Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped and positive-stranded RNA virus of Arteriviridae family, causes porcine reproductive and respiratory syndrome (PRRS). PRRS is responsible for over one billion dollar loss per year through direct and indirect costs in the US swine industry. Two entirely distinct genotypes of PRRSV circulate in European (genotype 1/PRRSV 1) and North American countries (genotype 2/PRRSV 2) and cause tremendous economic loss. PRRSV is transmitted through oral-nasal secretions and semen. The clinical signs include fever, anorexia, mild to severe respiratory problems, abortion and reproductive failures. It is the most common pathogen associated with porcine respiratory disease complex (PRDC).

Swine influenza (flu) constitutes another persistent health challenge to the global pig industry. Flu infection is caused by influenza A virus of Orthomyxoviridae family which has negative-sense, single-stranded, segmented RNA genome. Influenza virus is transmitted through direct contact with infected animals or contaminated fomites, aerosols and large droplets. The clinical signs of influenza infection include fever, anorexia, loss of weight gain and respiratory problems. Influenza associated economic losses are due to morbidity, loss of body weight gain, increased time to market, secondary infections, medication and veterinary expenses. Influenza of swine origin occasionally infect humans and can even lead to pandemics as of 2009.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) are enteric pathogens of young pigs. These viruses belong to Coronaviridae family and have positive-sense, single-stranded RNA genome. TGEV did serious economic damage to the swine industry in 1990s but with the advent of vaccines it has been largely controlled. PEDV still results in high morbidity and mortality in neonatal piglets with clinical signs like severe diarrhea, vomiting, dehydration and death. In 2013/14, PEDV outbreak in the US led to over a billion-dollar loss. Rotaviruses are double-stranded RNA viruses of Reoviridae family, cause enteric infections in pigs. Rotavirus of groups A, B, C, E and H are involved in porcine enteric infections. Some of these porcine rotaviruses also have zoonotic potential.

Foot and mouth disease (FMD) is another highly contagious, acute viral disease in pigs. The etiologic agent, FMD virus (FMDV), is a positive-sense, single-stranded RNA virus of Picornaviridae family. FMDV is transmitted through direct contact with infected animals or contaminated sources. Clinical signs include high fever, appearance of vesicular lesions on the extremities, salivation, lameness and death. FMDV causes frequent epizootics in many parts of the world resulting in severe economic loss, food insecurity and trade restrictions.

Classical swine fever (CSF) or hog cholera can result in high morbidity and mortality in pigs. It is caused by CSF virus (CSFV), an enveloped, positive-sense, single-stranded virus of Flaviviridae family. Transmission of CSFV occurs through oral-nasal routes after contact with infected pigs or contaminated resources and even vertically from infected sows to piglets. Clinical signs include fever, anorexia, respiratory problems, neurological disorders, reproductive failures and death. CSF is a notifiable disease to World Organization for Animal Health (OIE). The economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs. For example, the 1997/98 outbreak of CSFV in the Netherland resulted in death of 9 million pigs and economic losses of 2.3 billion dollars. United States is free of CSFV; however, this virus is endemic in many parts of the world including Central and South America, Africa and Asia.

Viral infection involves a large number of protein-protein interactions (PPIs) between virus and its targeted host. These interactions range from the initial binding of viral coat proteins to host membrane receptor to hijack the host transcription machinery by virus proteins. Various viral diseases are caused by infection with pathogenic viruses. For instance, Ebola virus disease is a highly contagious and fatal disease caused by infection with Ebola virus. During the 2014 Ebola epidemic, the world witnessed over 28,000 cases and over 11,000 deaths. So far, there is no specific vaccine or effective treatment for Ebola virus disease. Despite the increased number of known virus-host PPIs, viral infection mechanism is not fully understood. Thus, identifying interactions between virus proteins and host proteins helps understand the mechanism of viral infection and develop treatments and vaccines.

So far, many computational methods have been developed to predict PPIs. However, most of these methods predict PPIs within a single species and cannot be used to predict PPIs between different species because they do not distinguish interactions between proteins of the same species from those of different species. Recently, a few computational methods have been developed to predict virus-host PPIs using machine learning methods. For instance, a homology-based method predicts PPIs between H. sapiens and M. tuberculosis H37Rv. Support vector machine (SVM) models developed by Cui et al. and Kim et al. predicted PPIs between human and two types of viruses (hepatitis C virus and human papillomavirus). However, these methods are intended for PPIs between virus of a single type and host of a single type. Recent computational methods developed for predicting virus-host PPIs [6–8] are also limited to PPIs between human and the human immunodeficiency virus 1 (HIV-1) and cannot predict PPIs of new viruses or new hosts which have no known PPIs to the methods. A recent SVM model called DeNovo can exceptionally predict PPIs of new viruses with a shared host.

In this paper, we present a new method for predicting virus-host PPIs, which is applicable to new viruses or hosts using amino acid repeat patterns and composition. Proteins in a variety of species contain significant amino acid repeats, with more abundance of repeats in eukaryotic proteins than in prokaryotic proteins [10, 11]. It has been found that proteins with a large number of amino acid repeats have a greater number of interacting partners compared to those without. Experimental results of our method show that the repeat patterns and local composition of amino acids are simple, yet powerful features for predicting virus-host PPIs. The rest of this paper discusses the details of the method and its experimental results.

HMPV is thought to spread through direct or close contact with infected individuals or objects (fomites)

36. Symptoms and disease presentation of HMPV are similar to those of other respiratory viruses causing both upper and lower respiratory tract infections. Symptoms can include cough, rhinorrhea, sore throat, and fever as well as lower respiratory tract symptoms such as wheezing, difficulty breathing, and hypoxia

54,

75. The clinical diagnoses most commonly associated with HMPV are bronchiolitis and pneumonia

42.

The start of the twenty-first century has seen the discovery of several emerging or new respiratory pathogens causing human disease, including severe acute respiratory syndrome coronavirus and human metapneumovirus (HMPV). The metapneumoviruses are enveloped, non-segmented, negative-sense, single-stranded RNA viruses

1. They comprise a genus of two species: avian metapneumovirus and HMPV. The metapneumoviruses belong to the order

Mononegavirales and family

Pneumoviridae, which also includes respiratory syncytial virus (RSV)

2,

3.

Respiratory diseases are among the most devastating diseases in poultry industry because of their major economic losses. In most cases, there are more than one pathogen involving in the pathogenesis of the respiratory diseases.1 Among several avian viruses with predilection for the respiratory tract, infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) are the most important viruses of poultry worldwide. Similar respiratory signs of infectious bronchitis (IB) and Newcastle disease (ND) making differential diagnosis of these two diseases difficult.2

In broilers, IBV affects weight gain and feed efficiency, and, when complicated with bacterial infections like E. coli or S. aureus, it causes high mortality and increased condemnations.3-5 IBV, the causative agent of IB is a coronavirus readily undergoes mutation in chickens resulting in the emergence of new variant serotypes and genotypes.6 As new strains of IBV emerge, rapid detection of IBV is useful for implementation of control measures, research purposes, and understanding the epidemiology and evolution of IBVs.7

Newcastle disease classified as a list A disease by the Office Internationale des Epizooties (OIE), is caused by avian paramyxovirus 1 (APMV-1) or NDV.8 The virus is enveloped with a negative-sense, single stranded RNA genome of approximately 15 kb encoding six proteins (nucleoprotein, phosphorprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase protein, and large protein, respectively).9

Several laboratory methods such as virus isolation in embryonated eggs and organ cultures and serological tests are available for detecting and differentiating avian viral respiratory infections. However, these methods are time consuming and laborious.10-12 Molecular techniques such as reverse transcription-polymerase chain reaction (RT-PCR), sequencing and real time PCR, have been used for rapid and sensitive detection of IBV and NDV separately.13-17 However, those techniques detect only one specific pathogen at a time. The duplex PCR has the ability to amplify and differentiate multiple specific nucleic acids.18 The aim of the present study was to detect and differentiate two common avian viral pathogens using duplex RT-PCR for clinical diagnosis.

Coronaviruses (CoVs) are enveloped, single-stranded, positive-sense ribonucleic acid (RNA) viruses (1). They are widespread and can be found in many species of mice, horses, whales, birds, cats, dogs, pigs, and humans (1). Development of the infection, can lead to additional complications, including respiratory tract disease and organ dysfunction, particularly renal failure and immune suppression, enteric, neurologic or hepatic diseases (2, 3). The majority of patients have typical symptoms, such as fever with or without cough, and breathing difficulties (4). Middle east respiratory syndrome coronavirus (MERS-CoV) is a new and unknown origin respiratory virus with genotypic and phenotypic diversity; thus, this virus can mutate, increasing its virulence and even causing tissue tropism. There is a high-frequency mortality rate around > 50% and median age of the majority of identified cases affected with this virus is 56 years. Very little is known about its behavior (5, 6). Humans are known to maintain circulation of four different human coronaviruses (hCoVs) at a global population level. These are a part of the spectrum of agents that cause the common cold. The severe acute respiratory syndrome (SARS) CoV constitutes the fifth hCoV, which was in circulation for a limited time during 2002 and 2003, when a virus appeared in humans and caused an outbreak affecting at least 8,000 people. Symptoms matched the clinical picture of acute primary viral pneumonia. MERS-CoV, a novel coronavirus, was detected for the first time in September of 2012 in Saudi Arabia, an area heavily impacted by this virus at present. Between April and June 2013, 81 cases of infection by this hCoV were reported in Saudi Arabia. Of those, 49 patients died, so fatality rate is high (2). This virus (MERS-CoV) causes severe acute respiratory infection in humans (7). Subsequently MERS-CoV has been reported in other countries, including Tunisia, United Arab, Emirates, Italy, United Kingdom, Germany, France, and Qatar (2, 8-11). The first cases of MERS-CoV infections reported in Iran were two cases in Kerman, a city in southeast Iran. The virus killed one of them, a 53-year-old woman. With the appearance of new cases, in view of the risk of MERS-CoV transmission to humans (6), and because of severe infections that have been observed among the elderly, there is considerable concern about this virus (5). Although few cases have been reported annually (around 34 case as of 12 May 2013), the morbidity and mortality rate of this infection is alarming (7). Unfortunately, at present, there are no specific treatments or effective drugs for this deadly disease, and no vaccine. In the absence of an effective treatment, the appropriate infection controls include rapid diagnostics and isolation of patients, useful strategies for preventing further transmission and spread of this infectious agent (5). Currently, real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays are used for detection of MERS-CoV in respiratory, blood, and stool samples of patients. Real-time RT-PCR assay is highly sensitive and is able to detect viruses even in low copy numbers (12, 13).

Influenza pandemic are characterized by the worldwide spread of novel influenza strains for which most of the population lacks substantial immunity,. Pandemic viruses typically cause heightened morbidity and mortality. The continued circulation of highly pathogenic avian influenza (HPAI) viruses H5N1 has resulted in occasional coincident infections among humans. Since late 2003, when widespread H5N1 virus poultry outbreaks were reported in multiple countries in Asia, there have been 467 laboratory confirmed human cases in ten countries reported to the World Health Organization as of December 2009 with a mortality rate of about 60%,. Global public health concerns surrounding H5N1 viruses include not only individual transmission events between infected poultry and individual humans, but also their pandemic potential, should these viruses acquire genetic changes that result in sustained human-to-human transmission. To date, several case clusters of H5N1 infections have been reported and limited epidemiologic information has suggested person-to-person transmission of H5N1 in a few instances, usually involving family members. Of additional concern to both human and animal health, is the extensive geographic spread of HPAI H5N1 viruses in recent years and their isolation from multiple species of wild birds and mammals–[9]. Despite the recent emergence of the 2009 H1N1 pandemic, the pandemic threat from HPAI H5N1 viruses has not diminished.

Human H5N1 disease is clinically and pathologically distinct from that caused by seasonal human influenza A H3N2 or H1N1 viruses. The majority of confirmed human HPAI H5N1 virus infections have been characterized by a severe clinical syndrome including a rapid progression of lower respiratory tract disease, often requiring mechanical ventilation within days of admission to a hospital–[18]. In addition to pulmonary complications, other clinical manifestations of H5N1 virus infections may include severe lymphopenia, gastrointestinal symptoms, and liver and renal dysfunction,,,,. Reactive hemophagocytosis in multiple organs, and occasional detection of viral antigen or viral RNA in extrapulmonary organs suggest a broader tissue distribution of H5N1 viruses compared with seasonal viruses in fatal human cases,.

Patients with severe H5N1 disease have unusually higher serum concentrations of proinflammatory cytokines and chemokines. Levels of plasma macrophage attractant chemokines CXCL10 (IP-10), CXCL9 (MIG), and CCL-2 (monocyte chemoattractant protein 1, MCP-1) and of neutrophil attractant interleukin-8 (IL-8) were substantially higher in patients with H5N1 disease compared with those experiencing seasonal influenza virus and were significantly higher in H5N1 patients who died compared with those who recovered,. The elevation of plasma cytokine levels was positively correlated with pharyngeal viral load and may simply reflect more extensive viral replication, and consequently, direct viral pathology rather than being causative of the pathology observed in H5N1-infected patients. Compared with human H1N1 and H3N2 influenza viruses, infection of human primary macrophage cultures in vitro with H5N1 viruses also lead to the hyper-induction of proinflammatory cytokines.

Most studies on H5N1 pathology describe pulmonary features of human disease. Although H5N1 virus infection of humans is primarily one of the lower respiratory tract, more recent reports suggested that influenza A H5N1 may in rare, severe cases, disseminate beyond the lungs and infect brain,, intestines, and lymphoid tissues, and result in extra-pulmonary clinical manifestations including encephalopathy or encephalitis,. This extrapulmonary dissemination of HPAI H5N1 virus contrasts with seasonal influenza virus infection of humans which, even in fatal cases, is restricted to the respiratory tract. However, there have been relatively few reports describing histopathology and virus distribution in H5N1 cases,,,,,. To better understand the pathogenesis of human H5N1 virus infection, and investigate the route of virus dissemination in vivo, we report on the use of different techniques to detect virus distribution and infection of 5 organ systems in a laboratory confirmed fatal human H5N1 virus infection, and analyze the relationship between viral load in tissues and host response. Our results suggested that the virus can infect multi-organs besides pulmonary. High viral load is associated with increased host response though the viral load is significantly difference in various organs. Cells of immunologic system could not be excluded to play a role in dissemination of the virus.

Our aims in this research were to present two rRT-PCR assays for in-house rapid and sensitive diagnostic testing of MERS-CoV, detecting the regions upstream of the envelope gene (upE) and open reading frame (ORF) 1b, respectively, for initial screening and final confirmation of MERS-CoV infection (according to world health organization (WHO) recommendations).

Developing RT-PCR using vaccinal and reference strains of IBV and NDV. The specificity of duplex-RT-PCR was shown using IB88 and 793/B strains of IBV and two standard strains of NDV. The duplex-RT-PCR products visualize by gel electrophoresis was 433 bp for IBV and 121 bp for NDV (Fig. 1).

Application of developed duplex-RT-PCR for detection and differentiation of IBV and NDV in clinical samples. The applicability of developed duplex-RT-PCR assay for detection and differentiation of IBV and NDV in the diagnosis was validated examining 12 clinical samples as showed in Fig. 2. Among five positive clinical samples belonged to five different broiler farms, three farms were infected with only one virus and two farms were co-infected with IBV and NDV.

Rubella virus (RV) is an enveloped, single-stranded, positive-sense RNA virus in the Rubivirus genus, which has been recently moved from the Togaviridae to a new family, Matonaviridae. A total of 13 RV genotypes, which represent 2 clades, have been recognized, but 2 genotypes, 1E and 2B, are currently the most common worldwide. RV replicates at low levels and produces little cytopathology both in vitro and in vivo. A distinct feature of RV is the ability to persist in the placenta and fetus and in immune privileged body sites of immunologically competent individuals [2, 3]. Persistent RV infection is associated with a congenital rubella syndrome (CRS) and a number of less common pathologies such as rubella encephalitis and Fuchs uveitis [4, 5]. The live attenuated vaccine strain, RA27/3 (a virus from the likely extinct 1a genotype and a part of the MMR vaccine), is currently used in the US and globally. It has high immunogenicity, generates long-term immunity after a single dose, is effective in preventing clinical disease, and has a very low rate of adverse events. Worldwide, implementation of rubella vaccination programs has resulted in elimination of rubella and CRS from the Americas and significant reduction in the burden of disease in some developed countries. Similar to wild type RV, RA27/3 can persist in immunologically competent individuals for a limited time causing mild complications, such as transient arthralgia or arthritis in adult women. The vaccine virus involvement in the pathology of Fuchs uveitis is also suspected [5, 9]. The vaccine virus has not been associated with congenital defects, but asymptomatic persistent infections of the fetus have been reported after inadvertent vaccination of unknowingly pregnant women.

Primary immunodeficiency diseases (PID) are a group of hereditary disorders affecting different arms of the immune system. PID patients usually have increased susceptibility to infections and have difficulties eliminating pathogens. Live vaccines, including rubella vaccine, are contraindicated for individuals with severe antibody deficiency, T-cell deficiencies or innate immune defects because they may cause severe or chronic disease. Unfortunately, PID diagnosis often occurs after vaccination with MMR (usually given at the age of 12–15 months). Nevertheless, adverse outcomes related to MMR vaccination of children who are diagnosed with PID are thought to be rare.

Granuloma formation, a well-recognized disease in PID patients, is an accumulation of histiocytes and other immune cells near sites of chronic infection, which may persist for years sometimes resulting in significant pathology. The estimated granuloma prevalence in PID patients is 1–4% and thus ~4,000 individuals in the US are expected to be affected. RV antigen and RNA have been recently found in association with granulomas at various body sites (skin, liver, kidney, spleen, lung and bone periosteum) in children with a broad spectrum of PIDs [16–19]. RV positive cutaneous granulomas have been reported to develop 2–152 weeks (average 48 weeks) after MMR vaccination typically near the vaccination site, but can also appear at other body sites, e.g., face or legs, and then slowly spread. Prominent T cell deficiencies, often with concurrent antibody deficiencies, are common characteristics of PID patients with RV positive granulomas [17, 19]. Immunohistochemical analysis of granulomatous lesions revealed that M2 macrophages in the center of granulomas most commonly harbored RV antigen. Previously, mutated RA27/3 RNA was detected in a few cases but sequencing data were limited [16, 17]. As a result, little was known about the evolution of the vaccine virus during persistent infection in PID patients.

Our initial attempt to isolate infectious virus from the RV-positive skin granuloma of a single PID patient failed. Accumulated deleterious mutations in the vaccine virus after a 22-year-long persistence in this case may have caused loss of infectivity of that virus. However, it was unclear whether loss of infectivity is a common feature of RA27/3-derived viruses within PID patients or a characteristic of vaccine virus evolution within that particular patient.

Here we report the isolation of infectious immunodeficiency-related vaccine-derived rubella viruses (iVDRV) from the skin biopsies of four PID patients collected at different times after vaccination. We have determined full genomic sequences of these iVDRV and characterized the changes relative to the parental RA27/3 virus with the objective of characterizing the RA27/3 evolution during persistent infection in PID patients. The replicative and persistence properties of the recovered iVDRV were compared with those of RA27/3 and wild type RV (wtRV) in WI-38, the primary human fibroblasts used to culture RA27/3 during attenuation. This study also documents iVDRV detection in nasopharyngeal secretions raising the possibility of transmission of iVDRV strains to susceptible non-immune contacts.

The histopathologic features in different organs are shown in Fig. 2. Lung showed diffused alveolar damages including intra-alveolar edema, focal intra-alveolar hemorrhage, necrosis of alveolar line cells, focal desquamation of pneumocytes in alveolar spaces, interstitial mononuclear inflammatory cell infiltrates, and extensive hyaline membranes. Trachea showed focal denudation of the epithelium with edema, and mononuclear inflammatory cell infiltrates. Spleen showed depletion of lymphocytes with congestion and organized infarcts. Axillary lymph-node was congested with depletion of lymphocytes. The central nervous system showed extensive edema with focal neuronal necrosis in hippocampus. Diastem between Purkinje cells layer and particle cells layer showed focal augmentation in cerebellum. Liver was congested with edema and focal fatty degeneration. Kidney was congested with edema. Other selected tissues showed no significant histological changes.

Viruses are infective obligate parasites that can replicate only in the living cells of animals, plants, fungi, or bacteria. Although extremely small in size and simple in structure, viruses cause numerous diseases such as cancer, autoimmune disease, and immunodeficiency as well as organ-specific infectious diseases including the common cold, influenza, diarrhea, hepatitis, etc.,,,.

Recent progress in the formulation of antiviral therapies and vaccines has helped to prevent, shorten the duration, or decrease the severity of viral infection,,. Most antiviral agents are designed to target viral components, but mutations in the viral genome often result in drug resistance and immune evasion, creating a major hurdle for antiviral therapies and vaccine development. In addition, the continuous emergence of new infectious agents such as the Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) necessitate the advancement of novel therapeutic approaches. Accordingly, great attention has recently been drawn to the development of antivirals with broad-spectrum efficacy and immunomodulators which improve host resilience by increasing host resistance to the viral infection.

Korean ginseng (the root of Panax ginseng Meyer) is one of the most popular medicinal plants used in traditional medicine in East Asian countries including Korea. Ginseng contains various pharmacologically active substances such as ginsenosides, polysaccharides, polyacetylenes, phytosterols, and essential oils, and among those, ginsenosides are considered the major bioactive compounds. Korean Red Ginseng (KRG) is a heat-processed ginseng which is prepared by the repeated process of steaming and air-drying fresh ginseng. KRG has been shown to possess enhanced pharmacological activities and stability compared with fresh ginseng because of changes in its chemical constituents such as ginsenosides Rg2, Rg3 Rh1, and Rh2, which occur during the steaming process.

Currently, numerous studies have reported the beneficial effects of KRG on diverse diseases such as cancer, immune system disorder, neuronal disease, and cardiovascular disease,,,. In addition, KRG and its purified components have also been shown to possess protective activities against microbial infections. In this review, we summarize the current knowledge on the effects of KRG and its components on infections with human pathogenic viruses and discuss the therapeutic potential of KRG as an antiviral and vaccine adjuvant.

Japanese Encephalitis virus (JEV), a member of mosquito-borne flaviviruses, is an enveloped, positive-sense single-stranded RNA virus. Like other flaviviruses including dengue virus, West Nile virus, and Zika virus, JEV genome has an only open reading frame (ORF) encoding a polyprotein consisting of structural proteins (capsid (C), membrane (prM/M), and envelope (E)) and non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). C protein contains positively charged residues, enabling to bind viral RNA and form a ribonucleoprotein complex. The signal peptide of prM/M protein is sliced by a cell protease during maturation. E protein (E) is responsible for virus attachment and membrane fusion and is also the crucial antigen to inducing neutralizing antibodies. Non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, are responsible for viral replication. NS1 protein is associated with the initial negative-strand RNA synthesis in the early stage replication, secreted to modulate the host immune response. NS2A is a membrane associated protein that participates in virus replication and assembly. NS2B is a co-factor to activate the proteolytic activity of NS3 protein, which contains protease, helicase, and NTPase domains. A hydrophobic protein NS4A is also related to viral replication. NS4B and NS5 can block interferon signaling; moreover, NS5 is recognized as an RNA-dependent RNA polymerase and methyltransferase. JEV infection might lead to developing permanent neurologic or psychiatric sequelae, including memory loss, behavior disturbances, impaired cognition, convulsions and paralysis, and severe central nervous system damage. JEV is predominant in Southeast Asia and East Asia due to the distribution of Culex species. Although inactivated JEV genotype III vaccines have been invented and introduced in many countries, more than 30,000 JE cases including 10,000 deaths are annually reported in these areas. Recently, genotype I JEV has appeared in Vietnam, Taiwan, China, and Korea, potentially becoming the predominant genotype in these countries. Noticeably, the inactivated JEV genotype III vaccine elicited a low protective immunity against the JEV genotype I. Nowadays, there is still not an effective treatment yet for JE cases; the rapid and rational screening platforms are necessary for efficiently developing anti-JEV agents.

Replicons of positive-strand RNA viruses, such as hepatitis C virus, coronavirus, dengue virus, West Nile virus, and JEV are used to screen antiviral compounds and examine antiviral mechanisms. Replicons contain a partial genome, including cis-acting elements in the 5′- and 3′-ends as well as the genes of all non-structural proteins under the control of cytomegalovirus (CMV) immediate-early promoter. The CMV promoter driven viral RNA subgenomes enable self-replication in transfected cells but have no infectious particles. Moreover, reporter genes like green fluorescent protein (GFP) and firefly luciferase are in-frame cloned into viral subgenomes in the replicons, and quantitative analysis of the reporter expression becomes a measurable assay to monitor the virus replication. DNA-launched replicons are more convenient than RNA-launched replicons required producing viral RNA subgenomes using in vitro transcription assays. Because they are lacking the structure protein coding region, both types of virus replicons transiently self-replicate in cells post transfection and could not generate the infectious particles for analyzing the early stages of viral replication. Subsequently, DNA-launched replicons are transfected into the cells carrying the recombinant plasmid encoding the viral structural proteins, which express structural proteins and self-replicate viral subgenomes to generate single-round infectious particles (SRIP). Several flavivirus replicon-based SRIPs including JEV, dengue virus, West Nile virus, and tick-borne encephalitis virus have been generated, demonstrating the immunogenicity of virus-like particles.

Most reports recognize the replicon-based SRIPs as a suitable system for the development of safer vaccines against flavivirus infection. The replicon-based SRIPs containing the reporter genes of such fluorescent proteins could be more applicable as a rapid and real-time screening for the antiviral agents, as well as directly quantitate the antiviral activity under the condition without plaque-based assays. This study intends to establish a safe and reliable platform for screening antiviral agents and investigating antiviral mechanisms using JEV replicon-based SRIPs and flow cytometry (Figure 1). The reporter enhanced green fluorescence protein (EGFP) was fused with a foot-and-mouth disease virus (FMDV) 2A peptide, and then cloned and in-frame fused with NS1~5 coding regions in pBR322 plasmid-based JEV-EGFP replicon (named as pBR322-JEVrep-EGFP). Subsequently, JEV-EGFP SRIPs were generated in the cells co-transfected with pBR322-JEVrep-EGFP and pFlag-CMV3-CprME. The infectivity of JEV-EGFP SRIPs was characterized by the replication of viral subgenomes and the expression of EGFP as well as viral proteins using real-time PCR and fluorescent microscopy. Finally, the antiviral screening platform was performed according to comparing the fluorescence intensity of EGFP in SRIP-infected cells with and without treatment by flow cytometry. Accuracy and reproducibility of the half maximal inhibitory concentration (IC50) of anti-JEV agent MJ-47 by JEV-EGFP SRIP plus flow cytometry were further evaluated using plaque-reduction assay.

Single-stranded positive-sense RNA (ssRNA+) viruses constitute the largest group of viral agents (1). Even when not encapsidated by viral structural proteins, positive-sense RNA genomes are sufficient to initiate translation and viral replication upon introduction into permissive cells. The infectious nature of ssRNA+ viral genomes has made them amenable to reverse genetic manipulation for more than 30 years (2, 3). However, this property is a contributing factor in classifying many ssRNA+ viruses as select agents (SAs) under the Federal Select Agent Program (4). Moreover, many ssRNA+ viruses are high-priority biosafety level 3 and/or 4 (BSL-3/4) pathogens, with a subset listed as potential bioterrorism agents by the U.S. Department of Health and Human Services (5, 6). An additional complication is that full-length cDNAs from ssRNA+ viruses that are SAs can be used to create RNA and rescue/recover the pathogen. Thus, in some instances, large cDNAs, double-stranded DNAs (dsDNAs), or clones containing at least two-thirds of the genome may also be regulated as SAs.

Positive-sense RNA viruses span multiple virus families, and the infectious nature of these genomic RNAs coupled with SA/biosafety/biosecurity concerns inhibit rapid removal from BSL-3/4 containment (7) or transport and handling of RNA samples that are known to contain viral genomes from outbreak settings. This poses a challenge for timely sample processing and sequence analysis, which could significantly hamper responses during outbreak situations. Typically, all products generated from infectious material must be proven to no longer contain infectious viral particles or infectious genomic RNA prior to transfer to a BSL-2 space. Confirming loss of infectivity (LOI) typically occurs via blind infectivity testing, where a subset of the material is placed on a permissive cell line for at least three subsequent passages (8). For this reason, transferring cDNAs or other nucleic acids from a BSL-3/4 laboratory to a BSL-2 laboratory is a very difficult and time-consuming process for many laboratories. Some investigators have worked out procedures that have been approved by their institutional biosafety committee; however, these procedures vary from institution to institution. For example, some BSL-3 facilities work with mosquito-transmitted ssRNA+ viruses including West Nile virus (WNV) and Chikungunya virus (CHIKV) that are not considered SAs. Other BSL-3 facilities contain ssRNA+ viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Venezuelan equine encephalitis virus (VEEV) that are classified as SAs, but these facilities have safety protocols that allow transfer of genomic RNA or cDNA from BSL-3 to BSL-2 directly by scientists. Finally, there are BSL-3 facilities working with SAs such as foot-and-mouth disease virus (FMDV) at the Plum Island Animal Disease Center (PIADC). Here, only trained safety personnel are allowed to transfer cDNA samples from high-containment laboratories to BSL-2 laboratories after inactivation has been carried out under a validated protocol. For each of these scenarios, a robust, universal standard operating procedure (SOP) to eliminate infectious material while rapidly generating next-generation sequencing (NGS) libraries is critically needed.

FMDV illustrates the majority of the SA, biosafety, and biosecurity issues surrounding high-consequence RNA viruses (9). Among the foreign animal disease viruses, FMDV is the most contagious and has historically set standards for biosafety and biosecurity policies and procedures. The required biosafety level to carry out any infectious virus work with FMDV is biosafety level 3 agriculture (BSL-3Ag), and currently, the only facility authorized to work with FMDV in the United States is PIADC (BSL-3Ag safety considerations reviewed in references 10 and 11). The fact that the viral RNA and RNA derived from infected samples can be infectious when transfected or electroporated into susceptible cells results in strict regulation of any nucleic acid derived from FMDV-infected material. Currently, the only approved methodology to remove FMDV nucleic acids from the BSL-3Ag laboratory at PIADC involves harsh alkaline and thermal treatment that potentially has a deleterious effect on putative NGS libraries (12; M. McIntosh, personal communication). In addition, removal of material derived from diverse BSL-3/4 ssRNA+ pathogens out of containment requires time-consuming procedures (e.g., multiple blind infectivity passages) to rule out the presence of infectious material. This tremendously limits the capacity to conduct genomic research with viral samples, particularly the application of NGS techniques to understand viral pathogenesis, viral ecology, and vaccine development.

For the reasons presented, there is substantial need for a universal SOP to generate cDNA that rapidly and reproducibly inactivates BSL-3/4 viruses that can be easily assessed by institutional biosafety committees (IBC), which speeds the transfer of nucleic acids from high-containment laboratories to BSL-2 laboratories, and enables rapid introduction into a variety of NGS pipelines. Here, we present a robust SOP for generating high-quality, barcoded cDNAs directly from genomic RNA across multiple virus families. Families represented include Picornaviridae, Alphaviridae, Flaviviridae, and Coronaviridae, which have genome sizes from ~7 kb to 28 kb. The strategy builds upon established sequence-independent single-primer amplification (SISPA) methods (13-15). Our data prove that barcoded NGS sequencing libraries can be rapidly generated while simultaneously destroying both viral particle and genomic RNA infectivity. Our approach is scalable, highly adaptable, and sensitive. The SOP generates high-quality sequences spanning the entire genome, up to 288 pooled barcoded samples can be examined in a single NGS run, and the products of the SOP work on multiple NGS platforms (e.g., Illumina MiSeq, HiSeq, NextSeq, and Ion Torrent). The SOP works on starting material of purified virus, tissue culture samples, or tissue samples. We are able to detect virus-specific reads in samples where the input is fewer than 10 PFU and can identify viruses present in an unknown sample. Therefore, this application has potential for rapid sequencing of high-titer viral stocks as well as virus discovery and/or forensics. Finally, the nonspecific nature of the amplification makes this SOP adaptable to negative-strand RNA viruses (ssRNA−), double-strand RNA (dsRNA), single-strand DNA (ssDNA), or double-strand DNA (dsDNA) viruses.

Veterinary medicine considers prophylactic vaccines, in combination with strict bio-security, to be the most cost-effective tools for preventing viral infections. In general, veterinary viral vaccines aim to protect susceptible host animals from fatal infectious diseases by inducing a rapid and long-lasting immune response and by preventing the spread of such diseases among populations by reducing viral shedding from infected animals. Such vaccines must be cost-effective, stable, and easy to administer. Live attenuated virus and/or inactivated virus vaccines have been used for decades to prevent viral infectious diseases; however, many vaccines do not satisfy the requirements for an “ideal vaccine” in the field due to limited effectiveness and/or side effects. In addition, no vaccines are available for some infectious diseases.

Advances in recombinant DNA technology have made it possible to design new innovative genetically engineered vaccines with improved safety profiles and greater protective efficacy. These “next generation” vaccines include DNA vaccines, subunit or virus-like particle vaccines, genetically modified marker vaccines, and virus vectored vaccines. Of these, the latter are thought to be a promising tool for developing polyvalent or antigen delivery vaccines that express foreign antigen(s) derived from pathogens of economic importance from a veterinary and human perspective.

Many viruses have been used to develop virus vectored vaccines, which provide effective protective immunity against foreign antigens. The number of vectored vaccines licensed for veterinary and human use has increased over time. Initially, vectored vaccines were based on DNA viruses such as herpesviruses, animal poxviruses, and adenoviruses. Currently, with advances in reverse genetics approaches, many RNA viruses have been adapted for use as vectored vaccines. Such vaccine vectors include both positive-sense RNA viruses (picornaviruses, coronaviruses, and flaviviruses) and negative-sense RNA viruses (paramyxoviruses and orthomyxoviruses). In particular, Newcastle disease virus (NDV), a paramyxovirus that infects birds, is used as an important vaccine vector for development of bivalent vaccines against pathogens of economic importance to the poultry industry. Numerous studies demonstrate that NDV is a promising vector for delivery of protective antigens derived from pathogens infecting mammals and humans; indeed, NDV vectors are safer and more efficient than conventional whole virus vaccines. This article reviews recent developments in the field of NDV-vectored vaccines and the potential use of such vaccines in veterinary and human medicine.

Porcine circovirus 2 (PCV2), a single-stranded DNA virus of Circoviridae family, causes multi-systemic disease referred as porcine circovirus-associated disease (PCVAD). PCV2 is transmitted horizontally as well as vertically. Direct contact is the most efficient way of horizontal transmission of this virus. The clinical signs of PCV2 infection include poor weight gain, respiratory problems, dermatitis, enteritis, nephropathy and reproductive failures. Five genotypes of PCV2 (PCV2a to PCV2e) are identified and circulate with high prevalence in swine herds causing significant economic losses worldwide.

Porcine parvovirus (PPV) is the common cause of reproductive failure in swine herds. This single-stranded DNA virus of Parvoviridae family is transmitted through oral-nasal routes. Stillbirths, mummification, embryonic death, and infertility (SMEDI syndrome) are linked to PPV infection. Conventionally, PPV was considered genetically conserved but recent evidences suggest that several virulent strains have emerged due to its high mutation rate.

Aujeszky’s disease or pseudorabies in pigs is caused by Suid herpesvirus 1, a double stranded DNA virus belonging to Herpesviridae family. The causative agent is spread primarily through direct animal-to-animal (nose-to-nose or sexual) contact. Pseudorabies is characterized by nervous disorders, respiratory problems, weight loss, deaths in younger piglets and reproductive failures; and is one of the most devastating infectious diseases in pig industry [18, 19].

African Swine Fever (ASF) causes hemorrhagic infection with high morbidity and mortality. The etiologic agent, ASF virus (ASFV), is a double stranded DNA virus of Asfarviridae family. Virus transmission occurs through direct contact with infected animals, indirect contacts with fomites or through soft tick species of the genus Ornithodoros. Clinical disease may range from asymptomatic infection to death with no signs. Acute infections are characterized by high fever, anorexia, erythema, respiratory distress, reproductive failure in pregnant females and death. ASF is OIE notifiable disease. United States is free of ASFV, however, this virus is endemic in domestic and wild pig population in many parts of the world with possibility of transmission to the US and other nonendemic regions through animal trades. The economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs.

Besides the RNA and DNA viruses described above, many other emerging and re-emerging viruses such as porcine hepatitis E virus, porcine endogenous retrovirus, porcine sapovirus, Japanese encephalitis virus, encephalomyocarditis virus and others cause variable degree of impact in swine health and economic losses in pig industry globally [2, 21, 22].

The European Virus Bioinformatics Center (EVBC) was conceived of in 2017 to bring together experts in virology and virus bioinformatics in Europe. EVBC’s member numbers have increased steadily since then with currently 151 members from 78 research institutions distributed over 26 countries across Europe and internationally. This spring, the Annual Meeting of the EVBC was held for the third time (Table 1). The Third Annual Meeting of the EVBC attracted experts at all career stages to attend the two-day meeting in Glasgow in an inspiring and interactive scientific environment to promote discussion, exchange of ideas and collaboration and to inspire and suggest new research directions and opportunities.

Rotavirus is the leading cause of acute gastroenteritis in young children age ≤ 5 years. Two live oral rotavirus vaccines (Rotarix by GlaxoSmithKline, Unitied Kingdom, and RotaTeq by Merck, United States) are available, and the implementation of rotavirus vaccines in childhood immunization programs has significantly reduced the morbidity and mortality associated with Rotavirus infection. Nevertheless, there is no antiviral drug to treat rotavirus infection, and mostly, therapeutics involve the prevention of dehydration,.

In traditional medicine, ginseng has been known to improve gastrointestinal function and prevent gastrointestinal problems such as diarrhea. A recent study researched the active constituent in ginseng and reported that two pectic polysaccharides isolated from hot water extract of ginseng prevented cell death from viral infection. The polysaccharides, named GP50-dHR and GP50-her, did not have virucidal effects but inhibited viral attachment to the host cells thereby protecting them from virus-induced cell death. Given these results and an additional report that other pectin-type polysaccharides in ginseng inhibited the adherence of Helicobacter pylori to gastric epithelial cells and the ability of Porphyromonas gingivalis to agglutinate erythrocytes, further evaluation of the antimicrobial effects of acidic polysaccharides with the structure of pectin is merited.

Hepatitis C virus (HCV) is a globally prevalent human pathogen. More than 170 million people are chronically infected worldwide, among whom many will develop cirrhosis and hepatocellular carcinoma. HCV is an enveloped, single-stranded positive-sense RNA virus classified in the Hepacivirus genus within the Flaviviridae family. The 9.6 kb genome contains one open reading frame (ORF) that is flanked by non-translated regions, which are necessary for viral RNA translation and replication. A single polyprotein is translated from the ORF, which is co- and post-translationally processed by cellular and viral proteases to generate ten mature proteins: Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The structural proteins (Core, E1, and E2) are incorporated into virus particles, whereas the nonstructural proteins p7 to NS5B coordinate the intracellular processes of the virus life cycle. While p7 and NS2 are dispensable for genome replication, they are required for particle assembly. NS3 through NS5B are necessary and sufficient for HCV genome replication. As we will discuss extensively in this review, HCV replication is associated with the induction of host membrane alterations that are thought to support sites of viral RNA replication. The induction of altered host membranes for viral replication is characteristic of all positive sense RNA viruses. A negative sense replicative intermediate synthesized from the positive sense RNA genome serves as template for the generation of progeny positive sense RNA genomes. The newly-synthesized positive sense RNA can either enter a new translation/replication cycle or be packaged into virions. This review will summarize our current knowledge on HCV-induced membrane alterations, as well as the role of viral nonstructural proteins and host factors in this process.

The single stranded RNA viruses, such as negative-sense Ebola, Marburg, Lassa, and influenza and positive-sense human immunodeficiency viruses, are very important human pathogens in the world. Recent virus outbreaks with a large number of human deaths were caused by RNA viruses like Ebola, corona, Zika, and different strains of influenza A viruses. Among RNA viruses, the respiratory viruses are highly contagious and cause annually worldwide epidemics and occasional pandemic outbreaks. The influenza A virus even without a pandemic outbreak kills up to half million humans each year. A great number of diseases are attributable to human rhinoviruses (HRV) which are the major cause of the common cold. HRV infections are suffered by everyone. Recent reports suggest that HRV infection is associated with severe respiratory illness in children.

The main problem of antiviral therapy is drug resistance. Although the majority of common viral diseases are self-limited illnesses that do not require specific antiviral therapy, antiviral drugs help to prevent complications or shorten the severity and duration of viral diseases symptoms. Considerable economic losses from annual epidemics promote constant search for new antiviral agents, which become useless with time due to the high variability of viruses.

Usually, two groups of drugs are used for the treatment of influenza. The first group including rimantadine and its analogs inhibits the ion channel function of M2 protein, thereby inhibiting viral uncoating. The second one represented by oseltamivir inhibits the enzyme neuraminidase. Inducing adaptive immunity through the vaccination with inactivated or attenuated antigenic material of viruses helps to prevent influenza illness or to reduce its impact in individuals. There is no permanent vaccination against influenza virus and each year countries lose a large amount of money from their budget to prepare new vaccines and rescue the national public health and life. Widespread antiviral resistance due to the high mutation rate in viral genome limits the clinical impact of all types of medicinal products for the prevention and treatment of influenza infections.

Currently, there is no vaccine against HRV. The HRV has almost the highest number of serotypes (more than 100) among all respiratory viruses due to mutations of its genome. Antigenic heterogeneity of HRVs is regarded as a major barrier to effective vaccine development which has to be highly polyvalent and has resulted in little progress over 50 years [1, 4]. Other problems, which have impeded rhinoviral vaccine development, include the lack of convenient animal models and as expected the absence of great impact on respiratory morbidity and mortality while reducing the frequency of common colds

Today, there are no drugs that destroy viruses; all antiviral agents can only inhibit viral development. Therefore, degradation of the viral RNA seems to be a very promising approach of antiviral therapy. The earliest studies on the antiviral activity of RNases were performed using pancreatic RNase as an agent that quickly normalized the state and decreased the symptoms of meningitis and cerebrospinal pleocytosis in patients with tick borne encephalitis. Shortcoming of mammalian RNases is their affinity to cytosolic RNase inhibitor protein (RI) in human cells by which adventitious mammalian RNases are inhibited. In contrast, bacterial RNases are not inhibited by RI and can retain their catalytic activity in mammalian tissues. One of the well-studied bacterial RNases is binase, the guanyl-preferring low molecular weight RNase secreted by Bacillus pumilus.

We proposed earlier that mechanisms of antiviral activity of RNases include both the direct action on nucleic acid and indirect effects, that is, intervention into the RNA interference, immunomodulation, and induction of infected cell apoptosis (for review see). We have demonstrated that binase reduced the titer of pandemic influenza A/Hamburg/4/2009 (H1N1pdm), reovirus serotype 1 (Reo 1-Lang), herpes virus type I (pseudorabies), Middle East respiratory syndrome coronavirus (MERS-CoV), and human corona virus (HCoV-229E) in infected Madin-Darby canine kidney (MDCK) epithelial cells, African green monkey kidney (Vero) cells, Madin-Darby bovine kidney (MDBK) epithelial cells, human fetal lung fibroblast (MRC5) cells, and hepatocellular carcinoma (Huh7) cells, respectively [9–11]. Influenza viruses contain single stranded negative-sense RNA; reoviruses have a double stranded RNA genome, while herpes virus possesses genomic DNA. Because of the specific catalytic activity of binase towards RNA (mainly single stranded) and its inhibitory effect towards RNA- and DNA-containing viruses, it could be proposed that this enzyme should affect mRNA, which is synthesized by all viruses. The aim of our study was to prove that viral mRNA is a direct target of binase.

Lactate dehydrogenase-elevating virus (LDV) is a small, positive sense, single stranded RNA virus of the Arteriviridae family, related to coronaviruses such as the severe acute respiratory syndrome (SARS) virus,,,. It is a natural infectious agent of mice that causes very rapid lytic infections generally restricted to a minor subset of non-essential macrophages involved in scavenging extracellular lactate dehydrogenase,. The rapid loss of this subset results in the elevated lactate dehydrogenase levels for which the virus is named. Virus titers peak within the first day of infection as susceptible target cells are depleted, and then the infection is maintained at a much lower chronic level dependent on the replenishment of new macrophage targets. LDV establishes chronic infections regardless of mouse strain, age, sex or immune-status,,,. No clinical signs are typically associated with LDV infections, although co-infection with retroviruses can lead to CNS disease under certain circumstances,, and mice infected with LDV have suppressed immune responses,,,. We recently found that acute infection with LDV induced a state of partial and transient activation in the vast majority of splenic lymphocytes. Activation was characterized by high surface expression of the very early activation marker CD69. CD69 is the first surface marker known to be upregulated during the activation of lymphocytes and has recently been shown to interact with S1P1 to inhibit the egress of lymphocytes from lymphoid tissues. CD69 expression is upregulated by T cell receptor (TCR) ligation but is not dependent upon it and can be induced by inflammatory cytokines such as IFNα,.

Four distinct papillomavirus sequences were identified in two bat species, E. helvum (Eidolon helvum papillomavirus 8; EhPv-8) and T. perforatus (T. perforatus papillomavirus 1, 2 and 3; TpPv-1, -2, -3). Complete coding sequences were recovered for EhPv-8 (acc. no. KX434763, 6985 bp), TpPv-1 (acc. no. KX434764, 7180 bp) and TpPv-3 (acc. no. KX434767, 7083bp). EpPv-8 shared 63% aa similarity to E. helvum papillomavirus type 1 across the DNA helicase protein (E1). TpPv-1, TpPv-2, and TpPv-3 (acc. no. KX434766, partial genome) shared 44%, 47% and 44% aa similarity across the E1 protein with human papillomavirus 63, Miniopterus schrebersii papillomavirus 1, and Castor canadensis papillomavirus 1, respectively (Fig 7).

Infectious bronchitis virus (IBV) is a gammacoronavirus that infects poultry primarily, causing an infectious respiratory disease. In addition, infection results in substantial economic losses to the worldwide poultry industry as a result of reduced egg quality, egg production, and meat quality. Although vaccines are available, there is a large degree of variation between strains of IBV and poor cross-protection. As a result, novel strategies are required to develop more effective vaccines. To enable this, it is important to understand the interaction between the virus and the host cell, allowing future manipulation and targeted attenuation of the virus by reverse genetics to develop vaccine strains. A critical step in the replication of all positive sense single stranded RNA (+RNA) viruses is the induction of cellular membrane rearrangements, providing the virus with a platform for the assembly of replication complexes responsible for synthesizing viral RNA. It can be considered that a site for viral RNA synthesis would provide an enclosed environment that protects viral RNA from cellular detection, preventing host degradation of RNA and preventing cellular immune stimulation. However, the site must also allow for the exchange of material with the cytoplasm. Nucleotides and other cellular reagents must enter to allow RNA synthesis to occur and viral RNA must exit for translation and assembly of new virus particles. In broad terms, the type of membrane rearrangements induced by different +RNA viruses can be split into two groups: double membrane vesicles (DMVs) and spherules.1 Viruses that induce DMV type structures include poliovirus (PV) and hepatitis C virus (HCV). In PV infected cells, single membrane structures are induced early in infection, followed by double membrane structures.2 They seem to be derived from the cellular ER and viral replicase proteins have been found to localize to the cytoplasmic side of both single and double membrane structures.2,3 In the case of HCV, DMVs are derived from the endoplasmic reticulum (ER) and at early time points, the outer membrane remains attached to the ER. By later time points, the DMVs appear to bud and become free vesicles. The majority of DMVs were found to be sealed from the cytoplasm with only 10% having a visible pore from the interior.4 Viruses that induce spherule type structures, or invaginated vesicles, include Semliki Forest virus (SFV), Flock House virus (FHV), Brome Mosaic virus (BMV), and the Flaviviruses Dengue virus (DENV) and West Nile virus (WNV). For all of these viruses, the spherule-like structure is composed of an invagination of a single membrane derived from a cellular structure. SFV induces spherules at the plasma membrane early in infection, which later become internalized as part of the endo/lysosomal pathway.5 FHV induces spherules on the outer mitochondrial membrane6 and BMV, DENV, and WNV all induce spherules or invaginated vesicles on the ER.7-10 For each virus, replicase proteins and RNA have been found to localize to spherules5,8,9,11 and there is an 8–10 nm pore connecting the spherule interior with the cytoplasm.6,9,10,12

It has long been characterized that in continuous cell culture, coronaviruses induce the formation of double membrane vesicles (DMVs) and branching networks of membranes known as convoluted membranes.13-21 These structures have been identified in cells infected with three different betacoronaviruses: mouse hepatitis virus (MHV), severe acute respiratory syndrome coronavirus (SARS-CoV), and the recently identified Middle East respiratory syndrome (MERS)-CoV. DMVs provide an enclosed environment and have historically been proposed as the site of assembly of viral replication complexes. Some viral replicase proteins have been shown to be located inside DMVs or on their membranes.14-19 In addition, virus associated dsRNA, a potential replicative intermediate, is located on the interior of DMVs.18 In more recent work utilizing electron tomography allowing 3D reconstruction of membrane structures, it was demonstrated that in SARS-CoV infected continuous cell culture, these structures are derived from and remain connected to the cellular ER.18 In addition, using this technique Knoops et. al. were able to study in detail the membrane continuity of these vesicles. Interestingly, they were unable to find any pores or connections between the interior of the DMV and the cell cytoplasm.18 This raised the question, if DMVs are the site of RNA synthesis, how does newly synthesized RNA exit the compartment?

Other work has subsequently questioned of the role of dsRNA during coronavirus replication. Although at early time points post-infection dsRNA was found to co-localize with nascent RNA, at later time points this co-localization did not occur.22 This suggests that dsRNA cannot always be presumed to provide a marker for sites of active RNA synthesis and DMVs may provide a site to shield non-productive RNA from cellular detection, rather than provide a site for viral RNA synthesis.

The successful replication of a viral agent in a host is a complex process which consists of a number of interactions, most of them related to the coevolution of pathogen and host. This coevolution often leads to a species specificity of the virus and can make interspecies transmission difficult. Therefore, natural host range switches by viruses are rare events. However, when they occur the results can become severe because the viruses may then spread widely through non previously adapted, and therefore immunologically naïve host populations.

Upon transmission to a new host species, viruses must usually adapt to a new genetic and immunologic environment in order to replicate and spread to other individuals within the species. The high rates of mutation and replication of RNA viruses, such as human immunodeficiency virus (HIV) and influenza, facilitate the occurrence and fixation of those mutations that become beneficial under certain conditions. Viral adaptations to new hosts primarily manifest as amino acid substitutions which can allow more efficient virus cell entry into the new host, block interactions with detrimental host proteins or promote escape from both the new and the old host's immune responses.

Influenza A is the paradigm of a virus capable of interspecies and interclass transmission. Those viruses are found in humans as well as in other animals, including swine, horses and birds, waterfowl being considered the natural reservoir. Subtypes of Influenza A are distinguished by the two surface glycoproteins: haemagglutinin (HA) and neuraminidase (NA). Periodically a subtype of influenza can make the shift from aquatic birds to humans, possibly through an intermediate host, resulting in a widespread pandemic in an immunologically naïve population. These antigenic shifts can occur either through the transfer of an entire virus from one host to another or through a reassortment process where genomic segments of the avian virus mix with genomic segments of a virus currently circulating in humans.

A number of proteins have been implicated in determining host specificity of the virus. Influenza haemagglutinin binds to sialic acid linked to galactose on the surface of the targeted cell, and the differing nature of the sialic acid-galactose linkages in birds and humans provides an important barrier to host shift events. In this sense, a number of amino acid substitutions have been produced in influenza haemagglutinin to adjust to the different receptors. Neuraminidase, the protein responsible for cleaving the haemagglutinin from the receptor surface, also seems to be adapted to the particular sialic acid linkages. Proteins in the viral replication complex (PA, PB1, PB2 and NP) have also been implicated in limiting host range by restricting replication and intra-host spread in mammals (for a review see). In particular, a specific substitution in the PB2 gene has been identified as crucial for replication and intra-host spread in mammals.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a recently identified human coronavirus. The extremely high homology of the viral genomic sequences between the viruses isolated from humans (huSARS-CoV) and those of palm civet origin (pcSARS-CoV) suggested possible palm civet-to-human transmission. Genetic analysis revealed that the spike (S) protein of pcSARS-CoV and huSARS-CoV was subjected to the strongest positive selection pressure during transmission, and there were six amino acid residues within the receptor binding domain of the S protein that were potentially important for SARS progression and tropism. It has been demonstrated that the double substitution of two amino acid residues of pcSARS-CoV for those of huSARS-CoV made pcSARS-CoV capable of infecting human cells, suggesting that these two residues are involved in the palm civet-human transmission.

Under certain circumstances, even a genetically stable DNA virus can gain the mutation required to adapt to a new host. That is the case of canine parvovirus (CPV) which emerged in 1978 as the cause of new enteric and myocardial diseases in dogs. The new virus spread globally in a pandemic and has since remained endemic in dogs throughout the world. Phylogenetic analysis showed that all CPV isolates obtained so far, termed CPV type 2, descended from a single ancestor closely related to the feline panleukopenia virus (FPV) which infects cats, mink and raccoons, but not dogs or cultured dog cells. FPV and CPV type 2 isolates differ by as little as 0.5% in DNA sequence and it is possible that changes of only two amino acid residues in the capsid protein could have introduced the canine host range. During 1979 a CPV variant (CPV type 2a) emerged, spread worldwide within 1 year and replaced the CPV type 2 strain. CPV type 2a contained five substitutions in the capsid sequence compared to CPV type 2 and also infected and caused disease in cats. Therefore, the emergence of CPV seems to have been a multistep process, where a small number of mutations in the capsid protein gene allowed the virus to efficiently infect and spread within a new host order.

Viruses of lower vertebrates include a large number of viral agents, belonging to different viral families and genera, with RNA and DNA genomes, and displaying different host specificities. In fact, some viruses have a very narrow host range, whereas others are known to be able to infect a wide range of species. The wide host range suggests that, in any moment along the viral evolution, those viruses may have been involved in different host shift events. In the present review we will focus on well documented or hypothesized cases of host shift as well as variations in host range for the genera Ranavirus, Novirhabdovirus, Betanodavirus, Isavirus and several herpesvirus. However, the suspicion for interspecies transmission in other fish viruses remains.

Infection with negative-sense RNA viruses is associated with a broad spectrum of human diseases. Influenza and respiratory syncytial virus (RSV) are recognized as widespread global pathogens, while others, such as Ebola virus (EBOV), Lassa virus (LASV), or Crimean–Congo hemorrhagic fever virus (CCHFV) are known for sporadic outbreaks, their high epidemic potential, and their high mortality rates. Negative-sense RNA viruses are classified into segmented and non-segmented viruses. Segmented negative-sense RNA viruses are further subdivided into families that contain two single-stranded genome fragments such as the Arenaviridae (e.g., LASV), three fragments such as the order Bunyavirals (formerly the family Bunyaviridae, e.g., CCHFV), or multiple fragments such as the Orthomyxoviridae (e.g., influenza). Examples of families of non-segmented negative-sense RNA viruses with a single-stranded RNA genome (Mononegavirales) include the Filoviridae (e.g., EBOV), the Paramyxoviridae (e.g., Nipah virus (NiV)), and the Pneumoviridae (e.g., RSV).

Prevention and treatment of infection with negative-sense RNA viruses remain challenging. The 2014 Ebola virus disease (EVD) outbreak in West Africa caused approximately 28,000 cases and 11,310 deaths and is a sober reminder of an unmet medical need. Intensive research efforts during that outbreak led to the discovery and development of several vaccine and drug candidates. Promising experimental vaccines against EBOV are based on the envelope glycoprotein as the antigenic target. Monoclonal antibodies and nucleotide analogue inhibitors represent investigational therapies. ZMapp, a cocktail of three monoclonal antibodies that target the EBOV glycoprotein, was shown to reverse EVD in non-human primates. However, results from a randomized clinical trial were inconclusive. Like glycoprotein-based vaccines, the antibody cocktail is also not effective against all ebolavirus species. In contrast, several nucleotide analogues show antiviral activity against different ebolavirus species and also other negative- and positive-sense RNA viruses.

Favipiravir (T-705) is a nucleoside precursor that has been approved for treatment of pandemic influenza in Japan. Intracellular phosphoribosylation yields a triphosphate form (T-705-RTP) that is accommodated by the viral RNA-dependent RNA polymerase (RdRp) (Figure 1). Experiments with recombinant influenza RdRp show ambiguous base-pairing with both cytidine and uridine, which can lead to lethal mutagenesis. Incorporation of the T-705-RTP into the growing RNA chain can also cause moderate inhibition of RNA synthesis. Favipiravir is active against a large panel of RNA viruses in vitro, including EBOV. The clinical evaluation has been challenging and results of a multicenter non-randomized trial were largely inconclusive. The structure of the base moiety and proposed mechanism of action are reminiscent of ribavirin that has been used for over a decade to treat infection with the hepatitis C virus (HCV) (Figure 1). A structurally distinct broad-spectrum antiviral agent that is also active against EBOV in vitro is galidesivir (BCX4430). Galidesivir is an adenosine-like compound with a nitrogen-substituted sugar ring that likewise requires intracellular activation to the TP form (Figure 1). Biochemical experiments show that this compound serves as a substrate for the RdRp of HCV and causes chain termination following its incorporation.

Remdesivir (GS-5734) is a phosphoramidate prodrug of a 1′-cyano-substitued adenosine analogue that inhibits ebolaviruses with half maximal effective concentrations (EC50) in the submicromolar range, which is considerably lower than the values reported for favipiravir or galidesivir. The triphosphate form of remdesivir (remdesivir-TP) was shown to inhibit the RSV RdRp surrogate for EBOV RdRp. No significant inhibition was seen with human RNA Pol II and human mitochondrial RNA polymerase (h-mtRNAP). The biochemical data obtained with purified recombinant RSV RdRp and a recent study with NiV RdRp point to delayed chain termination as a possible mechanism of action. Delayed chain termination refers to inhibition of RNA synthesis a few residues downstream of the incorporated inhibitor. However, the inhibition results have yet to be translated in quantitative terms and data with recombinant EBOV RdRp are lacking. RSV, NiV, and EBOV are non-segmented viruses that share similar requirements for RNA synthesis. RdRp activity of RSV and NiV requires the multifunctional L protein and the phosphoprotein or P protein. While the L protein contains the polymerase active site, the P protein is necessary to form an active complex. We have recently expressed active EBOV RdRp that contains the L protein in complex with viral protein 35 (VP35), which is the functional equivalent of P proteins. In this study, we utilized the EBOV RdRp complex to study the mechanism of action of remdesivir. We demonstrate that incorporation of the nucleotide analogue at position i causes delayed chain termination predominantly at position i+5.