Douglas M. Watts

Failure of secondary infection with American genotype dengue 2 to cause dengue haemorrhagic fever

BACKGROUND Population-based epidemiological studies have shown that infection with dengue type 2 (DEN-2) virus in individuals previously infected with a different serotype of the virus is a major risk factor for dengue haemorrhagic fever and dengue shock syndrome. However, the western hemisphere was spared epidemics of these two syndromes, until the introduction of a southeast Asian DEN-2 genotype. Possibly American DEN-2 genotype strains lacked properties necessary to cause severe disease. We report on a major epidemic of DEN-2 in Peru in 1995, about 5 years after an epidemic of DEN-1 in the same population. METHODS In Iquitos, a city of 344,686 inhabitants in Peru, cases of dengue fever were studied prospectively from 1990. Acute phase of illness serum samples from patients were tested for virus in C6/36 cells, and virus isolates were identified by immunofluorescence. Isolates of dengue 2 virus obtained from patients during an outbreak of mild febrile illness in 1995 were sequenced to determine the genotype. Serological analysis of paired samples from the patients was done with an IgM capture ELISA and an indirect IgG ELISA. In addition, serum samples collected annually between 1993 and 1996 from a large cohort of students were tested for dengue IgG antibody by an ELISA. Serum samples from a random sample of 129 students from this cohort were tested for dengue neutralising antibodies to quantify the serotype specific infection rates. FINDINGS Among the 129 students (aged 7-20 years in 1993) who had serum samples available before and after the epidemic, 78 (60.5%) had a secondary DEN-2 virus infection. By extrapolation, 49,266 of the 81,479 children (aged 5-14 years) in Iquitos would have experienced such infections. From previous studies, between 887 and 10,247 cases of dengue haemorrhagic fever and dengue shock syndrome would have been expected. No cases were found. DEN-2 isolates were of the American genotype. INTERPRETATION This prospective study shows that secondary infection by the American DEN-2 genotype did not cause dengue haemorrhagic fever and dengue shock syndrome.

Crimean-Congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity.

Crimean-Congo hemorrhagic fever (CCHF) is the most important tick-borne viral disease of humans, causing sporadic cases or outbreaks of severe illness across a huge geographic area, from western China to the Middle East and southeastern Europe and throughout most of Africa. CCHFV is maintained in vertical and horizontal transmission cycles involving ixodid ticks and a variety of wild and domestic vertebrates, which do not show signs of illness. The virus circulates in a number of tick genera, but Hyalomma ticks are the principal source of human infection, probably because both immature and adult forms actively seek hosts for the blood meals required at each stage of maturation. CCHF occurs most frequently among agricultural workers following the bite of an infected tick, and to a lesser extent among slaughterhouse workers exposed to the blood and tissues of infected livestock and medical personnel through contact with the body fluids of infected patients. CCHFV is the most genetically diverse of the arboviruses, with nucleotide sequence differences among isolates ranging from 20% for the viral S segment to 31% for the M segment. Viruses with diverse sequences can be found within the same geographic area, while closely related viruses have been isolated in far distant regions, suggesting that widespread dispersion of CCHFV has occurred at times in the past, possibly by ticks carried on migratory birds or through the international livestock trade. Reassortment among genome segments during co-infection of ticks or vertebrates appears to have played an important role in generating diversity, and represents a potential future source of novel viruses. In this article, we first review current knowledge of CCHFV, summarizing its molecular biology, maintenance and transmission, epidemiology and geographic range. We also include an extensive discussion of CCHFV genetic diversity, including maps of the range of the virus with superimposed phylogenetic trees. We then review the features of CCHF, including the clinical syndrome, diagnosis, treatment, pathogenesis, vaccine development and laboratory animal models of CCHF. The paper ends with a discussion of the possible future geographic range of the virus. For the benefit of researchers, we include a Supplementary Table listing all published reports of CCHF cases and outbreaks in the English-language literature, plus some principal articles in other languages, with total case numbers, case fatality rates and all CCHFV strains on GenBank.

Development and Evaluation of Serotype- and Group-Specific Fluorogenic Reverse Transcriptase PCR (TaqMan) Assays for Dengue Virus

ABSTRACT Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60°C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Protective efficacy of oral whole-cell/recombinant-B-subunit cholera vaccine in Peruvian military recruits.

The cholera epidemic in South America has reinforced the need for safe and effective oral vaccines. In a randomised, double-blind, placebo-controlled efficacy trial among 1563 Peruvian military recruits we have investigated the protective efficacy of an oral inactivated whole-cell/recombinant-B-subunit (WC/rBS) cholera vaccine. Participants were given two oral doses of cholera vaccine or Escherichia coli K12 placebo, with an interval of 7-14 days. 1426 (91%) subjects received the two prescribed doses and were followed up for a mean of 18 weeks (median 21 weeks). After vaccination, Vibrio cholerae O1 El Tor Ogawa was isolated from 17 subjects with diarrhoea. 16 of the cholera cases occurred 2 weeks or longer after the second dose of vaccine (14 placebo recipients, 2 vaccinees). We also detected 14 symptomless infections (11 [7 placebo recipients, 4 vaccinees]) 2 weeks or longer after the second dose. The vaccine had significant protective efficacy against cholera (86% [95% CI 37-97], p < 0.01) but not against symptomless infection (42% [-96 to 85]). All cholera cases were in people of blood group O, who made up 76% of the study population (p < 0.01). Two doses of WC/rBS vaccine, given 1 to 2 weeks apart, provide rapid, short-term protection against symptomatic cholera in adult South Americans, who are predominantly of blood group O. Long-term efficacy studies in Peruvian adults and children are under way.

Effect of dengue-1 antibodies on American dengue-2 viral infection and dengue haemorrhagic fever

In Iquitos, Peru, no cases of dengue haemorrhagic fever have been recorded in individuals infected with dengue-1 virus followed by American genotype dengue-2 (American dengue-2) virus. We assayed serum samples collected in Iquitos that tested positive for antibodies of monotype dengue-1 and monotype dengue-2 using a plaque reduction neutralisation test to determine their ability to neutralise the infectivity of two dengue-1 viruses, two American dengue-2 viruses, and two Asian dengue-2 viruses. Sera positive for the dengue-1 antibody neutralised dengue-1 viruses and American dengue-2 viruses much more effectively than Asian dengue-2 viruses. Neutralisation of American dengue-2 virus by sera positive for dengue-1 antibodies may account for the absence of dengue haemorrhagic fever in individuals infected with dengue-1 in 1990-91 followed by American dengue-2 virus in 1995 in Iquitos, Peru.

Temporal and Geographic Patterns of Aedes aegypti (Diptera: Culicidae) Production in Iquitos, Peru

Abstract Large-scale longitudinal cohort studies are necessary to characterize temporal and geographic variation in Aedes aegypti (L.) (Diptera: Culicidae) production patterns and to develop targeted dengue control strategies that will reduce disease. We carried out pupal/demographic surveys in a circuit of ≈6,000 houses, 10 separate times, between January 1999 and August 2002 in the Amazonian city of Iquitos, Peru. We quantified the number of containers positive for Ae. aegypti larvae and/or pupae, containers holding pupae, and the absolute number of pupae by 4-mo sampling circuits and spatially by geographic area by using a geographic information system developed for the city. A total of 289,941 water-holding containers were characterized, of which 7.3% were positive for Ae. aegypti. Temporal and geographic variations were detected for all variables examined, and the relative importance of different container types for production of Ae. aegypti was calculated. Ae. aegypti larvae and pupae were detected in 64 types of containers. Consistent production patterns were observed for the lid status (lids: 32% wet containers, 2% pupal production), container location (outdoor: 43% wet containers, 85% pupal production), and method by which the container was filled with water (rain filled: 15% wet containers, 88.3% pupal production); these patterns were consistent temporally and geographically. We describe a new container category (nontraditional) that includes transient puddles, which were rare but capable of producing large numbers of pupae. Because of high variable pupal counts, four container categories (large tank, medium storage, miscellaneous, and nontraditional) should be targeted in addition to outdoor rain-filled containers that are not covered by a lid. The utility of targeted Ae. aegypti control is discussed, as well as the ability to achieve control objectives based on published but untested threshold values.

Epidemiology of Dengue Virus in Iquitos, Peru 1999 to 2005: Interepidemic and Epidemic Patterns of Transmission

Background Comprehensive, longitudinal field studies that monitor both disease and vector populations for dengue viruses are urgently needed as a pre-requisite for developing locally adaptable prevention programs or to appropriately test and license new vaccines. Methodology and Principal Findings We report the results from such a study spanning 5 years in the Amazonian city of Iquitos, Peru where DENV infection was monitored serologically among ∼2,400 members of a neighborhood-based cohort and through school-based absenteeism surveillance for active febrile illness among a subset of this cohort. At baseline, 80% of the study population had DENV antibodies, seroprevalence increased with age, and significant geographic variation was observed, with neighborhood-specific age-adjusted rates ranging from 67.1 to 89.9%. During the first 15 months, when DENV-1 and DENV-2 were co-circulating, population-based incidence rates ranged from 2–3 infections/100 person-years (p-years). The introduction of DENV-3 during the last half of 2001 was characterized by 3 distinct periods: amplification over at least 5–6 months, replacement of previously circulating serotypes, and epidemic transmission when incidence peaked at 89 infections/100 p-years. Conclusions/Significance Neighborhood-specific baseline seroprevalence rates were not predictive of geographic incidence patterns prior to the DENV-3 introduction, but were closely mirrored during the invasion of this serotype. Transmission varied geographically, with peak incidence occurring at different times among the 8 geographic zones in ∼16 km2 of the city. The lag from novel serotype introduction to epidemic transmission and knowledge of spatially explicit areas of elevated risk should be considered for more effective application of limited resources for dengue prevention.

A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys.

A DNA vaccine that expresses the premembrane/membrane (prM) and envelope (E) genes of dengue virus serotype-1 was tested for immunogenicity and protection against dengue-1 virus challenge in Aotus nancymae monkeys. The vaccine, in 1 mg doses, was administered intradermally (i.d.) to three monkeys and intramuscularly (i.m.) to three others. For controls, a 1 mg dose of vector DNA was administered i.d. to two monkeys and i.m. to one. All animals were primed and then boosted at one and five months post priming. Sera were collected monthly and analyzed for dengue-1 antibodies by enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). Dengue-1 antibodies were detectable in the sera from i.d. and i.m. vaccine inoculated animals one month after the first boost and peaked one month after the second boost. The antibody levels from sera of animals that received the vaccine via the i.d. route were twice those from sera of animals that received the vaccine via the i.m. route. Six months after the second boost all inoculated and two naive monkeys were challenged with 1.25x10(4) plaque forming units (PFU) of dengue-1 virus. Two vaccine immunized animals were protected from viremia while the others showed a reduction in viremia. The mean days of viremia were 1 and 1.3 for the animals that were immunized with the vaccine via the i.d. or i.m. route, respectively vs 4 and 2 mean days of viremia in the animals inoculated with control DNA. Naive animals were viremic for an average of 4 days. All of the three control monkeys that received control DNA inoculum by either the i.d. or i.m. route had an intermittent viremia pattern with one or more negative days interspersed between the positive days. This pattern was not observed in any of the vaccine recipients or the naïve control monkeys. These results demonstrate that DNA immunization is a promising approach for the development of dengue vaccines and that A. nancymae monkeys are suitable for dengue vaccine trials.

Detection of Dengue Viral RNA Using a Nucleic Acid Sequence-Based Amplification Assay

ABSTRACT Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The “gold standard” used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

Negevirus: a Proposed New Taxon of Insect-Specific Viruses with Wide Geographic Distribution

ABSTRACT Six novel insect-specific viruses, isolated from mosquitoes and phlebotomine sand flies collected in Brazil, Peru, the United States, Ivory Coast, Israel, and Indonesia, are described. Their genomes consist of single-stranded, positive-sense RNAs with poly(A) tails. By electron microscopy, the virions appear as spherical particles with diameters of ∼45 to 55 nm. Based on their genome organization and phylogenetic relationship, the six viruses, designated Negev, Ngewotan, Piura, Loreto, Dezidougou, and Santana, appear to form a new taxon, tentatively designated Negevirus. Their closest but still distant relatives are citrus leposis virus C (CiLV-C) and viruses in the genus Cilevirus, which are mite-transmitted plant viruses. The negeviruses replicate rapidly and to high titer (up to 1010 PFU/ml) in mosquito cells, producing extensive cytopathic effect and plaques, but they do not appear to replicate in mammalian cells or mice. A discussion follows on their possible biological significance and effect on mosquito vector competence for arboviruses.