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In Australia, the outbreak of pandemic (H1N1) 2009 began in Melbourne, Victoria; in the first 17 days, the Victorian Infectious Diseases Reference Laboratory detected 977 cases. Although the laboratory had a pandemic plan in place, a retrospective evaluation found 3 major variations from plan assumptions: 1) higher peak demand not limited by a case definition, 2) prolonged peak demand because containment attempts continued despite widespread influenza, and 3) unexpected influence of negative test results on public health actions. Although implementation of the plan was generally successful, the greatest challenges were limited availability of skilled staff and test reagents. Despite peak demand of 1,401 tests per day, results were provided within the usual 24 hours of specimen receipt; however, turnaround time seemed slower because of slow transport times (>3 days for 45% of specimens). Hence, effective laboratory capability might be enhanced by speeding transport of specimens and improving transmission of clinical data. [ABSTRACT FROM AUTHOR]

Background: Three patients who received visceral-organ transplants from a single donor on the same day died of a febrile illness 4 to 6 weeks after transplantation. Culture, polymerase-chain-reaction (PCR) and serologic assays, and oligonucleotide microarray analysis for a wide range of infectious agents were not informative. Methods: We evaluated RNA obtained from the liver and kidney transplant recipients. Unbiased high-throughput sequencing was used to identify microbial sequences not found by means of other methods. The specificity of sequences for a new candidate pathogen was confirmed by means of culture and by means of PCR, immunohistochemical, and serologic analyses. Results: High-throughput sequencing yielded 103,632 sequences, of which 14 represented an Old World arenavirus. Additional sequence analysis showed that this new arenavirus was related to lymphocytic choriomeningitis viruses. Specific PCR assays based on a unique sequence confirmed the presence of the virus in the kidneys, liver, blood, and cerebrospinal fluid of the recipients. Immunohistochemical analysis revealed arenavirus antigen in the liver and kidney transplants in the recipients. IgM and IgG antiviral antibodies were detected in the serum of the donor. Seroconversion was evident in serum specimens obtained from one recipient at two time points. Conclusions: Unbiased high-throughput sequencing is a powerful tool for the discovery of pathogens. The use of this method during an outbreak of disease facilitated the identification of a new arenavirus transmitted through solid-organ transplantation. N Engl J Med 2008;358:991-8. [ABSTRACT FROM AUTHOR]

Given the potential for laboratory-associated severe acute respiratory syndrome--associated coronavirus (SARS-CoV) infections, we must know which cell lines are susceptible to the virus. We investigated 21 cell lines routinely used for virus isolation or research. After infection with SARS-CoV, cells were observed for cytopathic effects, and quantitative real-time polymerase chain reaction was used to measure ongoing viral replication. An indirect immuno-fluorescence assay was also used as a confirmatory test. The study identified 10 new cell lines capable of supporting the replication of SARS-CoV and confirmed the susceptibility of 4 cell lines previously reported. This study shows that SARS-CoV can be isolated in several cell lines commonly used for diagnostic or research purposes. It also shows that SARS-CoV can achieve high titers in several cell lines, sometimes in the absence of specific cytopathic effects. [ABSTRACT FROM AUTHOR]

We characterised the antiviral phenotype and genotype of 41 herpes simplex virus (HSV) strains from patients clinically resistant to acyclovir (ACV). Our results confirm recognised mutational sites as being major determinants of thymidine kinase (tk)-associated ACV resistance, in particular insertions and/or deletions at homopolymer stretches of Gs and Cs (59% of all isolates). Previously described amino acid substitutions in functional sites of the tk were also identified (7% of all isolates). In addition, we identified several stop codons in novel locations on the amino acid sequence (7% of all isolates) and amino acid substitutions (15% of all isolates) likely to be directly responsible for conferring resistance to ACV. When there were no mutations detected in the tk gene (12% of all isolates), mutations in the DNA polymerase gene likely to be important in the generation of resistant virus were identified. [Copyright &y& Elsevier]

Although several clinical trials are now underway to test possible therapies, the worldwide response to the COVID-19 outbreak has been largely limited to monitoring/containment. We report here that Ivermectin, an FDA-approved anti-parasitic previously shown to have broad-spectrum anti-viral activity in vitro , is an inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 h post infection with SARS-CoV-2 able to effect ~5000-fold reduction in viral RNA at 48 h. Ivermectin therefore warrants further investigation for possible benefits in humans. • Ivermectin is an inhibitor of the COVID-19 causative virus (SARS-CoV-2) in vitro. • A single treatment able to effect ~5000-fold reduction in virus at 48 h in cell culture. • Ivermectin is FDA-approved for parasitic infections, and therefore has a potential for repurposing. • Ivermectin is widely available, due to its inclusion on the WHO model list of essential medicines. [ABSTRACT FROM AUTHOR]

Dengue neurological disease is an uncommon yet severe complication of dengue infection. It can manifest as encephalitis, encephalopathy, neuro-ophthalmic complications, or neuromuscular disorders. Severe infection can result in viral shedding across multiple body sites. We describe a case of severe neuro-ophthalmic dengue infection in an otherwise healthy returned traveller, presenting with prolonged multiple-body-site viral detections by PCR. The dengue virus (DENV) dynamics and serological response support a direct DENV neuropathogenicity. A retrospective review of the laboratory data at the Victorian Infectious Diseases Reference Laboratory (VIDRL) suggests that blood is the most frequent sample type with DENV detection (92% of all DENV-positive samples). Genotype variation is seen across different sample types. The similarity of CSF and nasopharyngeal DENV subtypes (genotype 1 and 3) suggests a possible correlation between nasopharyngeal replication and neurological complications. The case presented highlights the direct neuropathogenicity of DENV early in the course of infection, and a potential correlation between nasopharyngeal replication and neurological disease. [ABSTRACT FROM AUTHOR]

A letter to the editor about a traveler to Australia with chikungunya virus infection is presented.

This article reports on a case of clinical illness in a 40-year-old HIV-infected man who experienced 5 discrete episodes of "unexplained" multisystem illness over a 3-year period before developing acute respiratory distress syndrome and succumbing to pulmonary hemorrhage. The patient had a history of cutaneous Kaposisarcoma treated with liposomal doxorubicin therapy. He had been treated with HAART for 4 years; his CD4 cell count was stable, and his virus load was usually <400 copies/mL. The patient experienced four additional episodes of unexplained multisystem illness during the next 3 years.

While bats are often viewed as carriers of infectious disease agents, little research has been conducted on the effects these potential pathogens may have on the bat populations themselves. The southern bent-winged bat (Miniopterus orianae bassanii) is a critically endangered subspecies endemic to south-eastern Australia. Population numbers of this bat have been declining for the past 50 years, but the reasons for this are unclear. As part of a larger study to determine if disease could be a contributing factor to this decline, 351 southern bent-winged bats from four locations were captured, and oral swabs were collected and tested for the presence of potentially pathogenic viruses. Results were compared with those obtained from 116 eastern bent-winged bats (Miniopterus orianae oceanensis) from three different locations. The eastern bent-winged bat is a related but more common and widespread subspecies whose geographical range overlaps partly with southern bent-winged bats. Herpesviruses were detected in bent-winged bats from all seven locations. At least six novel herpesviruses (five betaherpesviruses and one gammaherpesvirus) were identified. The prevalence of herpesvirus infection was higher in eastern bent-winged bats (44%, 51/116), compared to southern bent-winged bats (27%, 95/351), although this varied across the locations and sampling periods. Adenoviruses and a range of different RNA viruses (lyssaviruses, filoviruses, coronaviruses and henipaviruses) were also tested for but not detected. The detected herpesviruses did not appear to be associated with obvious ill health, and may thus not be playing a role in the population decline of the southern bent-winged bat. The detection of multiple novel herpesviruses at a high prevalence of infection is consistent with our understanding of bats as hosts to a rich diversity of viruses. [ABSTRACT FROM AUTHOR]

Small-animal models have been used to obtain many insights regarding the pathogenesis and immune responses induced following infection with human respiratory syncytial virus (hRSV). Among those described to date, infections in cotton rats, mice, guinea pigs, chinchillas, and Syrian hamsters with hRSV strains Long and/or A2 have been well characterized, although clinical isolates have also been examined. Ferrets are also susceptible to hRSV infection, but the pathogenesis and immune responses elicited following infection have not been well characterized. Here, we describe the infection of adult ferrets with hRSV Long or A2 via the intranasal route and characterized virus replication, as well as cytokine induction, in the upper and lower airways. Virus replication and cytokine induction during the acute phase of infection (days 0 to 15 postinfection) were similar between the two strains, and both elicited high levels of F glycoprotein-specific binding and neutralizing antibodies following virus clearance (days 16 to 22 postinfection). Importantly, we demonstrate transmission from experimentally infected donor ferrets to cohoused naive recipients and have characterized virus replication and cytokine induction in the upper airways of infected contact animals. Together, these studies provide a direct comparison of the pathogenesis of hRSV Long and A2 in ferrets and highlight the potential of this animal model to study serological responses and examine interventions that limit transmission of hRSV. [ABSTRACT FROM AUTHOR]

Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibility among seven independent laboratories demonstrated less than 4.5% reproducibility relative standard deviation. With the exclusion of one laboratory, laboratories had appropriate technical competency, fully functional instrumentation, and suitable reagents to perform accurate ddPCR based DNA quantification measurements at the time of the study. The study results confirmed that NA008 High GC reference material is fit for the purpose of being used for quality control of ddPCR systems, consumables, instrumentation, and workflow. [ABSTRACT FROM AUTHOR]

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART ('HIV reservoir'). One such technique for HIV RNA quantitation, 'HIV transcription profiling', developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the 'HIV transcription profiling' technique to Qiagen's dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes. [ABSTRACT FROM AUTHOR]

• This comprehensive evaluation offers valuable information for laboratories on the performance characteristic of a broad range of diagnostic MPXV and OPX assays. • Assays evaluated demonstrated a range of Limits of Detection. • All assays demonstrated excellent intra-run variation and no cross-reactivity against a range of pathogens that may pose differential diagnoses, however some MPXV specific assays may detect vaccinia virus. • All but 1 assay demonstrated 100% clinical sensitivity. • Reliable and accurate laboratory diagnosis of MPXV and isolation is crucial to containing the spread of this current outbreak and informing public health interventions and response. The current global mpox virus (MPXV) outbreak has been declared a Public Health Emergency of International Concern by WHO, with more than 80,000 cases confirmed across multiple continents. Diagnosis is confirmed by PCR of viral DNA from vesicle and other swabs. The aim of this study was to assess commercial RT PCR assays for Orthopoxvirus (OPX) and MPXV for analytical sensitivity, and percent agreements and compare them to primer/probe sets employed at the Victorian Infectious Diseases Reference Laboratory (VIDRL), Centers for Disease Control andPrevention (CDC) and US Army Medical Research Institute of Infectious Diseases (USAMRIID). Limits of detection (LOD), intra-run variability, cross-reactivity and performance on forty clinical samples was assessed on eleven commercial assays and five primer/probe combinations used at VIDRL, CDC and USAMRIID. All assays were able to detect OPX and MPXV (LOD 57 to 14,495 copies/mL) with intra-run coefficients of variation between Cycle thresholds of 0.58 and 3.44, and there was no unexpected cross-reactivity. All assays demonstrated 100% negative percent agreement with clinical samples and all but one yielded 100% positive percent agreement. Variations in LOD between assays may be dependent on the platform used and sample type. Despite the overall comparable performance of the assays assessed, it is important that routine laboratories perform in-house validations before implementing RT PCR for OPX and/or MPXV as reliable and accurate laboratory diagnosis of MPXV and isolation is crucial to containing the spread of this current outbreak and informing public health interventions and response. [ABSTRACT FROM AUTHOR]

Background: National Influenza Centers (NICs) have played a crucial role in the surveillance of SARS‐CoV‐2. The FluCov project, covering 22 countries, was initiated to monitor the impact of the SARS‐CoV‐2 pandemic on influenza activity. Methods: This project consisted of an epidemiological bulletin and NIC survey. The survey, designed to assess the impact of the pandemic on the influenza surveillance system, was shared with 36 NICs located across 22 countries. NICs were invited to reply between November 2021 and March 2022. Results: We received 18 responses from NICs in 14 countries. Most NICs (76%) indicated that the number of samples tested for influenza decreased. Yet, many NICs (60%) were able to increase their laboratory testing capacity and the "robustness" (e.g., number of sentinel sites) (59%) of their surveillance systems. In addition, sample sources (e.g., hospital or outpatient setting) shifted. All NICs reported a higher burden of work following the onset of the pandemic, with some NICs hiring additional staff or partial outsourcing to other institutes or departments. Many NICs anticipate the future integration of SARS‐CoV‐2 surveillance into the existing respiratory surveillance system. Discussion: The survey shows the profound impact of SARS‐CoV‐2 on national influenza surveillance in the first 27 months of the pandemic. Surveillance activities were temporarily disrupted, whilst priority was given to SARS‐CoV‐2. However, most NICs have shown rapid adaptive capacity underlining the importance of strong national influenza surveillance systems. These developments have the potential to benefit global respiratory surveillance in the years to come; however, questions about sustainability remain. [ABSTRACT FROM AUTHOR]

Background: In animal, epidemiological, and human challenge studies, a pre-existing T-cell response to internal proteins of influenza A has been associated with improved virological and disease outcomes. The aim of this study was to assess whether inducing additional responses to conserved CD4 and CD8 T-cell antigens provides added benefit to standard influenza vaccination.Methods: We designed a phase 2b, randomised, placebo-controlled, double-blind trial of a recombinant viral-vectored vaccine (modified vaccinia Ankara expressing virus nucleoprotein and matrix protein 1; MVA-NP+M1), which has been shown to induce both CD4 and CD8 T cells, at eight outpatient clinical trial sites in Australia over two consecutive influenza seasons. We recruited non-immunosuppressed adults (≥18 years) who had received the 2019 quadrivalent influenza vaccine (QIV) vaccine within 28 days before study enrolment and randomisation (day 0). Participants were randomly assigned (1:1) according to a computer-generated random sequence to receive one dose of 1·5 × 108 plaque-forming units of MVA-NP+M1 or saline (placebo) intramuscularly. Randomisation was stratified by age (<65 years or ≥65 years). The patients and trial assessors were masked to treatment assignment. During the subsequent influenza seasons, participants with symptoms related to respiratory illness or influenza-like illness were to attend the clinic within 72 h of symptom onset for two nasal swabs for influenza testing by quantitative RT-PCR. The primary endpoint was the incidence rate of laboratory-confirmed influenza in the intention-to-treat (ITT) population. Safety (solicited adverse events within 7 days and unsolicited adverse events within 28 days after study vaccination, and serious adverse events for the study duration) was assessed in all randomly assigned participants who received at least one vaccination (according to the treatment received). The trial is registered with ClinicalTrials.gov, NCT03880474.Findings: Between April 2 and June 14, 2019, 2152 adults were randomly allocated and received MVA-NP+M1 (n=1077) or placebo (n=1075), comprising the efficacy (ITT) analysis set. Participants were followed up throughout the 2019 Australia influenza season (May 1 to Oct 15, 2019). 419 (19·5%) of 2152 participants were aged 65 years or older. The incidence of laboratory-confirmed influenza did not differ between the MVA-NP+M1 group (35 of 1077 participants; 3·25% [95% CI 2·31-4·44]) and the placebo group (23 of 1075; 2·14% [1·39-3·14]; Fisher's exact p=0·14). 23 severe solicited local injection site reactions were reported in 13 (0·6%) of 2152 participants, 22 of which were reported in the MVA-NP + M1 group (in 12 [1·1%] participants). 100 severe systemic events were reported in 45 (4·2%) MVA-NP + M1 recipients, and 20 were reported in 14 (1·3%) placebo recipients. Three unsolicited grade 3 events in three participants (two headache and one nausea, all in the MVA-NP+M1 group) were deemed vaccine related. 21 serious adverse events were reported in 18 (1·7%) of 1077 participants in the MVA-NP+M1 group and 25 serious adverse events were reported in 22 (2·0%) of 1075 participants in the placebo group; none were considered vaccine related. The trial was stopped after one season for futility on the recommendation of the data monitoring committee.Interpretation: MVA-NP+M1 was well tolerated with no vaccine-associated serious adverse events. A vaccine designed to induce moderate T-cell responses to the cross-reactive internal proteins of influenza A did not lead to improved incidence when given within 28 days after standard QIV immunisation. A greater magnitude of T-cell response with a different vaccine or regimen, or localisation in the lungs via alternative delivery, such as intranasal or aerosol, might be successful and require further investigation.Funding: Vaccitech. [ABSTRACT FROM AUTHOR]

Background. Although pandemic and avian influenza are known to be transmitted via human hands, there are minimal data regarding the effectiveness of routine hand hygiene (HH) protocols against pandemic and avian influenza. Methods. Twenty vaccinated, antibody-positive health care workers had their hands contaminated with 1 mL of 107 tissue culture infectious dose (TCID)50/0.1 mL live human influenza A virus (H1N1; A/New Caledonia/20/99) before undertaking 1 of 5 HH protocols (no HH [control], soap and water hand washing [SW], or use of 1 of 3 alcohol-based hand rubs [61.5% ethanol gel, 70% ethanol plus 0.5% chlorhexidine solution, or 70%isopropanol plus 0.5% chlorhexidine solution]). H1N1 concentrations were assessed before and after each intervention by viral culture and real-time reverse-transcriptase polymerase chain reaction (PCR). The natural viability of H1N1 on hands for >60 min without HH was also assessed. Results. There was an immediate reduction in culture-detectable and PCR-detectable H1N1 after brief cutaneous air drying—14 of 20 health care workers had H1N1 detected by means of culture (mean reduction, 103-4 TCID50/0.1 mL), whereas 6 of 20 had no viable H1N1 recovered; all 20 health care workers had similar changes in PCR test results. Marked antiviral efficacy was noted for all 4 HH protocols, on the basis of culture results (14 of 14 had no culturable H1N1; P < .002) and PCR results (P

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivatives that can withstand 100 °C for 90 min and 37 °C for four weeks and help eliminate cold-chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clinical trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID‐19. We propose a portable infrared spectrometer with purpose‐built transflection accessory for rapid point‐of‐care detection of COVID‐19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM‐IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS‐CoV‐2 by RT‐qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT‐qPCR. Herein, we demonstrate a proof‐of‐concept high throughput infrared COVID‐19 test that is rapid, inexpensive, portable and utilizes sample self‐collection thus minimizing the risk to healthcare workers and ideally suited to mass screening. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID‐19. We propose a portable infrared spectrometer with purpose‐built transflection accessory for rapid point‐of‐care detection of COVID‐19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM‐IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS‐CoV‐2 by RT‐qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT‐qPCR. Herein, we demonstrate a proof‐of‐concept high throughput infrared COVID‐19 test that is rapid, inexpensive, portable and utilizes sample self‐collection thus minimizing the risk to healthcare workers and ideally suited to mass screening. [ABSTRACT FROM AUTHOR]

Background Serological testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the coronavirus disease 2019 (COVID-19) pandemic response. It is important to understand the reliability of assays with different antigen or antibody targets to detect humoral immunity after SARS-CoV-2 infection and to understand how antibody (Ab) binding assays compare to those detecting neutralizing antibody (nAb), particularly as we move into the era of vaccines. Methods We evaluated the performance of 6 commercially available enzyme-linked immunosorbent assays (ELISAs), including a surrogate virus neutralization test (sVNT), for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or nAb. A result subset was compared with a cell culture–based microneutralization (MN) assay. We tested sera from patients with prior reverse transcription polymerase chain reaction–confirmed SARS-CoV-2 infection, prepandemic sera, and potential cross-reactive sera from patients with other non-COVID-19 acute infections. Results For sera collected >14 days post–symptom onset, the assay achieving the highest sensitivity was the Wantai total Ab at 100% (95% CI, 94.6%–100%), followed by 93.1% for Euroimmun NCP-IgG, 93.1% for GenScript sVNT, 90.3% for Euroimmun S1-IgG, 88.9% for Euroimmun S1-IgA, and 83.3% for Wantai IgM. Specificity for the best-performing assay was 99.5% for the Wantai total Ab, and for the lowest-performing assay it was 97.1% for sVNT (as per the Instructions for Use [IFU]). The Wantai Total Ab had the best agreement with MN at 98% followed by Euroimmun S1-IgA, Euro NCP-IgG, and sVNT (as per IFU) with 97%, 97% and 95%, respectively; Wantai IgM had the poorest agreement at 93%. Conclusions Performance characteristics of the SARS-CoV-2 serology assays detecting different antibody types are consistent with those found in previously published reports. Evaluation of the surrogate virus neutralization test in comparison to the Ab binding assays and a cell culture–based neutralization assay showed good result correlation between all assays. However, correlation between the cell-based neutralization test and some assays detecting Ab's not specifically involved in neutralization was higher than with the sVNT. This study demonstrates the reliability of different assays to detect the humoral immune response following SARS-CoV-2 infection, which can be used to optimize serological test algorithms for assessing antibody responses post–SARS-CoV-2 infection or vaccination. [ABSTRACT FROM AUTHOR]

• Antibody titres of healthcare workers were compared by prior vaccination history. • Healthcare workers in our study demonstrated protective antibody titres post-vaccination. • Different antibody responses observed for each influenza vaccine strain. • Greater post-vaccination responses observed among vaccine-naïve healthcare workers. • Repeated vaccination appears to attenuate NI antibody response to A(H3N2). Epidemiological studies suggest that influenza vaccine effectiveness decreases with repeated administration. We examined antibody responses to influenza vaccination among healthcare workers (HCWs) by prior vaccination history and determined the incidence of influenza infection. HCWs were vaccinated with the 2016 Southern Hemisphere quadrivalent influenza vaccine. Serum samples were collected pre-vaccination, 21–28 days and 7 months post-vaccination. Influenza antibody titres were measured at each time-point using the haemagglutination inhibition (HI) assay. Immunogenicity was compared by prior vaccination history. A total of 157 HCWs completed the study. The majority were frequently vaccinated, with only 5 reporting no prior vaccinations since 2011. Rises in titres for all vaccine strains among vaccine-naïve HCWs were significantly greater than rises observed for HCWs who received between 1 and 5 prior vaccinations (p < 0.001, respectively). Post-vaccination GMTs against influenza A but not B strains decreased as the number of prior vaccinations increased from 1 to 5. There was a significant decline in GMTs post-season for both B lineages. Sixty five (41%) HCWs reported at least one influenza-like illness episode, with 6 (4%) identified as influenza positive. Varying serological responses to influenza vaccination were observed among HCWs by prior vaccination history, with vaccine-naïve HCWs demonstrating greater post-vaccination responses against A(H3N2). [ABSTRACT FROM AUTHOR]

During a mouse plague in early 2021, a farmer from New South Wales, Australia, sought treatment for aseptic meningitis and was subsequently diagnosed with locally acquired lymphocytic choriomeningitis virus infection. Whole-genome sequencing identified a divergent and geographically distinct lymphocytic choriomeningitis virus strain compared with other published sequences. [ABSTRACT FROM AUTHOR]

Background Waning measles immunity among vaccinated individuals may result in an attenuated illness. This study compares the epidemiological, clinical, and laboratory profile of measles cases with waning immunity with other measles cases. Methods Polymerase chain reaction–positive (+) measles cases notified to Victoria's Department of Health and Human Services from 2008 to 2017 with immunoglobulin (Ig) M and IgG tested at diagnosis were classified according to serology at diagnosis: IgG negative (−) (nonimmune), IgM+/IgG+ (indeterminate), or IgM−/IgG+ (waning immunity). Results Between 2008 and 2017, 297 measles cases were notified, of whom 190 (64%) were included; 151 of 190 (79%) were nonimmune at diagnosis, 26 (14%) were indeterminate, and 13 (7%) had waning immunity. Between 2008–2013 and 2014–2017, the proportion of cases with waning immunity increased from 0 of 87 (0%) to 13 of 103 (13%) (P <.001) and the diagnostic sensitivity of initial IgM fell from 93% to 81% (P =.012), respectively. Seven (54%) waning immunity cases reported receiving measles-containing vaccines; 1 case had 2 documented doses. Compared with nonimmune and indeterminate cases, waning immunity cases were more likely to be male; less likely to report fever, coryza, and cough; and had lower burden of virus (higher cycle threshold values). Waning immunity cases had higher IgG titers than indeterminate cases (mean optical density values, 1.96 vs 0.71; P =.004). Onward transmission from 1 waning immunity case was documented. Conclusions Waning immunity among measles cases, associated with secondary vaccine failure and modified clinical illness, is emerging in Victoria with transmission potential. [ABSTRACT FROM AUTHOR]

Background: Genital herpes simplex infection affects more than 500 million people worldwide. We have previously shown that COR-1, a therapeutic HSV-2 polynucleotide vaccine candidate, is safe and well tolerated in healthy subjects. Objective: Here, we present a single center double-blind placebo-controlled, randomized phase I/IIa trial of COR-1 in HSV-2 positive subjects in which we assessed safety and tolerability as primary endpoints, and immunogenicity and therapeutic efficacy as exploratory endpoints. Methods: Forty-four HSV-2+ subjects confirmed by positive serology or pathology, and positive qPCR during baseline shedding, with a recurrent genital HSV-2 history of at least 12 months including three to nine reported lesions in 12 months prior to screening, aged 18 to 50 years females and males with given written informed consent, were randomized into two groups. Three immunizations at 4-week intervals and one booster immunization at 6 months, each of 1 mg COR-1 DNA or placebo, were administered intradermally as two injections of 500 μg each to either one forearm or both forearms. Results: No serious adverse events, life-threatening events or deaths occurred throughout the study. As expected, HSV-2 infected subjects displayed gD2-specific antibody titers prior to immunization. COR-1 was associated with a reduction in viral shedding after booster administration compared with baseline. Conclusions: This study confirms the previously demonstrated safety of COR-1 in humans and indicates a potential for use of COR-1 as a therapy to reduce viral shedding in HSV-2 infected subjects. [ABSTRACT FROM AUTHOR]

We report a case of Barmah Forest virus infection in a child from Central Province, Papua New Guinea, who had no previous travel history. Genomic characterization of the virus showed divergent origin compared with viruses previously detected, supporting the hypothesis that the range of Barmah Forest virus extends beyond Australia. [ABSTRACT FROM AUTHOR]

Recent parechovirus A3 (PeV-A3) outbreaks in Australia suggest lower population immunity compared with regions that have endemic PeV-A3 circulation. A serosurvey among populations in the Netherlands, the United States, and Australia before and after the 2013 Australia outbreak showed high PeV-A3 neutralizing antibody prevalence across all regions and time periods, indicating widespread circulation. [ABSTRACT FROM AUTHOR]

Epidemiological studies have observed that the seasonal peak incidence of influenza virus infection is sometimes separate from the peak incidence of human respiratory syncytial virus (hRSV) infection, with the peak incidence of hRSV infection delayed. This is proposed to be due to viral interference, whereby infection with one virus prevents or delays infection with a different virus. We investigated viral interference between hRSV and 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) in the ferret model. Infection with A(H1N1)pdm09 prevented subsequent infection with hRSV. Infection with hRSV reduced morbidity attributed to infection with A(H1N1)pdm09 but not infection, even when an increased inoculum dose of hRSV was used. Notably, infection with A(H1N1)pdm09 induced higher levels of proinflammatory cytokines, chemokines, and immune mediators in the ferret than hRSV. Minimal cross-reactive serological responses or interferon γ-expressing cells were induced by either virus ≥14 days after infection. These data indicate that antigen-independent mechanisms may drive viral interference between unrelated respiratory viruses that can limit subsequent infection or disease. [ABSTRACT FROM AUTHOR]

Control of measles was the focus of a national workshop held in 2015 in Sydney, Australia, bringing together stakeholders in disease control and immunisation to discuss maintaining Australia's measles elimination status in the context of regional and global measles control. The global epidemiology of measles was reviewed, including outbreaks in countries that have achieved elimination, such as the Disneyland outbreak in the United States and large outbreaks in Sydney, Australia. Transmission of measles between Australia and New Zealand occurs, but has not been a focus of control measures. Risk groups, the genetic and seroepidemiology of measles as well as surveillance, modelling and waning vaccine-induced immunity were reviewed. Gaps in policy, research and practice for maintaining measles elimination status in Australia were identified and recommendations were developed. Elimination of measles globally is challenging because of the infectiousness of measles and the need for 2-dose vaccine coverage rates in excess of 95% in all countries to achieve it. Until this occurs, international travel will continue to permit measles importation from endemic countries to countries that have achieved elimination. When measles cases are imported, failure to diagnose and isolate cases places the health system at risk of measles outbreaks. Vaccine funding models can result in gaps in vaccine coverage for adults and migrants. Australia introduced a whole-of-life immunisation register in 2016 and catch-up vaccination for at-risk communities, which will improve measles control. Research on diagnosis, immunology, case management and modelling of vaccination strategies are important to ensure continued control of measles. [ABSTRACT FROM AUTHOR]

Objective: It is recognized that human rhinovirus (HRV) infection is an important factor in asthma exacerbations requiring hospitalization in children. However, previous studies have disagreed on the differential impact of various HRV species. We sought to assess the impact of HRV species on the severity of asthma exacerbations in children and adolescents. We also examined whether the effect of HRV species on severity was modified by age and gender. Methods: Virus strain was determined for 113 children with HRV detectable at the time of admission for asthma exacerbation. Patient characteristics were collected on admission and exacerbation severity was scored using several validated scales. Results: HRV species by itself was not associated with moderate/severe vs. mild exacerbations. Boys with HRV-C infections were more likely (OR: 3.7, 95% CI: 1.2-13.4) to have a moderate/severe exacerbation than girls with HRV-C (p = 0.04 for interaction term). Higher odds were observed in younger boys (3 years old: OR: 9.1, 95% CI: 1.8-47.1 vs 5 years old: OR: 3.3, 95% CI: 0.9-11.8 vs 7 years old: OR: 1.2, 95% CI: 0.2-6.6). In contrast, children with HRV-C infection and sensitized to pollen during the pollen season were less likely to have moderate/severe exacerbations (p = 0.01 for the interaction term). Conclusion: Acute asthma exacerbations are more likely to be moderate/severe in boys under 5 years of age who had HRV-C infection on admission. The opposite was found in children with sensitization to pollen during pollen season. [ABSTRACT FROM AUTHOR]

Background Owing to limited availability of donor organs, previous solid organ transplant ( SOT) recipients are increasingly considered as potential organ donors. We report donor-derived transmission of herpes simplex virus type-2 ( HSV-2) to two clusters of SOT recipients with transmission from the original donor and an HSV-2-infected recipient who subsequently became a donor. Methods We reviewed medical records of the donors and recipients in both clusters. Pre-transplant serology and virological features of HSV-2 were characterized. Genotyping of HSV-2 isolates to determine potential for donor transmission of HSV-2 through transplantation of organs from prior organ recipients was performed. Results A kidney-pancreas recipient died day 9 post transplant. Following confirmation of brain death, the lungs and recently transplanted kidney were donated to two further recipients. The liver was not retrieved, but biopsy confirmed HSV-2 infection. Testing on the original donor showed negative HSV-2 polymerase chain reaction and HSV immunoglobulin (Ig)M, but positive HSV-2 IgG. The liver recipient from the original donor developed HSV-2 hepatitis and cutaneous infection that responded to treatment with intravenous acyclovir. In the second cluster, lung and kidney recipients both developed HSV-2 viremia that was successfully treated with antiviral therapy. Genotyping of all HSV-2-positive samples showed 100% sequence homology for three recipients. Conclusions Donor-derived HSV infection affected two clusters of recipients because of transplantation of organs from a prior organ recipient. HSV should be considered as a possible cause of illness in febrile SOT recipients in the immediate post-transplant period and may cause disseminated disease and re-infection in HSV-2-seropositive recipients. Testing of HSV serology and prophylaxis may be considered in SOT recipients not receiving cytomegalovirus prophylaxis. [ABSTRACT FROM AUTHOR]

• The QIAstat-Dx platform provides rapid results, may be performed at or near the point of care and can be used for syndromic testing. • Differential diagnoses of viral vesicular infections include chicken pox and disseminated herpes zoster caused by Varicella-zoster virus, herpes infections caused by herpes simplex virus 1, 2 and human herpesvirus 6; and Hand, foot and mouth disease caused by enterovirus. • Results of the QIAstat viral vesicular panel were highly sensitive and specific and demonstrated 99.6% concordance with laboratory developed tests. • The QIAstat-Dx viral vesicular panel, can play a significant role in rapid diagnosis of mpox and viral vesicular diseases, empowering fast and informed clinical decisions for patient centric treatment and public health response. The recent mpox outbreak has highlighted the need to rapidly diagnose the causative agents of viral vesicular disease to inform treatment and control measures. Common causes of vesicular disease include Monkeypox virus (MPXV), clades I and II, Herpes simplex viruses Type 1 and Type 2 (HSV-1, HSV-2), human herpes virus 6 (HHV-6), Varicella-zoster virus (VZV) and Enteroviruses (EVs). Here, we assessed a syndromic viral vesicular panel for rapid and simultaneous detection of these 7 targets in a single cartridge. The aim of this study was to evaluate the QIAStat-Dx ® viral vesicular (VV) panel and compare with laboratory developed tests (LDTs). Limit of detection, inter-run variability, cross-reactivity and specificity were assessed. Positive and negative percent agreement, and correlation between assays was determined using 124 clinical samples from multiple anatomical sites. The overall concordance between the QIAstat and LDTs was 96%. Positive percent agreement was 82% for HHV-6, 89% for HSV-1 and 100% for MPXV, HSV-2, EV and VZV. Negative percent agreement was 100% for all targets assessed. There was no cross-reactivity with Vaccinia, Orf, Molluscum contagiosum viruses, and a pooled respiratory panel. The QIAstat VV multi-target syndromic panel combine ease of use, rapid turnaround, good sensitivity and specificity for enhanced diagnosis, clinical care and public health responses. [ABSTRACT FROM AUTHOR]

Influenza C virus (ICV) is increasingly associated with community-acquired pneumonia (CAP) in children and its disease severity is worse than the influenza B virus, but similar to influenza A virus associated CAP. Despite the ubiquitous infection landscape of ICV in humans, little is known about its replication and pathobiology in animals. The goal of this study was to understand the replication kinetics, tissue tropism, and pathogenesis of human ICV (huICV) in comparison to the swine influenza D virus (swIDV) in guinea pigs. Intranasal inoculation of both viruses did not cause clinical signs, however, the infected animals shed virus in nasal washes. The huICV replicated in the nasal turbinates, soft palate, and trachea but not in the lungs while swIDV replicated in all four tissues. A comparative analysis of tropism and pathogenesis of these two related seven-segmented influenza viruses revealed that swIDV-infected animals exhibited broad tissue tropism with an increased rate of shedding on 3, 5, and 7 dpi and high viral loads in the lungs compared to huICV. Seroconversion occurred late in the huICV group at 14 dpi, while swIDV-infected animals seroconverted at 7 dpi. Guinea pigs infected with huICV exhibited mild to moderate inflammatory changes in the epithelium of the soft palate and trachea, along with mucosal damage and multifocal alveolitis in the lungs. In summary, the replication kinetics and pathobiological characteristics of ICV in guinea pigs agree with the clinical manifestation of ICV infection in humans, and hence guinea pigs could be used to study these distantly related influenza viruses. IMPORTANCE Similar to influenza A and B, ICV infections are seen associated with bacterial and viral co-infections which complicates the assessment of its real clinical significance. Further, the antivirals against influenza A and B viruses are ineffective against ICV which mandates the need to study the pathobiological aspects of this virus. Here we demonstrated that the respiratory tract of guinea pigs possesses specific viral receptors for ICV. We also compared the replication kinetics and pathogenesis of huICV and swIDV, as these viruses share 50% sequence identity. The tissue tropism and pathology associated with huICV in guinea pigs are analogous to the mild respiratory disease caused by ICV in humans, thereby demonstrating the suitability of guinea pigs to study ICV. Our comparative analysis revealed that huICV and swIDV replicated differentially in the guinea pigs suggesting that the type-specific genetic differences can result in the disparity of the viral shedding and tissue tropism. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to ascertain the clinical characteristics associated with herpes simplex virus (HSV) urethritis in men and to compare those with chlamydial urethritis.Methods: We compared clinical and laboratory data from men diagnosed with polymerase chain reaction confirmed HSV urethritis with those of men with chlamydial urethritis presenting to Melbourne Sexual Health Centre between 2000 and 2015.Results: Eighty HSV urethritis cases were identified: 55 (68%, 95% confidence interval, 58-78) were by HSV-1 and 25 (32%, 95% confidence interval, 22-42) by HSV-2. Compared with chlamydial urethritis, men with HSV urethritis were significantly more likely to report severe dysuria (20% vs 0%, P < 0.01) or constitutional symptoms (15% vs 0%, P < 0.01). Men with HSV urethritis were significantly more likely to have meatitis (62% vs 23%, P < 0.01), genital ulceration (37% vs 0%, P < 0.01), or inguinal lymphadenopathy (30% vs 0%, P < 0.01) but less likely to have urethral discharge (32% vs 69%, P < 0.01). There was no significant difference in the proportion of men who had raised (≥5) polymorphonuclear leukocytes per high-powered field between the two groups (P = 0.46).Conclusions: The clinical presentation of HSV urethritis in men may differ from those of chlamydial urethritis and guide testing for HSV in men presenting with non-gonococcal urethritis. [ABSTRACT FROM AUTHOR]

Background Influenza C is usually considered a minor cause of respiratory illness in humans with many infections being asymptomatic or clinically mild. Large outbreaks can occur periodically resulting in significant morbidity. Objectives This study aimed at analyzing the available influenza C clinical samples from two widely separated states of Australia, collected over a 7-year period and to compare them with influenza C viruses detected in other parts of the world in recent years. Patients/Methods Between 2008 and 2014, 86 respiratory samples that were influenza C positive were collected from subjects with influenza-like illness living in the states of Victoria and Western Australia. A battery of other respiratory viruses were also tested for in these influenza C-positive samples. Virus isolation was attempted on all of these clinical samples, and gene sequencing was performed on all influenza C-positive cultures. Results and conclusions Detections of influenza C in respiratory samples were sporadic in most years studied, but higher rates of infection occurred in 2012 and 2014. Many of the patients with influenza C had coinfections with other respiratory pathogens. Phylogenetic analysis of the full-length hemagglutinin-esterase-fusion ( HE) gene found that most of the viruses grouped in the C/Sao Paulo/378/82 clade with the remainder grouping in the C/Kanagawa/1/76 clade. [ABSTRACT FROM AUTHOR]

We describe the clinical presentations of 118 infants admitted to the hospital in New South Wales, Australia, during an epidemic of human parechovirus type 3. Infants presented with an acute sepsis-like syndrome with a high rate of intensive care admission.Background. Infections with human parechoviruses (HPeVs) are associated with a wide range of clinical presentations in children, ranging from mild or asymptomatic infections to severe sepsis-like presentations or meningoencephalitis.Methods. We reviewed medical records of infants admitted to 5 hospitals in New South Wales, Australia, during an outbreak of HPeV-3 infection. Data were collected on clinical presentation, laboratory markers, and outcome of infants with HPeV infection confirmed by reverse transcription polymerase chain reaction.Results. We identified 118 infected infants. Most presented with an acute sepsis-like syndrome with high fever, tachycardia, poor perfusion, and severe irritability. Other common features were erythrodermic rash, abdominal distension, edema, and hepatitis. The age range of infants was 4 days to 9.5 months; 75% were <2 months old, including all but 1 of the 30 infants (25%) admitted to intensive care units (ICUs), who as a group, were significantly younger than infants not admitted to ICUs. Only 4% of evaluable cerebrospinal fluid samples had pleocytosis, but HPeV was detected in 95%. Brain magnetic resonance imaging on a small number of children demonstrated white matter changes and diffusion restriction. Sequencing of the VP1 gene confirmed HPeV-3 in all samples tested. All children recovered without ongoing complications at last follow-up.Conclusions. We report the largest series of HPeV-3 infection in infants, and the first outbreak in Australia. Infants presented with a severe sepsis-like syndrome with a high rate of ICU admissions, but all recovered from the acute infection without complications. Long-term sequelae are unknown. [ABSTRACT FROM PUBLISHER]

Background: Acute respiratory tract infections (ARTIs) represent a serious global health burden. To date, few reports have addressed the prevalence of respiratory viruses (RVs) in adults with ARTIs attending an emergency department (ED). Therefore, the potential impact of respiratory virus infections on such patients remains unknown. Methodology/Principal Findings: To determine the epidemiological and clinical profiles of common and recently discovered respiratory viruses in adults with ARTIs attending an ED in Beijing, a 1-year consecutive study was conducted from May, 2010, to April, 2011. Nose and throat swab samples from 416 ARTI patients were checked for 13 respiratory viruses using multiple reverse transcription polymerase chain reaction(RT-PCR) assays for common respiratory viruses, including influenza viruses (Flu) A, B, and adenoviruses (ADVs), picornaviruses (PICs), respiratory syncytial virus (RSV), parainfluenza viruses (PIVs) 1-3, combined with real-time RT-PCR for human metapneumovirus (HMPV) and human coronaviruses (HCoVs, -OC43, -229E, -NL63, and -HKU1). Viral pathogens were detected in 52.88% (220/416) of patient samples, and 7.21% (30/416) of patients tested positive for more than one virus. PICs (17.79%) were the dominant agents detected, followed by FluA (16.11%), HCoVs (11.78%), and ADV (11.30%). HMPV, PIVs, and FluB were also detected (<3%), but not RSV. The total prevalence and the dominant virus infections detected differed significantly between ours and a previous report. Co-infection rates were high for HCoV-229E (12/39, 30.76%), PIC (22/74, 29.73%), ADV (12/47, 25.53%) and FluA (15/67, 22.39%). Different patterns of clinical symptoms were associated with different respiratory viruses. Conclusions: The pattern of RV involvement in adults with ARTIs attending an ED in China differs from that previously reported. The high prevalence of viruses (PIC, FluA, HCoVs and ADV) reported here strongly highlight the need for the development of safe and effective therapeutic approaches for these viruses. [ABSTRACT FROM AUTHOR]

Abstract: Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein–protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8–ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication. [Copyright &y& Elsevier]

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air–liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples. [ABSTRACT FROM AUTHOR]

• Clinical samples with 'neat' Ct values between 20 and 28 were all detected in pools of six. • Testing in a pool of six led to a mean drop of cycle threshold value of 2.9. • Samples with viral load around the assay's limit of detection may be missed by pooling. • All confirmed negative samples were also negative when tested in pools of 6. The COVID-19 pandemic has placed unprecedented global demand on laboratory supplies required for testing. Sample pooling has been investigated by laboratories as a strategy to preserve testing capacity. We evaluate the performance of Cepheid Xpert® Xpress SARS-CoV-2 RT-PCR assay for testing samples in pools of 4 and 6. Clinical samples containing SARS-CoV-2, and confirmed negative clinical samples were used to create sample pools. Clinical samples had 'neat' Xpert® E gene cycle threshold values ranging between 20 and 28 and all were detected qualitatively when contained in pools of 4 or 6 samples. For these samples, pooling had a median change in cycle threshold value of 2.0 in pools of 4, and of 2.9 in pools of 6. With the use of Cepheid Xpert® Xpress SARS-CoV-2 RT-PCR assay, pooling of 4 or 6 samples may be an effective strategy to increase testing capacity. [ABSTRACT FROM AUTHOR]

• Coronavirus pandemic has required rapid approval of diagnostic assays. • AusDiagnostics multiplex PCR allows integration of SARS-CoV-2 testing. • AusDiagnostics PCR has high specificity: 98.4% of positive samples confirmed. • AusDiagnostics PCR specificity: 2/7839 tests (0.02%) SARS-CoV-2 indeterminate. • AusDiagnostics multiplex tandem PCR is reliable for detection of COVID-19 virus. In the context of the pandemic, the rapid emergency use authorisation of diagnostic assays for SARS-CoV-2 has meant there are few peer-reviewed published studies of clinical performance of commercial assays. To evaluate the clinical performance of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2. We reviewed the results following implementation of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, and compared with an in-house RT-PCR assay at our State Reference Laboratory. Initial validation using AusDiagnostics coronavirus multiplex tandem PCR assay including SARS-CoV-2 demonstrated good concordance with the State Reference Laboratory. After implementing the AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, we tested 7839 samples. 127 samples in which SARS-CoV-2 was detected using the AusDiagnostics assay were referred for testing at the State Reference Laboratory, with concordant results in 118/127 (92.9%) of samples. After resolution of discrepancies, 125/127 (98.4%) of AusDiagnostics results were determined to be true positive results. Out of 7839 samples tested for SARS-CoV-2 during this period, only 2 tests (0.02%) were indeterminate results. The AusDiagnostics respiratory MT-PCR assay is a reliable assay for detection of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Influenza D virus is a novel influenza virus, which was first isolated from an ailing swine in 2011 and later detected in cattle, suggesting that these animals may be a primary natural reservoir. To date, few studies have been performed on human samples and there is no conclusive evidence on the ability of the virus to infect humans. The aim of this serological study was to assess the prevalence of antibodies against influenza D virus in human serum samples collected in Italy from 2005 to 2017. Serum samples were analysed by haemagglutination inhibition and virus neutralization assays. The results showed that the prevalence of antibodies against the virus increased in the human population in Italy from 2005 to 2017, with a trend characterized by a sharp increase in some years, followed by a decline in subsequent years. The virus showed the ability to infect and elicit an immune response in humans. However, prevalence peaks in humans appear to follow epidemics in animals and not to persist in the human population. [ABSTRACT FROM AUTHOR]

Human parechovirus (HPeV), particularly type 3 (HPeV3), is an important cause of sepsis-/meningitis-like illness in young infants. Laboratory records identified a total of ten HPeV-positive cases in Southeastern Australia between January and July 2019. The HPeV present in these cases were typed by Sanger sequencing of the partial viral capsid protein 1 (VP1) region and selected cases were further characterised by additional Sanger or Ion Torrent near-full length virus sequencing. In seven of the ten cases, an HPeV type 5 (HPeV5) was identified, and in the remaining three cases, an HPeV type 1 was identified. The HPeV5-positive cases were infants under the age of 3 months admitted to hospital with fever, rash, lethargy and/or sepsis-like clinical signs. Near full-length virus sequencing revealed that the HPeV5 was most likely a recombinant virus, with structural genes most similar to an HPeV5 from Belarus in 2018, and a polymerase gene most similar to an HPeV3 from Australia in 2013/14. While HPeV5 is not typically associated with severe clinical signs, the HPeV5 identified here may have been able to cause more severe disease in young infants through the acquisition of genes from a more virulent HPeV. [ABSTRACT FROM AUTHOR]

Murray Valley Encephalitis virus (MVEV) is a mosquito-borne Flavivirus. Clinical presentation is rare but severe, with a case fatality rate of 15–30%. Here we report a case of MVEV from the cerebrospinal fluid (CSF) of a patient in the Northern Territory in Australia. Initial diagnosis was performed using both MVEV-specific real-time, and Pan-Flavivirus conventional, Polymerase Chain Reaction (PCR), with confirmation by Sanger sequencing. Subsequent isolation, the first from CSF, was conducted in Vero cells and the observed cytopathic effect was confirmed by increasing viral titre in the real-time PCR. Isolation allowed for full genome sequencing using the Scriptseq V2 RNASeq library preparation kit. A consensus genome for VIDRL-MVE was generated and phylogenetic analysis identified it as Genotype 2. This is the first reported isolation, and full genome sequencing of MVEV from CSF. It is also the first time Genotype 2 has been identified in humans. As such, this case has significant implications for public health surveillance, epidemiology, and the understanding of MVEV evolution. [ABSTRACT FROM AUTHOR]

We describe a fatal case of Japanese encephalitis virus infection following short-term travel to Thailand. Viral RNA was detected in urine and whole blood out to 26 and 28 days, respectively, after the onset of symptoms. Live virus was isolated from a urine specimen from day 14. [ABSTRACT FROM AUTHOR]

Human parechovirus types 1-16 (HPeV1-16) are positive strand RNA viruses in the family Picornaviridae. We investigated a 2015 outbreak of HPeV3 causing illness in infants in Victoria, Australia. Virus genome was extracted from clinical material and isolates and sequenced using a combination of next generation and Sanger sequencing. The HPeV3 outbreak genome was 98.7% similar to the HPeV3 Yamagata 2011 lineage for the region encoding the structural proteins up to nucleotide position 3115, but downstream of that the genome varied from known HPeV sequences with a similarity of 85% or less. Analysis indicated that recombination had occurred, may have involved multiple types of HPeV and that the recombination event/s occurred between March 2012 and November 2013. However the origin of the genome downstream of the recombination site is unknown. Overall, the capsid of this virus is highly conserved, but recombination provided a different non-structural protein coding region that may convey an evolutionary advantage. The indication that the capsid encoding region is highly conserved at the amino acid level may be helpful in directing energy towards the development of a preventive vaccine for expecting mothers or antibody treatment of young infants with severe disease. [ABSTRACT FROM AUTHOR]

Whole genome phylogenetic analysis in this study resolved a total of five major genotypes among the 22 varicella-zoster virus (VZV) strains or isolates for which complete genomic sequences are available. Consistent with earlier publications we have designated these genotypes European 1 (E1), European 2 (E2), Japanese (J), mosaic 1 (M1), and mosaic 2 (M2). Single nucleotide polymorphism (SNP) analysis performed in a whole-genome alignment revealed that VZV isolates of all five genotypes can be accurately genotyped using SNPs from two amplicons: open reading frame 22 (ORF22) and either ORF21 or ORF50. This modified approach identifies all of the genotypes observed using any of the published genotyping protocols. Of 165 clinical varicella and zoster isolates from Australia and New Zealand typed using this approach, 67 of 127 eastern Australian isolates were E1, 30 were E2, 16 were J, 10 were M1, and 4 were M2; 25 of 38 New Zealand isolates were E1, 8 were E2, and 5 were M1. VZV strain diversity in eastern Australia is thus broader than has been described for any other region, including Europe, Africa, and North America. J strains were far more prevalent than previously observed in countries other than Japan. Two-amplicon typing was in complete accord with genotypes derived using SNP in multiple ORFs (ORFs 1, 21, 22, 38, 50, 54, and 62). Two additional minor genotypes, M3 and M4, could also be resolved using two-amplicon typing. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus (SARS-CoV) 7a protein, which is not expressed by other known coronaviruses, can induce apoptosis in various cell lines. In this study, we show that the overexpression of Bcl-XL, a prosurvival member of the Bcl-2 family, blocks 7a-induced apoptosis, suggesting that the mechanism for apoptosis induction by 7a is at the level of or upstream from the Bcl-2 family. Coimmunoprecipitation experiments showed that 7a interacts with Bcl-XL and other prosurvival proteins (Bcl-2, Bcl-w, Mcl-1, and A1) but not with the proapoptotic proteins (Bax, Bak, Bad, and Bid). A good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with Bcl-XL was observed, suggesting that 7a triggers apoptosis by interfering directly with the prosurvival function of Bcl-XL. Interestingly, amino acids 224 and 225 within the C-terminal transmembrane domain of Bcl-XL are essential for the interaction with the 7a protein, although the BH3 domain of Bcl-XL also contributes to this interaction. In addition, fractionation experiments showed that 7a colocalized with Bcl-XL at the endoplasmic reticulum as well as the mitochondria, suggesting that they may form complexes in different membranous compartments. [ABSTRACT FROM AUTHOR]