COVID‐19 and the gain of function debates: Improving biosafety measures requires a more precise definition of which experiments would raise safety concerns

The COVID‐19 pandemic has rekindled debates about gain‐of‐function experiments. This is an opportunity to clearly define safety risks and appropriate countermeasures. Subject Categories: Economics, Law & Politics, Microbiology, Virology & Host Pathogen Interaction, Science Policy & Publishing

The so‐called “gain of function” research has been recently debated in the context of viral research on coronaviruses and whether it is too risky to undertake such experiments. However, the meaning of “gain of function” or “GOF” in a science policy context has changed over time. The term was originally coined to describe two controversial research projects on H5N1 avian influenza virus and was later applied to specific experiments on coronavirus. Subsequent policies and discussions have attempted to define GOF in different ways, but no single definition has been widely accepted by the community. The fuzzy and imprecise nature of the term has led to misunderstandings and has hampered discussions on how to properly assess the benefit of such experiments and biosafety measures.

The fuzzy and imprecise nature of the term GOF has led to misunderstandings and has hampered discussions on how to properly assess the benefit of such experiments and biosafety measures

     

Coronavirus Infection and Diversity in Bats in the Australasian Region

Following the SARS outbreak, extensive surveillance was undertaken globally to detect and identify coronavirus diversity in bats. This study sought to identify the diversity and prevalence of coronaviruses in bats in the Australasian region. We identified four different genotypes of coronavirus, three of which (an alphacoronavirus and two betacoronaviruses) are potentially new species, having less than 90% nucleotide sequence identity with the most closely related described viruses. We did not detect any SARS-like betacoronaviruses, despite targeting rhinolophid bats, the putative natural host taxa. Our findings support the virus-host co-evolution hypothesis, with the detection of Miniopterus bat coronavirus HKU8 (previously reported in Miniopterus species in China, Hong Kong and Bulgaria) in Australian Miniopterus species. Similarly, we detected a novel betacoronavirus genotype from Pteropus alecto which is most closely related to Bat coronavirus HKU9 identified in other pteropodid bats in China, Kenya and the Philippines. We also detected possible cross-species transmission of bat coronaviruses, and the apparent enteric tropism of these viruses. Thus, our findings are consistent with a scenario wherein the current diversity and host specificity of coronaviruses reflects co-evolution with the occasional host shift.     

A molecular arms race between host innate antiviral response and emerging human coronaviruses

Coronaviruses have been closely related with mankind for thousands of years. Communityacquired human coronaviruses have long been recognized to cause common cold. However, zoonotic coronaviruses are now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses causing severe respiratory diseases. Infections by these emerging human coronaviruses are characterized by less robust interferon production. Treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. The mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. This review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. Particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against SARS and MERS coronavirus infection are discussed. On the other hand, the counter-measures evolved by SARS and MERS coronaviruses to circumvent host defense are also dissected. With a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived.

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Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft

Since the 2002–2003 severe acute respiratory syndrome(SARS) outbreak prompted a search for the natural reservoir of the SARS coronavirus, numerous alpha- and betacoronaviruses have been discovered in bats around the world. Bats are likely the natural reservoir of alpha- and betacoronaviruses, and due to the rich diversity and global distribution of bats, the number of bat coronaviruses will likely increase. We conducted a surveillance of coronaviruses in bats in an abandoned mineshaft in Mojiang County, Yunnan Province, China, from 2012–2013. Six bat species were frequently detected in the cave: Rhinolophus sinicus, Rhinolophus affinis, Hipposideros pomona, Miniopterus schreibersii, Miniopterus fuliginosus, and Miniopterus fuscus. By sequencing PCR products of the coronavirus RNA-dependent RNA polymerase gene(Rd Rp), we found a high frequency of infection by a diverse group of coronaviruses in different bat species in the mineshaft. Sequenced partial Rd Rp fragments had 80%–99% nucleic acid sequence identity with well-characterized Alphacoronavirus species, including Bt CoV HKU2, Bt CoV HKU8, and Bt CoV1,and unassigned species Bt CoV HKU7 and Bt CoV HKU10. Additionally, the surveillance identified two unclassified betacoronaviruses, one new strain of SARS-like coronavirus, and one potentially new betacoronavirus species. Furthermore, coronavirus co-infection was detected in all six bat species, a phenomenon that fosters recombination and promotes the emergence of novel virus strains. Our findings highlight the importance of bats as natural reservoirs of coronaviruses and the potentially zoonotic source of viral pathogens.     

Evolutionary insights into the ecology of coronaviruses

Although many novel members of the Coronaviridae have recently been recognized in different species, the ecology of coronaviruses has not been established. Our study indicates that bats harbor a much wider diversity of coronaviruses than any other animal species. Dating of different coronavirus lineages suggests that bat coronaviruses are older than those recognized in other animals and that the human severe acute respiratory syndrome (SARS) coronavirus was directly derived from viruses from wild animals in wet markets of southern China. Furthermore, the most closely related bat and SARS coronaviruses diverged in 1986, an estimated divergence time of 17 years prior to the outbreak, suggesting that there may have been transmission via an unknown intermediate host. Analysis of lineage-specific selection pressure also indicated that only SARS coronaviruses in civets and humans were under significant positive selection, also demonstrating a recent interspecies transmission. Analysis of population dynamics revealed that coronavirus populations in bats have constant population growth, while viruses from all other hosts show epidemic-like increases in population. These results indicate that diverse coronaviruses are endemic in different bat species, with repeated introductions to other animals and occasional establishment in other species. Our findings suggest that bats are likely the natural hosts for all presently known coronavirus lineages and that all coronaviruses recognized in other species were derived from viruses residing in bats. Further surveillance of bat and other animal populations is needed to fully describe the ecology and evolution of this virus family.     

Prevention of influenza in the general population

Background

Although all jurisdictions in Canada offer annual influenza immunization to people at high risk of complications, only Ontario has provided universal annual immunization of healthy adults and children. Use of chemotherapy (amantidine, neuraminidase inhibitors) to prevent influenza varies among provinces. We sought to systematically review the evidence for the prevention of influenza infection in the general population.

Methods

The interventions reviewed were influenza vaccination and prophylactic use of neuraminidase inhibitors. The health outcomes of interest were rates of laboratory-confirmed influenza infection, clinical definitions of influenza-like illness and work absenteeism. MEDLINE and Cochrane databases were searched for relevant articles published between 1966 and March 2003. Only randomized controlled trials (RCTs) were selected. Evidence was appraised using the methodology of the Canadian Task Force on Preventive Health Care.

Results

Eighteen trials involving more than 33 000 healthy adults were identified that met the inclusion criteria; of these, 15 showed that influenza vaccination with either live-attenuated and inactivated vaccines was efficacious. Eleven trials were considered to be of “good” quality, and 7 were considered to be of “fair” quality. The relative risk reduction (RRR) associated with influenza immunization in adults ranged from 0% to 91%. Fifteen RCTs involving more than 45 000 healthy children aged 6 months to 19 years were identified, of which 9 were considered to contain “good” evidence and 6 “fair” evidence. Results from 12 of these trials showed protection against influenza. The RRR ranged from 0% to 93%. There were 6 RCTs of “good” quality showing that neuraminidase inhibitors are effective in preventing influenza infection. Side effects from both influenza vaccination and neuraminidase inhibitor administration were mild.

Interpretation

There are numerous RCTs of good quality in large populations that have consistently shown that influenza vaccination, using inactivated or live-attenuated vaccines, is moderately effective in preventing influenza in the general population (healthy adults and children over 6 months of age). There is good evidence that neuraminidase inhibitor prophylaxis in contacts given within 36 to 48 hours of symptom onset of the household index case is effective; appropriate use of this prevention method requires access to rapid diagnostic methods. Decisions about introduction of routine immunization programs must take into account the cost and cost-effectiveness of a universal program and the burden of illness associated with influenza in each jurisdiction.Influenza virus causes yearly epidemics of respiratory illness of varying severity worldwide in people of all ages, and it may be the most important cause of medically attended acute respiratory illness.¹ In Canada influenza and pneumonia are the leading cause of death from infection and the sixth cause of death overall.² Rates of complications and death from influenza are high among adults over 65 years of age and people with cardiac or pulmonary disease or chronic medical conditions, and annual influenza immunization in this population is associated with lower frequency of hospital admissions because of respiratory disease, congestive heart failure and death from any cause.^3,4 Previously healthy young children are increasingly recognized as having hospital admission rates comparable to those among elderly people during influenza epidemics⁵ and up to 12-fold greater than rates among older children.⁶ Because influenza occurs yearly and because re-infections occur throughout the lifespan and affect up to 20% of the population each year, considerable attention has been directed to the prevention of influenza in healthy people. Although annual immunization programs are routinely offered to high-risk groups, only the province of Ontario routinely offers influenza immunization to healthy adults and children.We performed a systematic review of the literature to answer the following question: how effective are the influenza vaccine and prophylactic neuraminidase inhibitor antiviral agents for the prevention of influenza in healthy adults and children?     

Genetic diversity of coronaviruses in Miniopterus fuliginosus bats

Coronaviruses, such as severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, pose significant public health threats. Bats have been suggested to act as natural reservoirs for both these viruses, and periodic monitoring of coronaviruses in bats may thus provide important clues about emergent infectious viruses. The Eastern bent-wing bat Miniopterus fuliginosus is distributed extensively throughout China. We therefore analyzed the genetic diversity of coronaviruses in samples of M. fuliginosus collected from nine Chinese provinces during 2011–2013. The only coronavirus genus found was Alphacoronavirus. We established six complete and five partial genomic sequences of alphacoronaviruses, which revealed that they could be divided into two distinct lineages, with close relationships to coronaviruses in Miniopterus magnater and Miniopterus pusillus. Recombination was confirmed by detecting putative breakpoints of Lineage 1 coronaviruses in M. fuliginosus and M. pusillus(Wu et al., 2015), which supported the results of topological and phylogenetic analyses. The established alphacoronavirus genome sequences showed high similarity to other alphacoronaviruses found in other Miniopterus species, suggesting that their transmission in different Miniopterus species may provide opportunities for recombination with different alphacoronaviruses. The genetic information for these novel alphacoronaviruses will improve our understanding of the evolution and genetic diversity of coronaviruses, with potentially important implications for the transmission of human diseases.     

Ecoepidemiology and Complete Genome Comparison of Different Strains of Severe Acute Respiratory Syndrome-Related Rhinolophus Bat Coronavirus in China Reveal Bats as a Reservoir for Acute,Self-Limiting Infection That Allows Recombination Events

Despite the identification of severe acute respiratory syndrome-related coronavirus (SARSr-CoV) in Rhinolophus Chinese horseshoe bats (SARSr-Rh-BatCoV) in China, the evolutionary and possible recombination origin of SARSr-CoV remains undetermined. We carried out the first study to investigate the migration pattern and SARSr-Rh-BatCoV genome epidemiology in Chinese horseshoe bats during a 4-year period. Of 1,401 Chinese horseshoe bats from Hong Kong and Guangdong, China, that were sampled, SARSr-Rh-BatCoV was detected in alimentary specimens from 130 (9.3%) bats, with peak activity during spring. A tagging exercise of 511 bats showed migration distances from 1.86 to 17 km. Bats carrying SARSr-Rh-BatCoV appeared healthy, with viral clearance occurring between 2 weeks and 4 months. However, lower body weights were observed in bats positive for SARSr-Rh-BatCoV, but not Rh-BatCoV HKU2. Complete genome sequencing of 10 SARSr-Rh-BatCoV strains showed frequent recombination between different strains. Moreover, recombination was detected between SARSr-Rh-BatCoV Rp3 from Guangxi, China, and Rf1 from Hubei, China, in the possible generation of civet SARSr-CoV SZ3, with a breakpoint at the nsp16/spike region. Molecular clock analysis showed that SARSr-CoVs were newly emerged viruses with the time of the most recent common ancestor (tMRCA) at 1972, which diverged between civet and bat strains in 1995. The present data suggest that SARSr-Rh-BatCoV causes acute, self-limiting infection in horseshoe bats, which serve as a reservoir for recombination between strains from different geographical locations within reachable foraging range. Civet SARSr-CoV is likely a recombinant virus arising from SARSr-CoV strains closely related to SARSr-Rh-BatCoV Rp3 and Rf1. Such frequent recombination, coupled with rapid evolution especially in ORF7b/ORF8 region, in these animals may have accounted for the cross-species transmission and emergence of SARS.Coronaviruses can infect a wide variety of animals, causing respiratory, enteric, hepatic, and neurological diseases with different degrees of severity. On the basis of genotypic and serological characteristics, coronaviruses were classified into three distinct groups (2, 20, 54). Among coronaviruses that infect humans, human coronavirus 229E (HCoV-229E) and human coronavirus NL63 (HCoV-NL63) belong to group 1 coronaviruses and human coronavirus OC43 (HCoV-OC43), and human coronavirus HKU1 (HCoV-HKU1) belong to group 2 coronaviruses, whereas severe acute respiratory syndrome-related coronavirus (SARSr-CoV) has been classified as a group 2b coronavirus, distantly related to group 2a, and the recently discovered group 2c and 2d coronaviruses (6, 8, 10, 18, 31, 38, 43, 46, 49, 50). Recently, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses has proposed renaming the traditional group 1, 2, and 3 coronaviruses Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, respectively (http://talk.ictvonline.org/media/p/1230.aspx).Among all coronaviruses, SARSr-CoV has caused the most severe disease in humans, with over 700 fatalities since the SARS epidemic in 2003. Although the identification of SARSr-CoV in Himalayan palm civets and raccoon dogs in live animal markets in southern China suggested that wild animals could be the origin of SARS (11), the presence of the virus in only market or farmed civets, but not civets in the wild, and the rapid evolution of SARSr-CoV genomes in market civets suggested that these caged animals were only intermediate hosts (24, 39, 42, 52). Since bats are commonly found and served in wild animal markets and restaurants in Guangdong, China (47), we have previously carried out a study of bats from the region and identified a SARSr-CoV in Rhinolophus Chinese horseshoe bats (SARSr-Rh-BatCoV) (21). Similar viruses have also been found in three other species of horseshoe bats in mainland China (25), supporting the hypothesis that horseshoe bats are a reservoir of SARSr-CoV. Recently, viruses closely related to SARSr-Rh-BatCoV in China were also reported in Chaerophon bats from Africa, although only partial RNA-dependent RNA polymerase (RdRp) sequences were available (41). In addition, more than 10 previously unrecognized coronaviruses of huge diversity have since been identified in bats from China and other countries (1, 3, 5, 9, 22, 27, 32, 33, 40, 46, 51), suggesting that bats play an important role in the ecology and evolution of coronaviruses.As a result of the unique mechanism of viral replication, coronaviruses have a high frequency of recombination (20). Such a high recombination rate, coupled with the infidelity of the polymerases of RNA viruses, may allow them to adapt to new hosts and ecological niches (12, 48). Recombination in coronaviruses was first recognized between different strains of murine hepatitis virus (MHV) and subsequently in other coronaviruses, such as infectious bronchitis virus, between MHV and bovine coronavirus, and between feline coronavirus type I and canine coronavirus generating feline coronavirus type II (12, 16, 17, 23). Recently, by complete genome analysis of 22 strains of HCoV-HKU1, we have also documented for the first time that natural recombination events in a human coronavirus can give rise to three different genotypes (48).Although previous studies have attempted to study the possible evolutionary and recombination origin of SARSr-CoV, no definite conclusion can be made on whether the viruses from bats are the direct ancestor of SARSr-CoV in civets and humans, given the paucity of available strains and genome sequences. To better define the epidemiology and evolution of SARSr-Rh-BatCoV in China and their role as a recombination origin of SARSr-CoV in civets, we carried out a 4-year study on coronaviruses in Chinese horseshoe bats in Hong Kong and Guangdong Province of southern China. Bat tagging was also performed to study the migration pattern of bats and viral persistence. The complete genomes of 10 strains of SARSr-Rh-BatCoV obtained at different time were sequenced and compared to previously sequenced genomes. With the availability of this larger set of genome sequences for more accurate analysis, recombination and molecular clock analyses were performed to elucidate the evolutionary origin and time of interspecies transmission of SARSr-CoV.     

Unseasonal Transmission of H3N2 Influenza A Virus During the Swine-Origin H1N1 Pandemic

The initial wave of swine-origin influenza A virus (pandemic H1N1/09) in the United States during the spring and summer of 2009 also resulted in an increased vigilance and sampling of seasonal influenza viruses (H1N1 and H3N2), even though they are normally characterized by very low incidence outside of the winter months. To explore the nature of virus evolution during this influenza “off-season,” we conducted a phylogenetic analysis of H1N1 and H3N2 sequences sampled during April to June 2009 in New York State. Our analysis revealed that multiple lineages of both viruses were introduced and cocirculated during this time, as is typical of influenza virus during the winter. Strikingly, however, we also found strong evidence for the presence of a large transmission chain of H3N2 viruses centered on the south-east of New York State and which continued until at least 1 June 2009. These results suggest that the unseasonal transmission of influenza A viruses may be more widespread than is usually supposed.The recent emergence of swine-origin H1N1 influenza A virus (pandemic H1N1/09) in humans has heightened awareness of how the burden of morbidity and mortality due to influenza is associated with the appearance of new genetic variants (5) and of the genetic and epidemiological determinants of viral transmission (8). The emergence of pandemic H1N1/09 is also unprecedented in recorded history as it means that three antigenically distinct lineages of influenza A virus—pandemic H1N1/09 and the seasonal H1N1 and H3N2 viruses— currently cocirculate within human populations.Although the presence of multiple subtypes of influenza A virus may place an additional burden on public health resources, it also provides a unique opportunity to compare the patterns and dynamics of evolution in these viruses on a similar time scale. Indeed, one of the most interesting secondary effects of the current H1N1/09 pandemic has been an increased vigilance for cases of influenza-like illness and hence an intensified sampling of seasonal H1N1 and H3N2 viruses during the typical influenza “off-season” (i.e., spring-summer) in the northern hemisphere. Because the influenza season in the northern hemisphere generally runs from November through March, with a usual peak in January or February, influenza viruses sampled outside of this period are of special interest.The current model for the global spatiotemporal dynamics of influenza A virus is that the northern and southern hemispheres represent ecological “sinks” for this virus, with little ongoing viral transmission during the summer months (9). In contrast, more continual viral transmission occurs within the tropical “source” population (13) that is most likely centered on an intense transmission network in east and southeast Asia (10). However, the precise epidemiological and evolutionary reasons for this major geographic division, and for the seasonality of influenza A virus in general, remain uncertain (1, 4). Evidence for this “sink-source” ecological model is that viruses sampled from successive seasons in localities such as New York State do not usually form linked clusters on phylogenetic trees, indicating that they are not connected by direct transmission through the summer months (7). Similar conclusions can be drawn for the United States as a whole and point to multiple introductions of phylogenetically distinct lineages during the winter (6), followed by complex patterns of spatial diffusion (14). However, despite the growing epidemiological and phylogenetic data supporting this model, it is also evident that there is relatively little sequence data from seasonal influenza viruses that are sampled from April to October in the northern hemisphere. Hence, it is uncertain whether extended chains of transmission can occur during this time period, even though this may have an important bearing on our understanding of influenza seasonality.To address these issues, we examined the evolutionary behavior of seasonal H1N1 and H3N2 viruses as they cocirculated during a single time period—(late) April to June 2009—within a single locality (New York State). Not only are levels of influenza virus transmission in the northern hemisphere usually very low during this time period, but in this particular season the human host population was also experiencing the emerging epidemic of pandemic H1N1/09.     

The Role of Viral Population Diversity in Adaptation of Bovine Coronavirus to New Host Environments

The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were “selected” from a pre-existing pool rather than through de novo mutation and subsequent population fixation.     

How Are ‘Barack Obama’ and ‘President Elect’ Differentially Stored in the Brain? An ERP Investigation on the Processing of Proper and Common Noun Pairs

Background

One of the most debated issues in the cognitive neuroscience of language is whether distinct semantic domains are differentially represented in the brain. Clinical studies described several anomic dissociations with no clear neuroanatomical correlate. Neuroimaging studies have shown that memory retrieval is more demanding for proper than common nouns in that the former are purely arbitrary referential expressions. In this study a semantic relatedness paradigm was devised to investigate neural processing of proper and common nouns.

Methodology/Principal Findings

780 words (arranged in pairs of Italian nouns/adjectives and the first/last names of well known persons) were presented. Half pairs were semantically related (“Woody Allen” or “social security”), while the others were not (“Sigmund Parodi” or “judicial cream”). All items were balanced for length, frequency, familiarity and semantic relatedness. Participants were to decide about the semantic relatedness of the two items in a pair. RTs and N400 data suggest that the task was more demanding for common nouns. The LORETA neural generators for the related-unrelated contrast (for proper names) included the left fusiform gyrus, right medial temporal gyrus, limbic and parahippocampal regions, inferior parietal and inferior frontal areas, which are thought to be involved in the conjoined processing a familiar face with the relevant episodic information. Person name was more emotional and sensory vivid than common noun semantic access.

Conclusions/Significance

When memory retrieval is not required, proper name access (conspecifics knowledge) is not more demanding. The neural generators of N400 to unrelated items (unknown persons and things) did not differ as a function of lexical class, thus suggesting that proper and common nouns are not treated differently as belonging to different grammatical classes.     

Multiple Nucleic Acid Binding Sites and Intrinsic Disorder of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Implications for Ribonucleocapsid Protein Packaging

The nucleocapsid protein (N) of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genomic RNA and is crucial for viability. However, the RNA-binding mechanism is poorly understood. We have shown previously that the N protein contains two structural domains—the N-terminal domain (NTD; residues 45 to 181) and the C-terminal dimerization domain (CTD; residues 248 to 365)—flanked by long stretches of disordered regions accounting for almost half of the entire sequence. Small-angle X-ray scattering data show that the protein is in an extended conformation and that the two structural domains of the SARS-CoV N protein are far apart. Both the NTD and the CTD have been shown to bind RNA. Here we show that all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively. The effect can be explained by the “coupled-allostery” model, devised to explain the allosteric effect in a multidomain regulatory system. Although the N proteins of different coronaviruses share very low sequence homology, the physicochemical features described above may be conserved across different groups of Coronaviridae. The current results underscore the important roles of multisite nucleic acid binding and intrinsic disorder in N protein function and RNP packaging.Severe acute respiratory syndrome (SARS) is the first pandemic of the 21st century that spread to multiple nations, with a fatality rate of ca. 8%. The disease is caused by a novel SARS-associated coronavirus (SARS-CoV) closely related to the group II coronaviruses, which include the human coronavirus OC43 and murine hepatitis virus (6, 18). Traditional antiviral treatments have had little success against SARS during the outbreak, and vaccines have yet to be developed (35).Coronaviruses are positive-sense single-stranded RNA (ssRNA) viruses. The coronavirus genomic RNA is encapsidated into a helical capsid by the nucleocapsid (N) protein, which is one of the most abundant coronavirus proteins (19). The N protein has nonspecific binding activity toward nucleic acids, including ssRNA, single-stranded DNA, and double-stranded DNA (33). It can also act as an RNA chaperone (39). However, the mechanism of binding of the N protein to nucleic acids is poorly understood.The SARS-CoV N protein is a homodimer composed of 422 amino acids (aa) in each chain. The N protein can be divided into two structural domains interspersed with disordered (unstructured) regions (Fig. ​(Fig.1A)1A) (2). The N-terminal domain (NTD; also called RBD) serves as a putative RNA-binding domain, while the C-terminal domain (CTD; also called DD) is a dimerization domain (13, 36). Both the NTD and the CTD bind to nucleic acids through electropositive regions on their surfaces (3, 13, 32). All coronaviruses share similar domain architectures at both the sequence and structure levels. No structure of N protein or any of its domains in complex with nucleic acids is available.Open in a separate windowFIG. 1.(A) Schematic of the domain architecture of the SARS-CoV N protein. Structured domains are shown as balls, and unstructured regions are shown as lines. (B) Protein constructs used in the current study. Numbers represent the amino acid residue range relative to the full-length N protein (NP). Sumo-1-FL contains a Sumo-1 tag (shown as an oval), followed by the flexible linker of the N protein between residues 181 and 246.The functions of the disordered regions in the SARS-CoV N protein have not been clearly defined, although some evidence suggests that they are involved in protein-protein interactions between the N protein and other viral and host proteins (11, 20, 22, 38). A previous report has shown that part of the C-terminal disordered region with a polylysine sequence also binds to RNA (21). Unlike the structural domains, the disordered regions of the different coronaviruses share little sequence homology. However, they share a common physicochemical property: they are highly enriched in basic residues. Intrinsic disorder coupled with an abundance of positive charges leads to the possibility of nonspecific binding to nucleic acids (34). These findings prompted us to investigate the role of intrinsically disordered (ID) regions in the RNA-binding mechanism of the SARS-CoV N protein.Here we tested all three disordered regions of the SARS-CoV N protein and found that they are all involved in RNA binding. The central region, in particular, had a large impact on binding behavior as monitored by electrophoretic mobility shift assays (EMSA). Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) results show that this central region is a flexible linker (FL) that connects the two structural domains in an extended conformation. Our results provide new insights into the functional coupling of intrinsic disorder, RNA binding, and oligomerization.     

Proteasome Inhibition In Vivo Promotes Survival in a Lethal Murine Model of Severe Acute Respiratory Syndrome

Ubiquitination is a critical regulator of the host immune response to viral infection, and many viruses, including coronaviruses, encode proteins that target the ubiquitination system. To explore the link between coronavirus infection and the ubiquitin system, we asked whether protein degradation by the 26S proteasome plays a role in severe coronavirus infections using a murine model of SARS-like pneumonitis induced by murine hepatitis virus strain 1 (MHV-1). In vitro, the pretreatment of peritoneal macrophages with inhibitors of the proteasome (pyrrolidine dithiocarbamate [PDTC], MG132, and PS-341) markedly inhibited MHV-1 replication at an early step in its replication cycle, as evidenced by inhibition of viral RNA production. Proteasome inhibition also blocked viral cytotoxicity in macrophages, as well as the induction of inflammatory mediators such as IP-10, gamma interferon (IFN-γ), and monocyte chemoattractant protein 1 (MCP-1). In vivo, intranasal inoculation of MHV-1 results in a lethal pneumonitis in A/J mice. Treatment of A/J mice with the proteasome inhibitor PDTC, MG132, or PS-341 led to 40% survival (P < 0.01), with a concomitant improvement of lung histology, reduced pulmonary viral replication, decreased pulmonary STAT phosphorylation, and reduced pulmonary inflammatory cytokine expression. These data demonstrate that inhibition of the cellular proteasome attenuates pneumonitis and cytokine gene expression in vivo by reducing MHV-1 replication and the resulting inflammatory response. The results further suggest that targeting the proteasome may be an effective new treatment for severe coronavirus infections.Severe acute respiratory syndrome (SARS) was first introduced into the human population in the Guangdong Province in China and rapidly spread to several other countries (31). SARS is caused by infection with the SARS coronavirus (SARS-CoV) and is characterized by an atypical pneumonia and lymphopenia. Two-thirds of the SARS-infected patients developed a viral pneumonitis, of which 10% developed acute respiratory distress syndrome. During the outbreak in 2002 to 2003, 8,000 people were infected and 774 people died from respiratory failure (36; WHO, Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003 [http://www.who.int]). At present there are no effective treatments for SARS as well as other coronavirus infections. Finding an effective treatment for coronavirus infections could be protective in the event of a reemergent coronavirus outbreak (7).We have recently reported that a rodent model of SARS mimics many of the features of severe clinical SARS pathology (11, 12). Intranasal infection of A/J mice with strain 1 of murine hepatitis virus (MHV-1) causes a lethal form of pneumonitis, characterized by marked innate immune inflammatory cytokine production and replication of the virus in pulmonary macrophages (11, 12). MHV-1 infection is uniformly fatal in infected A/J mice; the resultant disease serves as a model to understand the pathology of the most severe SARS cases. In mice, the pulmonary damage is histologically similar to that seen in human SARS and is similarly associated with a marked upregulation of inflammatory mediators, including monocyte chemoattractant protein 1 (MCP-1), IP-10, MIG, gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-6 (11, 12, 25). These innate immune mediators are likely to play roles in human SARS and MHV-1 SARS-like pathogenesis.A critical aspect of the host innate immune response to viral illness is the upregulation of the antiviral type 1 IFN response. With respect to SARS, type 1 IFN responses have been reported to be suppressed by SARS-CoV in several models and in clinical cases (11, 39, 45, 52). In our model, MHV-1-infected A/J mice produce less type 1 IFN than resistant strains of mice and they respond poorly to IFN-β therapy (11). Type I IFN has been used clinically in the treatment of established SARS infections but has shown only limited efficacy (25). In the absence of an effective antiviral treatment, the innate immune pathways present a potential target for therapeutic intervention (7).Ubiquitination, the process by which cellular proteins are conjugated to the 7.5-kDa ubiquitin (Ub) protein, is a critical regulator of innate and adaptive immune pathways (40). There are several possible fates for ubiquitinated proteins: degradation by the 26S proteasome, trafficking to various subcellular sites, altered interactions with other proteins, and altered signal transduction functions (28). The fates of the ubiquitinated proteins, many of which overlap, can play a role in innate immunity. Since the first discovery that papillomavirus encodes an E3 ubiquitin ligase that targets p53, it has become widely appreciated that many viruses encode proteins that target or exploit ubiquitination pathways (37, 43). For example, Epstein-Barr virus and herpes simplex virus proteins interact with the host deubiquitinating (DUB) protein USP7 (13, 17). Ubiquitination of IRF3 has been implicated in the viral control of the innate immune system (22, 48, 49). DUB may also be important for viral functions, such as the assembly of viral replicase proteins with double-membrane vesicles at the site of replication, a process that parasitizes autophagy (39).All coronaviruses, including MHV (A59 and JHM), infectious bronchitis virus, and human CoV229E SARS coronavirus, encode one or more papain-like proteases (PLpros) (PL1pro and PL2pro) (3, 5, 19, 23, 50). One role for the PL2pro proteases is to cleave the coronavirus polyprotein into its component parts. This enzyme, isolated from the SARS-CoV, has also been shown to have DUB activity both in vitro and in HeLa cells (23), suggesting that it might also play a role in modulating the host ubiquitination pathways. PLpro proteases harbor an N-terminal Ub-like domain reported to mediate interactions between PLpro DUB activity and the cellular proteasome (35). Although there is no direct link between the proteasome and SARS-CoV DUB activity, the presence of the Ub1 domain and of SARS-CoV DUB activity suggests that the proteasome may be being exploited by the virus either to evade the immune response or to promote viral replication. These interactions also suggest that the ubiquitination system might be a target for antiviral therapeutic intervention.We explored the role of the cellular proteasome in MHV-1 replication and in the innate immune response to the virus by testing the effects of small-molecule proteasome inhibitors in both cell-based and murine models of SARS pneumonitis. We compared the results in the SARS model to a well-described model of lymphocytic choriomeningitis virus (LCMV) hepatitis in order to test for virus-specific effects. To control for nonspecific effects of the inhibitors, we used three different agents: pyrrolidine dithiocarbamate (PDTC), MG132, and PS-341 (bortezomib, Velcade). PDTC is a chelating agent that reversibly inhibits the proteasome complex, MG132 is a peptide aldehyde protease inhibitor, and PS-341 is a peptide boronic acid inhibitor (1, 20, 38). PS-341 is a clinically approved drug currently being used in the treatment of multiple myeloma.     

Searching PubMed during a Pandemic

Background

The 2009 influenza A(H1N1) pandemic has generated thousands of articles and news items. However, finding relevant scientific articles in such rapidly developing health crises is a major challenge which, in turn, can affect decision-makers'' ability to utilise up-to-date findings and ultimately shape public health interventions. This study set out to show the impact that the inconsistent naming of the pandemic can have on retrieving relevant scientific articles in PubMed/MEDLINE.

Methodology

We first formulated a PubMed search algorithm covering different names of the influenza pandemic and simulated the results that it would have retrieved from weekly searches for relevant new records during the first 10 weeks of the pandemic. To assess the impact of failing to include every term in this search, we then conducted the same searches but omitted in turn “h1n1,” “swine,” “influenza” and “flu” from the search string, and compared the results to those for the full string.

Principal Findings

On average, our core search string identified 44.3 potentially relevant new records at the end of each week. Of these, we determined that an average of 27.8 records were relevant. When we excluded one term from the string, the percentage of records missed out of the total number of relevant records averaged 18.7% for omitting “h1n1,” 13.6% for “swine,” 17.5% for “influenza,” and 20.6% for “flu.”

Conclusions

Due to inconsistent naming, while searching for scientific material about rapidly evolving situations such as the influenza A(H1N1) pandemic, there is a risk that one will miss relevant articles. To address this problem, the international scientific community should agree on nomenclature and the specific name to be used earlier, and the National Library of Medicine in the US could index potentially relevant materials faster and allow publishers to add alert tags to such materials.     

A human coronavirus evolves antigenically to escape antibody immunity

There is intense interest in antibody immunity to coronaviruses. However, it is unknown if coronaviruses evolve to escape such immunity, and if so, how rapidly. Here we address this question by characterizing the historical evolution of human coronavirus 229E. We identify human sera from the 1980s and 1990s that have neutralizing titers against contemporaneous 229E that are comparable to the anti-SARS-CoV-2 titers induced by SARS-CoV-2 infection or vaccination. We test these sera against 229E strains isolated after sera collection, and find that neutralizing titers are lower against these “future” viruses. In some cases, sera that neutralize contemporaneous 229E viral strains with titers >1:100 do not detectably neutralize strains isolated 8–17 years later. The decreased neutralization of “future” viruses is due to antigenic evolution of the viral spike, especially in the receptor-binding domain. If these results extrapolate to other coronaviruses, then it may be advisable to periodically update SARS-CoV-2 vaccines.     

Coexistence of Different Genotypes in the Same Bat and Serological Characterization of Rousettus Bat Coronavirus HKU9 Belonging to a Novel Betacoronavirus Subgroup

Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), a recently identified coronavirus of novel Betacoronavirus subgroup D, from Leschenault''s rousette, was previously found to display marked sequence polymorphism among genomes of four strains. Among 10 bats with complete RNA-dependent RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes sequenced, three and two sequence clades for all three genes were codetected in two and five bats, respectively, suggesting the coexistence of two or three distinct genotypes of Ro-BatCoV HKU9 in the same bat. Complete genome sequencing of the distinct genotypes from two bats, using degenerate/genome-specific primers with overlapping sequences confirmed by specific PCR, supported the coexistence of at least two distinct genomes in each bat. Recombination analysis using eight Ro-BatCoV HKU9 genomes showed possible recombination events between strains from different bat individuals, which may have allowed for the generation of different genotypes. Western blot assays using recombinant N proteins of Ro-BatCoV HKU9, Betacoronavirus subgroup A (HCoV-HKU1), subgroup B (SARSr-Rh-BatCoV), and subgroup C (Ty-BatCoV HKU4 and Pi-BatCoV HKU5) coronaviruses were subgroup specific, supporting their classification as separate subgroups under Betacoronavirus. Antibodies were detected in 75 (43%) of 175 and 224 (64%) of 350 tested serum samples from Leschenault''s rousette bats by Ro-BatCoV HKU9 N-protein-based Western blot and enzyme immunoassays, respectively. This is the first report describing coinfection of different coronavirus genotypes in bats and coronavirus genotypes of diverse nucleotide variation in the same host. Such unique phenomena, and the unusual instability of ORF7a, are likely due to recombination which may have been facilitated by the dense roosting behavior and long foraging range of Leschenault''s rousette.Coronaviruses infect a wide variety of animals in which they can cause respiratory, enteric, hepatic, and neurological diseases of various severities. Based on genotypic and serological characterization, coronaviruses were traditionally classified into three distinct groups, groups 1, 2, and 3 (3, 27, 59). Recently, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses has renamed the traditional group 1, 2, and 3 coronaviruses as Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, respectively (http://talk.ictvonline.org/media/p/1230.aspx). Coronaviruses are known to have a high frequency of recombination as a result of their unique mechanism of viral replication (27). Such tendency for recombination and high mutation rates may allow them to adapt to new hosts and ecological niches (24, 47, 52).The severe acute respiratory syndrome (SARS) epidemic has boosted interest in the study of coronaviruses in humans and animals (21, 34, 38, 41, 54). In the past few years, there has been a dramatic increase in the number of newly described human and animal coronaviruses (2, 4, 5, 8-10, 15-20, 23, 25, 28, 30, 32, 35, 36, 39, 43, 45, 50, 51, 53, 56, 58). Two novel human coronaviruses, human coronavirus NL63 (HCoV-NL63) and human coronavirus HKU1 (HCoV-HKU1), belonging to Alphacoronavirus and Betacoronavirus, respectively, have been discovered, in addition to the human coronavirus OC43 (HCoV-OC43), human coronavirus 229E (HCoV-229E), and SARS coronavirus (SARS-CoV) (17, 29, 45, 53, 55). We have also previously described the discovery of a diversity of novel coronaviruses in wild bats and birds in China, including SARSr-Rh-BatCoV, belonging to Betacoronavirus subgroup B, from Chinese horseshoe bats (30, 48, 56). Among these novel coronaviruses, three avian coronaviruses were found to belong to a novel subgroup of Gammacoronavirus (Gammacoronavirus subgroup C), while three bat coronaviruses were found to belong to two novel subgroups of Betacoronavirus (Betacoronavirus subgroups C and D) (48, 50). Based on the presence of the huge diversity of coronaviruses in Alphacoronavirus and Betacoronavirus among various bat species, bats are likely the reservoir for the ancestor of these two coronavirus genera (47).During our genome analysis of these novel coronaviruses, one of them, Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), belonging to Betacoronavirus subgroup D, which was identified in Leschenault''s rousette bats, was found to display marked nucleotide and amino acid sequence polymorphism among the four strains with complete genome sequences (50). In our study on HCoV-HKU1, it has been shown that such sequence polymorphisms may indicate the presence of different genotypes (52). By complete genome sequence analysis of the potentially different genotypes of HCoV-HKU1, we have demonstrated for the first time natural recombination in a human coronavirus, resulting in the generation of at least three genotypes (52). We have also recently shown that recombination between different strains of SARSr-Rh-BatCoV from different regions of China may have given rise to the emergence of civet SARSr-CoV (31). To investigate the presence of different genotypes of Ro-BatCoV HKU9, the complete RNA-dependent RNA polymerase (RdRp) (corresponding to nsp12), spike (S), and nucleocapsid (N) gene sequences of Ro-BatCoV HKU9 from 10 additional bats were determined. Since sequence analysis showed the possible coexistence of different genotypes in seven bat individuals, complete genome sequencing of these distinct genotypes from two bats was carried out to investigate for possible recombination events among the different genotypes. In addition, serological characterization of Ro-BatCoV HKU9 was also performed by Western blot and enzyme immunoassays using recombinant Ro-BatCoV HKU9 nucleocapsid proteins and recombinant nucleocapsid proteins of Betacoronavirus subgroup A, B, and C coronaviruses to determine possible cross-reactivity among the different Betacoronavirus subgroups and the seroepidemiology of Ro-BatCoV HKU9 in Leschenault''s rousette bats.     

Crystal Structure of the C-Terminal Cytoplasmic Domain of Non-Structural Protein 4 from Mouse Hepatitis Virus A59

Background

The replication of coronaviruses takes place on cytoplasmic double membrane vesicles (DMVs) originating in the endoplasmic reticulum (ER). Three trans-membrane non-structural proteins, nsp3, nsp4 and nsp6, are understood to be membrane anchors of the coronavirus replication complex. Nsp4 is localized to the ER membrane when expressed alone but is recruited into the replication complex in infected cells. It is revealed to contain four trans-membrane regions and its N- and C-termini are exposed to the cytosol.

Methodology/Principal Findings

We have determined the crystal structures of the C-terminal hydrophilic domain of nsp4 (nsp4C) from MHV strain A59 and a C425S site-directed mutant. The highly conserved 89 amino acid region from T408 to Q496 is shown to possess a new fold. The wild-type (WT) structure features two monomers linked by a Cys425-Cys425 disulfide bond in one asymmetric unit. The monomers are arranged with their N- and C-termini in opposite orientations to form an “open” conformation. Mutation of Cys425 to Ser did not affect the monomer structure, although the mutant dimer adopts strikingly different conformations by crystal packing, with the cross-linked C-termini and parallel N-termini of two monomers forming a “closed” conformation. The WT nsp4C exists as a dimer in solution and can dissociate easily into monomers in a reducing environment.

Conclusions/Significance

As nsp4C is exposed in the reducing cytosol, the monomer of nsp4C should be physiological. This structure may serve as a basis for further functional studies of nsp4.