Epidemiologic, Entomologic, and Virologic Factors of the 2014-15 Ross River Virus Outbreak, Queensland, Australia.

Australia experienced its largest recorded outbreak of Ross River virus (RRV) during the 2014-15 reporting year, comprising >10,000 reported cases. We investigated epidemiologic, entomologic, and virologic factors that potentially contributed to the scale of the outbreak in Queensland, the state with the highest number of notifications (6,371). Spatial analysis of human cases showed that notifications were geographically widespread. In Brisbane, human case notifications and virus detections in mosquitoes occurred across inland and coastal locations. Viral sequence data demonstrated 2 RRV lineages (northeastern genotypes I and II) were circulating, and a new strain containing 3 unique amino acid changes in the envelope 2 protein was identified. Longitudinal mosquito collections demonstrated unusually high relative abundance of Culex annulirostris and Aedes procax mosquitoes, attributable to extensive freshwater larval habitats caused by early and persistent rainfall during the reporting year. Increased prevalence of these mosquitoes probably contributed to the scale of this outbreak.

The effects of culture independent diagnostic testing on the diagnosis and reporting of enteric bacterial pathogens in Queensland, 2010 to 2014.

Changes in diagnostic laboratory testing procedures can impact on the number of cases notified and the public health surveillance of enteric pathogens. Culture independent diagnostic testing using a multiplex polymerase chain reaction (PCR) test was introduced for the rapid detection of bacterial enteric pathogens in pathology laboratories in Queensland, Australia, from late 2013 onwards. We conducted a retrospective descriptive study using laboratory data to assess the impact of the introduction of PCR testing on four common enteric pathogens, Salmonella, Campylobacter, Shigella and Yersinia, in Queensland between 2010 and 2014. The number of stool specimens tested and the proportion positive for each of the four pathogens increased in 2014 after the introduction of culture independent diagnostic testing. Among the specimens tested by both PCR and culture, 12% of Salmonella positive stools, 36% of Campylobacter positive stools, 74% of Shigella / enteroinvasive Escherichia coli positive stools and 65% of Yersinia positive stools were PCR positive only. Including those where culture was not performed, 19% of Salmonella positive stools, 44% of Campylobacter positive stools, 83% of Shigella positive stools and 79% of Yersinia positive stools had no cultured isolate available for further characterisation. The detection and tracking of foodborne and non-foodborne gastrointestinal outbreaks will become more difficult as culture independent diagnostic testing becomes more widespread. Until new techniques for characterisation of pathogens directly from clinical specimens have been developed, we recommend laboratories continue to culture specimens concurrently or reflexively with culture independent diagnostic tests.

Epidemiology of bacterial toxin-mediated foodborne gastroenteritis outbreaks in Australia, 2001 to 2013.

Bacterial toxin-mediated foodborne outbreaks, such as those caused by Clostridium perfringens, Staphylococcus aureus and Bacillus cereus, are an important and preventable cause of morbidity and mortality. Due to the short incubation period and duration of illness, these outbreaks are often under-reported. This is the first study to describe the epidemiology of bacterial toxin-mediated outbreaks in Australia. Using data collected between 2001 and 2013, we identify high risk groups and risk factors to inform prevention measures. Descriptive analyses of confirmed bacterial toxin-mediated outbreaks between 2001 and 2013 were undertaken using data extracted from the OzFoodNet Outbreak Register, a database of all outbreaks of gastrointestinal disease investigated by public health authorities in Australia. A total of 107 laboratory confirmed bacterial toxin-mediated outbreaks were reported between 2001 and 2013, affecting 2,219 people, including 47 hospitalisations and 13 deaths. Twelve deaths occurred in residents of aged care facilities. Clostridium perfringens was the most commonly reported aetiological agent (81 outbreaks, 76%). The most commonly reported food preparation settings were commercial food preparation services (51 outbreaks, 48%) and aged care facilities (42 outbreaks, 39%). Bacterial toxin outbreaks were rarely associated with food preparation in the home (2 outbreaks, 2%). In all outbreaks, the primary factor contributing to the outbreak was inadequate temperature control of the food. Public health efforts aimed at improving storage and handling practices for pre-cooked and re-heated foods, especially in commercial food preparation services and aged care facilities, could help to reduce the magnitude of bacterial toxin outbreaks.

Molecular evolution of lineage 2 West Nile virus.

Since the 1990s West Nile virus (WNV) has become an increasingly important public health problem and the cause of outbreaks of neurological disease. Genetic analyses have identified multiple lineages with many studies focusing on lineage 1 due to its emergence in New York in 1999 and its neuroinvasive phenotype. Until recently, viruses in lineage 2 were not thought to be of public health importance due to few outbreaks of disease being associated with viruses in this lineage. However, recent epidemics of lineage 2 in Europe (Greece and Italy) and Russia have shown the increasing importance of this lineage. There are very few genetic studies examining isolates belonging to lineage 2. We have sequenced the full-length genomes of four older lineage 2 WNV isolates, compared them to 12 previously published genomic sequences and examined the evolution of this lineage. Our studies show that this lineage has evolved over the past 300-400 years and appears to correlate with a change from mouse attenuated to virulent phenotype based on previous studies by our group. This evolution mirrors that which is seen in lineage 1 isolates, which have also evolved to a virulent phenotype over the same period of time.

Genetic divergence among members of the Kokobera group of flaviviruses supports their separation into distinct species.

The Kokobera virus group comprises mosquito-borne flaviviruses that cluster together phylogenetically. These viruses are unique to Australia and Papua New Guinea, and have been associated with a mild polyarticular disease in humans. Recent isolation of genetically diverse viruses within this group has prompted analysis of their genetic and phenotypic relationships. Phylogenetic analysis based on complete ORF, the envelope gene or the NS5/3' untranslated region supported the separation of the group into distinct species: Kokobera virus (KOKV), Stratford virus, New Mapoon virus, MK7979 and TS5273. Virulence studies in 3-week-old mice also provided the first evidence that a member of the KOKV group (MK7979) was neuroinvasive after intraperitoneal inoculation. In this context, our recent detection of KOKV group-specific antibodies in horses in the field suggests that these viruses should be considered in the epidemiology of flavivirus encephalitis in Australia.

Characterization of virulent West Nile virus Kunjin strain, Australia, 2011.

To determine the cause of an unprecedented outbreak of encephalitis among horses in New South Wales, Australia, in 2011, we performed genomic sequencing of viruses isolated from affected horses and mosquitoes. Results showed that most of the cases were caused by a variant West Nile virus (WNV) strain, WNV(NSW2011), that is most closely related to WNV Kunjin (WNV(KUN)), the indigenous WNV strain in Australia. Studies in mouse models for WNV pathogenesis showed that WNV(NSW2011) is substantially more neuroinvasive than the prototype WNV(KUN) strain. In WNV(NSW2011), this apparent increase in virulence over that of the prototype strain correlated with at least 2 known markers of WNV virulence that are not found in WNV(KUN). Additional studies are needed to determine the relationship of the WNV(NSW2011) strain to currently and previously circulating WNV(KUN) strains and to confirm the cause of the increased virulence of this emerging WNV strain.

Phylogeography of West Nile virus: from the cradle of evolution in Africa to Eurasia, Australia, and the Americas.

West Nile virus (WNV) is the most widely distributed of the encephalitic flaviviruses and is a major cause of encephalitis, with isolates obtained from all continents, apart from Antarctica. Subsequent to its divergence from the other members of the Japanese encephalitis virus complex, presumably in Africa, WNV has diverged into individual lineages that mostly correspond with geographic distribution. Here we elucidate the phylogeography and evolutionary history of isolates from lineage 1 of WNV. Interestingly, there are many examples of the same amino acid having evolved independently on multiple occasions. In Africa, WNV exists in an endemic cycle, whereas it is epidemic in Europe, being reintroduced regularly from Africa either directly (in western Europe) or via the Middle East (in eastern Europe). Significantly, introduction into other geographic areas has occurred on one occasion only in each region, leading to subsequent establishment and expansion of the virus in these areas. Only one endemic genotype each is present in India and Australia, suggesting that WNV was successfully introduced into these locations once only. Each introduction occurred many centuries ago, probably due to trade and exploration during the 19th century. Likewise, in the Americas, WNV was successfully introduced in 1999 and subsequently became endemic across most temperate regions of North America (NA). In contrast to previous suggestions, an isolate from the epidemic in Israel in 1998 was not the direct progenitor of the NA epidemic; rather, both epidemics originated from the same (unknown) location.

Evolution of new genotype of West Nile virus in North America.

Previous studies of North American isolates of West Nile virus (WNV) during 1999-2005 suggested that the virus had reached genetic homeostasis in North America. However, genomic sequencing of WNV isolates from Harris County, Texas, during 2002-2009 suggests that this is not the case. Three new genetic groups have been identified in Texas since 2005. Spread of the southwestern US genotype (SW/WN03) from the Arizona/Colorado/northern Mexico region to California, Illinois, New Mexico, New York, North Dakota, and the Texas Gulf Coast demonstrates continued evolution of WNV. Thus, WNV continues to evolve in North America, as demonstrated by selection of this new genotype. Continued surveillance of the virus is essential as it continues to evolve in the New World.

Determinants of attenuation in the envelope protein of the flavivirus Alfuy.

Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus endemic to Australia and Papua New Guinea. Most strains of MVEV cause potentially fatal cases of encephalitis in humans and horses, and have been shown to be highly neuroinvasive in weanling mice. In contrast, the naturally occurring subtype Alfuy virus (ALFV) has never been associated with human disease, nor is it neuroinvasive in weanling mice, even at high doses. To identify viral factors associated with ALFV attenuation, a chimeric infectious clone was constructed containing the structural genes premembrane (prM) and envelope (E) of ALFV swapped into the MVEV genome. The resulting virus (vMVEV/ALFVstr) was no longer neuroinvasive in mice, suggesting that motifs within prM-E of ALFV confer attenuation. To define these motifs further, mutants were constructed by targeting divergent sequences between the MVEV and ALFV E proteins that are known markers of virulence in other encephalitic flaviviruses. MVEV mutants containing a unique ALFV sequence in the flexible hinge region (residues 273-277) or lacking the conserved glycosylation site at position 154 were significantly less neuroinvasive in mice than wild-type MVEV, as determined by delayed time to death or increased LD(50). Conversely, when the corresponding MVEV sequences were inserted into the vMVEV/ALFVstr chimera, the mutant containing the MVEV hinge sequence was more neuroinvasive than the parental chimera, though not to the same level as wild-type MVEV. These results identify the hinge region and E protein glycosylation as motifs that contribute to the attenuation of ALFV.

Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines.

The plasma membrane Ca(2+) ATPase (PMCA) is an important regulator of free intracellular calcium, with dynamic regulation in the rat mammary gland during lactation. Recent studies suggest that Ca(2+) plays a role in cellular proliferation. To determine if PMCA expression is altered in tumorigenesis, we compared relative levels of PMCA1 mRNA. We found that the relative expression of PMCA1 mRNA is increased, by approximately 270% and 170%, in MCF-7 and MDA-MB-231 human breast cancer cell lines deprived of serum for 72 h, respectively, compared to the similarly treated MCF-10A human mammary gland epithelial cell line. Characterization of PMCA mRNA isoforms revealed that PMCA1b and PMCA4 mRNA are expressed in MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and BT-483 breast cancer cell lines. We also detected PMCA2 mRNA expression in all the breast cancer cell lines examined. However, PMCA3 mRNA was only detected in BT-483 cells. Our results suggest that PMCA expression may be altered in breast cancer cell lines, suggesting altered Ca(2+) regulation in these cell lines. Our results also indicate that breast cancer cell lines can express mRNAs for a variety PMCA isoforms.

Peroxisome proliferator-activated receptor beta expression in human breast epithelial cell lines of tumorigenic and non-tumorigenic origin.

Peroxisome proliferator-activated receptor beta (PPARbeta) is a member of the nuclear hormone receptor superfamily and is a ligand activated transcription factor, although the precise genes that it regulates and its physiological and pathophysiological role remain unclear. In view of the association of PPARbeta with colon cancer and increased mRNA levels of PPARbeta in colon tumours we sought in this study to examine the expression of PPARbeta in human breast epithelial cells of tumorigenic (MCF-7 and MDA-MB-231) and non-tumorigenic origin (MCF-10A). Using quantitative RT-PCR we measured PPARbeta mRNA levels in MCF-7, MDA-MB-231 and MCF-10A cells at various stages in culture. After serum-deprivation, MDA-MB-231 and MCF-10A cells had a 4.2- and 3.8-fold statistically greater expression of PPARbeta compared with MCF-7 cells. The tumorigenic cell lines also exhibited a significantly greater level of PPARbeta mRNA after serum deprivation compared with subconfluence whereas such an effect was not observed in non-tumorigenic MCF-10A cells. The expression of PPARbeta was inducible upon exposure to the PPARbeta ligand bezafibrate. Our results suggest that unlike colon cancer, PPARbeta overexpression is not an inherent property of breast cancer cell lines. However, the dynamic changes in PPARbeta mRNA expression and the ability of PPARbeta in the MCF-7 cells to respond to ligand indicates that PPARbeta may play a role in mammary gland carcinogenesis through activation of downstream genes via endogenous fatty acid ligands or exogenous agonists.

A new insect-specific flavivirus from northern Australia suppresses replication of West Nile virus and Murray Valley encephalitis virus in co-infected mosquito cells.

Recent reports of a novel group of flaviviruses that replicate only in mosquitoes and appear to spread through insect populations via vertical transmission have emerged from around the globe. To date, there is no information on the presence or prevalence of these insect-specific flaviviruses (ISFs) in Australian mosquito species. To assess whether such viruses occur locally, we used reverse transcription-polymerase chain reaction (RT-PCR) and flavivirus universal primers that are specific to the NS5 gene to detect these viruses in mosquito pools collected from the Northern Territory. Of 94 pools of mosquitoes, 13 were RT-PCR positive, and of these, 6 flavivirus isolates were obtained by inoculation of mosquito cell culture. Sequence analysis of the NS5 gene revealed that these isolates are genetically and phylogenetically similar to ISFs reported from other parts of the world. The entire coding region of one isolate (designated 56) was sequenced and shown to have approximately 63.7% nucleotide identity and 66.6% amino acid identity with its closest known relative (Nakiwogo virus) indicating that the prototype Australian ISF represents a new species. All isolates were obtained from Coquillettidia xanthogaster mosquitoes. The new virus is tentatively named Palm Creek virus (PCV) after its place of isolation. We also demonstrated that prior infection of cultured mosquito cells with PCV suppressed subsequent replication of the medically significant West Nile and Murray Valley encephalitis viruses by 10-43 fold (1 to 1.63 log) at 48 hr post-infection, suggesting that superinfection exclusion can occur between ISFs and vertebrate-infecting flaviviruses despite their high level of genetic diversity. We also generated several monoclonal antibodies (mAbs) that are specific to the NS1 protein of PCV, and these represent the first ISF-specific mAbs reported to date.

Multiple pathways to the attenuation of West Nile virus in south-east Texas in 2003.

West Nile virus (WNV) was first detected in Texas in 2002. During 2003, several isolates exhibiting significant attenuation of mouse neuroinvasiveness, and in some cases a small plaque and temperature sensitive phenotype when compared to other North American WNV isolates, were obtained from birds and mosquitoes in South-East Texas. To determine the attenuation markers of WNV, we have sequenced the genomes of three attenuated isolates and four temporally related virulent isolates and compared the amino acid substitutions in a total of 101 isolates, including three previously published genomes of attenuated strains, to identify mutations that are potentially involved in attenuation. Surprisingly, the attenuated strains fall into three separate "groups", suggesting that the attenuated phenotype evolved on three separate occasions in 2003. None of the groups share the same nucleotide changes or amino acid substitutions, and there are few mutations that can be clearly defined alone as being associated with attenuation.

Structure of yellow fever virus envelope protein domain III.

The structure of recombinant domain III of the envelope protein (rED3) of yellow fever virus (YFV), containing the major neutralization site, was determined using NMR spectroscopy. The amino acid sequence and structure of the YFV-rED3 shows differences from ED3s of other mosquito-borne flaviviruses; in particular, the partially surface-exposed BC loop where methionine-304 and valine-324 were identified as being critical for the structure of the loop. Variations in the structure and surface chemistry of ED3 between flaviviruses affect neutralization sites and may affect host cell receptor interactions and play a role in the observed variations in viral pathogenesis and tissue tropism.

Genetic variation of St. Louis encephalitis virus.

St. Louis encephalitis virus (SLEV) has been regularly isolated throughout the Americas since 1933. Previous phylogenetic studies involving 62 isolates have defined seven major lineages (I-VII), further divided into 14 clades. In this study, 28 strains isolated in Texas in 1991 and 2001-2003, and three older, previously unsequenced strains from Jamaica and California were sequenced over the envelope protein gene. The inclusion of these new sequences, and others published since 2001, has allowed better delineation of the previously published SLEV lineages, in particular the clades of lineage II. Phylogenetic analysis of 106 isolates identified 13 clades. All 1991 and 2001-2003 isolates from Nueces, Jefferson and Harris Counties (Texas Gulf Coast) group in clade IIB with other isolates from these counties isolated during the 1980s and 1990s. This lack of evidence for introduction of novel strains into the Texas Gulf Coast over a long period of time is consistent with overwintering of SLEV in this region. Two El Paso isolates, both from 2002, group in clade VA with recent Californian isolates from 1998-2001 and some South American strains with a broad temporal range. Overall, these data are consistent with multiple introductions of SLEV from South America into North America, and provide support for the hypothesis that in most situations, SLEV circulates within a locality, with occasional incursions from other areas. Finally, SLEV has much lower nucleotide (10.1 %) and amino acid variation (2.8 %) than other members of the Japanese encephalitis virus complex (maximum variation 24.6 % nucleotide and 11.8 % amino acid).

NMR assignments of the sylvatic dengue 1 virus envelope protein domain III.

Nearly complete backbone and side chain resonance assignments have been obtained for the third domain, residues M289-K400, of the envelope protein from the sylvatic strain (P72-1244) of the dengue 1 virus, containing mutations N336S and E370K, using double- and triple-resonance spectroscopy.

NMR assignments of the yellow fever virus envelope protein domain III.

Nearly complete backbone and sidechain resonance assignments have been obtained for the third domain, residues S288-K398, of the envelope protein from the Asibi strain of yellow fever virus using double- and triple-resonance spectroscopy.

Genetic and phenotypic differences between isolates of Murray Valley encephalitis virus in Western Australia, 1972-2003.

Murray Valley encephalitis virus (MVEV) is a medically important mosquito-borne flavivirus found in Australia and Papua New Guinea (PNG). Partial envelope gene nucleotide sequences of 28 isolates of MVEV from Western Australia (WA) between 1972 and 2003 were aligned and compared phylogenetically with the prototype MVE-1-51 from Victoria in 1951 and isolates from northern Queensland and PNG. Monoclonal antibody-binding patterns were also investigated. Results showed that the majority of isolates of MVEV from widely disparate locations in WA were genetically and phenotypically homogeneous. Furthermore, isolates of MVEV from WA and northern Queensland were almost identical, confirming results from earlier studies. Recent isolates of MVEV from Western Province in PNG were more similar to Australian isolates of MVEV than to isolates from PNG in 1956 and 1966, providing further evidence for the movement of flaviviruses between PNG and Australia. Additional representatives of a unique variant of MVEV (OR156) from Kununurra in the northeast Kimberley region of WA were also detected. This suggests that the OR156 lineage is still intermittently active but may be restricted to a small geographic area in northern WA, possibly due to altered biological characteristics.

Genetic stasis of dominant West Nile virus genotype, Houston, Texas.

The accumulation and fixation of mutations in West Nile virus (WNV) led to the emergence of a dominant genotype throughout North America. Subsequent analysis of 44 isolates, including 19 new sequences, from Houston, Texas, suggests that WNV has reached relative genetic stasis at the local level in recent years.

Biological, antigenic and phylogenetic characterization of the flavivirus Alfuy.

Alfuy virus (ALFV) is classified as a subtype of the flavivirus Murray Valley encephalitis virus (MVEV); however, despite preliminary reports of antigenic and ecological similarities with MVEV, ALFV has not been associated with human disease. Here, it was shown that ALFV is at least 10(4)-fold less neuroinvasive than MVEV after peripheral inoculation of 3-week-old Swiss outbred mice, but ALFV demonstrates similar neurovirulence. In addition, it was shown that ALFV is partially attenuated in mice that are deficient in alpha/beta interferon responses, in contrast to MVEV which is uniformly lethal in these mice. To assess the antigenic relationship between these viruses, a panel of monoclonal antibodies was tested for the ability to bind to ALFV and MVEV in ELISA. Although the majority of monoclonal antibodies recognized both viruses, confirming their antigenic similarity, several discriminating antibodies were identified. Finally, the entire genome of the prototype strain of ALFV (MRM3929) was sequenced and phylogenetically analysed. Nucleotide (73 %) and amino acid sequence (83 %) identity between ALFV and MVEV confirmed previous reports of their close relationship. Several nucleotide and amino acid deletions and/or substitutions with putative functional significance were identified in ALFV, including the abolition of a conserved glycosylation site in the envelope protein and the deletion of the terminal dinucleotide 5'-CU(OH)-3' found in all other members of the genus. These findings confirm previous reports that ALFV is closely related to MVEV, but also highlights significant antigenic, genetic and phenotypic divergence from MVEV. Accordingly, the data suggest that ALFV is a distinct species within the serogroup Japanese encephalitis virus.