East Central South African genotype as the causative agent in reemergence of Chikungunya outbreak in India

Chikungunya fever is an important arboviral infection prevalent throughout Africa and Southeast Asia. Recently, in 2006, it has reemerged in many parts of India, affecting more than a million persons. A detail serological, virological, and molecular investigation of this unprecedented outbreak was carried out by collecting and studying 540 samples from all the affected regions of India during this epidemic. An in-depth investigation revealed the presence of anti-Chikungunya antibodies in 68% of the samples and genomic RNA in 49% of them. In addition 32 Chikungunya viruses were isolated from 45 representative polymerase chain reaction-positive samples. The nucleotide sequences of partial E1 gene of 25 representative Chikungunya viruses were deciphered. The sequence analysis indicated that all the isolates of this epidemic belonged to the new Indian Ocean island clade of East Central South (ECS) African genotype. This study conclusively proved the genotype shift from Asian to ECS African as the major factor in the reemergence of Chikungunya in an unprecedented outbreak in India after a gap of 32 years.     

Imported cases of dengue fever in Russia during 2010-2013

Objective:To confirm dengue infection among Russian tourists returned from Southeast and Mexico in 2010-2013 with clinical signs of infection.Methods:Blood and serum samples from patients were collected.NSI antigen and human IgM/IgG antibodies to dengue virus were identified using commercial tests manufactured by "Standard Diagnostics,INC.",Korea.ELISA test was used for the quantitative analyses of human IgM/IgG antibodies to dengue virus( "Orgenics Ltd.",Israel).Viral RNA was delected using commercial real-lime PCR lesls manufactured by "Genome Diagnostics Pvt.Ltd.",India and "Vector",Russia.Genotypes of revealed dengue viruses were determined employing nucleotide sequencing and phylogenetic analysis of 5'-UTR of the viral genome.Results:A total of 98 collected blood samples were analyzed.Fifty samples were positive for at least one of four markers of dengue infection.IgM to dengue virus were revealed in 38 samples,in 25 samples IgM were combined with IgG.NSI antigen was detected in43 samples.22 serum samples were positive for dengue virus RNA.The majority of samples(12patients) from tourists returned from Thailand were positive for genotype 1 of dengue virus,2nd and 4lh genotype were identified each in 1 patient.Conclusions:Due to laboratory confirmed cases of imported dengue fever in Russia,the differential diagnosis of dengue is strictly recommended for tourists returning from endemic areas.     

Dengue virus infection during post-epidemic period in Delhi, India

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are major public health problems in India. During the period following an epidemic, a study was carried out using virological and serological tests for confirmation of suspected cases of dengue virus infection in fever cases presenting to the All India Institute of Medical Sciences. Serum samples of suspected DF/DHF cases were processed from January to December 1997. In 37 samples from patients with fever of less than 5-day duration, received on ice, virus isolation was attempted in C6/36 clone of Aedes albopictus cell line, followed by indirect fluorescent antibody staining with monoclonal antibodies to dengue viruses 1 to 4. One hundred and forty-three serum samples from patients with more than 5 days fever were tested for dengue specific IgM antibody by either MAC-ELISA or a rapid immunochromatographic assay. Dengue virus type 1 was demonstrated by culture in 8 (21.6%) of 37 serum samples and IgM antibody could be detected in 42 (29.4%) of the 143 serum samples by the serological methods. The peak of dengue virus infection was seen from September to November 1997.     

Emergence of an independent lineage of dengue virus type 1 (DENV-1) and its co-circulation with predominant DENV-3 during the 2006 dengue fever outbreak in Delhi.

OBJECTIVES: The sudden emergence of dengue virus type 1 (DENV-1) and its co-circulation with predominant DENV-3 was the hallmark of the 2006 dengue fever outbreak in Delhi. Viruses that circulated between 1996 and 2005 in the City have been well characterized, but the genomic diversity in 2006 strains is not known. The present study was undertaken to reveal the emerging molecular genotype(s) and evolutionary trend of the viruses responsible for the dengue fever outbreak in Delhi during 2006. STUDY DESIGN: The CprM gene junction of the DENV isolates from the 2006 Delhi dengue fever outbreak were subjected to nucleotide sequencing. Comparative phylogenetic analysis was done using DENV-1 and DENV-3 sequences retrieved from the global database. RESULTS: Multiple sequence alignment revealed only substitutions, with no insertions or deletions. A dendrogram indicated emergence of a distinct lineage of DENV-1 (having similarity with the Comoros/Singapore 1993 and Delhi 1982 strains, but quite different from the Delhi 2005 lineage) and microevolution of the pre-circulating DENV-3. These findings point towards the circulation of two independent lineages of DENV-1 in Delhi during 2005 and 2006. CONCLUSIONS: It is feared that the introduction of an independent lineage of the outbreak-associated strain of DENV-1 and its co-circulation with the deeply-rooted strain of DENV-3 in Delhi may result in yet another, possibly more severe outbreak in the near future.     

Molecular characterization and clinical evaluation of dengue outbreak in 2002 in Bangladesh

During the febrile illness epidemic in Bangladesh in 2002, 58 people died out of the 6,132 affected. Two hundred hospitalized patients were analyzed clinically, serologically and virologically to determine the features of this dengue infection. Among the 10- to 70-year-old age group of the 200 clinically suspected dengue patients, 100 (50%) were confirmed as dengue cases by virus isolation and dengue IgM-capture ELISA. Of the 100 dengue-confirmed cases, the mean age was 29.0 (+/-12.4). The possible dengue secondary infection rate determined by Flavivirus IgG-indirect ELISA was 78% in 2002. Eight dengue virus strains were isolated, representing the first dengue virus isolation in the country, and all of the strains were dengue virus type-3 (DEN-3). Sequence data for the envelope gene of the DEN-3 Bangladeshi isolates were used in a phylogenetic comparison with DEN-3 from other countries. A phylogenetic analysis revealed that all 8 strains of DEN-3 were clustered within a well-supported independent sub-cluster of genotype II and were closely related to the Thai isolates from the 1990s. Therefore, it is likely that the currently circulating DEN-3 viruses entered Bangladesh from neighboring countries.     

An outbreak of dengue fever in rural areas of northern India

During the past few decades, epidemics of dengue fever are causing concern in several South-East Asian countries including India. The rural areas of Hissar district of Haryana state, situated about 170 km North-West of Delhi, experienced an outbreak of febrile illness during July-August 1996. A total of 13 villages in eight affected primary health centres reported fever cases. The clinical, epidemiological and entomological findings indicated that the present episode was due to dengue fever. The aetiological agent of the current outbreak, the DEN-2 virus, was isolated from 12 acute-phase sera specimens. Though, in the recent past outbreaks have been reported from the rural areas of southern and western India, the present episode is the first outbreak being reported from the rural areas of northern India. The increasing frequency of dengue fever outbreaks in rural areas of various Indian states reflects the changing life style of the rural population as a result of urbanization process and calls for a suitable prevention and control policy based on strengthened surveillance, appropriate health education to the community coupled with proper training of health personnel.     

Outbreak of dengue fever in Palau,Western Pacific: risk factors for infection

Between January and June 1995, an outbreak of dengue fever occurred in Palau, an island nation of 32,000 inhabitants in the Western Pacific. To determine the magnitude of this outbreak and to determine modifiable risk factors to guide control strategies, we established active surveillance at the national hospital and private clinics, reviewed available clinical records, and conducted serologic and entomologic surveys. Between January 1 and July 1, 1995, 817 case-patients with acute febrile illness with body or joint aches and one of the following: headache, rash, nausea, vomiting, or hemorrhagic manifestations presented to health facilities in Palau. The epidemic peaked in the second week of April 1995. Of 338 case-patients tested, 254 (75%) had positive serologic results by an IgM capture enzyme-linked immunosorbent assay. Dengue 4 virus was isolated from 78 (51%) of 154 serum samples tested. Blood samples collected during a cross-sectional survey were tested for IgM antibody and yielded an attack ratio of 27% (95% confidence interval = 23-31%). Potential vectors included the introduced species Aedes aegypti and Ae. albopictus, and the native species Ae. hensilli. Significant risk factors (P < or = 0.05) for infection included age < 20 years, the presence of food or water pans for animals on the property, taro farming, the presence of Ae. aegypti on the property, and presence of Ae. scutellaris group mosquitoes (Ae. Hensilli, Ae. albopictus, and a native species). This was the first outbreak of dengue 4 virus in the Western Pacific, and the first documented epidemic of dengue in Palau since 1988.     

The seroprevalence and seroincidence of dengue virus infection in western Kenya

Epidemics of dengue fever have been documented throughout the African continent over the past several decades, however little is known about the prevalence or incidence of dengue virus infection in the absence of an outbreak. No studies have analyzed the prevalence of dengue infection in western Kenya to date. This study describes the seroincidence and seroprevalence of dengue infection in western Kenya. Banked sera obtained from 354 healthy, afebrile children ages 12–47 months from Kisumu District, Kenya, were analyzed for antibodies to dengue virus using an IgG indirect ELISA. We found a seroprevalence of 1.1% (4 of 354 samples) and incidence of 8.5 seroconversions per 1000 persons per year in this study population. This appears to be similar to that previously reported in coastal regions of the country outside of known epidemic periods. Since there has never been a reported dengue epidemic in western Kenya, continued investigation and evaluation in a patient population presenting with fever is necessary to further confirm this finding.     

First Isolation of Dengue Virus from the 2010 Epidemic in Nepal

Dengue is an emerging disease in Nepal and was first observed as an outbreak in nine lowland districts in 2006. In 2010, however, a large epidemic of dengue occurred with 4,529 suspected and 917 serologically-confirmed cases and five deaths reported in government hospitals in Nepal. The collection of demographic information was performed along with an entomological survey and clinical evaluation of the patients. A total of 280 serum samples were collected from suspected dengue patients. These samples were subjected to routine laboratory investigations and IgM-capture ELISA for dengue serological identification, and 160 acute serum samples were used for virus isolation, RT-PCR, sequencing and phylogenetic analysis. The results showed that affected patients were predominately adults, and that 10% of the cases were classified as dengue haemorrhagic fever/ dengue shock syndrome. The genetic characterization of dengue viruses isolated from patients in four major outbreak areas of Nepal suggests that the DENV-1 strain was responsible for the 2010 epidemic. Entomological studies identified Aedes aegypti in all epidemic areas. All viruses belonged to a monophyletic single clade which is phylogenetically close to Indian viruses. The dengue epidemic started in the lowlands and expanded to the highland areas. To our knowledge, this is the first dengue isolation and genetic characterization reported from Nepal.     

Comparative evaluation of various commercial assays for diagnosis of dengue fever

Dengue fever (DF) is endemic in India and dengue hemorrhagic fever (DHF) has been reported with increasing frequency in the last decade. We evaluated three commercial assays for detection of antibodies to dengue virus, to assess their performance in a diagnostic laboratory. Sera from 58 patients collected during a febrile outbreak in New Delhi in 1997 were studied. The methods evaluated were MRL Diagnostic Dengue Fever Virus IgM Capture ELISA, Pan Bio Dengue Duo IgM and IgG Capture ELISA and Pan Bio Rapid Immunochromatographic test. The MRL ELISA correctly identified 97.8% (43 of 44) of samples as dengue positive while the Pan Bio Duo ELISA and Pan Bio RIT identified 95.45% (42 of 44). The sensitivities of both Pan Bio Duo ELISA and Pan Bio RIT for primary dengue and secondary dengue were 100% and 93.54% respectively. The specificity of three assays were MRL IgM ELISA 100%, Pan Bio Duo ELISA 92.8% and Pan Bio RIT 85.7%.     

福建省1株登革Ⅱ型病毒的鉴定

目的对2005年福建省分离的1株登革病毒(DV)进行鉴定,并从分子水平追踪其可能的传染源。方法采用酶联免疫吸附试验(ELISA)检测疑似登革热患者血清中DV(IgM、IgG抗体;同时应用C6/36细胞、单克隆抗体间接免疫荧光(McAb-IFA)、逆转录(套式PCR法分别进行病毒的分离和鉴定,并对分离株的部分基因进行核苷酸序列分析。结果患者血清登革病毒特异性IgM抗体阳性、IgG抗体可疑,表明该患者在近期感染过登革病毒。患者血清接种C6/36细胞,观察到登革病毒特有的CPE。受感染的C6/36细胞能与登革病毒Ⅱ型单克隆抗体反应,表明分离的病毒株为登革Ⅱ型病毒。分离株的核酸提取物经RT(PCR扩增,登革病毒通用引物可扩增出511bp的特异性条带,型特异性引物扩增出119bp的特异性条带,进一步证实分离的病毒株为登革Ⅱ型病毒。分离株RT(PCR扩增产物的核苷酸序列与30株不同地域来源的登革Ⅱ型病毒相应序列构建的系统发生树表明,此毒株与东南亚地区的毒株比较接近。此次分离株的序列与1999年登革Ⅱ型病毒福建株的对应序列在亲缘关系上有一定程度距离。结合流行病学调查资料,进一步确定此病例为输入性感染病例。结论福建省首次从输入性登革热患者血清中分离出登革Ⅱ型病毒,该病毒来源于东南亚地区。     

Investigation of an outbreak of scrub typhus in the himalayan region of India

In Indira Gandhi Medical College, Himachal Pradesh, India, during autumn of 2003 (September-November), more than 100 cases of fever of unknown origin (FUO) were reported with 15 ensuing deaths. In addition to all routine investigations and cultures, the Weil-Felix test was incorporated for the investigation of these cases. Antigen was procured from the Central Research Institute, Kasauli. Forty-six percent (45/96) of the cases demonstrated a > or =1:80 titer of agglutinins against OXK antigen. A team from the National Institute of Communicable Diseases, New Delhi, confirmed the antibodies for scrub typhus in some of the serum samples tested for leptospirosis, dengue fever, and rickettsial infections. Twelve blood samples positive for OXK antigen were sent to the Defense Research Development Establishment, Gwalior, for polymerase chain reaction studies, but none of the samples were positive, as all of the patients were already on broad-spectrum antibiotics and had reported to our hospital after 7-10 days of fever. At our institute, the Weil-Felix test has now been rountinely introduced for the investigation of cases of FUO, and the results until April 2004 (150 cases) revealed the presence of other rickettsial infections prevalent in the region. To evaluate the epidemiology and magnitude of the problem, further prospective studies are required.     

感染登革病毒后抗体产生规律及应用于暴发疫情的血清学诊断

目的了解感染登革病毒后特异性抗体产生规律,指导基层实验室应用血清抗体检测方法对暴发疫情的早期病例作出正确诊断,为控制暴发提供实验室依据。方法采集福建省1次暴发登革热疫情中的发热病人和密切接触者血清标本441份,以及恢复期血清22份,用捕获法ELISA检测登革IgG和IgM抗体,并结合病例的流行病学资料进行分析。结果根据本研究规定的病例判定标准,441例中登革热感染者为228例。其中,IgM抗体检出226例,IgG抗体检出119例。226例IgM抗体阳性标本中,IgG抗体同时阳性的为118份;119例IgG阳性标本中,IgM抗体阴性1例;两者均阴性的标本中用RT-PCR方法检测出1例。22份恢复期血清两类抗体检测均为阳性。绝大部分病例集中在10～70岁,病例的职业分布较广,男女比例为1∶1.53。结论登革病毒感染后特异性IgM抗体在发病早期即可检出,并可维持至少一个半月,但个别病例发病早期IgM可能为阴性;IgG抗体随病程的延长,其检出率逐步增高,至发病后50 d,其检出率可达95%左右。本研究结果抗体产生规律同以往研究结果一致,可为基层实验室开展登革热血清学检测和病例诊断提供参考。     

Kinetics of antibodies in sera, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infections.

OBJECTIVES: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue infection were studied. DESIGN: Serum, saliva, and urine samples were collected from 22 patients with clinical and confirmed dengue 3 virus infection during the outbreak in Havana City in 2001. They were tested by capture IgM (MAC-ELISA), IgA (AAC-ELISA), and IgE (EAC-ELISA) and IgG ELISA inhibition method (EIM) to detect specific dengue antibodies. RESULTS: Similar kinetics were observed in IgM, IgA, and IgG antibodies in saliva and IgA and IgG in urine samples from secondary cases compared with kinetics in serum samples, although the values were lower. No IgG antibody was detected in saliva and urine samples in primary cases and IgM antibody was not detected in urine samples from either primary or secondary infection. All secondary cases were positive for IgG in saliva and urine samples at day 7. The kinetics of specific IgE antibodies in primary and secondary cases were different. CONCLUSIONS: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infection were studied for the first time, showing its behavior and usefulness in dengue virus diagnosis. The specific IgE could play a role as a serological marker in secondary infections.     

Virus role during intraepidemic increase in dengue disease severity

Dengue epidemics in Cuba have repeatedly demonstrated a month-to-month increase in clinical severity during secondary infections. The dengue 2 outbreak that occurred in Santiago de Cuba in 1997 was accompanied by the most severe intraepidemic increase in disease severity reported to date. It was initially proposed that the appearance of neutralization escape mutants during the course of the epidemic might explain this phenomenon. Recent studies have revealed that during the course of this epidemic, nucleotide substitutions appeared only in nonstructural (NS) genes, most of which were silent, except for one change in the NS1 gene. To study whether or not variation in the NS1 gene might be associated with increased disease severity during the epidemic, this gene was partially sequenced from 15 isolates obtained at different times during the 1997 epidemic. Early epidemic isolates differed from those obtained later by replacement only of threonine with serine at position 164 in the NS1 protein, an amino acid rarely found in any genotype of dengue 2 virus. All viruses isolated from patients located in Health Districts, where dengue 2 transmissions occurred late in the epidemic, contained Serine at position 164, indicating that this change was fixed within a few months. Here we argue that this single mutation contributes to viral survival or replication efficiency, resulting in enhanced infection in the presence of enhancing antibodies, a phenomenon that we term increased virus "fitness" in contrast to "virulence," an intrinsic property of the virus.     

An outbreak of Chandipura virus encephalitis in the eastern districts of Gujarat state, India

An outbreak of encephalitis with a case fatality rate of 78.3% was investigated among children in Gujarat State, India. Twenty-six cases were reported. Three patients had IgM antibodies to Chandipura virus. Virus was isolated from one patient with rhabdomyosarcoma in porcine stable cell lines and in suckling mice. Chandipura virus RNA was present in 9 of 20 acute-phase serum samples, and virus sequences from the present outbreak were closely related to prototype strain (1965) and Andhra Pradesh, India (2003) isolates. Serologic and molecular assays documented the absence of Japanese encephalitis virus, West Nile virus, dengue virus, and paramyxoviruses in clinical samples. The etiologic agent was Chandipura virus, which has become an important encephalitis-causing virus in India.     

First dengue virus detection in Aedes albopictus from Delhi, India: its breeding ecology and role in dengue transmission

Objective To report dengue virus and its disease transmission in Aedes albopictus in the National Capital Territory of Delhi, India. Methods Monthly Aedes surveys were carried out in 126 urban localities of Delhi in 2008 and 2009. Pools of all three species of Aedes mosquitoes were tested for Dengue virus (DENV) using an antigen‐capture enzyme‐linked immunosorbent assay. Results Aedes aegypti was the most prevalent species, breeding throughout the year. Aedes albopictus was found in 9.52% of surveyed localities including the central urban part of Delhi, in March and from August to October. Aedes albopictus and Aedes vittatus are adapting to breed in manmade containers in the urban areas of Delhi in addition to their natural habitats of bamboo bushes and rock pits. Of the 229 pools of Ae. aegypti and 34 pools of Ae. albopictus tested, 10.5% and 11.76% were positive for dengue virus, respectively. No dengue virus infection was recorded in Ae. vittatus. Conclusion This is the first report of dengue virus in Ae. albopictus from north India. Because DENV was detected in Ae. albopictus, which adapted to manmade containers, both its spread and transmission dynamics should be checked.     

Use of nucleotide sequencing of the genomic cDNA fragments of the capsid/premembrane junction region for molecular epidemiology of dengue type 2 viruses

The recent emergence of dengue hemorrhagic fever/dengue shock syndrome (DHF/ DSS) in India has been a source of concern. In the present study a quantitative comparison of 406 nucleotide long sequence from the capsid-premembrane junction region (C-PrM) of 9 dengue virus type 2 (DEN-2) isolates from Delhi with 10 DEN-2 isolates from diverse geographic areas provided sufficient information for estimating genetic relationships. The data indicated that the 1996 epidemic of DHF in Delhi was caused by genotype IV strains of DEN-2. This genotype, perhaps, displaced genotype V strains of DEN-2, which was circulating genotype in 1967. The period during which this displacement had occurred is not clear from the present study. Nonetheless, similar experience in four countries in Latin America and in Sri Lanka suggest that the introduction of new genotypes of DEN-2 displacing the circulating genotype may be associated with the appearance of DHF/DSS. More work is required to elucidate this hypothesis. Transitions at nucleotide positions 406 and 431 resulted in amino acid substitutions near (aa position 104, methionine --> valine) and at the hinge region (aa position 112, valine --> alanine) of C-PrM, respectively in all/most genotypes of group III and IV DEN-2 viruses analysed. Most of these virus strains have been isolated from DHF/DSS outbreaks. Significance of this observation is discussed. The data presented in this study suggest the utility of C-PrM sequence analysis for molecular epidemiology of dengue viruses.     

Dengue in Greece in 1927 and 1928 and the pathogenesis of dengue hemorrhagic fever: new data and a different conclusion

A massive outbreak of dengue, with a high incidence of hemorrhagic manifestations and a high death rate, occurred in Athens and neighboring areas of Greece in 1927 and 1928. For many years it was believed that the episode had been caused by dengue type 1 virus. Recently, it was claimed that dengue type 2 virus also was present in Athens in 1928, and this report is cited in support of the hypotheses that dengue hemorrhagic fever is almost always the result of sequential infection with different dengue serotypes and that infection with dengue type 2 following dengue type 1 is particularly pathogenic. In the present study of 258 Greek residents born from 1914 to 1938, it was also found that dengue type 2 had occurred in Greece--but after 1928. No evidence was found that that virus had occurred in the country during, or within 13 years before, the 1927-1928 epidemic.