Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Ross River virus disease in tropical Queensland: evolution of rheumatic manifestations in an inception cohort followed for six months

Objective: To describe the natural history of rheumatic manifestations of Ross River virus (RRV) disease. Design: Prospective longitudinal clinical review. Setting: North Queensland local government areas of Cairns, Douglas, Mareeba and Atherton during January to May 1998. Participants: General practice patients diagnosed with RRV disease on the basis of symptoms and a positive RRV IgM result. Main outcome measures: Rheumatic symptoms and signs assessed as soon as possible after disease onset and on two subsequent occasions (up to 6.5 months after onset). Results: 57 patients were recruited, 47 of whom were reviewed three times (at means of 1.1, 2.4 and 3.6 months after disease onset). Results are reported for these 47: 46 (98%) complained of joint pain at first review, with the ankles, wrists, fingers, knees and metacarpophalangeal joints (II-IV) most commonly involved. Prevalence of joint pain decreased progressively on second and third reviews, both overall (92% and 68% of patients, respectively), and in the five joints most commonly affected. The prevalence of other common rheumatic symptoms and signs, and use of non-steroidal anti-inflammatory drugs, also progressively declined over the three reviews. Conclusions: Earlier studies may have overestimated the prevalence and duration of symptoms in RRV disease. Progressive resolution over 3-6 months appears usual.

Development and evaluation of a species diagnostic polymerase chain reaction-restriction fragment-length polymorphism procedure for cryptic members of the Culex sitiens (Diptera : Culicidae) subgroup in Australia and the southwest Pacific

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

Enhancement or modulation of the vector competence of ochlerotatus vigilax (Diptera : Culicidae) for Ross River virus by temperature

Two different doses of Ross River virus (1111) were fed to Ochlerotatus vigilax (Skuse), the primary coastal vector in Australia; and blood engorged females were held at different temperatures up to 35 d. After ingesting 10(4.3) CCID50/Mosquito, mosquitoes reared at 18 and 25degreesC (and held at the same temperature) had higher body remnant and head and salivary gland titers than those held at 32degreesC, although infection rates were comparable. At 18, 25, and 32degreesC, respectively, virus was first detected in the salivary glands on days 3, 2, and 3. Based on a previously demonstrated 98.7% concordance between salivary gland infection and transmission, the extrinsic incubation periods were estimated as 5, 4, and 3 d, respectively, for these three temperatures. When Oc. vigilax reared at 18, 25, or 32degreesC were fed a lower dosage of 10(3.3) CCID50 RR/mosquito, and assayed after 7 d extrinsic incubation at these (or combinations of these) temperatures, infection rates and titers were similar. However, by 14 d, infection rates and titers of those reared and held at 18 and 32degreesC were significantly higher and lower, respectively. However, this process was reversible when the moderate 25degreesC was involved, and intermediate infection rates and titers resulted. These data indicate that for the strains of RR and Oc. vigilax used, rearing temperature is unimportant to vector competence in the field, and that ambient temperature variations will modulate or enhance detectable infection rates only after 7 d: extrinsic incubation. Because of the short duration of extrinsic incubation, however, this will do little to influence RR epidemiology, because by this time some Oc. vigilax could be seeking their third blood meal, the latter two being infectious.

Winter intervention against Aedes aegypti (Diptera : Culicidae) larvae in subterranean habitats slows surface recolonization in summer

At semiarid Charters Towers, north Queensland, Australia, the importance of Aedes aegypti (L.) in wells was assessed in relation to the colonization of surface habitats during the wet season. From April to July 1999, 10 wells (five positive for Ae. aegypti) were monitored to assess their status and larvae population numbers therein. All surface containers located within a 100 m radius of each well were removed, treated with s-methoprene or sealed to prevent the utilization of these containers by mosquitoes. These inner cores were surrounded by outer zones for a further 100 m in which surface containers were left untreated but all subterranean habitats were treated. Ovitraps were monitored monthly in the inner cores for 36 wk from August 1999 to April 2000 and differences in the proportions of ovitraps positive for Ae. aegypti and Ochlerotatus notoscriptus (Skuse) were analyzed by logistic regression. Analysis of the proportions of ovitraps positive for Ae. aegypti near positive wells indicated significantly greater colonization from November to March (the wet season), compared with those situated near Ae. aegypti negative wells. As Oc. notoscriptus were not produced from subterranean sites, comparisons of the proportions of ovitraps positive for Oc. notoscriptus in positive and negative inner cores provided an indication of the relative productivity of the uncontrolled surface containers in the outer zones. Differences in the utililization of ovitraps by Oc. notoscriptus among positive and negative cores were observed during only one month (March), when oviposition was greater in ovitraps in the negative cores, compared with the positive cores. Best subsets linear regression analysis of the proportion of ovitraps positive for Ae. aegypti against meteorological variables (rainfall, mean wind speed, mean relative humidity, mean minimum, and maximum temperature) during the week of ovitrapping indicated that minimum temperature and wind speed accounted for 63.4% of the variability. This study confirms that for semiarid towns such as Charters Towers, the practice of treating a relatively small number of key subterranean habitats during winter will significantly affect Ae. aegypti recolonization of surface container habitats during summer, the period of greatest risk for dengue.

Class benefits of AT1 antagonists in Type 2 diabetes with nephropathy

Diabetes mellitus has reached epidemic proportions in many countries and is the most common cause of end stage renal disease (ESRD). The angiotensin II receptor-1 (AT1) antagonists losartan and irbesartan have recently been evaluated as renoprotective agents in large clinical trials of patients with Type 2 diabetes and nephropathy. In the Reduction of End points in Non-insulin-dependent diabetes mellitus with the Angiotensin II Antagonist (RENAAL) study, losartan decreased the number of patients reaching the primary end point of a composite of measures of neuropathy. The relative risk reduction was ~ 15% with losartan and this was due to a reduction in both the doubling of creatinine concentration (25%) and of ESRD (28%) but not in death. In the Irbesartan Diabetic Nephropathy Trial (IDNT), the beneficial effect of irbesartan was mainly against the doubling of the baseline creatinine concentration (37% risk reduction) but there was also a 20% reduction in the onset of ESRD. Irbesartan had no effect on mortality. Beneficial effects occurred in addition to blood pressure being controlled by agents other than the AT1 antagonists. These clinical trials suggest that there may be a class renoprotective action with AT1 antagonists, although the mechanism is not clear. Patients with Type 2 diabetes and nephropathy should receive either an AT1 antagonist or the angiotensin converting enzyme inhibitor ramipril to ensure renoprotection.

Metformin & lifestyle intervention prevent Type 2 diabetes: lifestyle intervention has the greater effect

Diabetes mellitus is now occurring in epidemic proportions in many countries. Owing to the limited effectiveness of drug prophylaxis of diabetic complications after diabetes has developed, it may be more appropriate to investigate ways to prevent the onset of diabetes. A recent trial published by the Diabetes Prevention Programme Research Group investigated whether lifestyle changes or metformin were effective in delaying the onset of diabetes in subjects with impaired glucose tolerance. The goals of the intensive lifestyle intervention were to achieve and maintain a weight reduction of 7% through a low-calorie, low-fat diet and to engage in physical activity of moderate intensity, such as brisk walking, for at least 150 min/week. The effectiveness of the intensive lifestyle intervention on body weight was initially quite good but decreased over time. Metformin caused some weight loss but to a lesser extent than the intensive lifestyle intervention. Both therapies decreased the fasting plasma glucose levels to a similar extent initially. The intensive lifestyle intervention decreased plasma glycosylated haemoglobin levels to a greater extent than metformin. Both intensive lifestyle intervention and metformin reduced the incidence of diabetes, with the lifestyle intervention having the greater effect.

Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by similar to100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an similar to5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type lie residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus

A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) virus in vivo from a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN virus in weanling mice. KUN virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribed in vivo; however, no symptoms of virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 mug of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization data in vitro showing that serum from KUN DNA-immunized mice neutralized KUN and WN,viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN virus.

Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to West Nile virus in multiple avian species

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WW or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.11126 potentially discriminated between WW and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2132 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.676, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.

Epitope-blocking enzyme-linked immunosorbent assays for detection of West Nile virus antibodies in domestic mammals

We evaluated the ability of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) to detect West Nile virus (WNV) antibodies in domestic mammals. Sera were collected from experimentally infected horses, cats, and pigs at regular intervals and screened in ELISAs and plaque reduction neutralization tests. The diagnostic efficacies of these techniques were similar.

Telehealth responses to bio-terrorism and emerging infections

Emerging infectious diseases, such as severe acute respiratory syndrome (SARS), are of huge economic importance. They are difficult to predict. The World Health Organization has a Global Outbreak Alert and Response Network, which was involved at an early stage in the SARS outbreak in 2003. Three major lessons were learned as a result of the SARS epidemic in 2003, involving communication, evidence-based action and global partnerships. It is proposed that a series of broadband global response networks should be developed. At a technical level the networks are essentially in place, such as the Internet2 global network. Suitable peripheral devices also exist. What has not yet been created is the appropriate software to allow the use of these networks, although a number of commercial products are in the process of development.