人类病毒组——预测莫测之事，识别真正之敌

Tamoghna Ghosh, Pankaj Krishna, Prakash Singh Bisen

(1.印度医药城Avantor性能材料公司 研发诊断中心, 印度 德拉敦, 248011;2. 斋浦尔国立大学医学科学和研究中心研究所, 印度 斋浦尔，302017;3. 瓜廖尔大学生物技术研究学院, 印度 瓜廖尔, 474011)

Introduction

Zoonotic transfer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a pandemic with more than 16 883 695 infections and 662 480 mortality as of July 29th，2020[1-2]. Previously，sudden viral outbreaks like SARS, MERS, Ebola, Zika, and Nipah resulted in huge loss in health and economy due to unaware of countermeasures. The advent of biological databanks and its algorithms in health sciences aided in many environmental and pharmaceutical interventions, however, the limited datasets on human and zoonotic viruses (“biological dark matter”) poses significant hurdle on virus studies. Hence, an exhaustive virome research is required for the world to come up with a program for future endemics. From the discovery of Tobacco Mosaic Virus in 1892 and Foot and Mouth Virus in 1898, diverse virus species were discovered which are infectious, commensal and silent members of virome, analogous to bacterial counterpart of microbiome[3]. The viruses are interspersed in human genome and ubiquitously present in skin mucosa, gastrointestinal tract, respiratory and reproductive organs. They have perfectly adapted with the host and maintain an ideal equilibrium for persistence and transmission. Henceforth, thousands of orphan viruses are still being discovered. It has been established that these viruses provide a selective advantage to the host by participating in physiological processes and boosting immunity. Contrarily, endogenous viruses also invoke diverse immune response and secondary infections[4]. It has been demonstrated that more often, transcriptional patterns in human can be traced back to virome and microbiome interaction with host. Diversity and size of the endogenous viruses is quite astonishing. It is postulated that microbiome content is about 10 times of the human cells, and virome size is 10 times of the microbiome. Human feces contain 108to 109virus/g of its dry weight[5]. Almost 45% of the human genome is viral sequences, retro and DNA transposons. However, with high mutation frequency and evolution of new viruses, there is a constant update in the virome size. There are more than 220 virus species which can infect human and around 60% of them are from zoonotic sources[3, 6]. Examples include HIV (related to simian immunodeficiency virus) from African green monkey, SARS, MERS, SARS-2 from bat, pig, pangolin etc. , lyssaviruses of Rhabdoviridae (causes rabies) from dogs, cat, bat, ferrets etc[3, 7]. Even now, many of the mammalian virus are capable of crossing over the species barrier and infect human. It seems inevitable that new infectious viral species will soon emerge, and it is obligatory to look for similar characteristic motifs, domains and transmission parameters of the predecessors. However, rigid compliance to Koch′s postulate to certain pathogen may fail for some of the human endogenous and exogenous viruses[8]. Zoonotic transfers are predominate due to consumption of various wild and sea animals, expanded travels, mass farming, compromised ecosystem and climatic changes. With an idea of building a comprehensive virus platform, Global Virome Project and PREDICT Project were established in 2018 for forecasting possible leads in the future for resisting epidemics[9].

Culture dependent approaches fail to unfold most of the infectious and commensal viral agents, ergo the advent of deep sequencing technologies. Next generation sequencing using Pyrosequencing (Roche/454 Life Sciences), Nanopore technology (Oxford technologies), Illumina Genome Analyzer (Illumina Inc.), Solexa (SOLiD), and high-throughput screenings with global network of viral sequence data bank is successfully augmenting wide arrays of viral meta-data. Various human metagenomic and protein cluster databases are available for exploration[10-11]. Mining of the human viruses revealed their integration in the host germlines and extension over ages. The endogenous viral genomics can provide information related to variously related exogenous viruses[12]. Similarly, the cancer transcriptomics data provided numerous viral sequences which are currently being investigated for their correlation with cancer physiology[13-14]. Adequate epidemiological meta data from diseased and healthy individuals globally would strengthen metagenomics-based discoveries and disease related studies. Mutational and protein similarity studies will give a wider perspective for divergent and future infectious etiologies. Metagenomic data from various control groups should be carefully analyzed with the parameters like age, geographic provenance, and if possible general exposure to viruses (socio-demographics or occupation). Deep sequencing, transcriptomics and proteomics studies involving microbiome-virome-host interactions will also pave way for better understanding the cytopathology and transmission. Meanwhile, there are challenges in meta-analysis. Low sequence references, poor homology due to wide mutations, low-complexity sequence, short reading sequences and small portion of total nucleic acid in microbial communities are main obstacles in inclusive virome research[15]. Improvement of techniques associated with viral sequence enrichment and viral signal abundance will boost the study quality.

Members in virome

Human body is a reservoir for a wide range of viruses. Metagenomic analysis revealed a high percentage of phage sequences than eukaryotic viruses in oral cavity and respiratory organs. Phages are found predominantly from Siphoviridae, Myoviridae, and Podoviridae families. The localization of virus families in oral cavities is not homogenous in nature. Different virus sequences were observed in different parts of oral cavity (subgingival, supragingival biofilms, dental plaque, and saliva). Nasal aspirates revealed various viruses related to cytomegalovirus, α-, β-, and γ-Papillomaviruses, Lymphocryptovirus, Roseolovirus, herpes simplex virus, unclassified Papillomaviruses, Polyomavirus, Mastadenovirus, Alphatorquevirus and unclassified Anelloviruses families. Rhinoviruses are quite prevalent in the respiratory tract. The prevalence of rhinoviruses is frequently considered with the isolation rates of 2.3% in routine respiratory samples[16-17]. Studies from asymptomatic infants have detected numerous enterovirus in stool samples. Bacteriophage genotypes were discovered 2 to 5 times of 1 200-2 000 predicted bacterial genera in a stool community. Enteric viruses frequently found in stool are Astroviruses, Arenaviruses, Bocaviruses, Coronaviruses, Enteroviruses, Klasseviruses, Noroviruses and Polyomaviruses. Human Enterovirus (HEV) and Parechovirus (HPeV) undergo persistent or intermittent shedding in healthy individual[18]. Skin Viruses are consisted of both transient and resident viruses. Superficial layers of skin in most individuals are commonly consisted of cutaneous β- and γ-Human Paillomaviruses (β- and γ-HPVs). Based on sequencing data, HPVs predominate in skin samples, with at least 12 known species and many unclassified. HPV sequences are more abundant at the palm, forehead, retroauricular crease, and occiput sites[19-20]. The surface of the skin has revealed Circoviridae, Papillomaviridae and Polyomaviridae as the three predominant families during analysis of viral DNA sequences by NGS. The presence of numerous Circoviridae at the skin surface indicates the most likely cross-species transmission as it mainly infects animals but also is detected in the feces of primates including human[21]. Furthermore, Adenoviridae, Herpesviridae, Anelloviridae and Poxviridae are other eukaryotic viruses often found on the skin[16]. Other viruses like Torque teno viruses (TTV), TT-like mini viruses (TTMV), picobirnavirus, and HPeV types 1 and 6 are most frequently discovered in the body, whereas Aichi virus, adenovirus, astroviruses, human bocavirus (HBoV-1) and rotavirus are less frequent[22]. Plasma samples from febrile and afebrile children were found to contain anelloviruses. Well characterized human viruses of Herpesviridae include cytomegalovirus (CMV), Epstein- Barr virus (EBV), herpes simplex virus types 1 and 2, and HHV types 6/8 show high prevalence in human plasma. Some of these are leukotropic viruses and their DNA sequences can be readily identified in the circulating human leucocytes associated with either acute disease at the primary infection time or active replication of virus during immunosuppression or with lymphoma or sarcoma like pathological conditions (EBV and HHV8), encephalitis (HSV, CMV, EBV, and HHV6) or digestive symptoms (CMV and HHV6)[18, 23]. In most of the metagenomics studies, sequences classified as phages are much larger in proportion in comparison to those classified as eukaryotic viruses. The primary composition of phage communities includes Podoviridae, Myoviridae, Microviridae, and Siphoviridae (Order Caudovirales) along with a very low archaeal virus (mainly from Lipothrixviridae). Furthermore, Corticoviridae, Inoviridae and Tectiviridae families were also identified but less than 50% of phages are still unclassified[24-25]. RNA viruses reported are Caliciviridae, Picornaviridae (Enterovirus), Picobirnaviridae, and Reoviridae (Rotavirus)[26]. In blood samples of patients associated with cardiovascular diseases and HIV, a higher abundance of phage DNA has been reported when compared to healthy individuals, depicting abundance due to lower immunocompetence. Several strains of Lactobacillus phages exhibiting lytic and lysogenic phenotypes are mainly detected in vagina. The abundance and diversity of antibiotic resistance genes is reported to be increased in the mice gut phage communities after antibiotic treatment, displaying markers for antibiotic resistance in human microbiota[16]. Numerous viruses of the respiratory system such as HRVs, Human Rhinoviruses, Influenza, Coronaviruses and Respiratory Syncytial Virus (RSV) were found to be responsible for pathologies and classified as monophletic group of viruses under megavirales order. Giant viruses are comprised of seven viral families which include Ascoviridae, Asfaviridae, Iridiviridae, Poxviridae, Phycodnaviridae, Mimiviridae and Marseilleviridae infecting a wide range of eukaryotes including humans and phagocytic protists. Only two families, Poxviridae and Mimiviridae have been linked to disease in humans[27]. Viral infection outbreaks throughout the ages have resulted in discovery of various pathogenic viruses belonging to Paramyxoviridae, Coronaviridae, Togaviridae Flaviviridae, Filoviridae, Bunyaviridae, and Hepeviridae families. Some examples include BK virus (BKV), JC virus (JCV), hantavirus (HTNV), Sin Nombre virus (SNV) and Merkel cell polyomavirus (MCV or MCPyV)[7, 28].

Host-virus-microbiota interaction

Viruses confer a variety of inflammatory responses and are alternatively implicated in unexpected benefits. Various genera of viruses like Herpes, Noro and Polyoma fall in both commensal and pathogenic groups of human virome[29]. These viruses however mostly non-pathogenic in nature, contribute to host phenotype and their subsets sometimes act beneficial. Both RNA and DNA viruses trigger innate immune response followed by expression of interferons and cytokines. It also engages in antiviral gene expression and production of T cells and natural killer cells. The ramification primarily consequences in antiviral defense, although host becomes susceptible to complex diseases and secondary infections. For instance, RSV incites TH2 polarization in lungs, HIV potentially activates TH17 and HBV invokes low innate immune response by interfering with IFN-1 processes[30-31]. Viruses like Lymphocytic choriomeningitis (LCMV) and murine adenovirus induce suboptimal level of T cell response to irradiate competitions. More often there is a lethal consequence of viral immunomodulation. Recognition of HCV and HBV antigens and proteins with similar epitopes leads to liver cirrhosis and fibrosis. This also results in immunosuppression followed by secondary infections. Viral immunomodulation also has consequences for cancer. HPV causes hepatocellular carcinoma and Kaposi′s sarcoma-associated herpesvirus (KSHV) create favorable conditions for sarcomas and lymphoma. Similarly, Merkel polyomavirus has been implicated in Marek′s disease and Kaposi sarcoma[32]. Inflammation by various viruses has unknown etiology. Epstein-Barr virus (EBV) induces lymphocytes laden with high amount of affector tissues similar to cases of Systemic lupus erythematosus (SLE), Rheumatoid arthritis and Multiple Sclerosis[30, 33]. Likewise, molecular mimicry of virus and self-antigens occasionally causes bystander activation leading to autoimmune diseases. Virus induced immunomodulation also causes mutations and genetic abnormalities. It has been seen that in the persistent presence of Murine Norovirus (MNV) (a homologue for human norovirus), expression levels of beneficial genes associated with Inflammatory bowel disease (IBD) are significantly reduced in mice. MNV has also been known to induce intestinal inflammation[30]. The inflammation is often caused by gut microbiome-virome interaction. The virus-host genome interaction may also indicate the complexity of diseases. A lot of studies have been executed on gut virome and its diseases. Study on Type 1 diabetes (T1D) has shown enterovirus Coxsackie B to induce pancreatic cellular deprivation[34]. Infection by rotavirus increases auto antibodies in children for T1D[35]. Genome wide association study (GWAS) indicates adaptive immunity contributed to type 1 diabetes pathogenesis which subsequently indicates viral infection. SNPs in MDAS and IRF-7 gene related to viral immunity have been found to be associated with T1D. Lung infection by rhinoviruses in infants is seen to be corelated with Asthma susceptibility genes. Similarly, Sendai virus infection in infants leads to asthma and chronic obstructive pulmonary disease[5]. System biology studies have also shown that genetic variation in the host commensal viruses also affect transcription response of the immunomodulatory genes.

Alternatively, endogenous viruses are also found to aid in the physiological processes by eliminating viral pathogens by cross immunity, viral interference, expressing functionally useful proteins, killing pathogenic bacteria (phage) and virus-microbiome interaction. The virus and host susceptible genes also often cause beneficial complementation. Studies showed that LCMV prevents autoimmune diabetes by immune suppression[36]. γ-herpesvirus 68 protects from SLE and confers resistance toListeriamonocytogenesandYersiniapestisinfection by inducing IFN and macrophage production[37]. Endogenous virus also aids in resisting intestinal injuries resembling Crohn′s disease. MNV protects germ free mouse upon resisting localization of other pathogenic bacteria. Murine cytomegalovirus (MCMV) increases epithelial cell proliferation in multiple organs[30]. It has also been speculated that HPV could be a symbiont for keratinocytes proliferation during healing[18]. A native virus called gyrovirus harbor an apoptotic protein which showed cytotoxicity to cancer cells[38]. HIV and SIV lead to depletion of commensal bacteria in intestinal epithelium. MNV triggers IFN1 induction which leads to lethal response to E. coli. Endogenous retroviruses also contribute to the evolution of anti-viral gene expression and immunomodulation through cross reactivity[18]. For instance, ERV RNA expression induced by B and T cell interaction triggers antiviral gene expression[39]. ERV infection also triggers polyclonal proliferation, and causes immunomodulation against exogeneous viruses. The gut mucosa is dominated by endogenous phages which provide advantageous properties to the commensal bacteria[40]. These include bacterial virulence, pathogenesis and balancing the host microbiome homeostasis. The phages also provide a secondary layer on the mucosa which acts as a protective barrier. Role of phages in IBS and Crohn′s disease has also been postulated. Symbiotic activities between microbes and exogenous viruses have also been reported. For instance, it has been observed that Bacterial LPS assists some viruses to attach the host cell and GII 4 Human Norovirus (HNV) infect B cells only in presence of commensal bacteria[41-43]. Likewise, Enterobacter cloacae surface glycoprotein mediate polio virus attachment to the host cell. Some strains of MNV or norovirus are reported to alter the composition of bacterial microbiome[41, 44].

Conclusions

Endogenous viruses confer both complex deleterious and beneficial properties to the host depending upon location, genotype and host immunocompetence. Viruses also incorporate anomalies in coding genes due to integration and episome formation (viz. HIV, HBV, Herpes virus, HPV and adenovirus). The transcription patterns and frequencies of host functional genes also depend on virome and microbiota coexisting[45], which coevolve with different internal, external physiological parameters and time. Cataloging the mammalian virome, discovery of stable virus culture, and system level decoding will create effective measures for future countermeasures. Key concept of understanding the virome will be deciphering the nature of the viruses in different organs, stimulus for adaptive and innate immunity, interaction amongst trans-kingdom viruses and host microbiota and effect of virus consortia on overall physiology[46]. The virome projects undergoing should be able to postulate viral genotype/phenotype characteristics. There is a potential risk of infectious viral evolution both endogenously and exogenously. It has been observed that lower immunity, health hazards and epigenetic changes contribute to native virus turning infectious. On the other hand, communal mammals like primates, bovine, porcine, feline, bats, ferrets, civets and rodents are likely causes for zoonotic transmission. Many enteric viruses (e. g. , JCV) are found worldwide in high concentrations in urban sewage, and lead to water contamination in countries with poor resources. Recurring epidemics have necessitated the formulation of effective control measures. Limited consumption and domestication of wild and sea animals could be effective in minimizing the zoonotic transmission. The prevention of arthropod-borne infections (e. g. , DENV, CHIKV, and ZIKV) using vector control methods and effective sanitation has been partially successful[47]. Hence, identification and metagenomic study of both zoonotic and human virome are of utmost importance. Evolution of infectious viruses can be mapped through identification of conserved functional and structural proteins in human and zoonotic virome. However, challenging the present condition needs exhaustive studies and introduction of artificial intelligence and high throughput screening will mitigate the advent of future epidemics.