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Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet (\"airbome transmission\") between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza.

During the SARS epidemic in 2003/2004, a number of deletions were observed in ORF8 of SARS-CoV, and eventually deletion variants became predominant, leading to the hypothesis that ORF8 was an evolutionary hot spot for adaptation of SARS-CoV to humans. However, due to the successful control of the SARS epidemic, the importance of these deletions for the epidemiological fitness of SARS-CoV in humans could not be established. The emergence of multiple SARS-CoV-2 strains with ORF8 deletions, combined with evidence of a robust immune response to ORF8, suggests that the lack of ORF8 may assist with host immune evasion. In addition to providing a key insight into the evolutionary behavior of SARS-CoV-2 as the virus adapts to its new human hosts, the emergence of ORF8 deletion variants may also impact vaccination strategies. To date, limited genetic changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described. Here, we report a 382-nucleotide (nt) deletion in SARS-CoV-2 that truncates open reading frame 7b (ORF7b) and ORF8, removing the ORF8 transcription regulatory sequence (TRS) and eliminating ORF8 transcription. The earliest 382-nt deletion variant was detected in Singapore on 29 January 2020, with the deletion viruses circulating in the country and accounting for 23.6% (45/191) of SARS-CoV-2 samples screened in this study. SARS-CoV-2 with the same deletion has since been detected in Taiwan, and other ORF7b/8 deletions of various lengths, ranging from 62 nt to 345 nt, have been observed in other geographic locations, including Australia, Bangladesh, and Spain. Mutations or deletions in ORF8 of SARS-CoV have been associated with reduced replicative fitness and virus attenuation. In contrast, the SARS-CoV-2 382-nt deletion viruses showed significantly higher replicative fitness in vitro than the wild type, while no difference was observed in patient viral load, indicating that the deletion variant viruses retained their replicative fitness. A robust antibody response to ORF8 has been observed in SARS-CoV-2 infection, suggesting that the emergence of ORF8 deletions may be due to immune-driven selection and that further deletion variants may emerge during the sustained transmission of SARS-CoV-2 in humans. IMPORTANCE During the SARS epidemic in 2003/2004, a number of deletions were observed in ORF8 of SARS-CoV, and eventually deletion variants became predominant, leading to the hypothesis that ORF8 was an evolutionary hot spot for adaptation of SARS-CoV to humans. However, due to the successful control of the SARS epidemic, the importance of these deletions for the epidemiological fitness of SARS-CoV in humans could not be established. The emergence of multiple SARS-CoV-2 strains with ORF8 deletions, combined with evidence of a robust immune response to ORF8, suggests that the lack of ORF8 may assist with host immune evasion. In addition to providing a key insight into the evolutionary behavior of SARS-CoV-2 as the virus adapts to its new human hosts, the emergence of ORF8 deletion variants may also impact vaccination strategies.

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses.

An investigation into the transmissibility of the H7N9 influenza A virus in ferrets finds that although the virus has some determinants associated with human adaptation and transmissibility between mammals, the airborne transmission between ferrets is limited. Transmission of emerging H7N9 virus By 20 July 2013, there had been 134 laboratory-confirmed human cases of infection with avian influenza A H7N9 virus infection, including 43 deaths. Yoshihiro Kawaoka and colleagues characterize the biology of two recent isolates of the virus. They provide a wealth of data from infections in mice, pigs, macaques and ferrets. H7N9 virus is shown to be less sensitive to neuraminidase inhibitors than pandemic H1N1 virus, but equally susceptible to an experimental polymerase inhibitor. Terrence Tumpey and colleagues determine the capacity of two clinical H7N9 isolates to cause disease and transmit between mammals. They show that the virus can replicate in human airway cells and in the respiratory tract of ferrets to a higher level than can seasonal H3N2 virus, and show higher lethality in mice than genetically related H7N9 and H9N2 viruses. In transmission studies, the H7N9 virus showed limited transmission in ferrets by respiratory droplets. Ron Fouchier and colleagues investigate the transmissibility of H7N9 virus between ferrets. They show that airborne transmission can occur, but inefficiently. They also show that on passage in ferrets, virus variants that have higher avian receptor binding, higher pH of fusion and lower thermostability are selected, and they suggest that these characteristics may result in reduced transmissibility. Wild waterfowl form the main reservoir of influenza A viruses, from which transmission occurs directly or indirectly to various secondary hosts, including humans 1 . Direct avian-to-human transmission has been observed for viruses of subtypes A(H5N1), A(H7N2), A(H7N3), A(H7N7), A(H9N2) and A(H10N7) upon human exposure to poultry 2 , 3 , 4 , 5 , 6 , 7 , but a lack of sustained human-to-human transmission has prevented these viruses from causing new pandemics. Recently, avian A(H7N9) viruses were transmitted to humans, causing severe respiratory disease and deaths in China 8 . Because transmission via respiratory droplets and aerosols (hereafter referred to as airborne transmission) is the main route for efficient transmission between humans, it is important to gain an insight into airborne transmission of the A(H7N9) virus. Here we show that although the A/Anhui/1/2013 A(H7N9) virus harbours determinants associated with human adaptation and transmissibility between mammals, its airborne transmissibility in ferrets is limited, and it is intermediate between that of typical human and avian influenza viruses. Multiple A(H7N9) virus genetic variants were transmitted. Upon ferret passage, variants with higher avian receptor binding, higher pH of fusion, and lower thermostability were selected, potentially resulting in reduced transmissibility. This A(H7N9) virus outbreak highlights the need for increased understanding of the determinants of efficient airborne transmission of avian influenza viruses between mammals.

Avian A/H5N1 influenza viruses pose a pandemic threat. As few as five amino acid substitutions, or four with reassortaient, might be sufficient for mammal-to-mammal transmission through respiratory droplets. From surveillance data, we found that two of these substitutions are common in A/H5N1 viruses, and thus, some viruses might require only three additional substitutions to become transmissible via respiratory droplets between mammals. We used a mathematical model of within-host virus evolution to study factors that could increase and decrease the probability of the remaining substitutions evolving after the virus has infected a mammalian host. These factors, combined with the presence of some of these substitutions in circulating strains, make a virus evolving in nature a potentially serious threat. These results highlight critical areas in which more data are needed for assessing, and potentially averting, this threat.

Bats are reservoirs for several zoonotic pathogens, including filoviruses. Recent work highlights the diversity of bat borne filoviruses in Asia. High risk activities at the bat-human interface pose the threat of zoonotic virus transmission. We present evidence for prior exposure of bat harvesters and two resident fruit bat species to filovirus surface glycoproteins by screening sera in a multiplexed serological assay. Antibodies reactive to two antigenically distinct filoviruses were detected in human sera and to three individual filoviruses in bats in remote Northeast India. Sera obtained from Eonycteris spelaea bats showed similar patterns of cross-reactivity as human samples, suggesting them as the species responsible for the spillover. In contrast, sera from Rousettus leschenaultii bats reacted to two different virus glycoproteins. Our results indicate circulation of several filoviruses in bats and the possibility for filovirus transmission from bats to humans.

Despite initial findings indicating that SARS-CoV and SARS-CoV-2 are genetically related belonging to the same virus species and that the two viruses used the same entry receptor, angiotensin-converting enzyme 2 (ACE2), our data demonstrated that there is no detectable cross-neutralization by SARS patient sera against SARS-CoV-2. We also found that there are significant levels of neutralizing antibodies in recovered SARS patients 9-17 years after initial infection. These findings will be of significant use in guiding the development of serologic tests, formulating convalescent plasma therapy strategies, and assessing the longevity of protective immunity for SARS-related coronaviruses in general as well as vaccine efficacy.

[This corrects the article DOI: 10.1371/journal.pntd.0007733.].[This corrects the article DOI: 10.1371/journal.pntd.0007733.].

HPIVs are serologically and genetically grouped into four species that account for up to 10% of all hospitalizations due to acute respiratory infection in children under the age of five. Genetic and epidemiological data for the four HPIVs derived from two pediatric cohorts in Viet Nam are presented. Respiratory samples were screened for HPIV1–4 by real-time PCR. Demographic and clinical data of patients infected with different HPIV were compared. We used a hemi-nested PCR approach to generate viral genome sequences from HPIV-positive samples and conducted a comprehensive phylogenetic analysis. In total, 170 samples tested positive for HPIV. HPIV3 was most commonly detected in our cohort and 80 co-detections of HPIV with other respiratory viruses were found. Phylogenetic analyses suggest local endemic circulation as well as punctuated introductions of new HPIV lineages. Viral gene flow analysis revealed that Viet Nam is a net importer of viral genetic diversity. Epidemiological analyses imply similar disease severity for all HPIV species. HPIV sequences from Viet Nam formed local clusters and were interspersed with sequences from diverse geographic regions. Combined, this new knowledge will help to investigate global HPIV circulation patterns in more detail and ultimately define more suitable vaccine strains.

ABSTRACT The emergence of novel betacoronaviruses has posed significant financial and human health burdens, necessitating the development of appropriate tools to combat future outbreaks. In this study, we have characterized a human cell line, IGROV-1, as a robust tool to detect, propagate, and titrate betacoronaviruses SARS-CoV-2 and HCoV-OC43. IGROV-1 cells can be used for serological assays, antiviral drug testing, and isolating SARS-CoV-2 variants from patient samples. Using time-course transcriptomics, we confirmed that IGROV-1 cells exhibit a robust innate immune response upon SARS-CoV-2 infection, recapitulating the response previously observed in primary human nasal epithelial cells. We performed genome-wide CRISPR knockout genetic screens in IGROV-1 cells and identified Aryl hydrocarbon receptor (AHR) as a critical host dependency factor for both SARS-CoV-2 and HCoV-OC43. Using DiMNF, a small molecule inhibitor of AHR, we observed that the drug selectively inhibits HCoV-OC43 infection but not SARS-CoV-2. Transcriptomic analysis in primary normal human bronchial epithelial cells revealed that DiMNF blocks HCoV-OC43 infection via basal activation of innate immune responses. Our findings highlight the potential of IGROV-1 cells as a valuable diagnostic and research tool to combat betacoronavirus diseases.