Bruce Harrower

Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa

The neuraminidase inhibitors (NAIs) are an effective class of antiviral drugs for the treatment of influenza A and B infections. Until recently, only a low prevalence of NAI resistance (<1%) had been detected in circulating viruses. However, surveillance in Europe in late 2007 revealed significant numbers of A(H1N1) influenza strains with a H274Y neuraminidase mutation that were highly resistant to the NAI oseltamivir. We examined 264 A(H1N1) viruses collected in 2008 from South Africa, Oceania and SE Asia for their susceptibility to NAIs oseltamivir, zanamivir and peramivir in a fluorescence-based neuraminidase inhibition assay. Viruses with reduced oseltamivir susceptibility were further analysed by pyrosequencing assay. The frequency of the oseltamivir-resistant H274Y mutant increased significantly after May 2008, resulting in an overall proportion of 64% (168/264) resistance among A(H1N1) strains, although this subtype represented only 11.6% of all isolates received during 2008. H274Y mutant viruses demonstrated on average a 1466-fold reduction in oseltamivir susceptibility and 527-fold reduction in peramivir sensitivity compared to wild-type A(H1N1) viruses. The mutation had no impact on zanamivir susceptibility. Ongoing surveillance is essential to monitor how these strains may spread or persist in the future and to evaluate the effectiveness of treatments against them.

Detection of novel influenza A(H1N1) virus by real-time RT-PCR

Accurate and rapid diagnosis of novel influenza A(H1N1) infection is critical for minimising further spread through timely implementation of antiviral treatment and other public health based measures. In this study we developed two TaqMan-based reverse transcription PCR (RT-PCR) methods for the detection of novel influenza A(H1N1) virus targeting the haemagglutinin and neuraminidase genes. The assays were validated using 152 clinical respiratory samples, including 61 Influenza A positive samples, collected in Queenland, Australia during the years 2008 to 2009 and a further 12 seasonal H1N1 and H3N2 influenza A isolates collected from years 2000 to 2002. A wildtype swine H1N1 isolate was also tested. RNA from an influenza A(H1N1) virus isolate (Auckland, 2009) was used as a positive control. Overall, the results showed that the RT-PCR methods were suitable for sensitive and specific detection of novel influenza A(H1N1) RNA in human samples.

Leptospira kmetyi sp. nov., isolated from an environmental source in Malaysia

A single Leptospira strain (designated Bejo-Iso9(T)) was isolated from a soil sample taken in Johor, Malaysia. The isolate showed motility and morphology typical of the genus Leptospira under dark-field microscopy. Cells were found to be 10-13 microm in length and 0.2 microm in diameter, with a wavelength of 0.5 microm and an amplitude of approximately 0.2 microm. Phenotypically, strain Bejo-Iso9(T) grew in Ellinghausen-McCullough-Johnson-Harris medium at 13, 30 and 37 degrees C, and also in the presence of 8-azaguanine. Serologically, strain Bejo-Iso9(T) produced titres towards several members of the Tarassovi serogroup, but was found to be serologically unique by cross-agglutinin absorption test and thus represented a novel serovar. The proposed name for this serovar is Malaysia. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the genus Leptospira, with sequence similarities within the range 90.4-99.5% with respect to recognized Leptospira species. DNA-DNA hybridization against the three most closely related Leptospira species was used to confirm the results of the 16S rRNA gene sequence analysis. The G+C content of the genome of strain Bejo-Iso9(T) was 36.2 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Bejo-Iso9(T) represents a novel species of the genus Leptospira, for which the name Leptospira kmetyi sp. nov. is proposed. The type strain is Bejo-Iso9(T) (=WHO LT1101(T)=KIT Bejo-Iso9(T)).

Leptospira wolffii sp. nov., isolated from a human with suspected leptospirosis in Thailand

A single Leptospira strain (designated Khorat-H2(T)) was isolated from the urine of an adult male patient with suspected leptospirosis from the province of Nakornrachasima, Thailand. The isolate showed typical Leptospira motility and morphology under dark-field microscopy. Cells were 10-13 mum long and 0.2 mum in diameter, with a wavelength of 0.5 mum and an amplitude of approximately 0.3 mum. Phenotypically, strain Khorat-H2(T) did not grow at 13 degrees C but grew at 30 and 37 degrees C and in the presence of 8-azaguanine. Serological identification using the microscopic agglutination test revealed that strain Khorat-H2(T) had no cross-reaction with any recognized Leptospira serogroups. Phylogenetic analysis of the 16S rRNA gene sequence placed the novel strain within the radiation of the genus Leptospira, with sequence similarities of 88.1-97.7 % to recognized Leptospira species. DNA-DNA hybridization against the type strains of the three most closely related Leptospira species was used to confirm the results of the 16S rRNA sequence analysis. The G+C content of strain Khorat-H2(T) was 41.8 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Khorat-H2(T) represents a novel species of the genus Leptospira, for which the name Leptospira wolffii sp. nov. is proposed. The type strain is Khorat-H2(T) (=WHO LT1686(T) =KIT Khorat-H2(T)).

Detection of Australian bat lyssavirus using a fluorogenic probe

BACKGROUND Australian bat lyssavirus (ABLV) has been transmitted to humans following a scratch or bite from an infected bat in two cases. Following a scratch or bite to a person, the bat is usually submitted for testing and diagnosis is made using a direct fluorescent antibody test on a brain smear. A nested RT-PCR assay has also been utilised to confirm diagnosis. If positive for lyssavirus, post-exposure prophylaxis is administered. OBJECTIVES The TaqMan assay was developed to improve the diagnosis of ABLV infection, following problems encountered with the generation of spurious PCR products in the nested RT-PCR and also to reduce the high risk of contamination inherent with nested PCRs. STUDY DESIGN RNA was extracted from 161 bat brains and the samples were compared using a conventional RT-PCR and the TaqMan based assay. Samples from a patient with an ABLV infection collected antemortem and postmortem were also tested. RESULTS The sensitivity of the new TaqMan based PCR assay compared favourably with the nested PCR previously in use in our laboratory. This assay was able to detect RNA in samples collected antemortem and postmortem for the diagnosis of a human case of ABLV. CONCLUSIONS The major advantage of the TaqMan based assay was the speed of diagnosis with a result within minutes of completing the PCR (a result within 4 h of receiving the specimen). This test greatly reduces the chance of false positives through the elimination of second-round PCR and the requirement for agarose gels. The assay is sensitive and specific and should be invaluable for future antemortem and postmortem diagnosis of ABLV infection in humans.

Arboviruses isolated from mosquitoes collected from urban and peri-urban areas of eastern Australia.

Abstract To determine the presence of arboviruses in mosquito populations from major urban areas of eastern Australia, a total of 67,825 mosquitoes, representing ∼60 species, was collected and tested from Cairns, Brisbane, and Sydney between January 2005 and April 2008. Mosquito pools were screened by inoculation onto mosquito cell cultures and the detection of viral antigen using a panel of flavivirus and alphavirus monoclonal antibodies in an enzyme-linked immunosorbent assay. Suspect positive samples were confirmed using virus-specific real-time reverse transcriptase–polymerase chain reaction assays. No flaviviruses were detected, but 2 alphaviruses were isolated from mosquito pools collected from Cairns, with 1 Barmah Forest virus isolate from a pool of 100 Aedes vigilax and 1 Ross River virus isolate from a pool of 83 Verrallina carmenti. In addition, a single Aedes alternans collected from Sydney yielded an isolate most similar to Stretch Lagoon virus, a newly described virus from the genus Orbivirus. These results suggest that during the study, arboviruses were circulating at a low level in the areas sampled. The findings from this study will promote public health awareness of the risk of arboviruses in urban areas, leading to more informative public health campaigns to safeguard the Australian public.

Transmission of influenza A(H1N1) 2009 pandemic viruses in Australian swine

Please cite this paper as: Deng et al. (2012). Transmission of influenza A(H1N1) 2009 pandemic viruses in Australian swine. Influenza and Other Respiratory Viruses 6(3), e42–e47.

Public health surveillance for Australian bat lyssavirus in Queensland, Australia, 2000-2001.

From February 1, 2000, to December 4, 2001, a total of 119 bats (85 Megachiroptera and 34 Microchiroptera) were tested for Australian bat lyssavirus (ABLV) infection. Eight Megachiroptera were positive by immunofluorescence assay that used cross-reactive antibodies to rabies nucleocapsid protein. A case study of cross-species transmission of ABLV supports the conclusion that a bat reservoir exists for ABLV in which the virus circulates across Megachiroptera species within mixed communities.

A simple method for preparing synthetic controls for conventional and real-time PCR for the identification of endemic and exotic disease agents.

Abstract Medical and veterinary diagnostic and public health laboratories world-wide are increasingly being called upon to introduce molecular diagnostic tests for both endemic and exotic diseases. This demand has accelerated following increasing terrorism fears. Ironically these same concerns have lead to tightening of both import and export controls preventing many laboratories, particularly those outside of the United States, from gaining access to positive control material. This in turn has prevented many laboratories from introducing much needed molecular diagnostic tests. We describe here a generic approach for preparing synthetic DNA or RNA control material for use in either TaqMan or conventional PCR assays. The production of synthetic controls using this approach does not require cloning or special equipment or facilities beyond that found in any laboratory performing molecular diagnostics. The approach significantly reduces the possibility of contamination or erroneously reporting false-positive reactions due to contamination from positive control material. Synthetic controls produced using this approach have been employed in all molecular diagnostic tests performed in our laboratory and are used irrespective of whether we possess the organism or not.

Screening for H7N9 influenza A by matrix gene-based real-time reverse-transcription PCR.

Rapid detection of novel influenza A strains, including H7N9, is pivotal to ensuring appropriate public health-based responses and real-time reverse-transcription polymerase chain reaction (RT-PCR) methods are used typically for this purpose. However, the utility of such methods can be undermined by ongoing sequence variations, particularly when targeting the variable influenza A haemagglutinin (HA) and neuraminidase (NA) genes. This may often be a source of frustration for clinical laboratories that are implementing methods in preparation for potential pandemics as primers and probe targets may need to be checked regularly and updated. In this study, screening methods were developed for H7N9 influenza A strains based on the highly-conserved influenza A matrix gene. Three assays were developed and evaluated in parallel, and included two methods which simply involved inclusion of a single H7N9 probe sequence into an established influenza A and B multiplex RT-PCR (FluAB-PCR). The detection limits of the methods were compared using ten-fold dilutions of H7N9 RNA, and the specificity of the methods were tested using 32 influenza A RT-PCR-positive samples and a panel of 18 influenza A isolates, including representives of seasonal H3N2, seasonal H1N1, pandemic H1N1, H5N1, H5N3, H9N2 and H7N7. The detection limits of the three methods were the same, and no cross-reactions were observed with sH3N2, sH1N1, pH1N1 or H5N1. However, cross-reactions were observed with H5N3, H9N2 and H7N7. Overall, the results show that the methods are useful for front-line screening for H7N9.