Identifying and prioritizing potential human-infecting viruses from their genome sequences

Determining which animal viruses may be capable of infecting humans is currently intractable at the time of their discovery, precluding prioritization of high-risk viruses for early investigation and outbreak preparedness. Given the increasing use of genomics in virus discovery and the otherwise sparse knowledge of the biology of newly discovered viruses, we developed machine learning models that identify candidate zoonoses solely using signatures of host range encoded in viral genomes. Within a dataset of 861 viral species with known zoonotic status, our approach outperformed models based on the phylogenetic relatedness of viruses to known human-infecting viruses (area under the receiver operating characteristic curve [AUC] = 0.773), distinguishing high-risk viruses within families that contain a minority of human-infecting species and identifying putatively undetected or so far unrealized zoonoses. Analyses of the underpinnings of model predictions suggested the existence of generalizable features of viral genomes that are independent of virus taxonomic relationships and that may preadapt viruses to infect humans. Our model reduced a second set of 645 animal-associated viruses that were excluded from training to 272 high and 41 very high-risk candidate zoonoses and showed significantly elevated predicted zoonotic risk in viruses from nonhuman primates, but not other mammalian or avian host groups. A second application showed that our models could have identified Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a relatively high-risk coronavirus strain and that this prediction required no prior knowledge of zoonotic Severe Acute Respiratory Syndrome (SARS)-related coronaviruses. Genome-based zoonotic risk assessment provides a rapid, low-cost approach to enable evidence-driven virus surveillance and increases the feasibility of downstream biological and ecological characterization of viruses.

Surveillance of emerging viruses is one of the first steps to avoid the next pandemic. This study uses machine learning to identify many zoonotic viruses directly from their genomes. This allows rapid assessment of research priorities as soon as new viruses are discovered, focusing research and surveillance efforts on the viruses most likely to infect humans.     

Invertebrate Iridescent Virus 6, a DNA Virus,Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors

Insects are not only major vectors of mammalian viruses, but are also host to insect-restricted viruses that can potentially be transmitted to mammals. While mammalian innate immune responses to arboviruses are well studied, less is known about how mammalian cells respond to viruses that are restricted to infect only invertebrates. Here we demonstrate that IIV-6, a DNA virus of the family Iridoviridae, is able to induce a type I interferon-dependent antiviral immune response in mammalian cells. Although IIV-6 is a DNA virus, we demonstrate that the immune response activated during IIV-6 infection is mediated by the RIG-I-like receptor (RLR) pathway, and not the canonical DNA sensing pathway via cGAS/STING. We further show that RNA polymerase III is required for maximal IFN-β secretion, suggesting that viral DNA is transcribed by this enzyme into an RNA species capable of activating the RLR pathway. Finally, we demonstrate that the RLR-driven mammalian innate immune response to IIV-6 is functionally capable of protecting cells from subsequent infection with the arboviruses Vesicular Stomatitis virus and Kunjin virus. These results represent a novel example of an invertebrate DNA virus activating a canonically RNA sensing pathway in the mammalian innate immune response, which reduces viral load of ensuing arboviral infection.     

Chicken Interferon-Inducible Transmembrane Protein 3 Restricts Influenza Viruses and Lyssaviruses In Vitro

Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses both in vitro and in vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includes IFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. Chicken IFITM2 and -3 are constitutively expressed in all tissues examined, whereas IFITM1 is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections.     

Emergence of influenza A viruses.

Pandemic influenza in humans is a zoonotic disease caused by the transfer of influenza A viruses or virus gene segments from animal reservoirs. Influenza A viruses have been isolated from avian and mammalian hosts, although the primary reservoirs are the aquatic bird populations of the world. In the aquatic birds, influenza is asymptomatic, and the viruses are in evolutionary stasis. The aquatic bird viruses do not replicate well in humans, and these viruses need to reassort or adapt in an intermediate host before they emerge in human populations. Pigs can serve as a host for avian and human viruses and are logical candidates for the role of intermediate host. The transmission of avian H5N1 and H9N2 viruses directly to humans during the late 1990s showed that land-based poultry also can serve between aquatic birds and humans as intermediate hosts of influenza viruses. That these transmission events took place in Hong Kong and China adds further support to the hypothesis that Asia is an epicentre for influenza and stresses the importance of surveillance of pigs and live-bird markets in this area.     

Antiviral activity of ovotransferrin discloses an evolutionary strategy for the defensive activities of lactoferrin.

Ovotransferrin (formerly conalbumin) is an iron-binding protein present in birds. It belongs to the transferrin family and shows about 50% sequence homology with mammalian serum transferrin and lactoferrin. This protein has been demonstrated to be capable of delivering iron to cells and of inhibiting bacterial multiplication. However, no antiviral activity has been reported for ovotransferrin, although the antiviral activity of human and bovine lactoferrins against several viruses, including human herpes simplex viruses, has been well established. In this report, the antiviral activity of ovotransferrin towards chicken embryo fibroblast infection by Marek's disease virus (MDV), an avian herpesvirus, was clearly demonstrated. Ovotransferrin was more effective than human and bovine lactoferrins in inhibiting MDV infection and no correlation between antiviral efficacy and iron saturation was found. The observations reported here are of interest from an evolutionary point of view since it is likely that the defensive properties of transferrins appeared early in evolution. In birds, the defensive properties of ovotransferrin remained joined to iron transport functions; in mammals, iron transport functions became peculiar to serum transferrin, and the defensive properties towards infections were optimised in lactoferrin.     

Innate immunity against HIV: a priority target for HIV prevention research

The xenotropic/polytropic subgroup of mouse leukemia viruses (MLVs) all rely on the XPR1 receptor for entry, but these viruses vary in tropism, distribution among wild and laboratory mice, pathogenicity, strategies used for transmission, and sensitivity to host restriction factors. Most, but not all, isolates have typical xenotropic or polytropic host range, and these two MLV tropism types have now been detected in humans as viral sequences or as infectious virus, termed XMRV, or xenotropic murine leukemia virus-related virus. The mouse xenotropic MLVs (X-MLVs) were originally defined by their inability to infect cells of their natural mouse hosts. It is now clear, however, that X-MLVs actually have the broadest host range of the MLVs. Nearly all nonrodent mammals are susceptible to X-MLVs, and all species of wild mice and several common strains of laboratory mice are X-MLV susceptible. The polytropic MLVs, named for their apparent broad host range, show a more limited host range than the X-MLVs in that they fail to infect cells of many mouse species as well as many nonrodent mammals. The co-evolution of these viruses with their receptor and other host factors that affect their replication has produced a heterogeneous group of viruses capable of inducing various diseases, as well as endogenized viral genomes, some of which have been domesticated by their hosts to serve in antiviral defense.     

Temporal trends in the discovery of human viruses

On average, more than two new species of human virus are reported every year. We constructed the cumulative species discovery curve for human viruses going back to 1901. We fitted a statistical model to these data; the shape of the curve strongly suggests that the process of virus discovery is far from complete. We generated a 95% credible interval for the pool of as yet undiscovered virus species of 38-562. We extrapolated the curve and generated an estimate of 10-40 new species to be discovered by 2020. Although we cannot predict the level of health threat that these new viruses will present, we conclude that novel virus species must be anticipated in public health planning. More systematic virus discovery programmes, covering both humans and potential animal reservoirs of human viruses, should be considered.     

Viral adaptation to host: a proteome‐based analysis of codon usage and amino acid preferences

Viruses differ markedly in their specificity toward host organisms. Here, we test the level of general sequence adaptation that viruses display toward their hosts. We compiled a representative data set of viruses that infect hosts ranging from bacteria to humans. We consider their respective amino acid and codon usages and compare them among the viruses and their hosts. We show that bacteria‐infecting viruses are strongly adapted to their specific hosts, but that they differ from other unrelated bacterial hosts. Viruses that infect humans, but not those that infect other mammals or aves, show a strong resemblance to most mammalian and avian hosts, in terms of both amino acid and codon preferences. In groups of viruses that infect humans or other mammals, the highest observed level of adaptation of viral proteins to host codon usages is for those proteins that appear abundantly in the virion. In contrast, proteins that are known to participate in host‐specific recognition do not necessarily adapt to their respective hosts. The implication for the potential of viral infectivity is discussed.     

嗜肝病毒家族成员间进化关系的初步研究

比较分析了嗜肝病毒家族四成员间的全基因核苷酸序列,得出了它们之间的相对进化距离,DHBV 分野最早,GSHV,WHV 次之,HBV 最晚;其进化方式属趋 异性进化.进一步比较各 ORF 的保守性,发现各 ORF 间及同一 ORF 内的不同区域核苷酸发生替换的频率有明显差别;讨论了各 ORF 的特点及意义.     

Evolution and ecology of influenza A viruses.

In this review we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species and study the ecological features that permit the perpetuation of influenza viruses in aquatic avian species. Phylogenetic analysis of the nucleotide sequence of influenza A virus RNA segments coding for the spike proteins (HA, NA, and M2) and the internal proteins (PB2, PB1, PA, NP, M, and NS) from a wide range of hosts, geographical regions, and influenza A virus subtypes support the following conclusions. (i) Two partly overlapping reservoirs of influenza A viruses exist in migrating waterfowl and shorebirds throughout the world. These species harbor influenza viruses of all the known HA and NA subtypes. (ii) Influenza viruses have evolved into a number of host-specific lineages that are exemplified by the NP gene and include equine Prague/56, recent equine strains, classical swine and human strains, H13 gull strains, and all other avian strains. Other genes show similar patterns, but with extensive evidence of genetic reassortment. Geographical as well as host-specific lineages are evident. (iii) All of the influenza A viruses of mammalian sources originated from the avian gene pool, and it is possible that influenza B viruses also arose from the same source. (iv) The different virus lineages are predominantly host specific, but there are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. (v) The influenza viruses currently circulating in humans and pigs in North America originated by transmission of all genes from the avian reservoir prior to the 1918 Spanish influenza pandemic; some of the genes have subsequently been replaced by others from the influenza gene pool in birds. (vi) The influenza virus gene pool in aquatic birds of the world is probably perpetuated by low-level transmission within that species throughout the year. (vii) There is evidence that most new human pandemic strains and variants have originated in southern China. (viii) There is speculation that pigs may serve as the intermediate host in genetic exchange between influenza viruses in avian and humans, but experimental evidence is lacking. (ix) Once the ecological properties of influenza viruses are understood, it may be possible to interdict the introduction of new influenza viruses into humans.     

Influenza type A in humans,mammals and birds: determinants of virus virulence,host-range and interspecies transmission

The virulence of a virus is determined by its ability to adversely affect the host cell, host organism or population of host organisms. Influenza A viruses have been responsible for four pandemics of severe human respiratory disease this century. Avian species harbour a large reservoir of influenza virus strains, which can contribute genes to potential new pandemic human strains. The fundamental importance of understanding the role of each of these genes in determining virulence in birds and humans was dramatically emphasised by the recent direct transmission of avian influenza A viruses to humans, causing fatal infection but not community spread. An understanding of the factors involved in transmission between avian and mammalian species should assist in the development of better surveillance strategies for early recognition of influenza A virus strains having human pandemic potential, and possibly in the design of anti-viral strategies.     

A Preliminary Study of Viral Metagenomics of French Bat Species in Contact with Humans: Identification of New Mammalian Viruses

The prediction of viral zoonosis epidemics has become a major public health issue. A profound understanding of the viral population in key animal species acting as reservoirs represents an important step towards this goal. Bats harbor diverse viruses, some of which are of particular interest because they cause severe human diseases. However, little is known about the diversity of the global population of viruses found in bats (virome). We determined the viral diversity of five different French insectivorous bat species (nine specimens in total) in close contact with humans. Sequence-independent amplification, high-throughput sequencing with Illumina technology and a dedicated bioinformatics analysis pipeline were used on pooled tissues (brain, liver and lungs). Comparisons of the sequences of contigs and unassembled reads provided a global taxonomic distribution of virus-related sequences for each sample, highlighting differences both within and between bat species. Many viral families were present in these viromes, including viruses known to infect bacteria, plants/fungi, insects or vertebrates, the most relevant being those infecting mammals (Retroviridae, Herpesviridae, Bunyaviridae, Poxviridae, Flaviviridae, Reoviridae, Bornaviridae, Picobirnaviridae). In particular, we detected several new mammalian viruses, including rotaviruses, gammaretroviruses, bornaviruses and bunyaviruses with the identification of the first bat nairovirus. These observations demonstrate that bats naturally harbor viruses from many different families, most of which infect mammals. They may therefore constitute a major reservoir of viral diversity that should be analyzed carefully, to determine the role played by bats in the spread of zoonotic viral infections.     

Influenza virus A (H5N1): a pandemic risk?

Influenza A subtype H5N1 has represented a growing alarm since its recent identification in Asia. Previously thought to infect only wild birds and poultry, H5N1 has now infected humans, cats, pigs and other mammals in an ongoing outbreak, often with a fatal outcome. In order to evaluate the risk factors for human infection with influenza virus H5N1, here we summarize 53 case patients confirmed with H5N1 infection during 2006. The review also compares the mortality rate among human cases from late 2003 until 15 June 2006 in different countries. Neither how these viruses are transmitted to humans nor the most effective way to reduce the risk for infection is fully understood. The association between household contact with diseased poultry in human infection has been demonstrated. This association could possibly operate by 2 mechanisms. First, transmission may be by inhalation or conjunctival deposition of large infectious droplets which may travel only in short distances. Second, having infected poultry in the home and preparation of infected poultry for consumption may result in exposure to higher virus concentrations than other types of exposure. There is so far no significant evidence for repeated human to human transmission, yet some cases of human to human transmission among the family relatives in Indonesia, Azerbaijan, Iraq and Turkey have been described. Recent outbreaks of highly pathogenic avian influenza A virus (H5N1 subtype) infections in poultry and humans (through direct contact with infected birds) have raised concerns that a new influenza pandemic might occur in the near future.     

蝙蝠抗病毒天然免疫的研究进展

近年来,健康的蝙蝠体内检测到了很多与人类传染病相关的病毒,包括狂犬病病毒（Rabies virus）、埃博拉病毒（Ebola virus）、严重急性呼吸综合征病毒（SARS-CoV）以及最近新出现的新型冠状病毒（SARS-CoV-2）等。与其他哺乳动物不同的是,蝙蝠在感染了这些病毒后不会表现出明显的临床症状。因此,人类可以通过研究蝙蝠的免疫系统获得抗病毒免疫的新知识。本文综述了蝙蝠抗病毒天然免疫研究的最新进展,指出了蝙蝠在天然免疫方面的特殊性：蝙蝠独有的飞行能力可能导致其演化出一套独特的抗病毒免疫响应机制,同时具有一套独特的机制限制炎症反应。蝙蝠物种的多样性丰富（>1400种）,超过了哺乳动物的五分之一。因此对蝙蝠免疫基因的多样性研究,将促进对蝙蝠特殊免疫机制的理解,对人类传染病防治和畜牧业发展具有重要意义。     

Evidence of infection with H4 and H11 avian influenza viruses among Lebanese chicken growers

Human infections with H5, H7, and H9 avian influenza viruses are well documented. Exposure to poultry is the most important risk factor for humans becoming infected with these viruses. Data on human infection with other low pathogenicity avian influenza viruses is sparse but suggests that such infections may occur. Lebanon is a Mediterranean country lying under two major migratory birds flyways and is home to many wild and domestic bird species. Previous reports from this country demonstrated that low pathogenicity avian influenza viruses are in circulation but highly pathogenic H5N1 viruses were not reported. In order to study the extent of human infection with avian influenza viruses in Lebanon, we carried out a seroprevalence cross-sectional study into which 200 poultry-exposed individuals and 50 non-exposed controls were enrolled. We obtained their sera and tested it for the presence of antibodies against avian influenza viruses types H4 through H16 and used a questionnaire to collect exposure data. Our microneutralization assay results suggested that backyard poultry growers may have been previously infected with H4 and H11 avian influenza viruses. We confirmed these results by using a horse red blood cells hemagglutination inhibition assay. Our data also showed that farmers with antibodies against each virus type clustered in a small geographic area suggesting that unrecognized outbreaks among birds may have led to these human infections. In conclusion, this study suggests that occupational exposure to chicken is a risk factor for infection with avian influenza especially among backyard growers and that H4 and H11 influenza viruses may possess the ability to cross the species barrier to infect humans.     

Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt

ABSTRACT: BACKGROUND: Avian influenza virus H9N2 is a panzootic pathogen that affects poultry causing mild to moderate respiratory distress but has been associated with high morbidity and considerable mortality. Interspecies transmission of H9N2 from avian species to mammalian hosts does occur. The virus possesses human virus-like receptor specificity and it can infect humans producing flu-like illness. METHODS: Recently, mild influenza like symptoms were detected in H5N1 vaccinated flocks. Influenza A subtype H9N2 was isolated from the infected flock. The virus evolution was investigated by sequencing the viral genes to screen the possible virus recombination. The viral amino acid sequences from the isolated H9N2 strains were compared to other related sequences from the flu data base that were used to assess the robustness of the mutation trend. Changes in the species-associated amino acid residues or those that enabled virulence to mammals were allocated. RESULTS: Phylogenetic analyses of haemagglutinin and neuraminidase genes showed that the recently isolated Egyptian strain belonged to the H9N2 sub-lineage that prevails in Israel. The six internal segments of the isolated virus were found to be derived from the same sub-lineage with no new evidence of reassortment. The results demonstrated conserved genetic and biological constitution of H9N2 viruses in the Middle East. The recently isolated H9N2 virus from chicken in Egypt possessed amino acids that could enable the virus to replicate in mammals and caused severe disease in domestic chickens. CONCLUSION: The study highlights the importance of continuous monitoring of the mutations evolved in avian influenza viruses and its impact on virulence to avian species in addition to its importance in the emergence of new strains with the capacity to be a pandemic candidate.     

Spleen necrosis virus, an avian immunosuppressive retrovirus, shares a receptor with the type D simian retroviruses.

The reticuloendotheliosis viruses (REV) are a family of highly related retroviruses isolated from gallinaceous birds. On the basis of sequence comparison and overall genome organization, these viruses are more similar to the mammalian type C retroviruses than to the avian sarcoma/leukemia viruses. The envelope of a member of the REV family, spleen necrosis virus (SNV), is about 50% identical in amino acid sequence to the envelope of the type D simian retroviruses. Although SNV does not productively infect primate or murine cells, the receptor for SNV is present on a variety of human and murine cells. Moreover, interference assays show that the receptor for SNV is the same as the receptor for the type D simian retroviruses. We propose that adaptation of a mammalian type C virus to an avian host provided the REV progenitor.     

Molecular basis of efficient replication and pathogenicity of H9N2 avian influenza viruses in mice

H9N2 subtype avian influenza viruses (AIVs) have shown expanded host range and can infect mammals, such as humans and swine. To date the mechanisms of mammalian adaptation and interspecies transmission of H9N2 AIVs remain poorly understood. To explore the molecular basis determining mammalian adaptation of H9N2 AIVs, we compared two avian field H9N2 isolates in a mouse model: one (A/chicken/Guangdong/TS/2004, TS) is nonpathogenic, another one (A/chicken/Guangdong/V/2008, V) is lethal with efficient replication in mouse brains. In order to determine the basis of the differences in pathogenicity and brain tropism between these two viruses, recombinants with a single gene from the TS (or V) virus in the background of the V (or TS) virus were generated using reverse genetics and evaluated in a mouse model. The results showed that the PB2 gene is the major factor determining the virulence in the mouse model although other genes also have variable impacts on virus replication and pathogenicity. Further studies using PB2 chimeric viruses and mutated viruses with a single amino acid substitution at position 627 [glutamic acid (E) to lysine, (K)] in PB2 revealed that PB2 627K is critical for pathogenicity and viral replication of H9N2 viruses in mouse brains. All together, these results indicate that the PB2 gene and especially position 627 determine virus replication and pathogenicity in mice. This study provides insights into the molecular basis of mammalian adaptation and interspecies transmission of H9N2 AIVs.     

The Short Stalk Length of Highly Pathogenic Avian Influenza H5N1 Virus Neuraminidase Limits Transmission of Pandemic H1N1 Virus in Ferrets

H5N1 influenza viruses pose a pandemic threat but have not acquired the ability to support sustained transmission between mammals in nature. The restrictions to transmissibility of avian influenza viruses in mammals are multigenic, and overcoming them requires adaptations in hemagglutinin (HA) and PB2 genes. Here we propose that a further restriction to mammalian transmission of the majority of highly pathogenic avian influenza (HPAI) H5N1 viruses may be the short stalk length of the neuraminidase (NA) protein. This genetic feature is selected for when influenza viruses adapt to chickens. In our study, a recombinant virus with seven gene segments from a human isolate of the 2009 H1N1 pandemic combined with the NA gene from a typical chicken-adapted H5N1 virus with a short stalk did not support transmission by respiratory droplet between ferrets. This virus was also compromised in multicycle replication in cultures of human airway epithelial cells at 32°C. These defects correlated with a reduction in the ability of virus with a short-stalk NA to penetrate mucus and deaggregate virions. The deficiency in transmission and in cleavage of tethered substrates was overcome by increasing the stalk length of the NA protein. These observations suggest that H5N1 viruses that acquire a long-stalk NA through reassortment might be more likely to support transmission between humans. Phylogenetic analysis showed that reassortment with long-stalk NA occurred sporadically and as recently as 2011. However, all identified H5N1 viruses with a long-stalk NA lacked other mammalian adapting features and were thus several genetic steps away from becoming transmissible between humans.