Genetic Characteristics of Echovirus Type 6 Isolated from Hunan Province, China, 2009-2014 / 病毒学报

We wished to understand the genetic characteristics of enteric cytopathic human orphan (ECHO) virus type 6 (ECHO6) circulating in China. First, the partial VP1 coding region of six strains of the ECH-O6 virus isolated from cases of hand, foot and mouth diseases during routine surveillance in Hunan Province (China) from 2009 to 2014 were sequenced. Those sequences were analyzed along with 138 sequences of ECHO viruses covering five provinces of China and countries outside China retrieved from the GenBank database. A phylogenetic tree based on partial VPI was constructed, and it indicated that Chinese strains of the ECHO virus could form two distinct evolutionary branches: branch 1 and branch 2. All isolates of the ECHO virus from Hunan Province belonged to the 2c subranch, which revealed that they may share a common evolutionary origin. ECHO strains in branch 2 may be the predominant strains in China due to their wide geographic distribution and long period of circulation. We used nucleotide differences of >30%o as the basis of cluster division. ECHO, viruses could be divided into four clusters (A-D). Cluster D could be divided further into ten subclusters on the basis of nucleotide differences of 15%-30%. All ECHO6 isolates from Hunan Province belonged to the D7 subcluster. These data showed that the ECHO6 strains that circulated in Hunan Province in 2009-2014 were closely related to each other, and probably shared a common evolutionary origin. In addition, at least four distinct lineages of ECHO viruses have circulated in China.

Analysis of genetic characteristics of ECHO6 virus isolated from an epidemic outbreak of encephalitis in Longyan, China / 病毒学报

This study aimed to analyze the etiology of the encephalitis outbreak in Longyan, Fujian Province, China in 2010, in order to provide valuable information for this prevention and control of this disease. Pathogens were confirmed from cerebrospinal fluid samples with fluorescent RT-PCR, virus isolation (RD cells), and neutralization tests. Then, the VP1 fragments or whole genome nucleotide sequences were determined for four virus strains using PCR. Homology was assessed using the MegAlign software, and a phylogenetic evolutionary tree was drawn using Mega 4.0 software. The results confirmed that the etiology of the outbreak was the ECHO6 intestinal virus, and the nucleotide sequence of the VP1 segment indicated that the C2 subtype was responsible. The genome sequence consisted of 7407 nucleotides, and resembled the genome of other ECHO and CoxB viruses with homology levels of 78.5%-87.3%. The encephalitis outbreak in Longyan in 2010 was caused by the ECHO6 C2 subtype intestinal virus, and its complete genome sequence length is similar to the standard strain (U16283) with a sequence homology of 80.4%.

Description of a widespread outbreak of aseptic meningitis due to echovirus 30 in Rio de Janeiro state, Brazil

Echovirus 30 belongs to the genus Enterovirus and is widely associated with aseptic meningitis (AM) outbreaks. In Brazil epidemics due to this serotype were reported in several states but in Rio de Janeiro, before this study, it was only involved in sporadic episodes. We retrospectively collected data from AM notifications charts and enterovirus isolation database from Rio de Janeiro State Health Department (RJSHD) and Enterovirus Reference Laboratory in the year of 2005. An outbreak of AM was detected during March, April and May associated with a high cell culture isolation rate for echovirus 30 (17.4 percent). Male children with ages varying from 1 to 9 years were more affected. Of the 22 patients with confirmed echovirus 30 disease, clinical information was available in eight; fever, headache and vomiting were the most common manifestations. CSF analysis showed a typical pattern of viral infection with median of cellularity of 100 cells/mm³ and mononuclear cell predominance in 64.7 percent of the cases. The median of protein and glucose levels of 49 mg/dL and 56.5 mg/dL. The fatality rate was null. Despite its benign course and the lack of treatment options, aseptic meningitis surveillance is crucial for early identification of causative agents of outbreaks, which helps to avoid additional testing and inappropriate use of antimicrobials.

Echovirus 30 associated with cases of aseptic meningitis in state of Pará, Northern Brazil

Investigation of the aetiology of viral meningitis in Brazil is most often restricted to cases that occur in the Southern and Southeastern Regions; therefore, the purpose of this study is to describe the viral meningitis cases that occurred in state of Pará, Northern Brazil, from January 2005-December 2006. The detection of enterovirus (EV) in cerebrospinal fluid was performed using cell culture techniques, RT-PCR, nested PCR and nucleotide sequencing. The ages of the 91 patients ranged from < one year old to > 60 years old (median age 15.90 years). Fever (87.1 percent), headache (77.0 percent), vomiting (61.5 percent) and stiffness (61.5 percent) were the most frequent symptoms. Of 91 samples analyzed, 18 (19.8 percent) were positive for EV. Twelve were detected only by RT- PCR followed by nested PCR, whereas six were found by both cell culture and RT-PCR. From the last group, five were sequenced and classified as echovirus 30 (Echo 30). Phylogenetic analyses revealed that Echo 30 detected in Northern Brazil clustered within a unique group with a bootstrap value of 100 percent and could constitute a new subgroup (4c) according to the phylogenetic tree described by Oberste et al. (1999). This study described the first molecular characterization of Echo 30 in Brazil and this will certainly contribute to future molecular analyses involving strains detected in other regions of Brazil.

Brote epidémico de meningitis viral causado por echovirus tipo 4 en la provincia de Misiones / Outbreak of viral meningitis caused by echovirus type 4 in Misiones province

Se realizó un estudio retrospectivo a fin de describir un brote epidémico de meningitis causado por enterovirus, que comprometió a 143 niños de 1 mes a 14 años internados en el Hospital Pediátrico de Posadas (Misiones) con diagnóstico de meningitis aséptica, entre agosto y diciembre de 2005. Se observó un aumento de casos entre las semanas 33 a 50, con un pico máximo entre las semanas 47 y 48, lo que confirmó el brote. La mediana de edad de los niños afectados fue de 8 años y el 55,2% fueron varones. El 80% de los casos se observó entre escolares (5 a 14 años). El promedio del tiempo de internación fue de 4,5 ± 1,7 días, y no se registraron fallecidos. Los LCR se estudiaron mediante examen citoquímico y estudios bacteriológicos y virológicos (aislamiento viral, RT- PCR anidada e identificación molecular mediante secuenciación génica). Los recuentos de células en LCR variaron entre 6 y 5040 células /mm3, el 92% fueron inferiores a 500 células/mm3 y el 43,5% mostró predominio linfocitario. El 56% presentó concentraciones de glucosa normal, con proteínas ligeramente elevadas. El 28% de las muestras estudiadas por cultivo (17/60) mostró efecto citopático, compatible con enterovirus. La RT-PCR anidada permitió detectar enterovirus en un 73% de las muestras (43/59), con 6 casos que se tipificaron como echovirus tipo 4. El índice de positividad al combinar ambas técnicas alcanzó el 83%.

A descriptive retrospective study was carried out to describe an epidemic outbreak of enteroviral meningitis in Misiones. We reviewed records of 143 children from 1 month to 14 years of age who were hospitalized with aseptic meningitis in the Pediatric Hospital of Posadas from August to December 2005. Increased number of cases was observed between weeks 33 to 50 which reached a maximum peak in weeks 47 and 48, confirming an outbreak. The median of age was 8 years old, 55.2% were males. Eighty percent of cases were in 5 to 14 years old children. The average length of time spent in the hospital was 4.5±1.7 days, no deaths were reported. We performed cell counts, chemical and bacterial studies of CSF, and culture or RT-Nested/PCR for enteroviruses. Isolates were serotyped by RT-PCR amplification and genetic sequencing. Cell counts were from 6 to 5040 cells/mm3. Ninety two percent had less than 500 cells/mm3 and 43.5% had lymphocyte predominance. Glucose levels were normal with slightly elevated protein counts in 56% of cases. Of the cultured samples, 28% (17/60) showed cytopathic effect compatible with enterovirus. RT-n-PCR detected enterovirus in 73% (43/59) of the analyzed CSF. Echovirus type 4 was identified in 6 of them. The positive indicator obtained by combining both techniques was 83% (58/70).

Echovirus 6 associated to aseptic meningitis outbreak, in São Joaquim da Barra, São Paulo, Brazil

An aseptic meningitis outbreak occurred during a period from February to May 2004 in São Joaquim da Barra, a town in the northern region of São Paulo State. A total of 40 cases were reported to the Epidemiological Surveillance Center of São Paulo State. Cerebrospinal fluid samples obtained from 23 patients were sent to the Adolfo Lutz Institute for isolation of the virus. These samples were inoculated into RD, HEp2 and Vero cell lineages and those presenting a cytopathogenic effect were selected for analysis by indirect immunofluorescence assay (IFA), neutralization testing (Nt) and reverse transcriptase-polymerase chain reaction (RT-PCR). Cytopathogenic effects were observed in 52.2 percent (12/23) of these samples. All isolated viruses were identified as human enterovirus by IFA and RT-PCR and echovirus 6 was typed by IFA and Nt. Our results confirmed the participation and importance of echovirus as the etiological agent responsible for this outbreak and the serotype diversity of human enteroviruses circulating in São Paulo State.

Entre fevereiro e maio de 2004, em São Joaquim da Barra, Estado de São Paulo, foi notificado ao Centro de Vigilância Epidemiológica (CVE) um surto de meningite asséptica (MA) envolvendo 40 indivíduos. Foram enviadas ao Instituto Adolfo Lutz amostras de líquido cefalorraquidiano (LCR) de 23 pacientes com MA para tentativa de isolamento viral. Estas amostras foram inoculadas em 3 linhagens celulares: RD, HEp2 e Vero. Culturas celulares que apresentaram efeito citopático (ECP) foram submetidas a ensaio de Imunofluorescência Indireta (IFI), reação de Neutralização (Nt) e RT-PCR (Transcrição Reversa Reação em Cadeia da Polimerase). Em 52,2 por cento (12/23) das amostras foi observado ECP. Todos os vírus isolados foram identificados como gênero HEV por IFI e RT-PCR e o sorotipo echovirus 6 (E-6) por IFI e Nt. Nossos resultados confirmam a participação e importância dos echovirus como agente etiológico responsável pelo surto ocorrido e a diversidade de sorotipos circulantes no Estado de São Paulo.

RT-PCR for confirmation of echovirus 30 isolated in Belém, Brazil

Echovirus (Echo) 30 or human enterovirus B is the most frequent enterovirus associated with meningitis cases. Epidemics and outbreaks of this disease caused by Echo 30 have occurred in several countries. In Brazil, Echo 30 has been isolated from sporadic cases and outbreaks that occurred mainly in the south and southeast regions. We used RT-PCR to examine Echo 30 isolates from meningitis cases detected from March 2002 to December 2003 in Belém, state of Pará, in northern Brazil. The patients were attended in a Basic Health Unit (State Health Secretary of Pará), where cerebrospinal fluid (CSF) was collected and stored in liquid nitrogen. Weekly visits were made by technicians from Evandro Chagas Institute to the health unit and samples were stored at -70°C in the laboratory until use. HEp-2 and RD cell lines were used for viral isolation and neutralization with specific antisera for viral identification. RNA extraction was made using Trizol reagent. The RT-PCR was made in one step, and the total mixture (50 æL) was composed of: RNA, reaction buffer, dNTP, primers, Rnase inhibitor, reverse transcriptase, Taq polymerase and water. The products were visualized in agarose gel stained with ethidium bromide, visualized under UV light. Among the 279 CSF samples examined, 30 (10.7 percent) were EV positive, 29 being Echo 30 and one was Cox B. Nineteen Echo 30 were examined with RT-PCR; 18 tested positive (762 and 494 base pairs). The use of this technique permitted viral identification in less time than usual, which benefits the patient and is of importance for public-health interventions.

Study on an outbreak of echovirus type 6 of meningitis in Liu'an city, Anhui province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the epidemiological, clinical and etiological characteristics of an Echovirus type 6 meningitis outbreak in Jinzhai county, Liu'an city in Anhui, and to find out the proper way in controlling the aseptic meningitis outbreak.</p><p><b>METHODS</b>A surveillance system for aseptic meningitis was established in Jinzhai to confirm the case definition. Stool or cerebrospinal fluid (CSF) specimens from some cases were collected for entero-viruses isolation and identification. Case-control study was conducted. The case group involved patients while the controls would include: patients' classmate with same gender and the age difference was not over one year. Neutralization antibody in serum specimens were collected and tested in cases and in healthy people.</p><p><b>RESULTS</b>105 cases were distributed in 17 of the 30 towns in Jinzhai county while 41.0% of the cases were in Banzhuyuan town with an incidence rate of 203/10(5). Cases were clustered by school and classroom with age ranging from 3 to 15 years old and the highest as 10.9/10(5) in the 6 to 10 group. The incidence in males was 24.2/10(5) compared to 8.4/10(5) in females. The main clinic characteristics of cases were: fever, headache and vomiting. Echovirus type 6 from 25 of the 72 CSF samples (35%) was isolated. When comparing the cases group with control group, the OR of drinking home-made beverages was 4.1 (95% CI: 1.4-12.0), especially the beverages sacked by plastic bag: 3.3 (95% CI: 1.3-8.8). 6 out of 7 workers engaging in producing home-made beverages were detected to have carried Echovirus type 6 from their stool specimens. The Echovirus type 6 neutralization antibody positive rate in cases (73.5%) was significantly higher than that in 100 healthy people (46.0%) (X2 = 12. 526, P = 0.000).</p><p><b>CONCLUSION</b>This episode of meningitis outbreak was caused by Echovirus type 6. The proportion of drinking home-made beverages, especially the beverages sacked by plastic bag in cases group was higher than in control group.</p>

Molecular epidemiological analysis of echovirus 19 isolated from an outbreak associated with hand, foot, and mouth disease (HFMD) in Shandong Province of China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To elucidate the genetic characterization and molecular epidemiological features of Echovirus 19 (E19) isolates collected from an outbreak associated with hand, foot and mouth disease (HFMD) in Tai'an city of Shandong Province of China from July to September, 2003.</p><p><b>METHODS</b>Thirty seven Echovirus 19 isolates were isolated from stool specimens and throat swabs collected during the outbreak, then major capsid (VP1) genomic sequence was determined, and phylogenetic tree was done based on the VP1 sequences among these 37 and other E19 viruses deposited in the Genbank. Also a representative strain named CHN-SD03-TN12 was selected for sequencing of 5'-untranslated regions (5'-UTR).</p><p><b>RESULTS</b>The identity rate was about 98.9%-100% among all these 37 E19 viruses. The genetic relationships between these 37 E19 isolates and other strains reported were also depicted. The identity rate was about 78.4%-78.9% compared with E19 reference strain Burke. The substitutions in the sequence of 5'-UTR resulted in changes in the conjectural properties of 5'-UTR of E19 viruses.</p><p><b>CONCLUSION</b>The genetic features of E19 viruses isolated during the outbreak in Shandong Province in 2003 may be associated with a genetic and antigenic drift that changes the virulence of the Shandong isolates, but the molecular changes in Shandong E19 viruses contributing to their phenotype remain to be further illuminated. However, the sequences described in this paper substantiate the changes taken place in capsid VP1 and 5'UTR regions. These substitutions may contribute to their tropism and virulence, and play a significant role in pathogenesis and clinical manifestations of the disease.</p>

Contribution a l'etude virologique des souches d'enterovirus non-poliovirus isolees en Cote d'Ivoire : exemple des souches colligees de 1996 a 2004 a l'unite des virus du systeme nerveux de l'Instut Pasteur

OBJECTIF : La mise en place du programme mondial d'eradication de la poliomyelite et la disparition du Poliovirus pourrait entrainer une recrudescence des ENP dans la niche ecologique que ces virus occupaient. Ces virus sont responsables de flambees epidemiques de syndrome mains pieds bouche; de meningites a liquide claire et d'autres pathologies. Aussi devant la proportion importante d'ENP isolee de par le monde et surtout en Cote d'Ivoire; notre but est de decrire les caracteristiques virologiques et de montrer l'importance des ENP parmi les souches d'Enterovirus circulants dans notre pays. MATERIEL ET METHODES : Notre etude s'est deroulee de 1996 a 2004 a l'unite des virus du systeme nerveux de l'institut Pasteur. Elle a porte sur 286 souches d'ENP issues de 3092 echantillons (selles et LCR) soit 9;8pour cent. L'isolement viral s'est faite sur les cellules Hep-2C et RD et le typage par les immuns serums du kit RIVM. RESULTATS : Les sujets infectes par les ENP etaient ages de moins de l5ans (93pour cent) dont 66;1pour cent de 1 an a 4 ans. Une predominance masculine est observee avec un sexe ratio H/F=1;3. Ces ENP sevissent durant toute l'annee et sont predominant pendant les saisons de pluies (57pour cent) et en zone de foret (84;3pour cent). 54pour cent des souches ont ete isolees de cas de PFA. 62;9pour cent des souches se sont multiplies sur les cellules Hep-2C. Le serotype de 112 souches (39;2pour cent) a ete determine: il s'agit de Coxsackievirus B (10pour cent); Echovirus (19pour cent) et de souches non typables (71pour cent). Les Coxsackievirus B ont ete isoles a partir des echantillons de selles (100pour cent) et se sont multiplies sur les cellules Hep-2C (99pour cent). L'Echovirus 7(38pour cent) a ete isole chez les enfants de 0 a 4 ans et 1 Echovirus 33 a ete isole chez un malade hospitalise pour meningite. CONCLUSION : Devant la proportion importante de serotypes non typables; les laboratoires doivent s'investir dans les methodes de biologie moleculaire pour l'identification des ENP. Des collaborations doivent exister entre le laboratoire et les services hospitaliers afin que la recherche etiologique des pathologies infectieuses soit effectuee dans de meilleures conditions

Etiology of aseptic meningitis prevalent in Xuzhou / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To find the pathogenic agents of aseptic meningitis prevalent in Xuzhou of Jiangsu province in 2001.</p><p><b>METHODS</b>The enterovirus (EV) was cultured from CSF of the patients and identified with anti-serum by neutralization test. Neutralization titer of antibody in paired sera from meningitis children was determined. EV RNA was detected by RT-PCR.</p><p><b>RESULTS</b>Four strains of Coxsackievirus B5, 2 strains of Coxsackievirus B3 and 1 strain of Echovirus 7 were isolated from 22 CSF specimens. The isolation rate of virus was 31.8% (7/22), 21 CSF were tested by RT-PCR, the positive rate of EV RNA was 52.4% (11/21); 57.9% (11/19) of patients paired-sera had over 4 folds antibody rise or became seroconverted.</p><p><b>CONCLUSION</b>Enterovirus was the pathogenic agent of aseptic meningitis prevalent in Xuzhou of Jiangsu province, the main serotype of the virus was Coxsackievirus B5.</p>

Molecular identification and genetic analysis of an echovirus 19-associated epidemic of aseptic encephalitis / 中华流行病学杂志

<p><b>OBJECTIVE</b>To ascertain the pathogen of aseptic encephalitis epidemic in Long-Yan city in Fujian, and to find out the genetic characteristics of the virus.</p><p><b>METHODS</b>Rapid detection of enteroviral RNA by reverse transcription polymerasechain reaction (RT-PCR) was directly carried out in cerebrospinal fluid(CSF) to isolate and identify the viruses from CSF at the same time, and to detect the neutralization antibody in two serum specimens collected in acute and convalescence phase. Nucleotides of VP1 region was also analyzed by constructing phylogenetic tree.</p><p><b>RESULTS</b>ECHO 19 infection was rapidly diagnosed and sequence analysed by RT-PCR, and then echovirus type 19 from 16 of 30 CSF samples (53.33%) was isolated and detected using RD and Hep-2 cells simultaneity. The titer of ECHO 19 neutralization antibody became positive or increased by 4 times from acute to convalescence phase in 4 of the 5 patients. Phylogenetic analyses of the VP1 genes of these isolates showed that their nucleotides identity were 98.9% -100.0% which were different from those ECHO 19 from GeneBank database by 13.0%-22.4%.</p><p><b>CONCLUSION</b>The etiology of the epidemic of aseptic encephalitis was attributed to ECHO 19. The method of molecular identification not only provided rapid diagnosis of enterovirus infections, but also information about the genetic character of the viruses.</p>

Clonagem e expressão da principal proteína do capsídeo, VP1, do echovírus sorotipo 30 em células de eucarioto (BHK-21) / Clonagem and expression of the main capsid proteins, VP1, of echovirus serotype 30 in cells of eukaryote (BHK-21)

Os echovirus pertencem ao gênero Enterovirus, família Picornaviridae. Estes vírus apresentam um capsídeo icosaédrico constituído de quatro proteínas: VP1-VP4. Os epítopos responsáveis pela indução de anticorpos estão localizados principalmente em VP1, a proteína mais exposta da superfície viral. As principais síndromes clínicas relacionadas aos echovirus são: febre inespecífica branda, meningite asséptica, meningoencefalite, paralisia flácida aguda, síndrome de Guillain-Barré, miocardite, exantemas, encefalite e doenças respiratórias. O echovirus sorotipo 30 tem sido um dos enterovirus não-pólio mais freqüentemente associados a casos de meningite asséptica em várias regiões do mundo, incluindo o Brasil. Este estudo teve por objetivo a clonagem do gene VP1 de uma amostra de echovirus 30 isolada de um surto de meningite asséptica ocorrido no Brasil e a expressão da proteína correspondente em cultivos celulares. A proteína VP1 foi escolhida por conter os principais sítios antigênicos do vírion. O gene VP1 da amostra de echo 30 foi amplificado através da técnica de RT-PCR utilizando-se “primers” específicos contendo sítios para as enzimas de restrição Not I e Eco RI, e os códons de iniciação e término de tradução. Esta reação gerou um produto de 896 pb. O cDNA obtido foi clonado no plasmídeo pCR2.1 e subclonado em pcDNA3, um vetor de expressão em mamífero. Com a finalidade de se confirmar a identidade do inserto e averiguar possíveis erros durante o processo de clonagem, os clones obtidos foram seqüenciados. As seqüências foram alinhadas e comparadas com a cepa protótipo de echovirus 30 (Bastianni), exibindo uma homologia nucleotídica e de aminoácidos de 84.5 por cento e 94 por cento, respectivamente. Visando a expressão da proteína viral, células BHK-21 foram transfectadas transientemente utilizando-se o clone pc3/VP1E30. Imunofluorescência de localização citoplasmática foi observada após 24h em células BHK-21.

An outbreak of echovirus meningitis in children.

An outbreak of aseptic meningitis in children as evidenced by increase in the number of admissions in a tertiary care hospital is described. Clinical data and stool samples were collected from 25 hospitalized infants and young children. The stool samples were subjected to virological investigations. Fever and vomiting were the commonest symptoms. Cerebrospinal fluid (CSF) showed lymphocytic pleocytosis in majority of cases. Of the 25 stool samples, 14 showed an enterovirus specific cytopathogenic effect (CPE) in rhabdomyosarcoma (RD) cell line. All the 14 samples were positive for enterovirus RNA by reverse transcription-polymerase chain reaction (RT-PCR). Partial sequencing of the Virion protein 1 (VPI) region of the enterovirus genome carried out on the first 7 isolates revealed 5 isolates to be echovirus serotype 4 and one each to be echovirus serotypes 3 and 30. All children showed a rapid recovery and were discharged within 3 days of admission.

Establishment of an indirect enzyme-linked immunosorbent assay for detecting the specific IgM antibodies in patients with echovirus infection / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To provide a simple, specific and early serodiagnostic technique for the patients with aseptic meningitis caused by echovirus.</p><p><b>METHODS</b>An indirect enzyme-linked immunosorbent assay (ELISA) has been developed to detect echovirus-IgM and the specificity and availability of the assay were also examined.</p><p><b>RESULTS</b>In 78 cerebrospinal fluid (CSF) specimens which came from the children with aseptic meningitis, the positive rate was 17.9(14/78). In 64 CSF collected from non-aseptic meningitis (bacterial meningitis and cerebral trauma), the positive rate was 1.56(1/64). In 5 CSF specimens which were ELISA positive, the positive rate of neutralization test (NT) was 4/5, all the specimens which were ELISA negative were NT negative. In this assay there was no cross-reaction with poliovirus, Coxsackie virus B type 1-6 and A type 7. By blocking and destructive test of specific IgM, all CSF specimens with ELISA positive became negative.</p><p><b>CONCLUSIONS</b>The established indirect ELISA was specific and reliable. The te st was quick, simple and available, which is suitable for early and specific clinical diagnosis, and will be greatly significant to clinical treatment.</p>

Isolation and Identification of Respiratory Disease Virus in Pusan, 1998-1999 / 한국역학회지

This study was performed to monitor the causative agent of patient with respiratory disease in Pusan, 1998-1999. The results obtained were as follows. Among 1,320 cases of specimens from throat swab, influenzavirus infections were detected 232(89.6%), adenovirus infections were in 14(5.4%), coxsackievirus infections were in 11(4.2%), and echovirus infections were in 2(0.8%). The 222 strains out of 232 strains of influenzavirus showed A-type and the rest of them represented B-type. The distribution for sex- and age-groups is as follows. The male distribution was similar to the female distribution: male distribution, 47.1% and female distribution, 52.9%. Most of the patients was less than 10 years old. The monthly influenza distribution was consistent from Dec. 1998 to Apr. 1999. The 113 strains from the A-type isolates was A/Sydney/05/97(H3N2)-like, the 109 strains A/Beijing/262/95(H1N1)-like, and all of the 10 B-type isolates B/Harbin/07/94-like. Electron micrograph of negative-stained showed about 95 nm and about 71 nm with influenzavirus and adenovirus, respectively. Coxsackievirus and echovirus showed non-enveloped, isometric particle of about 30 nm diameter.