Individual bat viromes reveal the co-infection, spillover and emergence risk of potential zoonotic viruses (preprint)

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.

The enteric virome of cats with feline panleukopenia differs in abundance and diversity from healthy cats (preprint)

Feline panleukopenia (FPL) is a severe, often fatal disease caused by feline parvovirus (FPV). How infection with FPV might impact the composition of the entire eukaryotic enteric virome in cats has not been characterized. We used metatranscriptomic 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 were detected from 11 mammalian infecting viral families mostly belonging to Coronaviridae, Parvoviridae and Astroviridae . Among the healthy control cats the most abundant viruses were Feline coronavirus, Mamastrovirus 2 and Carnivore bocaparvovirus 3 (Feline bocavirus 1) with frequent co-infections of all three. Feline chaphamaparvovirus was only detected in healthy controls (6/36, 16.7%). Among the FPV-cases, in addition to FPV, the most abundant viruses were Mamastrovirus 2 , Feline coronavirus and Carnivore 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 high proportion of FPV-cases (11/23, 47.8%) compared to healthy controls (5/36, 13.9%, p=0.0067). Feline kobuvirus infections were also common among FPV-cases (9/23, 39.1%) and were not detected in any healthy control cats (p<0.0001). While abundant in both groups, astroviruses were more frequently present in FPV-cases (19/23, 82.6%) than in healthy controls (18/36, p=0.0142). The differences in eukaryotic virome composition found in this study indicate that further investigations to determine associations between enteric viral co-infections on clinical disease severity in cats with FPL are warranted.

Total virome characterizations of game animals in China reveals a spectrum of emerging viral pathogens (preprint)

Game animals are wildlife species often traded and consumed as exotic food, and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1725 game animals, representing 16 species and five mammalian orders, sampled across China. From this we identified 71 mammalian viruses, with 45 described for the first time. Eighteen viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high risk viruses. We identified the transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We similarly identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence.

Total Infectomes Characterization of Respiratory Infections in pre-COVID-19 Wuhan, China (preprint)

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 - SARS-CoV-2. Herein, 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. Consequently, we identified 37 pathogen species, comprising 15 RNA viruses, 3 DNA viruses, 16 bacteria and 3 fungi, often at high abundance and including multiple co-infections (12.8%). However, 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.

Co-Infecting Pathogens Can Contribute to Inflammatory Responses and Severe Symptoms in COVID-19 (preprint)

Background: The current pandemic of COVID-19 is posing a major challenge to public health on a global scale. While it is generally believed 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 disease in severe COVID-19 cases to reveal 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 disease.Methods: We considered 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) taken in each case. Total RNA sequencing (meta-transcriptomics) was performed to simultaneously reveal pathogen diversity and abundance, as well as host immune responses, within each sample. A Bio-Plex method was used to measure serum cytokine and chemokine levels.Findings: Eight pathogens were identified in these COVID-19 patients - Aspergillus fumigatus, Mycoplasma orale, Myroides odorantus, Acinetobacter baumannii, Candida tropicalis, herpes simplex virus and human cytomegalovirus - that appeared at different stages of disease course. Notably, the dynamics of inflammatory mediators in the serum as well as respiratory tract were better associated with the dynamics of the infectome as a whole rather than SARS-CoV-2 alone. Correlation analysis revealed that pulmonary injury was directly associated with cytokine levels, which in turn was associated with the proliferation of SARS-CoV-2 and the co-infecting pathogens.Interpretation: The cytokine storm that resulted in aggravated acute lung injury and death involved the highly complex and dynamic entire infectome of each patient, of which SARS-CoV-2 was a component. These results call for a precision-medicine approach to investigating both the infection and the host response on a daily basis as a standard means of infectious disease characterization.Funding: Guangzhou Institute of Respiratory Health Open Project (Funds provided by China Evergrande Group) - Project No. (2020GIRHHMS01), Guangdong Province “Pearl River Talent Plan” Innovation and Entrepreneurship Team Project (2019ZT08Y464), Macao Science and Technology Development Fund (0042/2020/A), Science research project of the Guangdong Province (2019B030316028), Special Project for Scientific and Technological Development and Emergency Response in COVID-19 Prevention and Control of Guangdong Province (2020A111129028), Special Project for Research and Promotion of Prevention and Control Techniques of COVID-19 and Emergency Response in Dongguan City (202071715001114), Jack Ma Foundation (2020-CMKYGG-02), Guangzhou Medical University High-level University Clinical Research and Cultivation Program ([2017] 159 and 160) and ARC Australian Laureate Fellowship (FL170100022).Declaration of Interests: We declare no competing interests.Ethics Approval Statement: The ethics committee of the FAHGMU (Ethics No. 2020-85) and Dongguan’s People’s Hospital (KYKT2020-005-A1) approved the sampling procedure and the use of patient samples for this study. Informed consent was obtained from each patient.

Risk factors for the critical illness in SARS-CoV-2 infection: a multicenter retrospective cohort study (preprint)

Background: Prior studies reported that 5~32% COVID-19 patients were critically ill, a situation that poses great challenge for the management of the patients and ICU resources. We aim to identify independent risk factors to serve as prediction markers for critical illness of SARS-CoV-2 infection. Methods Fifty-two critical and 200 non-critical SARS-CoV-2 nucleic acid positive patients hospitalized in 15 hospitals outside Wuhan from January 19 to March 6, 2020 were enrolled in this study. Multivariable logistic regression and LASSO logistic regression were performed to identify independent risk factors for critical illness. Results Age older than 60 years, dyspnea, respiratory rate > 24 breaths per min, leukocytosis >9.5 X10 9 /L, neutrophilia >6.3 X10 9 /L, lymphopenia <1.1 X10 9 /L, neutrophil-to-lymphocyte ratio >3.53, fibrinogen >4g/L, d-dimer >0.55 µg/mL, blood urea nitrogen >7.1 mM, elevated aspartate transaminase, elevated alanine aminotransferase, total bilirubin >21 µM, and Sequential Organ Failure Assessment (SOFA) score ≥2 were identified as risk factors for critical illness. LASSO logistic regression identified the best combination of risk factors as SOFA score, age, dyspnea, and leukocytosis. The Area Under the Receiver-Operator Curve values for the risk factors in predicting critical illness were 0.921 for SOFA score, 0.776 for age, 0.764 for dyspnea, 0.658 for leukocytosis, and 0.960 for the combination of the four risk factors. Conclusions Our findings advocate the use of risk factors SOFA score ≥2, age >60, dyspnea and leukocytosis >9.5 X10 9 /L on admission, alone or in combination, to determine the optimal management of the patients and health care resources.