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Background Mycoplasma genitalium was recently added to the CDC’s antimicrobial resistance threats ‘watch list’, as it has rapidly become resistant to mainstay treatments. In Australia, treatment failure with fluoroquinolones remain commonplace, even when Sanger sequencing fails to identify evidence of resistance mutations. Methods Suspecting that Sanger sequencing may miss low-load mixed infections, we applied three additional PCR-based approaches (allele-specific primer-based PCR, probe-based PCR and amplicon deep sequencing) to detect mutations associated with fluoroquinolone susceptibility/resistance. We focused on resistance mutations at amino acid positions 83 and 87 of parC, as these were previously shown to be common in Australia. Results Our results showed evidence of mixtures of fluoroquinolone-susceptible and -resistant strains in up to 27/423 samples (6.4%). These included 1 sample that was indicated to be mixed by Sanger sequencing and all three additional PCR methods, 6 samples detected by two or more of the additional PCRs but not by Sanger sequencing and finally 20 samples that were detected by only one of the additional PCR methods. A key question was whether Sanger sequencing failed to detect fluoroquinolone resistance in any samples; overall, we observed that Sanger sequencing failed to detect fluoroquinolone resistance in up to 3.8% (16/423) of samples. Conclusions The presence of mixed susceptibility infections may have important implications for clinical patient management and stresses the need for appropriate detection of resistance and selection of antimicrobials to ensure appropriate treatment of M. genitalium infections.

An ongoing outbreak of syphilis in Australia, first reported in the state of Queensland in 2011, has led to increasing cases of congenital syphilis, including several deaths. Here, we applied multi-locus sequence typing (MLST) on available Treponema pallidum PCR-positive samples from the state of Queensland from the beginning of the outbreak to July 2020. In total, 393 samples from 337 males and 56 females were genotyped. Of 36 different Treponema pallidum sequence types (ST) observed, the two most common STs, ST 1 (also reported to be a dominant strain in various other countries) and ST 100 (the latter differing from ST 1 by only one single nucleotide polymorphism (SNP) based on the MLST scheme), together comprised 69% (271/393) of all samples, including the majority of samples in females (79%; 44/56). ST 1 was prevalent throughout the entire study period. Both strains remained the most common STs during the year 2020 where social distancing and other measures were implemented due to the COVID-19 pandemic. Both STs had high male-to-female ratios and included male rectal infections, therefore suggestive of occurrence primarily among men-who-have-sex-with-men (MSM). Hence, bridging from MSM to heterosexual networks may potentially contribute to infections among females, but further studies are needed to confirm this. Overall, there was considerable diversity of Treponema pallidum genotypes observed throughout the study period, but the fact that two key strains accounted for the majority of infections, including among females, stresses the need for further investigations into the transmission of these strains, and potentially a need for targeted public health interventions to better control the spread of syphilis in Queensland.

This research was funded by the Biotechnology and Biological Sciences Research Council (BB/M009343/1 to D.J.E), an ISSF award from The Wellcome Trust to the BSRC, University of St Andrews, and a Royal Society Newton Advanced Fellowship (NA160353 to Y.F.C). The APC was funded by The University of St Andrews. Positive-strand RNA virus evolution is partly attributed to the process of recombination. Although common between closely genetically related viruses, such as within species of the Enterovirus genus of the Picornaviridae family, inter-species recombination is rarely observed in nature. Recent studies have shown recombination is a ubiquitous process resulting in a wide range of recombinant genomes and progeny viruses. While not all recombinant genomes yield infectious virus progeny, their existence and continued evolution during replication has critical implications for evolution of the virus population. In this study we utilised an in vitro recombination assay to demonstrate inter-species recombination events between viruses from four enterovirus species’, A-D. We show that inter-species recombinant genomes are generated in vitro with polymerase template-switching events occurring within the virus polyprotein coding region. However, these genomes did not yield infectious progeny virus. Analysis and attempted recovery of a constructed recombinant cDNA revealed a restriction in positive- but not negative -strand RNA synthesis, indicating a significant block in replication. This study demonstrates the propensity for inter-species recombination at the genome level but suggests that significant sequence plasticity would be required in order to overcome blocks in the virus life cycle and allow for the production of infectious virus. Publisher PDF

Mycoplasma genitalium is an important sexually transmitted pathogen that is commonly associated with nongonococcal urethritis in men and pelvic inflammatory disease in women ([1][1]). Over the past decade, treatment of M. genitalium has become increasingly challenging due to the fact that this

Dengue virus (DENV) is the most important mosquito-borne viral pathogen of humans, comprising four serotypes (DENV-1 to -4) with a myriad of genotypes and strains. The kinetics of DENV replication within the mosquito following ingestion of a blood meal influence the pathogen&rsquo, s ability to reach the salivary glands and thus the transmission potential. The influence of DENV serotype and strain diversity on virus kinetics in the two main vector species, Aedes aegypti and Ae. albopictus, has been poorly characterized. We tested whether DENV replication kinetics vary systematically among serotypes and strains, using Australian strains of the two vectors. Mosquitoes were blood fed with two strains per serotype, and sampled at 3, 6, 10 and 14-days post-exposure. Virus infection in mosquito bodies, and dissemination of virus to legs and wings, was detected using qRT-PCR. For both vectors, we found significant differences among serotypes in proportions of mosquitoes infected, with higher numbers for DENV-1 and -2 versus other serotypes. Consistent with this, we observed that DENV-1 and -2 generally replicated to higher RNA levels than other serotypes, particularly at earlier time points. There were no significant differences in either speed of infection or dissemination between the mosquito species. Our results suggest that DENV diversity may have important epidemiological consequences by influencing virus kinetics in mosquito vectors.

Between 1996 and 1998, two clades (B and C; genotype I) of dengue virus type 1 (DENV-1) appeared in Myanmar (Burma) that were new to that location. Between 1998 and 2000, a third clade (A; genotype III) of DENV-1, which had been circulating at that locality for at least 25 years, became extinct. These changes preceded the largest outbreak of dengue recorded in Myanmar, in 2001, in which more than 95% of viruses recovered from patients were DENV-1, but where the incidence of severe disease was much less than in previous years. Phylogenetic analyses of viral genomes indicated that the two new clades of DENV-1 did not arise from the, now extinct, clade A viruses nor was the extinction of this clade due to differences in the fitness of the viral populations. Since the extinction occurred during an inter-epidemic period, we suggest that it was due to a stochastic event attributable to the low rate of virus transmission in this interval.

Outbreaks of dengue due to dengue virus type 1 (DENV-1) occurred almost simultaneously in 2001 in Myanmar and at multiple sites almost 10,000 km away in the Pacific. Phylogenetic analyses of the E protein genes of DENV-1 strains recovered from Asia and the Pacific revealed three major viral genotypes (I, II, and III) with distinct clades within each. The majority of strains from the Pacific and Myanmar, and a number of other Asian strains fell into genotype I. Genotype II comprised a smaller set of Asian and Pacific strains, while genotype III contained viruses from diverse geographical localities. These analyses suggested that the continuing outbreak of dengue in the Pacific has been due to multiple, direct, introductions of dengue viruses from a variety of locations in Asia followed by local transmission. There was no evidence that the introduction of these viruses into the Pacific was associated with any adaptive changes in the E protein of the viruses.

In 2001, Myanmar (Burma) had its largest outbreak of dengue-15,361 reported cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), including 192 deaths. That year, 95% of dengue viruses isolated from patients were serotype 1 viruses belonging to two lineages that had diverged from an earlier, now extinct, lineage sometime before 1998. The ratio of DHF to DSS cases in 2001 was not significantly different from that in 2000, when 1,816 cases of DHF/DSS were reported and dengue 1 also was the most frequently isolated serotype. However, the 2001 ratio was significantly higher than that in 1998 (also an outbreak year) and in 1999, when all four serotypes were detected and serotypes 1, 2, and 3 were recovered in similar numbers. The large number of clinical cases in 2001 may have been due, in part, to a preponderance of infections with dengue 1 viruses.

Articles also free to read on publisher website Abstract Dengue and dengue hemorrhagic fever re-emerged in Bangladesh in 2000 and 2001 and nearly all viruses isolated were dengue type 3. Phylogenetic analyses of the envelope genes of examples of these viruses indicated that they were most closely related to recently emerged dengue type 3 viruses from neighboring Thailand and Myanmar but distinct from those from India and Sri Lanka. Since this strain of dengue virus type 3 had not been associated with unusual patterns of disease in Thailand or Myanmar, it suggested that the outbreak in Bangladesh was due to local factors after the introduction of viruses from countries to the east rather than to the evolution of an unusually virulent strain of virus in Bangladesh.

Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes four viral interferon regulatory factors (vIRF-1–4). We investigated the mechanism and consequences of vIRF-2-mediated inhibition of interferon-response element signalling following type I interferon (IFN) induction. Western blot and electrophoretic mobility-shift assays identified the interferon-stimulated gene factor-3 (ISGF-3) components STAT1 and IRF-9 as the proximal targets of vIRF-2 activity. The biological significance of vIRF-2 inhibition of ISGF-3 was demonstrated by vIRF-2-mediated rescue of the replication of the IFN-sensitive virus encephalomyocarditis virus. This study provides both a mechanism and evidence for KSHV vIRF-2-mediated suppression of the consequences of type 1 IFN-induced signalling.

Ross River virus (RRV) is a mosquito-borne member of the genus Alphavirus that causes epidemic polyarthritis in humans, costing the Australian health system at least US$10 million annually. Recent progress in RRV vaccine development requires accurate assessment of RRV genetic diversity and evolution, particularly as they may affect the utility of future vaccination. In this study, we provide novel RRV genome sequences and investigate the evolutionary dynamics of RRV from time-structured E2 gene datasets. Our analysis indicates that, although RRV evolves at a similar rate to other alphaviruses (mean evolutionary rate of approx. 8x10(-4) nucleotide substitutions per site year(-1)), the relative genetic diversity of RRV has been continuously low through time, possibly as a result of purifying selection imposed by replication in a wide range of natural host and vector species. Together, these findings suggest that vaccination against RRV is unlikely to result in the rapid antigenic evolution that could compromise the future efficacy of current RRV vaccines.

Article also free to read on publisher website Australian mosquitoes from which Japanese encephalitis virus (JEV) has been recovered (Culex annulirostris, Culex gelidus, and Aedes vigilax) were assessed for their ability to be infected with the ChimeriVax-JE vaccine, with yellow fever vaccine virus 17D (YF 17D) from which the backbone of ChimeriVax-JE vaccine is derived and with JEV-Nakayama. None of the mosquitoes became infected after being fed orally with 6.1 log(10) plaque-forming units (PFU)/mL of ChimeriVax-JE vaccine, which is greater than the peak viremia in vaccinees (mean peak viremia = 4.8 PFU/mL, range = 0-30 PFU/mL of 0.9 days mean duration, range = 0-11 days). Some members of all three species of mosquito became infected when fed on JEV-Nakayama, but only Ae. vigilax was infected when fed on YF 17D. The results suggest that none of these three species of mosquito are likely to set up secondary cycles of transmission of ChimeriVax-JE in Australia after feeding on a viremic vaccinee.

Dengue and dengue hemorrhagic fever re-emerged in Bangladesh in 2000 and 2001 and nearly all viruses isolated were dengue type 3. Phylogenetic analyses of the envelope genes of examples of these viruses indicated that they were most closely related to recently emerged dengue type 3 viruses from neighboring Thailand and Myanmar but distinct from those from India and Sri Lanka. Since this strain of dengue virus type 3 had not been associated with unusual patterns of disease in Thailand or Myanmar, it suggested that the outbreak in Bangladesh was due to local factors after the introduction of viruses from countries to the east rather than to the evolution of an unusually virulent strain of virus in Bangladesh.

In 2001, dengue virus type 1 (DENV-1) populations in humans and mosquitoes from Myanmar acquired a stop-codon mutation in the surface envelope (E) protein gene. Within a year, this stop-codon strain had spread to all individuals sampled. The presence of truncated E protein species within individual viral populations, along with a general relaxation in selective constraint, indicated that the stop-codon strain represents a defective lineage of DENV-1. We propose that such long-term transmission of defective RNA viruses in nature was achieved through complementation by coinfection of host cells with functional viruses.