EV71:TLLcho virus murine model of enterovirus A71 neurological disease does not exhibit neurogenic pulmonary oedema.

Small animal models play an important role in investigating and revealing the molecular determinants and mechanisms underlying neuro-virulence of enterovirus A71 (EV-A71). In our previous study, we successfully developed two mouse cell-line replication competent EV-A71 strains (EV71:TLLm and EV71:TLLmv) which were capable of inducing neuro-invasion in BALB/c mice. The more virulent EV71:TLLmv exhibited ability to induce acute encephalomyelitis accompanied by neurogenic pulmonary oedema. EV71:TLLcho virus strain was generated from EV71:TLLm by a series of passages in CHO-K1 cells. EV71:TLLcho demonstrated a broader range of infectivity across various mammalian cell lines and exhibited complete cytopathic effects (CPE) within 48 hours post-inoculation in comparison to EV71:TLLm or EV71:TLLmv. EV71:TLLcho consistently yielded higher levels of viral replication at all time points examined. In comparison to EV71:TLLm, EV71:TLLcho consistently induced more severe disease and increased mortality in one-week old BALB/c mice. However, unlike mice challenged with EV71:TLLmv, none of the mice challenged with EV71:TLLcho progressed to severe acute encephalomyelitis and developed neurogenic pulmonary oedema.

Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression.

Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection.

Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression

Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were Ser[326]Leu, Ser[340]Leu at the deduced E protein, Ile[250]Thr at NS1 protein, and Thr[41]Ser at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection.

A comparative evaluation of dengue diagnostic tests based on single-acute serum samples for laboratory confirmation of acute dengue.

A prospective study was carried out to evaluate the sensitivity of dengue NS1 antigen-capture ELISA in comparison with dengue virus isolation, conventional RT-PCR and real-time RT-PCR for laboratory confirmation of acute dengue based on single-acute serum samples. Four primary healthcare centres were involved to recruit patients with clinical diagnosis of dengue illness. Patient's demographic, epidemiological and clinical information were collected on a standardized data entry form and 5 ml of venous blood was collected upon consent. In the laboratory, six types of laboratory tests were performed on each of the collected acute serum sample. Of the 558 acute serum samples collected from 558 patients with clinical diagnosis of dengue from mid-August 2006 to March 2009, 174 serum samples were tested positive by the dengue NS1 antigen-capture ELISA, 77 by virus isolation, 92 by RT-PCR and 112 by real-time RT-PCR. A total of 190 serum samples were tested positive by either one or a combination of the four methods whereas, only 59 serum samples were tested positive by all four methods. Thus, based on single-acute serum samples, 190 of the 558 patients (34.1%) were laboratory-confirmed acute dengue. The overall test sensitivity was 91.6%, 40.5%, 48.4% and 58.9% for dengue NS1 antigen-capture ELISA, virus isolation, conventional RT-PCR and real-time RT-PCR respectively. Statistically, dengue NS1 antigen-capture ELISA was the most sensitive and virus isolation was the least sensitive test for the laboratory confirmation of acute dengue based on single-acute serum specimens. Real-time RT-PCR was significantly more sensitive than the conventional RT-PCR.

Field evaluation on the effectiveness of a modified approach of chemical fogging against the conventional fogging in controlling dengue outbreak.

A field evaluation on the effectiveness of a modified approach of chemical fogging of insecticides against the conventional method was carried out in the Seremban district within the state of Negeri Sembilan, Malaysia from 7th February 2003 to 7th September 2003. In the 3 months period, November 2002 to January 2003, prior to institution of modified approach of chemical fogging, 27 of 42 (64.3%) dengue outbreaks were successfully controlled within the stipulated time frame of 14 days by the conventional approach of thermal chemical fogging. However, during the period when the modified approach of chemical fogging was instituted, 25 of 27 (92.6%) dengue outbreaks within the same district were successfully controlled within the 14-days time-line. Statistically, the modified approach of chemical fogging significantly improved the success rate of achieving dengue outbreak control within the stipulated time frame (chi2 = 5.65, p = 0.01745). The modified approach of chemical fogging also appeared to reduce the number of dengue cases recorded in the same district. This small pilot study shows that the modified approach of chemical fogging reduced cost in carrying out each fogging activity to control dengue outbreak. It also substantially reduced the required time taken to complete each fogging activity in comparison to the conventional approach. Thus, it enabled similar number of workers to cover more localities simultaneously affected by the outbreaks. In addition, the modified approach reduced the exposure time to hazardous insecticides for each worker doing hand-held thermal fogging.

Broad reactive monoclonal antibodies for rapid identification of enteroviruses show cross-reactivity with chikungunya virus infected cells.

In the past decade, enterovirus 71 (EV71) and chikungunya (CHIK) virus have re-emerged periodically causing serious public health problems in Malaysia, since their first emergence in 1997 and 1998 respectively. This study demonstrates that CHIK virus causes similar patterns of cytopathic effect in cultured Vero cells as some enteroviruses. They also show positive cross-reaction on direct immunofluorescence staining using monoclonal antibodies meant for typing enteroviruses. Without adequate clinical and epidemiological information for correlation, CHIK virus isolated from patients with acute febrile rash can be wrongly reported as untypeable enterovirus due to its cross-reactivity with commercial pan-enterovirus monoclonal antibodies. This is due to the diagnostic laboratory being unaware of such cross-reactions as it has not been reported previously. Final identification of the virus could be determined with specific antibodies or molecular typing using specific oligonucleotide primers for the CHIK virus.

Epidemiology of chikungunya in Malaysia: 2006-2009.

This is a retrospective cross-sectional study based on the database of clusters of patients with clinical diagnosis of chikungunya (CHIK) that were referred to the National Public Health Laboratory for diagnostic investigations from January 2006 to December 2009. Of the 13,759 referred patients, a total of 6314 (45.9%) patients were laboratory confirmed to have CHIK and 7445 (54.1) patients were considered as clinical cases of CHIK by epidemiological link. Epidemic curves plotted using date of onset of illness for all referred clusters of cases showed that there were three unrelated outbreaks of CHIK in Malaysia from 2006 to 2009. There were two small outbreaks that occurred within the state of Perak in 2006. The cluster of cases in 2008 and 2009 were of related outbreak which started in Johor state and subsequently spread to various parts of Malaysia. The mean age of the patients was 37.0 years old and those patients in the laboratory confirmed group were significantly younger than those in the epidemiological linked group. The main presenting clinical features recorded in this study were fever, arthralgia, myalgia and rashes. Those patients in the laboratory confirmed group had a significant higher incidence of fever, arthralgia and rash than those in the epidemiological linked group.

Epidemiological and clinical features of dengue versus other acute febrile illnesses amongst patients seen at government polyclinics.

Classical dengue fever is characterized by the clinical features of fever, headache, severe myalgia and occasionally rash, which can also be caused by a number of other viral and bacterial infections. Five hundred and fifty eight patients who fulfilled the criteria of clinical diagnosis of acute dengue from 4 government outpatient polyclinics were recruited in this prospective field study. Of the 558 patients, 190 patients were categorized as acute dengue fever, 86 as recent dengue and 282 as non-dengue febrile illnesses based on the results of a number of laboratory tests. Epidemiological features of febrile patients showed that the mean age of patients in the dengue fever group was significantly younger in comparison with patients in the non-dengue group. There was no significant difference between the two groups with respect to gender but there was significant ethnic difference with foreign workers representing a higher proportion in the dengue fever group. Patients with acute dengue fever were more likely to have patient-reported rash and a history of dengue in family or neighbourhood but less likely to have respiratory symptoms, sore-throat and jaundice in comparison to patients with non-dengue febrile illnesses. As with patients with dengue fever, patients in the recent dengue group were more likely to have history of patient-reported rash and a history of dengue contact and less likely to have respiratory symptoms in comparison to patients with non-dengue febrile illnesses. In contrast to patients with dengue fever, patients in the recent dengue group were more likely to have abdominal pain and jaundice in comparison to non-dengue febrile patients. The finding strongly suggests that a proportion of patients in the recent dengue group may actually represent a subset of patients with acute dengue fever at the late stage of illness.

Molecular epidemiology of chikungunya virus in Malaysia since its first emergence in 1998.

Malaysia experienced the first outbreak of chikungunya (CHIK) in Klang in late 1998 due to CHIK virus of Asian genotype. The CHIK virus of Asian genotype reemerged causing outbreak in Bangan Panchor, Perak in March 2006. CHIK virus of Central/East African genotype was first detected from a patient who returned from India in August 2006. In December 2006, CHIK virus of Central/East African genotype was re-introduced into Malaysia from India and caused an outbreak in Kinta district, Perak but was successfully controlled following an early detection and institution of intensive vector control measures. In late April 2008, CHIK virus of Central/East African genotype was laboratory confirmed as the cause of CHIK outbreak in Johore which spread to other parts of Malaysia by August 2008. Phylogenetic analysis based on the 254-bp fragment of the virus envelope protein gene as the genetic marker showed that three different strains of CHIK virus of Central/East African genotype were introduced into Malaysia on three separate occasions from 2006 to 2008. The strain that was introduced into Johor state was responsible for its subsequent spread to other parts of Malaysia, inclusive of Sarawak.

A fatal case of chikungunya virus infection with liver involvement.

Recovery from chikungunya is previously considered universal and mortality due to the virus is rare and unusual. Findings from recent chikungunya outbreaks occurred in Reunion Island and India have since challenged the conventional view on the benign nature of the illness. Malaysia has experienced at least of 4 outbreaks of chikungunya since 1998. In the present on-going large outbreak due to chikungunya virus of Central/East African genotype, a previous healthy sixty six years gentleman without co-morbidity was noted to have severe systemic infection by the virus and involvement of his liver. He subsequently passed away due to cardiovascular collapse after 5 days of illness.

Epidemiology, surveillance and control of Nipah virus infections in Malaysia.

The outbreak of Nipah virus, affecting pigs and pig-farm workers, was first noted in September 1998 in the north-western part of peninsular Malaysia. By March 1999, the outbreak had spread to other pig-farming areas of the country, inclusive of the neighbouring country, Singapore. A total of 283 human cases of viral encephalitis with 109 deaths were recorded in Malaysia from 29 September 1998 to December 1999. During the outbreak period, a number of surveillances under three broad groups; Surveillance in Human Health Sector, Surveillance in Animal Health Sector, and Surveillance for the Reservoir Hosts, were carried out to determine the prevalence, risk of virus infections and transmission in human and swine populations as well as the source and reservoir hosts of Nipah virus. Surveillance data showed that the virus spread rapidly among pigs within infected farms and transmission was attributed to direct contact with infective excretions and secretions. The spread of the virus among pig farms within and between states of peninsular Malaysia was due to movement of pigs. The transmission of the virus to humans was through close contact with infected pigs. Human to human transmission was considered a rare event though the Nipah virus could be isolated from saliva, urine, nasal and pharyngeal secretions of patients. Field investigations identified fruitbats of the Pteropid species as the natural reservoir hosts of the viruses. The outbreak was effectively brought under control following the discovery of the virus and institution of correct control measures through a combined effort of multi-ministerial and multidisciplinary teams working in close co-operation and collaboration with other international agencies.

Risk factors, prevention and communication strategy during Nipah virus outbreak in Malaysia.

An outbreak of acute febrile encephalitis affecting pig-farm workers and owners was recognized in peninsular Malaysia as early as September 1998. The outbreak was initially thought to be due to Japanese encephalitis (JE) virus and thus very intensive prevention, control and communication strategies directed at JE virus were undertaken by the Ministry of Health and Ministry of Agriculture of Malaysia. There was an immediate change in the prevention, control and communication strategies with focus and strategies on infected pigs as the source of infections for humans and other animals following the discovery of Nipah virus. Information and understanding the risks of Nipah virus infections and modes of transmission strengthened the directions of prevention, control and communication strategies. A number of epidemiological surveillances and field investigations which were broadly divided into 3 groups covering human health sector, animal health sector and reservoir hosts were carried out as forms of risk assessment to determine and assess the factors and degree of risk of infections by the virus. Data showed that there was significant association between Nipah virus infection and performing activities involving close contact with pigs, such as processing of piglets, administering injection or medication to pigs, assisting in the birth of piglets, assisting in pig breeding, and handling of dead pigs in the affected farms. A complex process of anthropogenic driven deforestation, climatic changes brought on by El Niño-related drought, forest fire and severe haze, and ecological factors of mixed agro-pig farming practices and design of pig-sties led to the spillovers of the virus from its wildlife reservoir into pig population.

Isolation of measles virus from clinical specimens using B95a and Vero/hSLAM cell-lines.

The clinical presentation of acute measles is normally quite typical, especially in the presence of Koplik's spots, that laboratory test is seldom required to confirm the diagnosis. However, with wide measles vaccination coverage and the extensive use of immunosuppressive chemotherapy, the diagnosis of atypical manifestations of acute measles may require laboratory confirmation. When compared with B95a cell-line, this study shows that the Vero/hSLAM cell-line is sensitive and is recommended for use in the primary isolation of wild-type measles virus from clinical specimens. Throat swab and urine specimens are the clinical specimens of choice and both are recommended for optimal isolation of measles virus from patients suspected of acute measles virus infection.

Performance of a commercial rapid dengue NS1 antigen immunochromatography test with reference to dengue NS1 antigen-capture ELISA.

The performance of a commercial immunochromatography test for rapid detection of dengue NS1 antigen present in serum or plasma of patients was evaluated against a commercial dengue NS1 antigen-capture ELISA. The rapid immunochromatography test gave an overall sensitivity of 90.4% with a specificity of 99.5%. The sensitivity was highest for serum samples from which virus was isolated (96.3%) and lowest for those from which virus was not isolated and RT-PCR was negative (76.4%). The sensitivity was significantly higher for serum samples from patients with acute primary dengue (92.3%) than those from patients with acute secondary dengue (79.1%). The positive predictive value and negative predictive value of this commercial immunochromatography test were 99.6% and 87.9% respectively.

Acute dengue in a neonate secondary to perinatal transmission.

We report a newborn baby girl with acute dengue due to vertical transmission. A 31 year old factory worker of 38+ week gestation, gravida 5 para 3+1, developed acute dengue fever two days prior to delivery. She delivered a normal term baby girl by spontaneous vaginal delivery and recovered uneventfully without peripartum haemorrhage despite the presence of thrombocytopenia. The baby girl developed low grade fever on day four of post-natal life and except for the transient thrombocytopenia, also recovered uneventfully following three days of mild illness. The clinical diagnosis of acute dengue virus infection was confirmed by laboratory tests.

A comparative study on the performance of two commercial anti-dengue IgM assay kits.

The performance of a commercial rapid immunochromatographic dengue IgG/IgM assay device was evaluated against an in-place dengue IgM-capture ELISA in the National Public Health laboratory. Of the 239 serum samples from patients with clinical diagnosis of acute dengue illness, 140 and 99 samples were tested positive and negative respectively for anti-dengue IgM by the in-placed ELISA. Comparatively, 72 and 76 samples were tested positive and negative respectively, and 91 samples gave equivocal results by the rapid dengue test device. The rapid immunochromatographic assay device gave a relative sensitivity of 49.3% and a relative specificity of 62.6%. Though the rapid immunochromatographic assay device has the advantages of rapid testing which simultaneously detects both IgG and IgM and can also be performed with whole blood, serum or plasma, the user has to exercise extreme caution with the interpretation of the test result.

Evaluating the sensitivity of a commercial dengue NS1 antigen-capture ELISA for early diagnosis of acute dengue virus infection.

INTRODUCTION: The aim of this report is to establish an accurate diagnosis of acute dengue virus infection early, in order to provide timely information for the management of patients and early public health control of dengue outbreak. METHODS: 224 serum samples from patients with a clinical diagnosis of acute dengue infection, which were subsequently confirmed by laboratory tests, were used to evaluate the performance of a commercially-available dengue NS1 antigen-capture ELISA kit. RESULTS: The dengue NS1 antigen-capture ELISA gave an overall sensitivity rate of 93.3 percent (209/224). The sensitivity rate was significantly higher in acute primary dengue (97.4 percent) than in acute secondary dengue (68.8 percent). In comparison, the virus isolation gave an overall positive isolation rate of 64.7 percent, with a positive rate of 70.8 percent and 28.1 percent, for acute primary dengue and acute secondary dengue, respectively. Molecular detection of dengue RNA by RT-PCR gave an overall positive detection rate of 63.4 percent, with a positive rate of 62.5 percent and 68.8 percent, for acute primary dengue and acute secondary dengue, respectively. Of the 224 acute serum samples from patients with laboratory-confirmed acute dengue infection, dengue IgM was detected in 88 specimens, comprising 68 acute primary dengue specimens and 20 acute secondary dengue specimens. NS1 antigen-capture ELISA kit gave an overall sensitivity rate of 88.6 percent in the presence of anti-dengue IgM and 96.3 percent in the absence of anti-dengue IgM. CONCLUSION: Of the 224 acute serum samples, the sample ages of 166 acute serum samples are known. The positive detection rate of dengue NS1 antigen-capture ELISA, on the whole, was higher than the other three established diagnostic test methods for laboratory diagnosis of acute dengue infection.

Full-length genome sequence and genetic relationship of two paramyxoviruses isolated from bat and pigs in the Americas.

Mapuera virus (MPRV) was isolated from a fruit bat in Brazil in 1979, but its host range and disease-causing potential are unknown. Porcine rubulavirus (PoRV) was identified as the aetiological agent of disease outbreaks in pigs in Mexico during early 1980s, but the origin of PoRV remains elusive. In this study, the completed genome sequence of MPRV was determined, and the complete genome sequence of PoRV was assembled from previously published protein-coding genes and the non-coding genome regions determined from this study. Comparison of sequence and genome organization indicated that PoRV is more closely related to MPRV than to any other members of the genus Rubulavirus. In the P gene coding region of both viruses, there is an ORF located at the 5' end of the P gene overlapping with the P protein coding region, similar to the C protein ORF present in most viruses of the subfamily Paramyxovirinae, but absent in other known rubulaviruses. Based on these findings, we hypothesise that PoRV may also originate from bats, and spillover events from bats to pigs, either directly or via an intermediate host, were responsible for the sporadic disease outbreaks observed in Mexico.

Molecular phylogeny of modern coxsackievirus A16.

A phylogenetic analysis of VP1 and VP4 nucleotide sequences of 52 recent CVA16 strains demonstrated two distinct CVA16 genogroups, A and B, with the prototype strain being the only member of genogroup A. CVA16 G-10, the prototype strain, showed a nucleotide difference of 27.7-30.2% and 19.9-25.2% in VP1 and VP4, respectively, in relation to other CVA16 strains, which formed two separate lineages in genogroup B with nucleotide variation of less than 13.4% and less than 16.3% in VP1 and VP4, respectively. Lineage 1 strains circulating before 2000 were later displaced by lineage 2 strains.

Outbreak of chikungunya due to virus of Central/East African genotype in Malaysia.

Chikungunya is an acute febrile illness caused by an alphavirus which is transmitted by infective Aedes mosquitoes. Two previous outbreaks of chikungunya in Malaysia were due to chikungunya virus of Asian genotype. The present outbreak involved two adjoining areas in the suburb of Ipoh city within the Kinta district of Perak, a state in the northern part of Peninsular Malaysia. Thirty seven residents in the main outbreak area and two patients in the secondary area were laboratory confirmed to be infected with the virus. The index case was a 44-year Indian man who visited Paramakudi, Tamil Naidu, India on 21st November 2006 and returned home on 30th of November 2006, and subsequently developed high fever and joint pain on the 3rd of December 2006. A number of chikungunya virus isolates were isolated from both patients and Aedes albopictus mosquitoes in the affected areas. Molecular study showed that the chikungunya virus causing the Kinta outbreak was of the Central/East African genotype which occurred for the first time in Malaysia.