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An array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.

The mutation profile of the SARS-CoV-2 Omicron (lineage BA.1) variant posed a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2 ancestral isolate (Australia/VIC01/2020, VIC01) to protect against disease caused by BA.1. We established that BA.1 infection in naïve Syrian hamsters resulted in a less severe disease than a comparable dose of the ancestral virus, with fewer clinical signs including less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of BA.1 50 days after an initial infection with ancestral virus. These data provide evidence that convalescent immunity against ancestral SARS-CoV-2 is protective against BA.1 in the Syrian hamster model of infection. Comparison with published pre-clinical and clinical data supports consistency of the model and its predictive value for the outcome in humans. Further, the ability to detect protection against the less severe disease caused by BA.1 demonstrates continued value of the Syrian hamster model for evaluation of BA.1-specific countermeasures.

The mutation profile of the SARS-CoV-2 Omicron variant poses a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2, 99.99% identical to Wuhan-Hu-1, to protect against disease caused by the Omicron variant. We established that infection with Omicron in naïve Syrian hamsters resulted in a less severe disease than a comparable dose of prototype SARS-CoV-2 (Australia/VIC01/2020), with fewer clinical signs and less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of Omicron 50 days after an initial infection with Australia/VIC01/2020. The data provide evidence for immunity raised against prototype SARS-CoV-2 being protective against Omicron in the Syrian hamster model. Further investigation is required to conclusively determine whether Omicron is less pathogenic in Syrian hamsters and whether this is predictive of pathogenicity in humans.

LamPORE is a novel diagnostic platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyze thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against reverse transcriptase PCR (RT-PCR) using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 10 genome copies/μl of extracted RNA, which is above the limit achievable by RT-PCR, but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among them, LamPORE had a diagnostic sensitivity of 99.1% (226/228; 95% confidence interval [CI], 96.9% to 99.9%). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279; 98.0% to 100.0%). Overall, 1.4% (7/514; 0.5% to 2.9%) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494; 94.8% to 98.1%). LamPORE has a similar performance as RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients and offers a promising approach to high-throughput testing.

The implementation of pneumococcal conjugate vaccines (PCVs) has led to a decline in vaccine-type disease. However, there is evidence that the epidemiology of non-typeable Haemophilus influenzae (NTHi) carriage and disease can be altered as a consequence of PCV introduction. We explored the epidemiological shifts in NTHi carriage using whole genome sequencing over a 5-year period that included PCV13 replacement of PCV7 in the UK's National Immunization Programme in 2010. Between 2008/09 and 2012/13 (October to March), nasopharyngeal swabs were taken from children5 years of age. Significantly increased carriage post-PCV13 was observed and lineage-specific associations with Streptococcus pneumoniae were seen before but not after PCV13 introduction. NTHi were characterized into 11 discrete, temporally stable lineages, congruent with current knowledge regarding the clonality of NTHi. The increased carriage could not be linked to the expansion of a particular clone and different co-carriage dynamics were seen before PCV13 implementation when NTHi co-carried with vaccine serotype pneumococci. In summary, PCV13 introduction has been shown to have an indirect effect on NTHi epidemiology and there exists both negative and positive, distinct associations between pneumococci and NTHi. This should be considered when evaluating the impacts of pneumococcal vaccine design and policy.

Recently, a new variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) designated VOC 202012/01 (or B.1.1.7 lineage) has become highly prevalent in several countries, after first being described in the United Kingdom (UK). Its rate of transmission has been estimated to be increased compared to other lineages. In the present study, we show the emergence, dominance and the rapid spread of the B.1.1.7 lineage in Lebanon.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5–15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease., SARS-CoV-2 induces mild infection in ferret model. Here, Ryan et al. characterise optimal infection dosage inducing upper respiratory tract (UTR) viral shedding, progression time of viral shedding, and pathology in ferrets and finally provide evidence for protection after re-challenge.

In December 2019 an outbreak of coronavirus disease (COVID-19) emerged in Wuhan, China. The causative agent was subsequently identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread worldwide causing a pandemic. Currently there are no licensed vaccines or therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in development and repurposed drugs are being tested in the clinic. There is a vital need for authentic COVID-19 animal models to further our understanding of pathogenesis and viral spread in addition to pre-clinical evaluation of candidate interventions.Here we report a dose titration study of SARS-CoV-2 to determine the most suitable infectious dose to use in the ferret model. We show that a high (5×106 pfu) and medium (5×104 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral RNA shedding in both groups of six challenged animals, whilst a low dose (5×102 pfu) resulted in only one of six displaying signs of URT viral RNA replication. The URT shedding lasted up to 21 days in the high dose animals with intermittent positive signal from day 14. Sequential culls revealed distinct pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were observed although animals did present with a consistent post-viral fatigue lasting from day 9-14 in the medium and high dose groups. After virus shedding ceased, re-challenged ferrets were shown to be fully protected from acute lung pathology. The endpoints of URT viral RNA replication in addition to distinct lung pathology and post viral fatigue were observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 14-21 parallel observations reported in a minority of clinical cases.

The airway macrophage is crucial for immune surveillance and pathogen clearance, however, this function is impaired in chronic respiratory diseases such as COPD and asthma. This may lead to colonization of the airways by Nontypeable Haemophilus influenzae (NTHi). NTHi is a heterogeneous organism and this heterogeneity could result in strain-dependent differences in macrophage responses, leading to airway persistence of certain NTHi strains. The aim of this work was to investigate macrophage responses to different clinical strains of NTHi. Strain diversity assessment of eight NTHi clinical isolates was performed using ParSNP. Subsequently, three strains (ST14, 201 and 408) were chosen to infect monocyte-derived macrophages (MDM) isolated from the blood of healthy volunteers to investigate expression of inflammatory pathways by PCR or ELISA. Measurement of NTHi hel gene expression revealed increased presence of ST14 and ST408 compared to ST201 (p This work suggests ST14 elicits a differential MDM inflammatory response compared to ST201 and ST408. These differences highlight that NTHi heterogeneity must be further explored in order to comprehend the role of NTHi in chronic respiratory disease.

The implementation of pneumococcal conjugate vaccines (PCVs) has led to a decline in vaccine-type disease. However, there is evidence that the epidemiology of non-typeable Haemophilus influenzae (NTHi) carriage and disease can be altered as a consequence of PCV introduction. We explored the epidemiological shifts in NTHi carriage using whole genome sequencing over a 5-year period that included PCV13 replacement of PCV7 in the UK’s National Immunization Programme in 2010. Between 2008/09 and 2012/13 (October to March), nasopharyngeal swabs were taken from children

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen of the respiratory tract and the greatest contributor to invasive Haemophilus disease. Additionally, in children, NTHi is responsible for the majority of otitis media (OM) which can lead to chronic infection and hearing loss. In adults, NTHi infection in the lungs is responsible for the onset of acute exacerbations in chronic obstructive pulmonary disease (COPD). Unfortunately, there is currently no vaccine available to protect against NTHi infections. Areas covered: NTHi uses an arsenal of adhesins to colonise the respiratory epithelium. The adhesins also have secondary roles that aid in the virulence of NTHi, including mechanisms that avoid immune clearance, adjust pore size to avoid antimicrobial destruction, form micro-colonies and invoke phase variation for protein mediation. Bacterial adhesins can also be ideal antigens for subunit vaccine design due to surface exposure and immunogenic capabilities. Expert commentary: The host-pathogen interactions of the NTHi adhesins are not fully investigated. The relationship between adhesins and the extracellular matrix (ECM) play a part in the success of NTHi colonisation and virulence by immune evasion, migration and biofilm development. Further research into these immunogenic proteins would further our understanding and enable a basis for better combatting NTHi disease.

H. haemolyticus is often misidentified as NTHi due to their close phylogenetic relationship. Differentiating between the two is important for correct identification and appropriate treatment of infective organism and to ensure any role of H. haemolyticus in disease is not being overlooked. Speciation however is not completely reliable by culture and PCR methods due to the loss of haemolysis by H. haemolyticus and the heterogeneity of NTHi. Haemophilus isolates from COPD as part of the AERIS study (ClinicalTrials - NCT01360398) were speciated by analysing sequence data for the presence of molecular markers. Further investigation into the genomic relationship was carried out using average nucleotide identity and phylogeny of allelic and genome alignments. Only 6.3% were identified as H. haemolyticus. Multiple in silico methods were able to distinguish H. haemolyticus from NTHi. However, no single gene target was found to be 100% accurate. A group of omp2 negative NTHi were observed to be phylogenetically divergent from H. haemolyticus and remaining NTHi. The presence of an atypical group from a geographically and disease limited set of isolates supports the theory that the heterogeneity of NTHi may provide a genetic continuum between NTHi and H. haemolyticus.