20 CBA/J mice chronically infected with E. coli CFT073. 23 total outer membrane antigens, in which a unique iron compound receptor is included, are reacted with antisera and were identified by mass spectrometry. These antigens comprises of proteins with known functions in UPEC pathogenesis such as, ChuA, IroN, IreA, Iha, IutA, and FliC. These all information and data elaborate that these factors are associated with virulence during UTI are directed by antibody response. We also represents that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent among UPEC strains than among fecal-commensal E. coli isolates. Therefore we concluded that, the outer membrane antigens are identified in this study are conserved, could be reflective part for the UTI vaccine generated to induce protective immunity against UPEC infections.

Characterization of non-polio enterovirus isolates from acute flaccid paralysis children in Pakistan reflects a new genotype of EV-107

Enteroviruses comprise a group of genetically diverse RNA viruses that usually cause respiratory illnesses, myocarditis, enteritis, meningitis, encephalitis and poliomyelitis. In this study, 23 non-polio enteroviruses, isolated during September–October 2009 from stool specimens of children suffering from acute flaccid paralysis (AFP) were subjected to serotype determination by using RIVM antiserum pools. Twenty-two isolates were identified as members of human enterovirus B (HEV-B) species except one that remained untypeable. Phylogenetic analysis of the untypeable isolate (PAKNIH-2009/23) based on the sequence of 5′UTR, capsid and 2A regions confirmed that this isolate is closely related to TN94-0349 strain of EV-107 serotype with 70.4% nucleotide similarity in VP1/2A junction, 79.4% in the capsid coding region and 89.1% in 5′UTR region respectively. Our data strongly suggests that PAKNIH-2009/23 is a genetically distinct strain of EV-107 currently circulating in Pakistan. This study also highlights the probable role of non-polio viral etiologies associated with AFP and needs to plan new strategies especially during the post polio eradication era.

Detection of non‐polio enteroviruses from 17 years of virological surveillance of acute flaccid paralysis in the Philippines

Acute flaccid paralysis (AFP) surveillance has been conducted as part of the World Health Organization (WHO) strategy on poliomyelitis eradication. Aside from poliovirus, which is the target pathogen, isolation, and identification of non-polio enteroviruses (NPEVs) is also done by neutralization test using pools of antisera which can only identify limited number of NPEVs. In the Philippines, despite the significant number of isolated NPEVs, no information is available with regard to its occurrence, diversity, and pattern of circulation. In this study, a total of 790 NPEVs isolated from stool samples submitted to the National Reference Laboratory from 1992 to 2008 were analyzed; neutralization test was able to type 55% (442) of the isolates. Of the remaining 356 isolates, which were untyped by using neutralization test, 348 isolates were analyzed further by RT-PCR targeting the VP1 gene. A total of 47 serotypes of NPEV strains were identified using neutralization test and molecular typing, including 28 serotypes of human enterovirus B (HEV-B), 12 serotypes of HEV-A, and 7 of HEV-C. The HEV-B group (625/790; 79%) constituted the largest proportion of isolates, followed by HEV-C (108/790; 13.7%), HEV-A (57/790; 7.2%), and no HEV-D. Coxsackievirus (CV) B, echovirus (E)6, E11, and E13 were the most frequent isolates. E6, E11, E13, E14, E25, E30, E33, CVA20, and CVA24 were considered as endemic strains, some NPEVs recurred and few serotypes existed only for 1–3 years during the study period. Despite some limitations in this study, plural NPEVs with multiple patterns of circulation in the Philippines for 17 years were identified. J. Med. Virol. 84:624–631, 2012. © 2011 Wiley Periodicals, Inc.

Passive Immunization with a Polyclonal Antiserum to the Hemoglobin Receptor of Haemophilus ducreyi Confers Protection against a Homologous Challenge in the Experimental Swine Model of Chancroid

Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

Biological characters of a coxsackievirus B3 variant strain isolated from a hand-foot-mouth disease patient with severe clinical symptoms

To analyze the genetic and biological characters of a new isolate of coxsackievirus B3 (CoxB3), i.e. FY-19 strain, and investigate its mechanistic role in causing different clinical symptoms of hand-foot-mouth disease (HFMD). FY-19 strain, isolated from a patient with severe clinical symptoms from Fuyang, China in 2008, was identified by the serological parameters via the Lim Benyesh-Melnick (LBM) antiserum pools. Its genotype was further characterized by sequencing the whole genome. And its biological characters were also examined by proliferation kinetic and pathogenetic analysis. FY-19 strain was identified as CoxB3 showing 23.0%, 16.5% and 32.1% difference with Nancy strain in 3'-, 5'-noncoding and coding regions respectively. FY-19 also showed a high homology with other HFMD-related CoxB3 isolates in China. But its homology with non-HFMD-related CoxB3 isolates was lower (13.5% and 25.0% difference in 3'-NCR and coding region respectively). The viral replication kinetic analysis suggested that the FY-19 proliferation increased rapidly and peaked at 14 hours post-infection. In pathological analysis, FY-19 strain induced mortal pathology in sucking mice. Differences in genetic and biological characters exist between FY-19 and Nancy strains. Further analysis on the pathogenesis of this variant may aid in elucidating the mechanisms of HFMD.

Synthesis, Conjugation, and Immunological Evaluation of the Serogroup 6 Pneumococcal Oligosaccharides

The first synthesis of the newly discovered oligosaccharide of pneumococcal serotype 6C and its spacer-containing analogue is reported. Conjugation of the spacer-containing oligosaccharides of pneumococcal saccharides 6A, 6B, 6C and derivatives thereof with bovine serum albumin (BSA) protein carrier was carried out by using squaric-acid approach to obtain the oligosaccharide-protein conjugates in excellent yields. The conjugates have been tested with a rabbit antiserum pool (Pool B) used for pneumococcal serotyping. The results showed that synthetic carbohydrate conjugates express epitopes found in native capsular polysaccharides of serotypes 6A, 6B, and 6C.

Disruption of a Plasmodium falciparum Multidrug Resistance-associated Protein (PfMRP) Alters Its Fitness and Transport of Antimalarial Drugs and Glutathione

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC50 levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPΔ) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPΔ parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.

Diagnosis of human metapneumovirus by immunofluorescence staining with monoclonal antibodies in the North-East of England

Background Since its discovery in 2001 human metapneumovirus (hMPV) has been shown to be a significant cause of human respiratory disease, responsible for 5–8% of respiratory infections in hospitalised children. Diagnosis hitherto has been largely carried out by reverse tanscriptase polymerase chain reaction (RT-PCR) but immunofluorescence staining of cells from nasopharyngeal secretions (IF) offers advantages for some laboratories and may produce a more rapid result in urgent cases. We have recently demonstrated that IF with a rabbit antiserum gave sensitivity equal to that of RT-PCR. However, monoclonal antibodies offer a more plentiful, uniform IF reagent. Objectives Here we have evaluated a pool of anti-hMPV monoclonal antibodies in the routine diagnosis of respiratory infections in hospitalised infants and children. Study design Eight hundred and fifty-seven routine respiratory specimens were tested by IF with rabbit polyclonal antiserum and monoclonal antibody pool in parallel. A further 1003 specimens were tested with the monoclonal antibody pool alone. All specimens were also tested for a panel of other respiratory viruses by IF. Results Both rabbit polyclonal antiserum and monoclonal antibody pool gave positive results in 56 and negative results in 797 specimens. The rabbit polyclonal antibody detected virus in a further two specimens which were negative when tested with the monoclonal pool giving a concordance of 96.6% and a specificity of 100% for the monoclonal antibody pool. Overall hMPV was detected in 5% of specimens whilst 18.4% were positive for hRSV. Conclusions The monoclonal antibody pool-based IF is a robust assay suitable for routine use with a sensitivity only slightly less than that of the other major diagnostic methodologies available.

Uropathogenic Escherichia coli Outer Membrane Antigens Expressed during Urinary Tract Infection

Uncomplicated urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) represents a prevalent and potentially severe infectious disease. In this study, we describe the application of an immunoproteomics approach to vaccine development that has been used successfully to identify vaccine targets in other pathogenic bacteria. Outer membranes were isolated from pyelonephritis strain E. coli CFT073 cultured under conditions that mimic the urinary tract environment, including iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells. To identify antigens that elicit a humoral response during experimental UTI, outer membrane proteins were separated by two-dimensional gel electrophoresis and probed using pooled antisera from 20 CBA/J mice chronically infected with E. coli CFT073. In total, 23 outer membrane antigens, including a novel iron compound receptor, reacted with the antisera and were identified by mass spectrometry. These antigens also included proteins with known roles in UPEC pathogenesis, such as ChuA, IroN, IreA, Iha, IutA, and FliC. These data demonstrate that an antibody response is directed against these virulence-associated factors during UTI. We also show that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent (P < 0.01) among UPEC strains than among fecal-commensal E. coli isolates. Thus, we suggest that the conserved outer membrane antigens identified in this study could be rational candidates for a UTI vaccine designed to elicit protective immunity against UPEC infection.

Epidemiology and clinical findings associated with enteroviral acute flaccid paralysis in Pakistan

BackgroundEnteroviruses are among the most common viruses infecting humans worldwide and they are associated with diverse clinical syndromes. Acute flaccid paralysis (AFP) is a clinical manifestation of enteroviral neuropathy, transverse myelitis, Guillian-Barre Syndrome, Traumatic neuritis and many other nervous system disorders. The objective of this study was to understand the role of Non-Polio Enteroviruses (NPEV) towards this crippling disorder.MethodsStool specimens of 1775 children, aged less than 15 years, suffering from acute flaccid paralysis were collected after informed consent within 14 days of onset of symptoms during January 2003 to September 2003. The specimens were inoculated on RD and L20B cells using conventional tube cell culture while micro-neutralization test was used to identify the non-polio enterovirus (NPEV) serotypes. Detailed clinical information and 60-days follow-up reports were analyzed for NPEV-associated AFP cases.ResultsNPEV were isolated from 474 samples. The male to female ratio was 1.4:1. The isolation of NPEV decreased significantly with the increase in age. Cases associated with fever at the onset of NPEV-associated AFP were found to be 62%. The paralysis was found asymmetrical in 67% cases, the progression of paralysis to peak within 4 days was found in 72% cases and residual paralysis after 60 days of paralysis onset was observed in 39% cases associated with NPEV. A clinical diagnosis of Guillian-Barre syndrome was made in 32% cases. On Microneutralization assay, echo-6 (13%) and coxsackievirus B (13%) were the most commonly isolated serotypes of NPEV along with E-7, E-13, E-11, E-4 and E-30. The isolates (n = 181) found untypable by the antiserum pools were confirmed as NPEV by PCR using Pan-Enterovirus primers.ConclusionThe present study suggests that NPEV are a dominant cause of AFP and different serotypes of NPEV are randomly distributed in Pakistan. The untypable isolates need further characterization and analysis in order to determine their association with clinical presentation of a case.

Isolation and initial identification of pathogens causing an aseptic meningitis outbreak in north area of Jiangsu province

目的 分离并初步鉴定引起2003年我国苏北地区无菌性脑膜炎暴发的病原体.方法来自70例患者的66份脑脊液和4份粪便标本,用3种细胞系(MRC-5、Vero、Hep-2)进行病毒分离,并通过电镜、逆转录聚合酶链反应(RT-PCR)和中和试验等方法,分别从形态学、分子生物学和血清学的角度对分离毒株进行鉴定.结果得到22株阳性分离株病毒,18株分离自脑脊液(分离率27.3%),4株分离自粪便标本.经电镜、肠道病毒通用引物RT-PCR以及肠道病毒组合及单价抗血清的中和试验鉴定,所有分离毒株均为Echo30型肠道病毒.结论 Echo30是本次脑膜炎暴发流行的病因病毒。

The Lim Benyesh-Melnick antiserum pools for serotyping human enterovirus cell culture isolates—still useful, but may fail to identify current strains of echovirus 18

Neutralisation tests are often used as a second line method of serotyping human enterovirus cell culture isolates after failure of indirect immunofluorescence. We reviewed the results of 85 consecutive neutralisation tests performed routinely on presumptive enterovirus isolates using the Lim Benyesh-Melnick antiserum pools after failure of immunofluorescence on most of them, in order to assess the value of these antiserum pools. Confirmation of serotype was determined by partial VP1 genome sequencing. Forty isolates were serotyped successfully and 45 isolates were untypable by the LBM pools, although 16 of the latter would not be expected to be typable by this means. Of eighteen echovirus 18 isolates tested, only two were typable by neutralisation. Of 15 coxsackievirus A16 isolates only nine were typable by neutralisation. Neutralisation with the LBM pools remains a useful second line method of serotyping human enteroviruses despite difficulties with at least two serotypes.

Enteroviruses in Tunisia: virological surveillance over 12 years (1992–2003)

This report is an overview of enterovirus epidemiology in Tunisia during a 12-year period from 1992 to 2003. A total of 4700 clinical samples were collected as part of the national poliovirus surveillance programme and the routine diagnostic programme for aseptic meningitis. Enterovirus detection was performed by isolation on cell culture according to World Health Organization recommended protocols. Serotype identification was performed by seroneutralization of the cytopathic effect using pools of specific antisera and sequencing in the VP1 region of the genome. Poliovirus isolates were assessed for their wild or vaccine-related origin by standard World Health Organization recommended methods (PCR, probe hybridization and ELISA). The results confirm the interruption of wild poliovirus circulation since 1995. A total of 236 non-polio enterovirus (NPEV) strains were isolated; seroneutralization allowed typing of 93 % (219 out of 236) of them. The antisera used allowed the identification of the most common enterovirus serotypes. The remaining 17 isolates were sequenced; 16 of them belonged to enterovirus serotypes that were not targeted by the antisera pools used. A total of 29 different serotypes of NPEV were detected in the country during the study period. Echoviruses of serotypes 6, 11 and 30 were the most frequently isolated, almost every year; other serotypes had a cyclic occurrence and others were detected during a limited period with very few isolates. The NPEV isolation rate varied from year to year but was steadily under 10 %, suggesting a relatively low prevalence of these viruses in comparison to that in other developing countries. A seasonal variation was also noted; the high transmission period starts in March and peaks in September-November. This study is the first report of the epidemiology of NPEV in Tunisia. These viruses are associated with various diseases and epidemiological data may help to clarify their impact on human health.

Immunocytochemical localization of antigens recognized by human antisera in infective larvae of Wuchereria bancrofti.

Ultrathin sections of L3 of Wuchereria bancrofti embedded in hydrophilic resin were incubated with antisera pools from individuals (1) asymptomatic microfilaremic with different microfilaria (mf) densities (1-100, 101-500, and >1,000 mf/ml); (2) chronic with hydrocele or lymphedema; and (3) with no evidence of microfilaremia or clinical filariasis but residing in an endemic area. The groups of microfilaremic subjects studied presented differences relative to the intensity of labeling, with the density of gold particles per square micrometer proportional to microfilaremia. Incubation of ultrathin sections of W. bancrofti L3 larvae in the presence of antisera from patients exhibiting chronic obstructive lymphatic pathology of hydrocele and from individuals with clear clinical evidence of lymphedema exhibited a strong reaction in the same tissues. Except for the endemic normal group, all groups studied showed reactivity against epitopes in all tissues of infective larvae of W. bancrofti. The cuticle presented an intense labeling, suggesting a possible target structure for immune response.

The bradykinin B2-receptor in human decidua.

Bacterial infection of the amniotic cavity is one of the most frequent causes of preterm delivery. Bacterial products activate a network of autocrine and paracrine mediators in fetal membranes and decidua, with prostaglandins finally inducing contractions of the myometrium. Bradykinin and its B2-receptor (B2R) seem to be part of this network. In cultured decidua-derived cells, bradykinin stimulates the release of arachidonic acid, interleukin-6 (IL-6), and interleukin-8 (IL-8). These effects are prevented by the specific B2R antagonist Hoe 140. Using a pooled antiserum against peptide sequences of the B2R protein, the receptor can be visualized immunocytochemically. The cells contain mRNA for the B2R, as shown by reverse transcriptase polymerase chain reaction (RT-PCR). Binding studies reveal specific and saturable binding sites for bradykinin with characteristics of the B2R. Binding of bradykinin to the cells is enhanced by the inflammatory mediator interleukin-1beta. In summary, human decidua-derived cells express the B2R, its expression is upregulated in response to interleukin-1beta, and bradykinin stimulates the secretion of further mediators by these cells. Thus, bradykinin and the B2R could play a central role in decidual activation. If so, B2R antagonists would add to established tocolytic therapies that are applied together with antibiotics in cases of chorioamnionitis at low gestational age.

Protection against development of otitis media induced by nontypeable Haemophilus influenzae by both active and passive immunization in a chinchilla model of virus-bacterium superinfection.

Three separate studies, two involving active-immunization regimens and one involving a passive-transfer protocol, were conducted to initially screen and ultimately more fully assess several nontypeable Haemophilus influenzae outer membrane proteins or their derivatives for their relative protective efficacy in chinchilla models of otitis media. Initial screening of these antigens (P5-fimbrin, lipoprotein D, and P6), delivered singly or in combination with either Freund's adjuvant or alum, indicated that augmented bacterial clearance from the nasopharynx, the middle ears, or both anatomical sites could be induced by parenteral immunization with P5-fimbrin combined with lipoprotein D, lipoprotein D alone, or the synthetic chimeric peptide LB1 (derived from P5-fimbrin), respectively. Data from a second study, wherein chinchillas were immunized with LB1 or lipoprotein D, each delivered with alum, again indicated that clearance of nontypeable H. influenzae could be augmented by immunization with either of these immunogens; however, when this adjuvant was used, both antibody titers in serum and efficacy were reduced. A third study was performed to investigate passive delivery of antisera directed against either LB1, lipoprotein D, nonacylated lipoprotein D, or a unique recombinant peptide designated LPD-LB1(f)2,1,3. The last three antiserum pools were generated by using the combined adjuvant of alum plus monophosphoryl lipid A. Passive transfer of sera specific for LB1 or LPD-LB1(f)2,1,3 to adenovirus-compromised chinchillas, prior to intranasal challenge with nontypeable H. influenzae, significantly reduced the severity of signs and incidence of otitis media which developed (P </= 0.001). Collectively, these data indicate the continued merit of further developing LB1 and LPD-LB1(f)2,1,3 as components of vaccines for otitis media.

A new variant of echovirus 4 associated with a large outbreak of aseptic meningitis.

A large outbreak of aseptic meningitis which began in April 1997 involved hundreds of cases in all geographical regions of Israel and the Palestinian Authority, peaked between June and September, and lasted until December. We have investigated the virus associated with the outbreak to determine its serotype and molecular type and to establish epidemiological links. Virus strains isolated from 210 clinical samples were serotyped by neutralization using LBM and WHO antiserum pools and two echovirus 4 (EV4)-specific antisera, and by immunofluorescence using a monoclonal antibody. RNA was extracted and a 435 base long fragment derived from the 5'UTR of the genome was amplified by RT-PCR using common primers, and sequenced. Sequences were compared to echoviruses 4, 6 and 7 prototypes from ATCC, and to other echoviruses sequences from the EMBL/Genbank data base. The outbreak isolates were identified by the EV4 type-specific antisera and the monoclonal antibody but not with the WHO pools. Very few isolates could be typed by the LBM pools. The EV4 isolates accounted for 68% of all enterovirus isolates in our laboratory in 1997. The age distribution of the patients was: 0-11 month, 11.2%; 1-4 years, 16.1%; 5-9 years, 31.8%; 10-14 years, 9.9%; 15-20 years, 9.5%; 21-44 years, 21.5%; and > 45 years, 0%. Males between 1 and 14 years of age were affected more frequently than females of the same age. The sequences of 25 of 28 EV4 isolates analyzed were closely related to each other (> 95% homology) and the remaining three isolates had < 95% homology to the others and to each other. Interestingly, the outbreak strains were less closely related to the EV4 prototype, than to several other echoviruses. Three closely related subgroups were identified which correlated with geographical distribution but the temporal distribution did not reveal links leading to the source of the outbreak. The outbreak was caused by a variant of EV4 which apparently did not circulate in the area before and thus was capable of causing a widespread infection.

A genotypic characterization of enteroviral antigenic variants isolated in eastern Canada

Antigenic variation within serotypes of enteroviruses can have a significant impact on the effectiveness of routine diagnosis by neutralization assays. The focus of this particular study was to initiate a genetic characterization of echovirus type 9 (E9) antigenic variants and nontypeable strains isolated in Canada from 1991 to 1993. All variant strains were initially identified by the serological parameter of neutralization ‘breakthrough’ during conventional serotyping using the Lim–Benyesh–Melnick antiserum pools and by assessing neutralization endpoints using micro-neutralization methodology. Both E9 variant and non-variant isolates were further characterized by sequencing amplicons generated from the VP2 capsid protein-coding region of these particular strains. Variants from the provinces of Ontario and New Brunswick were shown to include a number of genotypically distinct strains and all the variant strains were significantly different from non-variant E9 isolates when nucleotide sequences were compared. A similar genetic analysis of two completely non-typeable isolates from Quebec showed that these viruses seemed to belong to a genetic cluster of enteroviruses that included coxsackievirus A16 and enterovirus 71 serotypes. The use of genetic typing by sequence analysis provides a molecular tool for determining the genotypic diversity of variant and non-typeable isolates and their possible relatedness to other enteroviral strains.

An epidemic of aseptic meningitis caused by echoviruses in Fukuoka Prefecture during April 1997 to August 1998

An epidemic of aseptic meningitis in Fukuoka Prefecture during April 1997 to August 1998 was studied to determine the serotype of viruses isolated in Fukuoka Prefecture. In Fukuoka Prefecture, bimodal peaks were seen in July and December 1997. Monthly changes of reported aseptic meningitis patients and period of virus isolation revealed that epidemics of the earlier part in 1997 was caused by echovirus 9 (E 9) and the latter part due to echovirus 30 (E 30). E 9 was isolated mainly in Chikugo Area from June to October 1997 but, E 30 was isolated all in areas of Fukuoka Prefecture. Isolation of E 30 continued after January 1998 in Fukuoka Prefecture. Isolation of echovirus 18 started in June 1998. The main serotypes of isolates are changing. The E 30 isolates are serotyped by neutralization with the aid of antiserum pools for enterovirus type differentiation, but serotyping was difficult with commercially available antiserum. The result of neutralization tests with standard serum and an immune albino rabbit serum prepared in our laboratory with the E 30 isolates indicated that the isolates in Fukuoka Prefecture was an antigenic variant.