Pathogenesis and transmission of SARS-CoV-2 in golden hamsters.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus with high nucleotide identity to SARS-CoV and to SARS-related coronaviruses that have been detected in horseshoe bats, has spread across the world and had a global effect on healthcare systems and economies1,2. A suitable small animal model is needed to support the development of vaccines and therapies. Here we report the pathogenesis and transmissibility of SARS-CoV-2 in golden (Syrian) hamsters (Mesocricetus auratus). Immunohistochemistry assay demonstrated the presence of viral antigens in nasal mucosa, bronchial epithelial cells and areas of lung consolidation on days 2 and 5 after inoculation with SARS-CoV-2, followed by rapid viral clearance and pneumocyte hyperplasia at 7 days after inoculation. We also found viral antigens in epithelial cells of the duodenum, and detected viral RNA in faeces. Notably, SARS-CoV-2 was transmitted efficiently from inoculated hamsters to naive hamsters by direct contact and via aerosols. Transmission via fomites in soiled cages was not as efficient. Although viral RNA was continuously detected in the nasal washes of inoculated hamsters for 14 days, the communicable period was short and correlated with the detection of infectious virus but not viral RNA. Inoculated and naturally infected hamsters showed apparent weight loss on days 6-7 post-inoculation or post-contact; all hamsters returned to their original weight within 14 days and developed neutralizing antibodies. Our results suggest that features associated with SARS-CoV-2 infection in golden hamsters resemble those found in humans with mild SARS-CoV-2 infections.

Avian Influenza A(H5N1) Neuraminidase Inhibition Antibodies in Healthy Adults after Exposure to Influenza A(H1N1)pdm09.

We detected high titers of cross-reactive neuraminidase inhibition antibodies to influenza A(H5N1) virus clade 2.3.4.4b in 96.8% (61/63) of serum samples from healthy adults in Hong Kong in 2020. In contrast, antibodies at low titers were detected in 42% (21/50) of serum samples collected in 2009. Influenza A(H1N1)pdm09 and A(H5N1) titers were correlated.

Reduced Pathogenicity and Transmission Potential of Omicron BA.1 and BA.2 Sublineages Compared with the Early Severe Acute Respiratory Syndrome Coronavirus 2 D614G Variant in Syrian Hamsters.

BACKGROUND: The epidemiological advantage of Omicron variant is evidenced by its rapid spread and the ability to outcompete prior variants. Among Omicron sublineages, early outbreaks were dominated by BA.1, while BA.2 has gained dominance since February 2022. The relative pathogenicity and transmissibility of BA.1 and BA.2 have not been fully defined. METHODS: We compared viral loads and clinical signs in Syrian hamsters after infection with BA.1, BA.2, or D614G variant. A competitive transmission model and next-generation sequencing were used to compare the relative transmission potential of BA.1 and BA.2. RESULTS: BA.1 and BA.2 caused no apparent clinical signs, while D614G caused more than 10% weight loss. Higher viral loads were detected in nasal wash samples and nasal turbinate and lung tissues from BA.1-inoculated hamsters compared with BA.2-inoculated hamsters. No aerosol transmission was observed for BA.1 or BA.2 under the experimental condition in which D614G transmitted efficiently. BA.1 and BA.2 were able to transmit among hamsters via direct contact; however, BA.1 transmitted more efficiently than BA.2 under the competitive transmission model. No recombination was detected from direct contacts exposed simultaneously to BA.1 and BA.2. CONCLUSIONS: Omicron BA.1 and BA.2 demonstrated attenuated pathogenicity and reduced transmission potential in hamsters compared with early SARS-CoV-2 strains.

Transmission of SARS-CoV-2 delta variant (AY.127) from pet hamsters to humans, leading to onward human-to-human transmission: a case study.

BACKGROUND: Transmission of SARS-CoV-2 from humans to other mammals, including pet animals, has been reported. However, with the exception of farmed mink, there is no previous evidence that these infected animals can infect humans, resulting in sustained human-to-human transmission. Following a confirmed SARS-CoV-2 infection of a pet shop worker, animals in the shop and the warehouse supplying it were tested for evidence of SARS-CoV-2 infection. METHODS: In this case study, viral swabs and blood samples were collected from animals in a pet shop and its corresponding warehouse in Hong Kong. Nasal swab or saliva samples from human COVID-19 patients epidemiologically linked to the pet shop and from subsequent local cases confirmed to be infected by SARS-CoV-2 delta variant were collected. Oral swabs were tested by quantitative RT-PCR (RT-qPCR) for SARS-CoV-2 and blood samples were serologically tested by a surrogate virus neutralisation test and plaque reduction neutralisation test. The SARS-CoV-2 RT-qPCR positive samples were sequenced by next generation viral full genome sequencing using the ISeq sequencing platform (Illumina), and the viral genomes were phylogenetically analysed. FINDINGS: Eight (50%) of 16 individually tested Syrian hamsters in the pet shop and seven (58%) of 12 Syrian hamsters in the corresponding warehouse were positive for SARS-CoV-2 infection in RT-qPCR or serological tests. None of the dwarf hamsters (n=75), rabbits (n=246), guinea pigs (n=66), chinchillas (n=116), and mice (n=2) were confirmed positive for SARS-CoV-2 in RT-qPCR tests. SARS-CoV-2 viral genomes deduced from human and hamster cases in this incident all belong to the delta variant of concern (AY.127) that had not been circulating locally before this outbreak. The viral genomes obtained from hamsters were phylogenetically related with some sequence heterogeneity. Phylogenetic dating suggests infection in these hamsters occurred around Oct 14, 2021 (95% CI Sept 15 to Nov 9, 2021). Multiple zoonotic transmission events to humans were detected, leading to onward human-to-human transmission. INTERPRETATION: Pet hamsters can be naturally infected with SARS-CoV-2. The virus can circulate among hamsters and lead to human infections. Both genetic and epidemiological results strongly suggest that there was more than one hamster-to-human transmission event in this study. This incident also led to onward human transmission. Importation of SARS-CoV-2-infected hamsters was a likely source of this outbreak. FUNDING: US National Institutes of Health, Research Grants Council of Hong Kong, Food and Health Bureau, and InnoHK.

A(H1N1)pdm09 Influenza Viruses Replicating in Ferret Upper or Lower Respiratory Tract Differed in Onward Transmission Potential by Air.

BACKGROUND: A(H1N1)pdm09 influenza viruses replicate efficiently in respiratory epithelia and are transmitted via respiratory droplets and aerosols expelled by infected hosts. The relative onward transmission potential of influenza viruses replicating in the upper and lower respiratory epithelial cells has not been fully defined. METHODS: Wild-type and barcoded A(H1N1)pdm09 viruses that differed by 2 synonymous mutations per gene segment were inoculated into ferrets via intranasal and intratracheal routes. Naive recipients were exposed to the exhaled breath of inoculated donors for 8 hours on day 2 postinoculation. Onward transmission potential of wild-type and barcoded genotypes were monitored by next generation sequencing. RESULTS: Transmissible airborne particles were respired from the upper but not the lower respiratory epithelial cells of donor ferrets. There was limited mixing of viral populations replicating in the upper and lower respiratory tissues. CONCLUSIONS: The ferret upper respiratory epithelium was mapped as the anatomic site that generated influenza virus-laden particles mediating onward transmission by air. Our results suggest that vaccines and antivirals should aim to reduce viral loads in the upper respiratory tract for prevention of influenza transmission.

Reducing Influenza Virus Transmission: The Potential Value of Antiviral Treatment.

Prompt antiviral treatment has the potential to reduce influenza virus transmission to close contacts, but rigorous data on the magnitude of treatment effects on transmission are limited. Animal model data indicate that rapid reductions in viral replication after antiviral treatment reduce the risk of transmission. Observational and clinical trial data with oseltamivir and other neuraminidase inhibitors indicate that prompt treatment of household index patients seems to reduce the risk of illness in contacts, although the magnitude of the reported effects has varied widely across studies. In addition, the potential risk of transmitting drug-resistant variants exists with all approved classes of influenza antivirals. A controlled trial examining baloxavir treatment efficacy to reduce transmission, including the risk of transmitting virus with reduced baloxavir susceptibility, is currently in progress. If reduced transmission risk is confirmed, modeling studies indicate that early treatment could have major epidemiologic benefits in seasonal and pandemic influenza.

Determining Existing Human Population Immunity as Part of Assessing Influenza Pandemic Risk.

Zoonotic influenza infections continue to threaten human health. Ongoing surveillance and risk assessment of animal viruses are needed for pandemic preparedness, and population immunity is an important component of risk assessment. We determined age-stratified hemagglutinin inhibition seroprevalence against 5 swine influenza viruses circulating in Hong Kong and Guangzhou in China. Using hemagglutinin inhibition seroprevalence and titers, we modeled the effect of population immunity on the basic reproduction number (R0) if each virus were to become transmissible among humans. Among 353 individual serum samples, we reported low seroprevalence for triple-reassortant H1N2 and Eurasian avian-like H1N1 influenza viruses, which would reduce R0 by only 18%-20%. The smallest R0 needed to cause a pandemic was 1.22-1.24, meaning existing population immunity would be insufficient to block the spread of these H1N1 or H1N2 variants. For human-origin H3N2, existing population immunity could suppress R0 by 47%, thus reducing pandemic risk.

Phenotypic and Functional Characteristics of a Novel Influenza Virus Hemagglutinin-Specific Memory NK Cell.

Immune memory represents the most efficient defense against invasion and transmission of infectious pathogens. In contrast to memory T and B cells, the roles of innate immunity in recall responses remain inconclusive. In this study, we identified a novel mouse spleen NK cell subset expressing NKp46 and NKG2A induced by intranasal influenza virus infection. These memory NK cells specifically recognize N-linked glycosylation sites on influenza hemagglutinin (HA) protein. Different from memory-like NK cells reported previously, these NKp46+ NKG2A+ memory NK cells exhibited HA-specific silence of cytotoxicity but increase of gamma interferon (IFN-γ) response against influenza virus-infected cells, which could be reversed by pifithrin-µ, a p53-heat shock protein 70 (HSP70) signaling inhibitor. During recall responses, splenic NKp46+ NKG2A+ NK cells were recruited to infected lung and modulated viral clearance of virus and CD8+ T cell distribution, resulting in improved clinical outcomes. This long-lived NK memory bridges innate and adaptive immune memory response and promotes the homeostasis of local environment during recall response.IMPORTANCE In this study, we demonstrate a novel hemagglutinin (HA)-specific NKp46+ NKG2A+ NK cell subset induced by influenza A virus infection. These memory NK cells show virus-specific decreased cytotoxicity and increased gamma interferon (IFN-γ) on reencountering the same influenza virus antigen. In addition, they modulate host recall responses and CD8 T cell distribution, thus bridging the innate immune and adaptive immune responses during influenza virus infection.

Cellular tropism of SARS-CoV-2 in the respiratory tract of Syrian hamsters and B6.Cg-Tg(K18-ACE2)2Prlmn/J transgenic mice.

Several animal models have been developed to study the pathophysiology of SARS-CoV-2 infection and to evaluate vaccines and therapeutic agents for this emerging disease. Similar to infection with SARS-CoV-1, infection of Syrian hamsters with SARS-CoV-2 results in moderate respiratory disease involving the airways and lung parenchyma but does not lead to increased mortality. Using a combination of immunohistochemistry and transmission electron microscopy, we showed that the epithelium of the conducting airways of hamsters was the primary target for viral infection within the first 5 days of infection, with little evidence of productive infection of pneumocytes. At 6 days postinfection, antigen was cleared but parenchymal damage persisted, and the major pathological changes resolved by day 14. These findings are similar to those previously reported for hamsters with SARS-CoV-1 infection. In contrast, infection of K18-hACE2 transgenic mice resulted in pneumocyte damage, with viral particles and replication complexes in both type I and type II pneumocytes together with the presence of convoluted or cubic membranes; however, there was no evidence of virus replication in the conducting airways. The Syrian hamster is a useful model for the study of SARS-CoV-2 transmission and vaccination strategies, whereas infection of the K18-hCE2 transgenic mouse results in lethal disease with fatal neuroinvasion but with sparing of conducting airways.

Evaluation of a SARS-CoV-2 Surrogate Virus Neutralization Test for Detection of Antibody in Human, Canine, Cat, and Hamster Sera.

Surrogate neutralization assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can be done without biosafety level 3 containment and in multiple species are desirable. We evaluate a recently developed surrogate virus neutralization test (sVNT) in comparison to 90% plaque reduction neutralization tests (PRNT90) in human, canine, cat, and hamster sera. With PRNT90 as the reference, sVNT had sensitivity of 98.9% and specificity of 98.8%. Using a panel of immune sera corresponding to other coronaviruses, we confirm the lack of cross-reactivity to other coronaviruses in SARS-CoV-2 sVNT and PRNT90, except for cross-reactivity to SARS-CoV-1 in sVNT.

1'-Ribose cyano substitution allows Remdesivir to effectively inhibit nucleotide addition and proofreading during SARS-CoV-2 viral RNA replication.

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. Remdesivir is one effective inhibitor for SARS-CoV-2 viral RNA replication. It supersedes other NTP analogues because it not only terminates the polymerization activity of RNA-dependent RNA polymerase (RdRp), but also inhibits the proofreading activity of intrinsic exoribonuclease (ExoN). Even though the static structure of Remdesivir binding to RdRp has been solved and biochemical experiments have suggested it to be a "delayed chain terminator", the underlying molecular mechanisms is not fully understood. Here, we performed all-atom molecular dynamics (MD) simulations with an accumulated simulation time of 24 microseconds to elucidate the inhibitory mechanism of Remdesivir on nucleotide addition and proofreading. We found that when Remdesivir locates at an upstream site in RdRp, the 1'-cyano group experiences electrostatic interactions with a salt bridge (Asp865-Lys593), which subsequently halts translocation. Our findings can supplement the current understanding of the delayed chain termination exerted by Remdesivir and provide an alternative molecular explanation about Remdesivir's inhibitory mechanism. Such inhibition also reduces the likelihood of Remdesivir to be cleaved by ExoN acting on 3'-terminal nucleotides. Furthermore, our study also suggests that Remdesivir's 1'-cyano group can disrupt the cleavage site of ExoN via steric interactions, leading to a further reduction in the cleavage efficiency. Our work provides plausible and novel mechanisms at the molecular level of how Remdesivir inhibits viral RNA replication, and our findings may guide rational design for new treatments of COVID-19 targeting viral replication.

Defining the sizes of airborne particles that mediate influenza transmission in ferrets.

Epidemics and pandemics of influenza are characterized by rapid global spread mediated by non-mutually exclusive transmission modes. The relative significance between contact, droplet, and airborne transmission is yet to be defined, a knowledge gap for implementing evidence-based infection control measures. We devised a transmission chamber that separates virus-laden particles by size and determined the particle sizes mediating transmission of influenza among ferrets through the air. Ferret-to-ferret transmission was mediated by airborne particles larger than 1.5 µm, consistent with the quantity and size of virus-laden particles released by the donors. Onward transmission by donors was most efficient before fever onset and may continue for 5 days after inoculation. Multiple virus gene segments enhanced the transmissibility of a swine influenza virus among ferrets by increasing the release of virus-laden particles into the air. We provide direct experimental evidence of influenza transmission via droplets and fine droplet nuclei, albeit at different efficiency.

Lack of cross-transmission of SARS-CoV-2 between passenger's cabins on the Diamond Princess cruise ship.

An outbreak of COVID-19 occurred on the Diamond Princess cruise ship in January and February 2020 in Japan. We analysed information on the cases of infection to infer whether airborne transmission of SARS-CoV-2, the causative agent of COVID-19, had occurred between cabins. We infer from our analysis that most infections in passengers started on 28 January and were completed by 6 February, except in those who shared a cabin with another infected passenger. The distribution of the infected cabins was random, and no spatial cluster of the infected can be identified. We infer that the ship's central air-conditioning system for passenger's cabins did not play a role in SARS-CoV-2 transmission, i.e. airborne transmission did not occur between cabins during the outbreak, suggesting that the sufficient ventilation was provided. We also infer that the ship's cabin drainage system did not play a role. Most transmission appears to have occurred in the public areas of the cruise ship, likely due to crowding and insufficient ventilation in some of these areas.

Detection of Influenza and Other Respiratory Viruses in Air Sampled From a University Campus: A Longitudinal Study.

BACKGROUND: Respiratory virus-laden particles are commonly detected in the exhaled breath of symptomatic patients or in air sampled from healthcare settings. However, the temporal relationship of detecting virus-laden particles at nonhealthcare locations vs surveillance data obtained by conventional means has not been fully assessed. METHODS: From October 2016 to June 2018, air was sampled weekly from a university campus in Hong Kong. Viral genomes were detected and quantified by real-time reverse-transcription polymerase chain reaction. Logistic regression models were fitted to examine the adjusted odds ratios (aORs) of ecological and environmental factors associated with the detection of virus-laden airborne particles. RESULTS: Influenza A (16.9% [117/694]) and influenza B (4.5% [31/694]) viruses were detected at higher frequencies in air than rhinovirus (2.2% [6/270]), respiratory syncytial virus (0.4% [1/270]), or human coronaviruses (0% [0/270]). Multivariate analyses showed that increased crowdedness (aOR, 2.3 [95% confidence interval {CI}, 1.5-3.8]; P < .001) and higher indoor temperature (aOR, 1.2 [95% CI, 1.1-1.3]; P < .001) were associated with detection of influenza airborne particles, but absolute humidity was not (aOR, 0.9 [95% CI, .7-1.1]; P = .213). Higher copies of influenza viral genome were detected from airborne particles >4 µm in spring and <1 µm in autumn. Influenza A(H3N2) and influenza B viruses that caused epidemics during the study period were detected in air prior to observing increased influenza activities in the community. CONCLUSIONS: Air sampling as a surveillance tool for monitoring influenza activity at public locations may provide early detection signals on influenza viruses that circulate in the community.

Avian Influenza Virus Detection Rates in Poultry and Environment at Live Poultry Markets, Guangdong, China.

We report the use of environmental samples to assess avian influenza virus activity in chickens at live poultry markets in China. Results of environmental and chicken samples correlate moderately well. However, collection of multiple environmental samples from holding, processing, and selling areas is recommended to detect viruses expected to have low prevalence.

Close contact behavior in indoor environment and transmission of respiratory infection.

Close contact was first identified as the primary route of transmission for most respiratory infections in the early 20th century. In this review, we synthesize the existing understanding of the mechanisms of close contact transmission. We focus on two issues: the mechanism of transmission in close contact, namely the transmission of the expired particles between two people, and the physical parameters of close contact that affect the exposure of particles from one individual to another, or how the nature of close contact plays a role in transmission. We propose the existence of three sub-routes of transmission: short-range airborne, large droplets, and immediate body-surface contact. We also distinguish a "body contact," which is defined with an interpersonal distance of zero, from a close contact. We demonstrate herein that the short-range airborne sub-route may be most common. The timescales over which data should be collected to assess the transmission risk during close contact events are much shorter than those required for the distant airborne or fomite routes. The current paucity of high-resolution data over short distances and timescales makes it very difficult to assess the risk of infection in these circumstances.

Influenza H5/H7 Virus Vaccination in Poultry and Reduction of Zoonotic Infections, Guangdong Province, China, 2017-18.

We compared the detection frequency of avian influenza H7 subtypes at live poultry markets in Guangdong Province, China, before and after the introduction of a bivalent H5/H7 vaccine in poultry. The vaccine was associated with a 92% reduction in H7 positivity rates among poultry and a 98% reduction in human H7N9 cases.

Ferrets as Models for Influenza Virus Transmission Studies and Pandemic Risk Assessments.

The ferret transmission model is extensively used to assess the pandemic potential of emerging influenza viruses, yet experimental conditions and reported results vary among laboratories. Such variation can be a critical consideration when contextualizing results from independent risk-assessment studies of novel and emerging influenza viruses. To streamline interpretation of data generated in different laboratories, we provide a consensus on experimental parameters that define risk-assessment experiments of influenza virus transmissibility, including disclosure of variables known or suspected to contribute to experimental variability in this model, and advocate adoption of more standardized practices. We also discuss current limitations of the ferret transmission model and highlight continued refinements and advances to this model ongoing in laboratories. Understanding, disclosing, and standardizing the critical parameters of ferret transmission studies will improve the comparability and reproducibility of pandemic influenza risk assessment and increase the statistical power and, perhaps, accuracy of this model.

CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo.

Human infections with influenza viruses exhibit mild to severe clinical outcomes as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin domain family 5 member A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of proinflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced levels of tumor necrosis factor alpha (TNF-α) and IP-10 but elevated alpha interferon (IFN-α) compared to those of wild-type mice. The heightened type I IFN response in the macrophages of CLEC5A-deficient mice was associated with upregulated TLR3 mRNA after treatment with double-stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of proinflammatory cytokines, decreased immune cell infiltration in the lungs, and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections. IMPORTANCE: Multiple pattern recognition receptors work in synergy to sense viral RNA or proteins synthesized during influenza replication and mediate host responses for viral control. Well-orchestrated host responses may help to maintain the inflammatory response to minimize tissue damage while inducing an effective adaptive immune response for viral clearance. We identified that CLEC5A, a C-type lectin receptor which has previously been reported to mediate flavivirus-induced inflammatory responses, enhanced induction of proinflammatory cytokines and chemokines in myeloid cells after influenza infections. CLEC5A-deficient mice infected with influenza virus showed reduced inflammation in the lungs and improved survival compared to that of the wild-type mice despite comparable viral loads. The survival difference was more prominent at a lower dose of inoculum. Collectively, our results suggest that dampening CLEC5A-mediated inflammatory responses in myeloid cells reduces immunopathogenesis after influenza infections.