Hand foot and mouth disease due to enterovirus 71 in Malaysia

Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth.The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents.Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae.EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD),aseptic meningitis,encephalitis and poliomyelitis-like acute flaccid paralysis.A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997,and caused 41deaths amongst young children.In late 2000,a recurrence of an outbreak of HFMD occurred in Malaysia with S fatalities in peninsular Malaysia.Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities.A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006.The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010.As with other HFMD outbreaks in Malaysia,both EV71 and CA16 were the main aetiological viruses isolated.In similarity with the HFMD outbreak in 2005,the isolation of CA16 preceded the appearance of EV71.Based on the VP 1 gene nucleotide sequences,4 sub-genogroups of EV71 (C1,C2,B3 and B4) co-circulated and caused the outbreak of hand,foot and mouth disease in peninsular Malaysia in 1997.Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak.EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003.In the 2005 outbreak,besides the EV71 strains of sub-genogroup C1,EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003.The four EV71 strains isolated from clinical specimens of patients with hand,foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5.Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years.In each of the past outbreaks,more than one sub-genogroup of the virus co-circulate.     

Complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand,foot and mouth disease outbreak in Singapore (2000)

Enterovirus 71 (EV71) is a major aetiological agent of hand, foot and mouth disease (HFMD). In recent years, several outbreaks in East Asia were associated with neurological complications and numerous deaths. An outbreak in Singapore in October 2000 afflicted thousands of children, resulting in four fatal cases from three of whom EV71 was isolated. The genomes of two representative EV71 strains isolated from a fatal case and a surviving patient were completely sequenced, and their nucleotide and amino acid sequences compared with known EV71 strains. The two outbreak strains were classified under genogroup B, together with those previously isolated in Singapore, Malaysia and Japan. Comparative sequence analysis of the two Singapore strains revealed 99% nucleotide similarity, while their deduced amino acid sequences were almost identical except for residue 1506 in the 3A non-structural region. Given that the outbreak involved closely related genetic variants of EV71, the broad spectrum of disease severity may be attributed to critical factors such as varying viral inoculation doses or differing host immune responses following infection, but is less likely to be due to the emergence of EV71 strains with heightened virulence.     

Real-time multiplex PCR for rapid detection of enteroviruses, adenoviruses and hepatitis A virus in clinical specimens

Real-time multiplex polymerase chain reaction (PCR) with internal positive control (IPC) was developed for simultaneous detection of adenoviruses (AV), enteroviruses (EV) and hepatitis A virus (HAV). Primes and probes labeled with different fluorophores (FAM, R6G, ROX, and Cy5) and able to detect up to four viral RNAs (DNAs) with high specificity in a single tube in real-time PCR were designed. Sensitivity and specificity of the method was estimated using cultural strains of 8 serotypes of EV, 5 serotypes of AV and 2 clinical specimens containing HAV. Sensitivity of the method for detection of polioviruses types 1, 2, and 3 (Sabin vaccine strains) was 0.5--1 TCID50 per reaction mixture. Thirty clinical specimens were analyzed by the multiplex PCR with and without IPC, and by mono-specific PCR. Comparison of these methods with cultural one revealed results agreement in 86.7% in case of multiplex PCR with IPC and in 100% in case of multiplex PCR without IPC and mono-specific PCR. This method can be used for rapid diagnostics of enteric viral infections as well as for determination of viral contamination level of water. As intermediate results of the study the methods for quantitative assessment of HAV, AV, and EV nucleic acids were developed which are convenient tools for the control of antiviral therapy effectiveness.     

Detection and ecology of leptospirosis in Iowa wildlife

To gain additional information on the extent of leptospirosis in wildlife following a human outbreak in Iowa, wild mammals and lower forms of life were collected. Isolation, darkfield microscopic, serologic and pathologic procedures were used to identify past or present evidence of leptospiral infection. Leptospires were isolated from 7 of 75 (9%) mammals. Serotype grippotyphosa was isolated from three raccoons (procyon lotor) and one Western Harvest Mouse (Reithrodontomys megalotis). Serotype ballum was isolated from three opossums (Didelphis marsupialis). Leptospires, unidentified to date, were isolated from frog (Rana pipiens) kidneys. Other positive serologic and pathologic tests gave evidence of infection or previous infection. Utilization of Darkfield microscopic and silver staining techniques did not detect all cases of leptospiral infection. Macroscopic and microscopic serologic methods failed to identify evidence of leptospirosis in all mammals from which leptospires were isolated. Pathologic lesions could only be considered presumptive evidence for leptospirosis. These findings indicate that detection of leptospirosis in wildlife cannot be limited to a single diagnostic test. A combination of diagnostic procedures and clinical evaluation is necessary. Although serotype pomona was implicated as the predominant infecting leptospire in the human cases and domestic animals and was isolated from water at a swimming site, only serotypes grippotyphosa, ballum and ICF (frog isolate) were isolated from wild mammals and lower forms of life in the same vicinity.     

Assessment of an enterovirus sewage surveillance system by comparison of clinical isolates with sewage isolates from milwaukee, wisconsin, collected august 1994 to december 2002

The quantity and serotypes of enteroviruses (EVs) in the influent of a local sewage treatment plant were compared to local clinical EV cases to determine if testing of sewage is adequate for an EV surveillance system. The study was carried out from August 1994 to December 2002. Monthly influent specimens were processed by organic flocculation, and dilutions of concentrate were inoculated onto a number of different cell types for virus isolation. EVs were detected in 88 of 100 monthly influent samples. Sewage EV titers were calculated by using software provided by the U.S. Environmental Protection Agency for most-probable-number determination. All 1,068 sewage EV isolates were further grouped (echovirus, coxsackievirus B, coxsackievirus A, or poliovirus) by cell culture host range analysis (growth pattern of isolates on passage to seven cell lines), and 39.0% of the 1,022 EV isolates categorized as non-poliovirus EVs were specifically serotyped. For clinical cases, primary virus isolation tests were performed on specimens submitted by local hospitals and EV isolates submitted by hospitals were serotyped. Clinical EVs were documented for 81 of the 100 months studied. In all, 694 EV isolates from clinical cases were serotyped. Annually, between 4 and 11 different serotypes of non-poliovirus EVs were identified in sewage and from 9 to 19 different non-poliovirus EV serotypes were identified from clinical specimens. Usually, the most commonly detected sewage EV serotypes were similar to the most commonly detected clinical serotypes; e.g., for 1997, echovirus 6 accounted for 53.1% of the typed sewage isolates and 39.4% of the clinical infections, while in 1998, echovirus 30 accounted for 50.0 and 46.1%, respectively. In 1999, 60.3% of the EVs from clinical cases and 79.7% of the sewage isolates were echovirus 11; in 2000, 33.3% of the EVs from clinical cases and 40.7% of the sewage isolates were coxsackievirus B5; and in 2001, 44.1% of the EVs from clinical cases and 36.2% of the sewage isolates were echovirus 13. Annual peaks of both sewage EV titers and clinical cases occurred in late summer or early fall. In some years, early spring sewage EVs portended some of the EVs that would predominate clinically during the following summer.     

Assessment of an Enterovirus Sewage Surveillance System by Comparison of Clinical Isolates with Sewage Isolates from Milwaukee, Wisconsin, Collected August 1994 to December 2002

The quantity and serotypes of enteroviruses (EVs) in the influent of a local sewage treatment plant were compared to local clinical EV cases to determine if testing of sewage is adequate for an EV surveillance system. The study was carried out from August 1994 to December 2002. Monthly influent specimens were processed by organic flocculation, and dilutions of concentrate were inoculated onto a number of different cell types for virus isolation. EVs were detected in 88 of 100 monthly influent samples. Sewage EV titers were calculated by using software provided by the U.S. Environmental Protection Agency for most-probable-number determination. All 1,068 sewage EV isolates were further grouped (echovirus, coxsackievirus B, coxsackievirus A, or poliovirus) by cell culture host range analysis (growth pattern of isolates on passage to seven cell lines), and 39.0% of the 1,022 EV isolates categorized as non-poliovirus EVs were specifically serotyped. For clinical cases, primary virus isolation tests were performed on specimens submitted by local hospitals and EV isolates submitted by hospitals were serotyped. Clinical EVs were documented for 81 of the 100 months studied. In all, 694 EV isolates from clinical cases were serotyped. Annually, between 4 and 11 different serotypes of non-poliovirus EVs were identified in sewage and from 9 to 19 different non-poliovirus EV serotypes were identified from clinical specimens. Usually, the most commonly detected sewage EV serotypes were similar to the most commonly detected clinical serotypes; e.g., for 1997, echovirus 6 accounted for 53.1% of the typed sewage isolates and 39.4% of the clinical infections, while in 1998, echovirus 30 accounted for 50.0 and 46.1%, respectively. In 1999, 60.3% of the EVs from clinical cases and 79.7% of the sewage isolates were echovirus 11; in 2000, 33.3% of the EVs from clinical cases and 40.7% of the sewage isolates were coxsackievirus B5; and in 2001, 44.1% of the EVs from clinical cases and 36.2% of the sewage isolates were echovirus 13. Annual peaks of both sewage EV titers and clinical cases occurred in late summer or early fall. In some years, early spring sewage EVs portended some of the EVs that would predominate clinically during the following summer.     

A One-Step,Triplex, Real-Time RT-PCR Assay for the Simultaneous Detection of Enterovirus 71, Coxsackie A16 and Pan-Enterovirus in a Single Tube

The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001–0.04 TCID₅₀/ml, 0.02 TCID₅₀/ml, and 0.001 TCID₅₀/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.     

Naturally Acquired Picornavirus Infections in Primates at the Dhaka Zoo

The conditions in densely populated Bangladesh favor picornavirus transmission, resulting in a high rate of infection in the human population. Data suggest that nonhuman primates (NHP) may play a role in the maintenance and transmission of diverse picornaviruses in Bangladesh. At the Dhaka Zoo, multiple NHP species are caged in close proximity. Their proximity to other species and to humans, both zoo workers and visitors, provides the potential for cross-species transmission. To investigate possible interspecies and intraspecies transmission of picornaviruses among NHP, we collected fecal specimens from nine NHP taxa at the Dhaka Zoo at three time points, August 2007, January 2008, and June 2008. Specimens were screened using real-time PCR for the genera Enterovirus, Parechovirus, and Sapelovirus, and positive samples were typed by VP1 sequencing. Fifty-two picornaviruses comprising 10 distinct serotypes were detected in 83 fecal samples. Four of these serotypes, simian virus 19 (SV19), baboon enterovirus (BaEV), enterovirus 112 (EV112), and EV115, have been solely associated with infection in NHP. EV112, EV115, and SV19 accounted for 88% of all picornaviruses detected. Over 80% of samples from cages housing rhesus macaques, olive baboons, or hamadryas baboons were positive for a picornavirus, while no picornaviruses were detected in samples from capped langurs or vervet monkeys. In contrast to our findings among synanthropic NHP in Bangladesh where 100% of the picornaviruses detected were of human serotypes, in the zoo population, only 15% of picornaviruses detected in NHP were of human origin. Specific serotypes tended to persist over time, suggesting either persistent infection of individuals or cycles of reinfection.     

Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71

Human enterovirus species A (HEV-A) consists of at least 16 members of different serotypes that are known to be the causative agents of hand, foot, and mouth disease (HFMD), herpangina, and other diseases, such as respiratory disease and polio-like flaccid paralysis. Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of HFMD. CVA5, CVA6, CVA10, and CVA12 mainly cause herpangina or are occasionally involved with sporadic cases of HFMD. We have previously shown that human scavenger receptor class B, member 2 (SCARB2) is a cellular receptor for EV71 and CVA16. Using a large number of clinical isolates of HEV-A, we explored whether all clinical isolates of EV71 and other serotypes of HEV-A infected cells via SCARB2. We tested this possibility by infecting L-SCARB2 cells, which are L929 cells expressing human SCARB2, by infecting human RD cells that had been treated with small interfering RNAs for SCARB2 and by directly binding the viruses to a soluble SCARB2 protein. We showed that all 162 clinical isolates of EV71 propagated in L-SCARB2 cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains. In addition, CVA7, CVA14, and CVA16, which are most closely related to each other, also utilized SCARB2 for infection. EV71, CVA14, and CVA16 are highly associated with HFMD, and EV71 and CVA7 are occasionally associated with neurological diseases, suggesting that SCARB2 plays important roles in the development of these diseases. In contrast, another group of viruses, such as CVA2, CVA3, CVA4, CVA5, CVA6, CVA8, CVA10, and CVA12, which are relatively distant from the EV71 group, is associated mainly with herpangina. None of these clinical isolates infected via the SCARB2-dependent pathway. HEV-A viruses can be divided into at least two groups depending on the use of SCARB2, and the receptor usage plays an important role in developing the specific diseases for each group.     

Genital co-infection with herpes simplex viruses type 1 and 2: comparison of real-time PCR assay and traditional viral isolation methods

We report the clinical case of a genital outbreak with both Herpes Simplex Type 1 (HSV-1) and Herpes Simplex Type 2 (HSV-2) during pregnancy. Herpes was presumptively identified by clinical presentation of lesion and Tzanck smear while serotypes were identified by cell culture and polymerase chain reaction (PCR). This case report highlights the need for increased surveillance of both serotypes in genital infection of pregnant women for effective disease management and reduced risk of transmission. Increasing rates of genital infection with HSV-1, the possibility of genital co-infection with HSV-1 and HSV-2 and the non-specificity and lack of sensitivity of traditional viral isolation methods may lead to under-diagnosis of genital HSV-1 infections unless molecular diagnostic methods, such as polymerase chain reaction (PCR) are routinely deployed in the clinical setting.     

Co-Circulation and Genomic Recombination of Coxsackievirus A16 and Enterovirus 71 during a Large Outbreak of Hand,Foot, and Mouth Disease in Central China

A total of 1844 patients with hand, foot, and mouth disease (HFMD), most of them were children of age 1–3-year-old, in Central China were hospitalized from 2011 to 2012. Among them, 422 were infected with coxsackievirus A16 (CVA16), 334 were infected with enterovirus 71 (EV71), 38 were co-infected with EV71 and CVA16, and 35 were infected with other enteroviruses. Molecular epidemiology analysis revealed that EV71 and CVA16 were detected year-round, but EV71 circulated mainly in July and CVA16 circulated predominantly in November, and incidence of HFMD was reduced in January and February and increased in March. Clinical data showed that hyperglycemia and neurologic complications were significantly higher in EV71-infected patients, while upper respiratory tract infection and C-reactive protein were significantly higher in CVA16-associated patients. 124 EV71 and 80 CVA16 strains were isolated, among them 56 and 68 EV71 strains were C4a and C4b, while 25 and 55 CVA16 strains were B1a and B1b, respectively. Similarity plots and bootscan analyses based on entire genomic sequences revealed that the three C4a sub-genotype EV71 strains were recombinant with C4b sub-genotype EV71 in 2B–2C region, and the three CVA16 strains were recombinant with EV71 in 2A–2B region. Thus, CVA16 and EV71 were the major causative agents in a large HFMD outbreak in Central China. HFMD incidence was high for children among household contact and was detected year-round, but outbreak was seasonal dependent. CVA16 B1b and EV71 C4b reemerged and caused a large epidemic in China after a quiet period of many years. Moreover, EV71 and CVA16 were co-circulated during the outbreak, which may have contributed to the genomic recombination between the pathogens. It should gain more attention as there may be an upward trend in co-circulation of the two pathogens globally and the new role recombination plays in the emergence of new enterovirus variants.     

Prevalence of Nonpolio Enteroviruses in the Sewage of Guangzhou City,China, from 2009 to 2012

The human-pathogenic viruses in urban sewage have been extensively monitored to obtain information on circulating viruses in human communities. Enteroviruses (EVs) excreted by patients who present with diverse clinical syndromes can remain infectious in the environment for several weeks, and limited data on circulating environmental EVs are available. A 4-year (2009 to 2012) surveillance study was conducted to detect nonpolio enteroviruses (NPEVs) in the urban sewage of Guangzhou city, China. After the viruses in the sewage samples were concentrated and isolated, molecular identification was used to detect and type the NPEVs. During the 4-year study, 17 different NPEV serotypes were identified in the sewage of Guangzhou city. The most common serotypes were echovirus 11 (ECHO11), ECHO6, ECHO7, and ECHO12 and coxsackie group B viruses 5 (CVB5) and CVB3. The predominant serotypes were influenced by spatial and temporal factors and differed each year. CVB5 was commonly detected in 2009 and 2010 but was rarely isolated in 2011 and 2012. In contrast, CVB3 was not observed in 2009 and 2010 but was increasingly detected in 2011 and 2012. Our study provides an overview of the serotype distribution and circulation patterns of NPEVs in the sewage of Guangzhou, China. In the absence of a systematic EV disease surveillance system, the detection and characterization of sewage-borne NPEVs will help us better understand the changes in EV disease trends and the epidemic background of circulating EVs, which could help interpret the EV trends and warn of future outbreaks in this area.     

Prevalence of Salmonella serotypes in environmental waters and their relationships with indicator organisms

The incidence of serotypes of Salmonella in three types of environmental water (sea, river and fresh reservoirs) from north-east Spain was investigated. The study was performed at specific sampling locations during the summer for a period of five years (1992–1996). A total of 823 strains were isolated and 55 different serotypes were identified, 42 were recovered from sea water, 32 from river water and 12 from freshwater reservoirs. The most frequently isolated serotypes coincided with those involved in clinical cases in the area studied. Salmonella enteritidis was the most common (111 isolates), it was found in all types of water, although most predominantly in sea water (16.1% of the isolates). This serotype, together with S. hadar, significantly increased in frequency during the five year study period. The most frequent serotypes in river water and freshwater reservoirs were S. virchow (9.5%) and S. mikawasima (23.8%) respectively. Significant differences were assessed in the indicator organism densities between the samples with serotypes of clinical significance (S. enteritidis, S. infantis, S. typhimurium, S. virchow and S. paratyphi B) and those without clinical significance. Therefore their presence in all environmental waters may be of epidemiological significance.     

An outbreak of Legionella infection in Armavir

In March-April 1987 an outbreak of Legionella infection was registered among 236 workers at a rubber factory in Armavir. An ARD-like syndrome and acute alveolitis were the main variants of the clinical course of the infection. Legionella infection was diagnosed on the basis of a fourfold and greater increase in the titers of antibodies to L. pneumophila, serogroup 1, as determined in the indirect immunofluorescence test and enzyme immunoassay (EIA). In urine samples obtained from patients Legionella antigen was determined with the use of EIA techniques. The culture of L. pneumophila, serogroup 1, was isolated from the recycling-type water supply system of the factory. The outbreak was stopped by a complex of sanitary, hygienic and epidemic control measures.     

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.     

Human SCARB2-mediated entry and endocytosis of EV71

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.     

Genome sequence of the Bacteroides fragilis phage ATCC 51477-B1

Background

Co-circulation of multiple dengue virus serotypes has been reported from many parts of the world including India, however concurrent infection with more than one serotype of dengue viruses in the same individual is rarely documented. An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) occurred in and around Delhi in 2006. This is the first report from India with high percentage of concurrent infections with different dengue virus serotypes circulating during one outbreak.

Results

Acute phase sera from patients were tested for the presence of dengue virus RNA by RT-PCR assay. Of the 69 samples tested for dengue virus RNA, 48 (69.5%) were found to be positive. All the four dengue virus serotypes were found to be co-circulating in this outbreak with DENV-3 being the predominant serotype. In addition in 9 of 48 (19%) dengue virus positive samples, concurrent infection with more than one dengue virus serotype were identified.

Conclusion

This is the first report in which concurrent infections with different dengue virus serotypes is being reported during an outbreak from India. Delhi is now truly hyperendemic for dengue.     

Isolation and Identification of Hepatitis Contagiosa Canis Virus (Hcc Virus) from an Enzootic Outbreak in a Dog Kennel

Using a continuous cell line of canine kidney (MDCK-cell line) cells, HCC virus was isolated from 4 of 6 puppies affected in an outbreak of the disease in a dog kennel. The virus was most easily, and rapidly, isolated in primary cultures from the liver samples, but also from other organs such as the spleen and lung, although isolation from these organs could require 1 or 2 passages in addition to the primary culture. Attention is therefore drawn to the necessity of using a sufficient number of passages when organs other than the liver are available. Among the bacteria identified in addition to the virus infection, were Staphylococcus aureus, Bordetella bronchiseptica and β-hemolytic streptococci. Emphasis is made on the significance of isolation and identification of the virus in pure culture, thus enabling its various properties to be studied in detail, and possible new serotypes to be recognized.     

Outbreaks of pseudotuberculosis and intestinal yersiniosis among Soviet specialists and members of their families in the Mongolian People's Republic

In summer and winter of 1986 two outbreaks of alimentary enteric diseases occurred among Soviet specialists and members of their families in Ulan Bator. These diseases were identified as Yersinia infections registered in Mongolia for the first time. In July 114 children in a kindergarten fell ill after being fed with salad prepared from vegetables and spring onions supplied from a nearby state farm. 20 Y. pseudotuberculosis cultures, serovar 1, were isolated, and in 25 persons antibodies to the isolated microorganisms were detected; altogether 32% of cases were confirmed by laboratory methods. During the December outbreak 187 persons were affected, among them 90% of children, through the consumption of imported oranges and tangerines, simultaneous infection with Y. enterocolitica, serovars 05.27 and 09, and hepatitis A virus being observed. The fact of associated infection was registered after the simultaneous detection of the markers of viral hepatitis A and Yersinia infection in 61 patients. In the kindergarten the disease produced a typical clinical picture of Far Eastern scarlatiniform fever, and in winter the abdominal forms of the disease prevailed. In cases of combined Yersinia and viral infection a specific variant of acute hepatitis developed; as regards this variant, the authors present heretofore unknown information on its epidemiology, clinical features and outcome.