Evaluation of Illumina® COVIDSeq™ as a tool for Omicron SARS-CoV-2 characterisation.

The continued emergence and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires ongoing genetic surveillance to support public health responses. The expansion of reliable next generation sequence (NGS) platforms has enabled the rapid characterisation of the constant emergence of new SARS-CoV-2 variants using nasopharyngeal swab specimens. Several studies have assessed the ability of COVIDSeq to type earlier SARS-CoV-2 strains (pre-Delta) rapidly and successfully, however, there is limited data showing suitability against Omicron variants. In the present study, we evaluated the performance of the Illumina COVIDSeq Assay as a streamlined amplicon-based NGS platform for detection and typing of Omicron variants. Our results demonstrate the high performance of SARS-CoV-2 sequencing using the COVIDSeq approach, with good repeatability, reproducibility and sensitivity for samples approaching CT 31. The COVIDSeq approach was 100% concordant with samples previously characterized by sequencing methods. The quick library preparation process and high throughput kit made it ideal for reflex testing, with a total time required for sequencing and analysis of approximately two days. This study demonstrates the effectiveness and versatility of the amplicon-based NGS characterisation method for SARS-CoV-2, providing a foundation for further research and development of custom-designed amplicon panels targeting different microorganisms.

Application of the ViroKey® SQ FLEX assay for detection of cytomegalovirus antiviral resistance.

BACKGROUND: Cytomegalovirus (CMV) is a viral infection which establishes lifelong latency, often reactivating and causing disease in immunosuppressed individuals, including haematopoietic stem cell transplant (HSCT) recipients. Treatment can be problematic due to antiviral resistance which substantially increases the risk of patient mortality. Diagnostic testing capabilities for CMV antiviral resistance in Australia and elsewhere have traditionally relied on gene-specific Sanger sequencing approaches, however, are now being superseded by next generation sequencing protocols. OBJECTIVE: Provide a snapshot of local mutations and explore the feasibility of the ViroKeyࣨ® SQ FLEX Genotyping Assay (Vela Diagnostics Pty Ltd) by examining sequencing success. METHOD: Performed sequencing on adult (n = 38) and paediatric (n = 81) plasma samples, over a large range of viral loads (above and below the assay recommended threshold of ≥1,000 International Units (IU)/mL; noting most of our paediatric samples have loads <1,000 IU/mL). RESULTS: Eleven test runs (including three repeat runs; 14 to 15 samples per run) were conducted, and four runs were deemed valid. The overall individual sample success rate for the four evaluable test runs was 71.2% (42/59 samples); 80.4% (37/46) samples ≥1,000 IU/mL were valid. Ten clinically important antiviral resistance mutations were detected, the most common being A594V in the UL97 gene, found in 6 (5%) samples. CONCLUSIONS: A range of technical issues were experienced, however with improvement this platform could be a useful addition to routine pathology workflows, providing timely antiviral resistance results for patients undergoing HSCT.

Cytomegalovirus in children undergoing haematopoietic stem cell transplantation: a diagnostic and therapeutic approach to antiviral resistance.

Cytomegalovirus (CMV) is a ubiquitous virus which causes a mild illness in healthy individuals. In immunocompromised individuals, such as children receiving haematopoietic stem cell transplantation, CMV can reactivate, causing serious disease and increasing the risk of death. CMV can be effectively treated with antiviral drugs, but antiviral resistance is an increasingly common complication. Available therapies are associated with adverse effects such as bone marrow suppression and renal impairment, making the choice of appropriate treatment challenging. New agents are emerging and require evaluation in children to establish their role. This review will discuss established and emerging diagnostic tools and treatment options for CMV, including antiviral resistant CMV, in children undergoing haematopoietic stem cell transplant.

A universal reagent for detection of emerging diseases using bioengineered multifunctional yeast nanofragments.

Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction.

A Multiplexed SERS Microassay for Accurate Detection of SARS-CoV-2 and Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 variants play an important role in predicting patient outcome during postinfection, and with growing fears of COVID-19 reservoirs in domestic and wild animals, it is necessary to adapt detection systems for variant detection. However, variant-specific detection remains challenging. Surface-enhanced Raman scattering is a sensitive and multiplexing technique that allows the simultaneous detection of multiple targets for accurate identification. Here we propose the development of a multiplex SERS microassay to detect both the spike and nucleocapsid structural proteins of SARS-CoV-2. The designed SERS microassay integrates gold-silver hollow nanobox barcodes and electrohydrodynamically induced nanomixing which in combination enables highly specific and sensitive detection of SARS-CoV-2 and the S-protein epitopes to delineate between ancestral prevariant strains with the newer variants of concern, Delta and Omicron. The microassay allows detection from as low as 20 virus/µL and 50 pg/mL RBD protein and can clearly identify the virus among infected versus healthy nasopharyngeal swabs, with the potential to identify between variants. The detection of both S- and N-proteins of SARS-CoV-2 and the differentiation of variants on the SERS microassay can aid the early detection of COVID-19 to reduce transmission rates and lead into adequate treatments for those severely affected by the virus.

SARS-CoV-2 RT-PCR to Screen for B.1.617.2 (Delta) Variant of Concern.

The continuous transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has required that diagnostic capabilities be constantly monitored and updated as new variants emerge and prior variants disappear. Although whole genome sequencing provides full characterisation of SARS-CoV-2 directly from patient samples, this has limited throughput and requires sufficient resources. To enhance screening for circulating variants, we designed a rapid in-house RT-PCR assay to target a spike mutation (D950N) in Delta variants, which is not detected in the remaining variants of concern (VOCs). Assay sensitivity for detecting Delta variants was 93% and specificity was 100% using a sequenced sample bank of several lineages. As the D950N mutation is prevalent in >95% of the global Delta variant sequences deposited in GISAID, this assay has the potential to provide rapid results to determine if the samples are presumptively Delta variants and can support clinicians in timely clinical decision-making for effective treatments and surveillance.

Two Treponema pallidum strains account for the majority of syphilis infections, including among females, in Queensland, Australia.

Abstract: 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.

Isolation and Identification of Inter-Species Enterovirus Recombinant Genomes.

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 progeny virus, their existence and continued evolution during replication have critical implications for the 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-strand 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 viruses.

Mycoplasma genitalium infections can comprise a mixture of both fluoroquinolone-susceptible and fluoroquinolone-resistant strains.

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.

Effect of Serotype and Strain Diversity on Dengue Virus Replication in Australian Mosquito Vectors.

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'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.

Identification of the source of blood meals in mosquitoes collected from north-eastern Australia.

BACKGROUND: More than 70 arboviruses have been identified in Australia and the transmission cycles of most are poorly understood. While there is an extensive list of arthropods from which these viruses have been recovered, far less is known about the non-human hosts that may be involved in the transmission cycles of these viruses and the relative roles of different mosquito species in cycles of transmission involving different hosts. Some of the highest rates of human infection with zoonotic arboviruses, such as Ross River (RRV) and Barmah Forest (BFV) viruses, occur in coastal regions of north-eastern Australia. METHODS: Engorged mosquitoes collected as a part of routine surveillance using CO2-baited light traps in the Rockhampton Region and the adjoining Shire of Livingstone in central Queensland, north-eastern Australia, were analysed for the source of their blood meal. A 457 or 623 nucleotide region of the cytochrome b gene in the blood was amplified by PCR and the amplicons sequenced. The origin of the blood was identified by comparing the sequences obtained with those in GenBank®. RESULTS: The most common hosts for the mosquitoes sampled were domestic cattle (26/54) and wild birds (14/54). Humans (2/54) were an infrequent host for this range of mosquitoes that are known to transmit arboviruses causing human disease, and in an area where infections with human pathogens like RRV and BFV are commonly recorded. The blood meals identified in the most abundant vector analysed, Culex annulirostris, were from 10 different vertebrate hosts. The notable detection of chimpanzee blood in two mosquitoes, presumably obtained from a nearby zoo, extends the known range of hosts for this species. Culex quinquefasciatus and Cx. sitiens fed almost exclusively on a variety of bird species. CONCLUSIONS: While human-mosquito-human transmission of arboviruses like RRV can occur, this study highlights the potential importance of zoonotic cycles of transmission, including avian species, of arboviruses that are indigenous to Australia. Further studies on larger samples of blood-engorged mosquitoes are required to validate the trends observed herein. Moreover, serological and virological evidence that the hosts on which the mosquitoes are feeding are being infected with arboviruses of interest are required.

Recombination in enteroviruses is a biphasic replicative process involving the generation of greater-than genome length 'imprecise' intermediates.

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of 'evolution by duplication' in the positive-strand RNA viruses.

Defective interfering viral particles in acute dengue infections.

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.

Lineage replacement accompanying duplication and rapid fixation of an RNA element in the nsP3 gene in a species of alphavirus.

A sequence of thirty-six nucleotides in the nsP3 gene of Ross River virus (RRV), coding for the amino acid sequence HADTVSLDSTVS, was duplicated some time between 1969 and 1979 coinciding with the appearance of a new lineage of this virus and with a major outbreak of Epidemic Polyarthritis among residents of the Pacific Islands. This lineage of RRV continues to circulate throughout Australia and both earlier lineages, which lacked the duplicated element, now are extinct. Multiple copies of several other elements also were observed in this region of the nsP3 gene in all lineages of RRV. Multiple copies of one of these, coding for the amino acid sequence P*P*PR, were detected in the C-terminal region of the nsP3 protein of all alphaviruses except those of African origin. The fixation of duplications and insertions in 3' region of nsP3 genes from all lineages of alphaviruses, suggests they provide some fitness advantage.

Molecular evolutionary dynamics of Ross River virus and implications for vaccine efficacy.

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.

Multiple recombinant dengue type 1 viruses in an isolate from a dengue patient.

Between 2000 and 2004, dengue virus type 1 (DENV-1) genotypes I and II from Asia were introduced into the Pacific region and co-circulated in some localities. Envelope protein gene sequences of DENV-1 from 12 patients infected on the island of New Caledonia were obtained, five of which carried genotype I viruses and six, genotype II viruses. One patient harboured a mixed infection, containing viruses assigned to both genotypes I and II, as well as a number of inter-genotypic recombinants. This is the first report of a population of dengue viruses isolated from a patient containing both parental and recombinant viruses.

Experimental infection of Culex annulirostris, Culex gelidus, and Aedes vigilax with a yellow fever/Japanese encephalitis virus vaccine chimera (ChimeriVax-JE).

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.

Origin of dengue type 3 viruses associated with the dengue outbreak in Dhaka, Bangladesh, in 2000 and 2001.

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.