Pathogenicity, tissue tropism and potential vertical transmission of SARSr-CoV-2 in Malayan pangolins.

Malayan pangolin SARS-CoV-2-related coronavirus (SARSr-CoV-2) is closely related to SARS-CoV-2. However, little is known about its pathogenicity in pangolins. Using CT scans we show that SARSr-CoV-2 positive Malayan pangolins are characterized by bilateral ground-glass opacities in lungs in a similar manner to COVID-19 patients. Histological examination and blood gas tests are indicative of dyspnea. SARSr-CoV-2 infected multiple organs in pangolins, with the lungs the major target, and histological expression data revealed that ACE2 and TMPRSS2 were co-expressed with viral RNA. Transcriptome analysis indicated that virus-positive pangolins were likely to have inadequate interferon responses, with relative greater cytokine and chemokine activity in the lung and spleen. Notably, both viral RNA and viral proteins were detected in three pangolin fetuses, providing initial evidence for vertical virus transmission. In sum, our study outlines the biological framework of SARSr-CoV-2 in pangolins, revealing striking similarities to COVID-19 in humans.

Zoonotic risk assessment among farmed mammals.

Farmed mammals may act as hosts for zoonotic viruses that can cause disease outbreaks in humans. This SnapShot shows which farmed mammals, and to what extent, are of particular risk of harboring and spreading viruses from viral families that are commonly associated with zoonotic disease. It also discusses genome surveillance methods and biosafety measures. To view this SnapShot, open or download the PDF.

Nonsystematic Reporting Biases of the SARS-CoV-2 Variant Mu Could Impact Our Understanding of the Epidemiological Dynamics of Emerging Variants.

Developing a timely and effective response to emerging SARS-CoV-2 variants of concern (VOCs) is of paramount public health importance. Global health surveillance does not rely on genomic data alone to identify concerning variants when they emerge. Instead, methods that utilize genomic data to estimate the epidemiological dynamics of emerging lineages have the potential to serve as an early warning system. However, these methods assume that genomic data are uniformly reported across circulating lineages. In this study, we analyze differences in reporting delays among SARS-CoV-2 VOCs as a plausible explanation for the timing of the global response to the former VOC Mu. Mu likely emerged in South America in mid-2020, where its circulation was largely confined. In this study, we demonstrate that Mu was designated as a VOC ∼1 year after it emerged and find that the reporting of genomic data for Mu differed significantly than that of other VOCs within countries, states, and individual laboratories. Our findings suggest that nonsystematic biases in the reporting of genomic data may have delayed the global response to Mu. Until they are resolved, the surveillance gaps that affected the global response to Mu could impede the rapid and accurate assessment of future emerging variants.

The Ecology of Viral Emergence.

The coronavirus disease 2019 (COVID-19) pandemic has had a profound impact on human health, economic well-being, and societal function. It is essential that we use this generational experience to better understand the processes that underpin the emergence of COVID-19 and other zoonotic diseases. Herein, I review the mechanisms that determine why and how viruses emerge in new hosts, as well as the barriers to this process. I show that traditional studies of virus emergence have an inherent anthropocentric bias, with disease in humans considered the inevitable outcome of virus emergence, when in reality viruses are integral components of a global ecosystem characterized by continual host jumping with humans also transmitting their viruses to other animals. I illustrate these points using coronaviruses, including severe acute respiratory syndrome coronavirus 2, as a case study. I also outline the potential steps that can be followed to help mitigate and prevent future pandemics, with combating climate change a central component.

Human-to-human transmission of Chlamydia psittaci in China, 2020: an epidemiological and aetiological investigation.

BACKGROUND: Chlamydia psittaci can infect a wide range of avian species, occasionally causing psittacosis (also known as parrot fever) in humans. Most human psittacosis cases are associated with close contact with pet birds or poultry. In December, 2020, an outbreak of severe community-acquired pneumonia of unknown aetiology was reported in a hospital in Shandong province, China, and some of the patients' close contacts had respiratory symptoms. Our aims were to determine the causative agent of this epidemic and whether there had been human-to-human transmission. METHODS: For this epidemiological and aetiological investigation study, we enrolled patients who had community-acquired pneumonia confirmed by chest CT at two local hospitals in Shandong Province in China. We collected sputum, bronchoalveolar lavage fluid, and nasopharyngeal swab samples from participants and detected pathogens by surveying for 22 target respiratory microbes using a commercial assay, followed by metagenomic next-generation sequencing, specific nested PCR, and qPCR tests. We excluded individuals who were C psittaci-negative on both tests. We recruited close contacts of the C psittaci-positive patients, and tested nasopharyngeal swabs from the close contacts and samples from ducks from the processing plant where these patients worked. We then integrated the epidemiological, clinical, and laboratory data to reveal the potential chain of transmission of C psittaci that characterised this outbreak. FINDINGS: Between Dec 4 and 29, 2020, we used metagenomic next-generation sequencing and different PCR-based approaches to test 12 inpatients with community-acquired pneumonia, of whom six (50%) were workers at a duck-meat processing plant and two (17%) were unemployed people, who were positive for C psittaci and enrolled in this study. We contacted 61 close contacts of the six patients who worked at the duck-meat processing plant, of whom 61 (100%) were enrolled and tested, and we determined that the community-acquired pneumonia outbreak was caused by C psittaci. Within the outbreak cluster, 17 (77%) of 22 participants had confirmed C psittaci infections and five (23%) of 22 participants were asymptomatic C psittaci carriers. The outbreak had begun with avian-to-human transmission, and was followed by secondary and tertiary human-to-human transmission, which included transmission by several asymptomatic carriers and by health-care workers. In addition, some of the participants with confirmed C psittaci infection had no identified source of infection, which suggested cryptic bacterial transmission. INTERPRETATION: Our study data might represent the first documented report of human-to-human transmission of C psittaci in China. Therefore, C psittaci has the potential to evolve human-to-human transmission via various routes, should be considered an elevated biosecurity and emergent risk, and be included as part of the routine diagnosis globally, especially for high-risk populations. FUNDING: Academic Promotion Programme of Shandong First Medical University, National Science and Technology Major Project, ARC Australian Laureate Fellowship.

Faecal virome of the Australian grey-headed flying fox from urban/suburban environments contains novel coronaviruses, retroviruses and sapoviruses.

Bats are important reservoirs for viruses of public health and veterinary concern. Virus studies in Australian bats usually target the families Paramyxoviridae, Coronaviridae and Rhabdoviridae, with little known about their overall virome composition. We used metatranscriptomic sequencing to characterise the faecal virome of grey-headed flying foxes from three colonies in urban/suburban locations from two Australian states. We identified viruses from three mammalian-infecting (Coronaviridae, Caliciviridae, Retroviridae) and one possible mammalian-infecting (Birnaviridae) family. Of particular interest were a novel bat betacoronavirus (subgenus Nobecovirus) and a novel bat sapovirus (Caliciviridae), the first identified in Australian bats, as well as a potentially exogenous retrovirus. The novel betacoronavirus was detected in two sampling locations 1375 km apart and falls in a viral lineage likely with a long association with bats. This study highlights the utility of unbiased sequencing of faecal samples for identifying novel viruses and revealing broad-scale patterns of virus ecology and evolution.

Zoonotic disease and virome diversity in bats.

The emergence of zoonotic viral diseases in humans commonly reflects exposure to mammalian wildlife. Bats (order Chiroptera) are arguably the most important mammalian reservoir for zoonotic viruses, with notable examples including Severe Acute Respiratory Syndrome coronaviruses 1 and 2, Middle East Respiratory Syndrome coronavirus, henipaviruses and lyssaviruses. Herein, we outline our current knowledge on the diversity of bat viromes, particularly through the lens of metagenomic next-generation sequencing and in the context of disease emergence. A key conclusion is that although bats harbour abundant virus diversity, the vast majority of bat viruses have not emerged to cause disease in new hosts such that bats are better regarded as critical but endangered components of global ecosystems.

The Huanan Seafood Wholesale Market in Wuhan was the early epicenter of the COVID-19 pandemic.

Understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 is critical to preventing future zoonotic outbreaks before they become the next pandemic. The Huanan Seafood Wholesale Market in Wuhan, China, was identified as a likely source of cases in early reports, but later this conclusion became controversial. We show here that the earliest known COVID-19 cases from December 2019, including those without reported direct links, were geographically centered on this market. We report that live SARS-CoV-2-susceptible mammals were sold at the market in late 2019 and that within the market, SARS-CoV-2-positive environmental samples were spatially associated with vendors selling live mammals. Although there is insufficient evidence to define upstream events, and exact circumstances remain obscure, our analyses indicate that the emergence of SARS-CoV-2 occurred through the live wildlife trade in China and show that the Huanan market was the epicenter of the COVID-19 pandemic.

The molecular epidemiology of multiple zoonotic origins of SARS-CoV-2.

Understanding the circumstances that lead to pandemics is important for their prevention. We analyzed the genomic diversity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) early in the coronavirus disease 2019 (COVID-19) pandemic. We show that SARS-CoV-2 genomic diversity before February 2020 likely comprised only two distinct viral lineages, denoted "A" and "B." Phylodynamic rooting methods, coupled with epidemic simulations, reveal that these lineages were the result of at least two separate cross-species transmission events into humans. The first zoonotic transmission likely involved lineage B viruses around 18 November 2019 (23 October to 8 December), and the separate introduction of lineage A likely occurred within weeks of this event. These findings indicate that it is unlikely that SARS-CoV-2 circulated widely in humans before November 2019 and define the narrow window between when SARS-CoV-2 first jumped into humans and when the first cases of COVID-19 were reported. As with other coronaviruses, SARS-CoV-2 emergence likely resulted from multiple zoonotic events.

Co-infection with SARS-CoV-2 Omicron and Delta variants revealed by genomic surveillance.

Co-infections with different variants of SARS-CoV-2 are a key precursor to recombination events that are likely to drive SARS-CoV-2 evolution. Rapid identification of such co-infections is required to determine their frequency in the community, particularly in populations at-risk of severe COVID-19, which have already been identified as incubators for punctuated evolutionary events. However, limited data and tools are currently available to detect and characterise the SARS-CoV-2 co-infections associated with recognised variants of concern. Here we describe co-infection with the SARS-CoV-2 variants of concern Omicron and Delta in two epidemiologically unrelated adult patients with chronic kidney disease requiring maintenance haemodialysis. Both variants were co-circulating in the community at the time of detection. Genomic surveillance based on amplicon- and probe-based sequencing using short- and long-read technologies identified and quantified subpopulations of Delta and Omicron viruses in respiratory samples. These findings highlight the importance of integrated genomic surveillance in vulnerable populations and provide diagnostic pathways to recognise SARS-CoV-2 co-infection using genomic data.

The enteric virome of cats with feline panleukopenia differs in abundance and diversity from healthy cats.

Feline panleukopenia (FPL) is a severe, often fatal disease caused by feline panleukopenia virus (FPV). How infection with FPV might impact the composition of the entire eukaryotic enteric virome in cats has not been characterized. We used meta-transcriptomic and viral particle enrichment metagenomic approaches to characterize the enteric viromes of 23 cats naturally infected with FPV (FPV-cases) and 36 age-matched healthy shelter cats (healthy controls). Sequencing reads from mammalian infecting viral families largely belonged to the Coronaviridae, Parvoviridae and Astroviridae. The most abundant viruses among the healthy control cats were feline coronavirus, Mamastrovirus 2 and Carnivore bocaparvovirus 3 (feline bocavirus), with frequent coinfections of all three. Feline chaphamaparvovirus was only detected in healthy controls (6 out of 36, 16.7%). Among the FPV-cases, in addition to FPV, the most abundant viruses were Mamastrovirus 2, feline coronavirus and C. bocaparvovirus 4 (feline bocaparvovirus 2). The latter and feline bocaparvovirus 3 were detected significantly more frequently in FPV-cases than in healthy controls. Feline calicivirus was present in a higher proportion of FPV-cases (11 out of 23, 47.8%) compared to healthy controls (5 out of 36, 13.9%, p = 0.0067). Feline kobuvirus infections were also common among FPV-cases (9 out of 23, 39.1%) and were not detected in any healthy controls (p < .0001). While abundant in both groups, astroviruses were more frequently present in FPV-cases (19 out of 23, 82.6%) than in healthy controls (18 out of 36, p = .0142). The differences in eukaryotic virome composition revealed here indicate that further investigations are warranted to determine associations between enteric viral co-infections on clinical disease severity in cats with FPL.

Off-season RSV epidemics in Australia after easing of COVID-19 restrictions.

Human respiratory syncytial virus (RSV) is an important cause of acute respiratory infection with the most severe disease in the young and elderly. Non-pharmaceutical interventions and travel restrictions for controlling COVID-19 have impacted the circulation of most respiratory viruses including RSV globally, particularly in Australia, where during 2020 the normal winter epidemics were notably absent. However, in late 2020, unprecedented widespread RSV outbreaks occurred, beginning in spring, and extending into summer across two widely separated regions of the Australian continent, New South Wales (NSW) and Australian Capital Territory (ACT) in the east, and Western Australia. Through genomic sequencing we reveal a major reduction in RSV genetic diversity following COVID-19 emergence with two genetically distinct RSV-A clades circulating cryptically, likely localised for several months prior to an epidemic surge in cases upon relaxation of COVID-19 control measures. The NSW/ACT clade subsequently spread to the neighbouring state of Victoria and to cause extensive outbreaks and hospitalisations in early 2021. These findings highlight the need for continued surveillance and sequencing of RSV and other respiratory viruses during and after the COVID-19 pandemic, as mitigation measures may disrupt seasonal patterns, causing larger or more severe outbreaks.

Diagnosis and analysis of unexplained cases of childhood encephalitis in Australia using metatranscriptomic sequencing.

Encephalitis is most often caused by a variety of infectious agents identified through diagnostic tests utilizing cerebrospinal fluid. We investigated the clinical characteristics and potential aetiological agents of unexplained encephalitis through metagenomic sequencing of residual clinical samples from multiple tissue types and independent clinical review. Forty-three specimens were collected from 18 encephalitis cases with no cause identified by the Australian Childhood Encephalitis study. Samples were subjected to total RNA sequencing ('metatranscriptomics') to determine the presence and abundance of potential pathogens, and to describe the possible aetiologies of unexplained encephalitis. Using this protocol, we identified five RNA and two DNA viruses associated with human infection from both non-sterile and sterile sites, which were confirmed by PCR. These comprised two human rhinoviruses, two human seasonal coronaviruses, two polyomaviruses and one picobirnavirus. Human rhinovirus and seasonal coronaviruses may be responsible for five of the encephalitis cases. Immune-mediated encephalitis was considered likely in six cases and metatranscriptomics did not identify a possible pathogen in these cases. The aetiology remained unknown in nine cases. Our study emphasizes the importance of respiratory viruses in the aetiology of unexplained child encephalitis and suggests that non-central-nervous-system sampling in encephalitis clinical guidelines and protocols could improve the diagnostic yield.

Replacement of the Gamma by the Delta variant in Brazil: Impact of lineage displacement on the ongoing pandemic.

The coronavirus disease 2019 (COVID-19) epidemic in Brazil was driven mainly by the spread of Gamma (P.1), a locally emerged variant of concern (VOC) that was first detected in early January 2021. This variant was estimated to be responsible for more than 96 per cent of cases reported between January and June 2021, being associated with increased transmissibility and disease severity, a reduction in neutralization antibodies and effectiveness of treatments or vaccines, and diagnostic detection failure. Here we show that, following several importations predominantly from the USA, the Delta variant rapidly replaced Gamma after July 2021. However, in contrast to what was seen in other countries, the rapid spread of Delta did not lead to a large increase in the number of cases and deaths reported in Brazil. We suggest that this was likely due to the relatively successful early vaccination campaign coupled with natural immunity acquired following prior infection with Gamma. Our data reinforce reports of the increased transmissibility of the Delta variant and, considering the increasing concern due to the recently identified Omicron variant, argues for the necessity to strengthen genomic monitoring on a national level to quickly detect the emergence and spread of other VOCs that might threaten global health.

Co-infecting pathogens can contribute to inflammatory responses and severe symptoms in COVID-19.

Background: The current COVID-19 pandemic is posing a major challenge to public health on a global scale. While it is generally believed that severe COVID-19 results from over-expression of inflammatory mediators (i.e., a "cytokine storm"), it is still unclear whether and how co-infecting pathogens contribute to disease pathogenesis. To address this, we followed the entire course of the disease in cases with severe or critical COVID-19 to determine the presence and abundance of all potential pathogens present-the total "infectome"-and how they interact with the host immune system in the context of severe COVID-19. Methods: We examined one severe and three critical cases of COVID-19, as well as a set of healthy controls, with longitudinal samples (throat swab, whole blood, and serum) collected from each case. Total RNA sequencing (meta-transcriptomics) was performed to simultaneously investigate pathogen diversity and abundance, as well as host immune responses, in each sample. A Bio-Plex method was used to measure serum cytokine and chemokine levels. Results: Eight pathogens, SARS-CoV-2, Aspergillus fumigatus (A. fumigatus), Mycoplasma orale (M. orale), Myroides odoratus (M. odoratus), Acinetobacter baumannii (A. baumannii), Candida tropicalis, herpes simplex virus (HSV) and human cytomegalovirus (CMV), identified in patients with COVID-19 appeared at different stages of the disease. The dynamics of inflammatory mediators in serum and the respiratory tract were more strongly associated with the dynamics of the infectome compared with SARS-CoV-2 alone. Correlation analysis revealed that pulmonary injury was directly associated with cytokine levels, which in turn were associated with the proliferation of SARS-CoV-2 and co-infecting pathogens. Conclusions: For each patient, the cytokine storm that resulted in acute lung injury and death involved a dynamic and highly complex infectome, of which SARS-CoV-2 was a component. These results indicate the need for a precision medicine approach to investigate both the infection and host response as a standard means of infectious disease characterization.

Surveillance of Rodent Pests for SARS-CoV-2 and Other Coronaviruses, Hong Kong.

We report surveillance conducted in 217 pestiferous rodents in Hong Kong for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We did not detect SARS-CoV-2 RNA but identified 1 seropositive rodent, suggesting exposure to a virus antigenically similar to SARS-CoV-2. Potential exposure of urban rodents to SARS-CoV-2 cannot be ruled out.

COVID-19-lessons for zoonotic disease.

Disease emergence is driven by human-animal contact in a global viral ecosystem.

Assessment of Coronavirus Disease 2019 Intervention Strategies in the Nordic Countries Using Genomic Epidemiology.

We explored how the duration, size, and number of virus transmission clusters, defined as country-specific monophyletic groups in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) phylogenetic tree, differed among the Nordic countries of Norway, Sweden, Denmark, Finland, and Iceland. Our results suggest that although geographical connectivity, population density, and openness influence the spread and the size of SARS-CoV-2 transmission clusters, the different country-specific intervention strategies had the largest impact. We also found a significant positive association between the size and duration of transmission clusters in the Nordic countries, suggesting that the rapid deployment of contact tracing is a key response measure in reducing virus transmission.

Total infectome characterization of respiratory infections in pre-COVID-19 Wuhan, China.

At the end of 2019 Wuhan witnessed an outbreak of "atypical pneumonia" that later developed into a global pandemic. Metagenomic sequencing rapidly revealed the causative agent of this outbreak to be a novel coronavirus denoted SARS-CoV-2. To provide a snapshot of the pathogens in pneumonia-associated respiratory samples from Wuhan prior to the emergence of SARS-CoV-2, we collected bronchoalveolar lavage fluid samples from 408 patients presenting with pneumonia and acute respiratory infections at the Central Hospital of Wuhan between 2016 and 2017. Unbiased total RNA sequencing was performed to reveal their "total infectome", including viruses, bacteria and fungi. We identified 35 pathogen species, comprising 13 RNA viruses, 3 DNA viruses, 16 bacteria and 3 fungi, often at high abundance and including multiple co-infections (13.5%). SARS-CoV-2 was not present. These data depict a stable core infectome comprising common respiratory pathogens such as rhinoviruses and influenza viruses, an atypical respiratory virus (EV-D68), and a single case of a sporadic zoonotic pathogen-Chlamydia psittaci. Samples from patients experiencing respiratory disease on average had higher pathogen abundance than healthy controls. Phylogenetic analyses of individual pathogens revealed multiple origins and global transmission histories, highlighting the connectedness of the Wuhan population. This study provides a comprehensive overview of the pathogens associated with acute respiratory infections and pneumonia, which were more diverse and complex than obtained using targeted PCR or qPCR approaches. These data also suggest that SARS-CoV-2 or closely related viruses were absent from Wuhan in 2016-2017.