Interspecies exchange mutagenesis of the first epidermal growth factor-like domain of human factor VII.

The first epidermal growth factor-like (EGF1) domain of human factor VII (FVII) is essential for binding to tissue factor (TF). We hypothesized that the previously observed increased coagulant activity of rabbit plasma (i.e. FVII) with human TF might be explained by the five non-conserved amino acids in the rabbit vs. the human FVII EGF1 domain. Accordingly, we 'rabbitized' the human FVII EGF1 domain either by exchanging the entire EGF1 domain creating human FVII(rabEGF1) or by the single amino acid substitutions S53N, K62E, P74A, A75D and T83K. After transient expression in HEK293 cells, the recombinant FVII (rFVII) mutant proteins were analyzed for biological activity and binding affinity to human TF by competitive enzyme-linked immunosorbent assay (ELISA). Biological activity of the unpurified rFVII mutant proteins was either depressed or statistically unchanged vs. rFVII(WT). However, three of six rFVII mutant proteins had increased affinity for human TF in the rank order rFVII(rabEGF1) (3.3-fold) > rFVII(K62E) (2.9-fold) > rFVII(A75D) (1.7-fold). The mutant protein rFVII(K62E) was then permanently expressed and purified. Fully activated, purified rFVIIa(K62E) had a twofold greater clotting activity and 2.8-fold greater direct FVIIa amidolytic activity when compared with rFVIIa(WT). Quantitation of the affinity of TF binding by surface plasmon resonance indicated that the KD of purified rFVII(K62E) for human soluble TF (sTF) was 1.5 nM compared with 7.5 nM for rFVII(WT), i.e. fivefold greater affinity. We conclude that substitution of selected amino acid residues of the FVII EGF1 domain facilitated the creation of human rFVII chimeric proteins with both enhanced biological activity and increased affinity for TF.

Emerging viral diseases of Southeast Asia and the Western Pacific.

Over the past 6 years, a number of zoonotic and vectorborne viral diseases have emerged in Southeast Asia and the Western Pacific. Vectorborne disease agents discussed in this article include Japanese encephalitis, Barmah Forest, Ross River, and Chikungunya viruses. However, most emerging viruses have been zoonotic, with fruit bats, including flying fox species as the probable wildlife hosts, and these will be discussed as well. The first of these disease agents to emerge was Hendra virus, formerly called equine morbillivirus. This was followed by outbreaks caused by a rabies-related virus, Australian bat lyssavirus, and a virus associated with porcine stillbirths and malformations, Menangle virus. Nipah virus caused an outbreak of fatal pneumonia in pigs and encephalitis in humans in the Malay Peninsula. Most recently, Tioman virus has been isolated from flying foxes, but it has not yet been associated with animal or human disease. Of nonzoonotic viruses, the most important regionally have been enterovirus 71 and HIV.

Entomological investigations of an outbreak of Japanese encephalitis virus in the Torres Strait, Australia, in 1998.

Japanese encephalitis (JE) virus first appeared in Australia in 1995, when three clinical cases (two fatal) were diagnosed in residents on Badu Island in the Torres Strait, northern Queensland. More recently, two confirmed human JE cases were reported in the Torres Strait Islands and Cape York Peninsula, in northern Queensland in 1998. Shortly after JE virus activity was detected in humans and sentinel pigs on Badu Island in 1998, adult mosquitoes were collected using CO2 and octenol-baited CDC light traps; 43 isolates of JE virus were recovered. Although Culex sitiens group mosquitoes yielded the majority of JE isolates (42), one isolate was also obtained from Ochlerotatus vigilax (Skuse). Four isolates of Ross River virus and nine isolates of Sindbis (SIN) virus were also recovered from members of the Culex sitiens group collected on Badu Island in 1998. In addition, 3,240 mosquitoes were speciated and pooled after being anesthetized with triethylamine (TEA). There was no significant difference in the minimum infection rate of mosquitoes anesthetized with TEA compared with those sorted on refrigerated tables (2.8 and 1.6 per 1,000 mosquitoes, respectively). Nucleotide analysis of the premembrane region and an overlapping region of the fifth nonstructural protein and 3' untranslated regions of representative 1998 Badu Island isolates of JE virus reveled they were identical to each other. Between 99.1% and 100% identity was observed between 1995 and 1998 isolates of JE from Badu Island, as well as isolates of JE from mosquitoes collected in Papua New Guinea (PNG) in 1997 and 1998. This suggests that the New Guinea mainland is the likely source of incursions of JE virus in Australia.

Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia.

In response to an outbreak of Japanese encephalitis (JE) virus on Cape York Peninsula, Australia, in 1998, mosquitoes were collected using CO2 and octenol-baited Centers for Disease Control and Prevention light traps. A total of 35,235 adult mosquitoes, comprising 31 species, were processed for virus isolation. No isolates of JE virus were recovered from these mosquitoes. However, 18 isolates of Kokobera virus, another flavivirus were obtained from Culex annulirostris. Twelve isolates were from western Cape York (minimum infection rate (MIR) of 0.61: 1,000 mosquitoes) and 6 were from the Northern Peninsula Area (MIR of 1.0:1,000). Potential explanations for the failure to detect JE virus in mosquitoes collected from Cape York Peninsula include the timing of collections, the presence of alternative bloodmeal hosts, differences in pig husbandry, asynchronous porcine seroconversion, and the presence of other flaviviruses.

An epidemic of dengue 3 in far north Queensland, 1997-1999.

OBJECTIVES: To describe an epidemic of dengue type 3 that occurred in far north Queensland in 1997-1999 and its influence on the further development of dengue prevention and control strategies. DESIGN: Epidemiological and laboratory investigation of cases, entomological surveys and phylogenetic analysis of dengue virus isolates. MAIN OUTCOME MEASURES: Numbers and characteristics of confirmed cases; Breteau Index (BI; number of containers breeding Aedes aegypti per 100 premises); effect of control measures on mosquito populations; genetic homology of epidemic virus with other dengue virus isolates. RESULTS: The epidemic lasted 70 weeks and comprised 498 confirmed cases in three towns (Cairns, Port Douglas and Mossman); 101 patients (20%) were admitted to hospital. Median interval between symptom onset and notification was seven days (range, 0-53 days), and cumulative duration of viraemia of public health significance was 2,072 days. BIs in affected areas were high, particularly in Mossman (45) and Port Douglas (31). Control measures significantly reduced mosquito populations (assessed as number of ovitraps containing Ae. aegypti eggs and mean number of eggs per trap [P< 0.05 for both]). However, transmission persisted in several foci, in part due to undetected waterfilled containers breeding Ae. aegypti. The epidemic virus belonged to serotype 3; phylogenetic analysis suggested it was imported from Thailand. CONCLUSIONS: The epidemic had greater morbidity than other recent Queensland epidemics of dengue and was harder to control, necessitating substantial revision of the Dengue Fever Management Plan for North Queensland. The epidemic's severity supports the hypothesis that dengue viruses from South East Asia are more virulent than others.

The appearance of a second genotype of Japanese encephalitis virus in the Australasian region.

In mid-January 2000, the reappearance of Japanese encephalitis (JE) virus activity in the Australasian region was first demonstrated by the isolation of JE virus from 3 sentinel pigs on Badu Island in the Torres Strait. Further evidence of JE virus activity was revealed through the isolation of JE virus from Culex gelidus mosquitoes collected on Badu Island and the detection of specific JE virus neutralizing antibodies in 3 pigs from Saint Pauls community on Moa Island. Nucleotide sequencing and phylogenetic analyses of the premembrane and envelope genes were performed which showed that both the pig and mosquito JE virus isolates (TS00 and TS4152, respectively) clustered in genotype I, along with northern Thai, Cambodian, and Korean isolates. All previous Australasian JE virus isolates belong to genotype II, along with Malaysian and Indonesian isolates. Therefore, for the first time, the appearance and transmission of a second genotype of JE virus in the Australasian region has been demonstrated.

Japanese encephalitis in north Queensland, Australia, 1998.

OBJECTIVE: To describe the circumstances of two cases of Japanese encephalitis (JE) in north Queensland in 1998, including one acquired on the Australian mainland. DESIGN: Serological surveillance of sentinel pigs for JE virus activity; serological surveys of humans and pigs and viral cultures of mosquito collections. SETTING: Islands in the Torres Strait and communities in the Northern Peninsula Area (NPA) and near the mouth of the Mitchell River in Cape York, Queensland, in the 1998 wet season (December 1997-May 1998). RESULTS: Sentinel pigs in the Torres Strait began to seroconvert to JE virus in February 1998, just before onset of JE in an unvaccinated 12-year-old boy on Badu island. By mid-April, most sentinel pigs had seroconverted. Numerous JE viruses were isolated from Culex annulirostris mosquitoes collected on Badu. In early March, a person working at the mouth of the Mitchell River developed JE. Serological surveys showed recent JE virus infection in 13 young pigs on a nearby farm, but not in 488 nearby residents. In NPA communities, sentinel pigs seroconverted slowly and JE viruses were isolated from three, but none of 604 residents showed evidence of recent infection. Nucleotide sequencing showed that 1998 JE virus isolates from the Torres Strait were virtually identical not only to the 1998 isolate from an NPA pig, but also to previous (1995) Badu isolates. CONCLUSIONS: JE virus activity was more widespread in north Queensland in the 1998 wet season than in the three previous wet seasons, but ecological circumstances (e.g., less intensive pig husbandry, fewer mosquitoes) appear to have limited transmission on the mainland. Nucleotide sequencing indicated a common source for the 1995 and 1998 JE viruses. Circumstantial evidence suggests that cyclonic winds carried infected mosquitoes from Papua New Guinea.