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.

Viruses, which consist of nucleic acid encased in a protein shell, are parasites of host organisms. The term ‘virus’ comes from the Latin word ‘venom’, which means poison, because a virus is generally considered to be a causative agent like a poison that causes infectious diseases. These tiny, living entities have considerable import, because they can cause substantial damage to humans and non‐human animals and other living organisms. The relationship between humankind and viruses has a long history. For example, the earliest evidence of smallpox was found in 3000‐year‐old Egyptian mummies, who had smallpox‐like eruptions on their skins.1 The overall mortality rate of smallpox was around 30%,2 making it one of the most feared infectious diseases. In 1918–1919, during World War I, influenza A virus caused the Spanish flu pandemic, resulting in infection of approximately 500 million people and more than 20–40 million death worldwide.3

Since the initial isolation of viruses in the 19th century, scientists have identified and characterised a wide variety of viruses, and the field of virology has progressed remarkably since then, enabling us to combat the frequently deadly effects of these viruses. One of the greatest achievements is the complete eradication of smallpox. Although smallpox was once rampant in the world, vaccination of the entire population has eradicated this disease.1 Similarly, the poliovirus vaccine has significantly reduced the incidence of poliomyelitis.4 Despite the progress of virology, we still have many unconquered viral diseases and we are confronted with the problem of emerging infectious diseases, which are caused by newly identified species or strains. For example, Ebola virus disease and acquired immunodeficiency syndrome emerged in 1976 and 1981, respectively,5, 6, 7, 8, 9 and more recently, severe acute respiratory syndrome (SARS), highly pathogenic avian influenza viruses and Middle East respiratory syndrome (MERS) have appeared in human society.10, 11, 12, 13, 14, 15 Therefore, it is important to continue studying the mechanisms of viral replication and pathogenicity.

Yet, these negative aspects of viruses do not tell the whole story since the relationships between hosts and viruses are multitudinous, and virus infections do not necessarily lead to disease symptoms in hosts. Rather, recent studies suggest that there are viruses that are beneficial to the biological functions and/or evolution of their hosts. Recently, we established a research consortium, designated as ‘Neo‐virology’, which is supported by Grants‐in‐Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan. In this consortium, we define a virus as a component of the global ecosystem. Our aim was to elucidate the roles of viruses in host organisms and the global ecosystem, in contrast to traditional virology research, which tends to focus on pathogenic viruses that cause diseases in their hosts. This research project is expected to develop into an important scientific field that examines the interactions between the global ecosystem and viruses. In this brief review, we give some insights into the positive side of viruses.

HBV is the prototype virus of the Hepadnaviridae family—small spherical viruses with icosahedral symmetry that combine a partial double-stranded (ds) DNA genome and virus-encoded RT. Within the Baltimore virus classification system, which classifies viruses based on their genomic composition and replication cycles,29 the Hepadnaviridae are classified as group VII (sometimes referred to as pararetroviruses)—they are the only animal viruses of this group. Until recently, the family was divided into 2 genera: the Orthohepadnavirus species (which infect mammals, including primates and bats) and the Avihepadnavirus species (which infect birds). However, the recent discovery of putative hepadnaviruses that infect fish30 and amphibians31 indicates that the viral family might be larger than initially believed (Figure 1A).32, 33 Based on sequence diversity, HBV is divided into 9 genotypes and 1 putative genotype (Figure 1B). Hepadnaviruses have some of the smallest known viral genomes, ranging from 3.0 to 3.3 kb; the HBV genome is approximately 3.2 kb34 (Figure 1C).

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.

Rabies is an ancient neurological disease caused mainly by the rabies virus (RABV) and is almost invariably fatal once clinical symptoms develop. Currently, rabies continues to pose a serious public health threat in most areas of the world, especially in the developing countries of Asia and Africa. It has been estimated by the World Health Organization (WHO) that more than 55,000 annual human deaths are caused by rabies, through bites of rabid animals, worldwide. After the virus has entered the periphery site after exposure, it subsequently spreads into the central nervous system (CNS), causing neuronal dysfunction, which is most likely the main cause of the fatal outcome of rabies.

The causative agent RABV is the type species of the genus Lyssavirus in the family Rhabdoviridae. The RABV genome is a single-stranded, negative-sense RNA of approximately 12 kb, which encodes five structural proteins in the order (3′ to 5′) nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L). The negative-sense RNA genome is tightly encapsidated by N, P, and L proteins to form a ribonucleoprotein complex that is responsible for virus replication in the cytoplasm within infected cells. The RABV G protein is the only viral protein exposed on the surface of the virus and is not only the major determinant of viral pathogenicity, but also the major protective antigen responsible for inducing protective immunity against rabies.

Fortunately, rabies can be a vaccine-preventable disease, provided that post-exposure prophylaxis (PEP) is given promptly and correctly. Protection against rabies correlates with the presence of rabies-specific virus-neutralizing antibodies (VNAs). According to the WHO, VNA titers greater than 0.5 international units per mL serum can reliably provide protection to humans and animals. Currently, rabies-infected dogs are the major reason for the high incidence of human rabies, and therefore vaccinating dogs has been shown to be the most cost-effective strategy for preventing rabies in humans. As reported by the WHO, vaccination coverage of 70% of the canine population can efficiently reduce virus transmission and prevent human rabies. However, despite the fact that efficacious vaccines are readily available, rabies still has a high death rate, mainly due to the cost and accessibility of proper PEP treatment. The current PEP schedule not only requires multiple injections but is also time-consuming, a problem that is even more pronounced due to the fact that RABV-specific immunoglobulin (RIG), which is both expensive and often in short supply, is required to treat severe exposure. This creates a particular burden for rural regions of developing countries that suffer from the highest incidence rates of rabies. Therefore, the development of alternative, cost-effective vaccines that would induce sustained immunity after a single dose inoculation and could ideally clear virus infection from the CNS is warranted.

The advent of the reverse genetics technique has revolutionized the study of RABV, as well as other negative-strand RNA viruses, which has greatly advanced our understanding of the biology of these viruses and profoundly accelerated the development of novel vaccines against various pathogens. While a number of excellent reviews have been written focusing on reverse genetics of negative-strand RNA viruses, the biology of RABV, rabies vaccines, and the pathology and prophylaxis of rabies, this review rectifies the lack of focus on current strategies that have been evaluated for prevention or as PEP of rabies. This review places particular emphasis on the most promising approaches using live-attenuated and/or recombinant vaccine platforms for preventive vaccinations. Other innovative modalities, such as monoclonal antibody-based platforms and small interfering RNAs (siRNAs) interfering with virus replication, which may deserve future research for rabies treatment, will also be briefly introduced.

Emerging infectious diseases (EIDs) have exerted a significant burden on public health and global economies,. During the past decade, novel viruses, particularly those causing severe acute respiratory syndrome (SARS) and avian influenza A H5N1, have attracted international concern. These diseases represent only part of a rich tapestry of pathogens that have emerged to pose public health threats in recent years. Clearly, there is a pressing need for rapid and accurate identification of viral etiological agents. The development of Next Generation Sequencing (high throughput sequencing) technology provides a possible solution to this problem; indeed several recent studies have used these techniques to identify novel viral agents,,,,. Palacios et al. identified a novel and deadly arenavirus by employing 454-pyrosequencing technology, the results of which were later confirmed by PCR. Recent studies, have identified a novel strain of Ebola virus which caused a hemorrhagic fever epidemic in Uganda, and dengue virus type 1 (DENV-1) sequences in laboratory reared mosquitoes experimentally infected with DENV-1. Using de novo next generation sequencing, Makoto Kuroda et al. showed that the etiologic agent identified in a deceased pneumonia patient was, in fact, the pandemic influenza A H1N1 virus, rather than that originally assumed to be pneumococcus.

These studies highlight the power and feasibility of high throughput sequencing techniques for detection of unsuspected or novel etiologic agents. The sequencing technologies offer distinct advantages over traditional viral detection and surveillance methods that generally require prior knowledge of the etiologic agents, as well as depending on virus-specific primers, probes or antibodies. These traditional techniques are, therefore, unsuitable in situations where the causative agent of an outbreak is entirely novel, or is a pathogen variant with several mutations to key priming regions. Hence, high throughput sequencing techniques provide a powerful new opportunity for surveillance and discovery of novel pathogens. The techniques provide a cost-effective mechanism for massive parallel sequencing generating extreme sequencing depth, whilst providing multiplex analyses for etiologic agent identification.

Mosquito-borne infectious diseases have been emerging and re-emerging in many areas of the world, especially in tropical and subtropical areas where agents such as West Nile virus (WNV), dengue virus (DENV), chikungunya virus (CHIKV) and yellow fever virus (YFV) are present. Surveillance of infectious agents carried by mosquitoes is important for predicting the risk of vector-borne infectious disease outbreaks. Recently, a new strategy based on small interfering RNA (siRNA) immunity to virus infection was proposed for detecting novel RNA viruses in laboratory reared drosophilae and mosquitoes, as well as RNA/DNA viruses in plants using high throughput sequencing techniques,. Prompted by these results (in laboratory reared insects and plants by deep sequencing and assembly of small RNAs isolated from the host organisms), we explored the feasibility of using this approach to identify viruses from wild-caught mosquitoes. Our findings show for the first time that high throughput sequencing of small RNAs can detect both RNA- and DNA viruses in wild-caught insects, thus supporting the feasibility of employing this approach for surveillance purposes.

Very few topics in Virology relate so closely to the general concept of biological complexity as the emergence and re-emergence of viral disease. In the introduction to their classic book, Solé and Goodwin define the sciences of complexity as “the study of those systems in which there is no simple and predictable relationship between levels, between the properties of parts and of wholes”. The emergence of viral disease involves several levels of complexity. The underlying level stems from the population structure of viral populations as they replicate in their standard hosts. Model studies of plaque-to-plaque transfers (bottleneck passages) of foot-and-mouth disease virus (FMDV) in BHK-21 cells (Fig. 1) showed that the pattern of fitness decay of the virus followed a Weibull distribution. This type of statistical distribution suggests that the mutations fixed in the viral genome at each transfer produced a cascade of perturbations in the virus-host interactions that were sensed in the form of a change in virus yield. The unpredictability of the effect of mutations is further reinforced by the increasing evidence that viral proteins are multi-functional so that mutations can alter one or more of the interactions between viral and host components that determine the viral yield per cell.

The second level of complexity results from a network of environmental, ecologic, and sociologic influences that affect the probability that a potentially pathogenic virus comes into contact with a new host. A good number of such influences are subjected to indetermination. To give a specific example, a change in temperature and humidity in a large geographical area may alter flora and fauna, and, as a result, the distribution of viral vectors (arthropods, birds, mammals). However, which vectors and how they will be affected by weather or climate conditions are usually difficult to predict since they depend on sets of ecological interactions. Finally, another level of complexity intervenes in the epidemiological outcome of a viral disease, once it has emerged. Severe acute respiratory syndrome (SARS) faded months after its emergence, while Acquired Immune Deficiency Syndrome (AIDS) has become a severe pandemics, to last for many decades or forever. The difference can be attributed to the pattern of virus-host interaction and the capacity of the virus to be transmitted from an infected host individual into a susceptible individual. Again, these processes fall into the domain of biological complexity. Advances in molecular virology and the development of computational tools for viral surveillance have greatly improved our knowledge of the factors that underlie the emergence and re-emergence of viral disease, and, interestingly and paradoxically, we understand some of the reasons behind the unpredictability of emergences.

Clinical virology and virus discovery in the 20th century focused chiefly on the identification of viruses through microscopy, serology, and cell or animal infection studies (1). With the advent of nucleic acid amplification, a wide range of molecular approaches for virus detection became available: PCR (2), consensus PCR (cPCR) and multiplex PCR systems (3–10), differential display (11), representational difference analysis (12, 13), subtractive cloning (14), domain-specific differential display (15), cDNA cloning (16–18), cDNA immunoscreening (19, 20), microarrays (21, 22), and, most recently, high-throughput sequencing (HTS). HTS has enabled unbiased pathogen discovery and facilitated virome analyses that have enhanced our understanding of the origin, evolution, and ecology of known and novel viruses (1). However, HTS has not been widely implemented in clinical diagnostic laboratories largely due to operational complexity, cost, and insensitivity with respect to agent-specific PCR assays.

Strategies to increase the sensitivity of HTS have focused on the enrichment of viral template through subtraction of host nucleic acid via nuclease digestion and depletion of rRNA. Although they are helpful, none has achieved the sensitivity required for clinical applications. To address this challenge, we have established a positive selection probe capture-based system to enrich sequence libraries for viral sequences. Here, we describe the virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) and demonstrate its potential utility as a sensitive and specific HTS-based platform for clinical diagnosis and virome analysis.

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.

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].

In May 2013, a devastating outbreak of epidemic diarrhea in young piglets commenced in swine farms of the United States, causing immense economic concerns. The mortality can reach up to 100% in piglets less than 10 days of age, with a recorded loss of at least 8 million neonatal pigs since 2013 (1, 2). Enteric viruses, such as swine enteric coronaviruses (SECoVs), porcine epidemic diarrhea virus (PEDV), and porcine deltacoronavirus (PDCoV), were isolated from these outbreaks (3, 4) and characterized (5). However, despite intensive biosecurity measures adopted to prevent the spread of SECoV in many farms and the use of two U.S. Department of Agriculture (USDA) conditionally licensed vaccines against PEDV, the outbreaks continue and have now spread to many other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, and Ecuador in the Americas (6) and Ukraine (7). Repeated outbreaks have also been reported on the same farms that were previously infected with PEDV. In June 2014, the USDA issued a federal order to report, monitor, and control swine enteric coronavirus disease (SECD) (8). In our efforts to understand the seemingly uncontrollable porcine epidemic diarrhea outbreaks, we discovered a novel mammalian orthoreovirus type 3 (MRV3) in feces of pigs from these outbreaks and ring-dried swine blood meal (RDSB). We have also reproduced severe diarrhea and acute gastroenteritis in neonatal pigs experimentally infected with purified MRV3 strains.

The family Reoviridae comprises 15 genera of double-stranded RNA (dsRNA) viruses (9). Orthoreoviruses with 10 discrete RNA segments have been isolated from a wide variety of animal species, including bats, civet cats, birds, reptiles, pigs, and humans (10, 11). Most orthoreoviruses are recognized to cause respiratory infections, gastroenteritis, hepatitis, myocarditis, and central nervous system disease in humans, animals, and birds (11); orthoreovirus genomes are prone to genetic reassortment and intragenic rearrangement (11, 12). The exchange of RNA segments between viruses could lead to molecular diversity and evolution of viruses with increased virulence and host range (13, 14). MRV serotypes 1 to 3 were associated with enteritis, pneumonia, or encephalitis in swine around the world, including China and South Korea (15–18). The zoonotic potential of MRV3 has been reported recently (19–21). However, porcine orthoreovirus infection of pigs was unknown previously in the United States.

Human diseases causing RNA viruses include Orthomyxoviruses, Hepatitis C Virus (HCV), Ebola disease, SARS, influenza, polio measles and retrovirus including adult Human T-cell lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus (HIV). RNA viruses have RNA as genetic material, that may be a single-stranded RNA or a double stranded RNA. Viruses may exploit the presence of RNA-dependent RNA polymerases for replication of their genomes or, in retroviruses, with two copies of single strand RNA genomes, reverse transcriptase produces viral DNA which can be integrated into the host DNA under its integrase function. Studies showed that endogenous retroviruses are long-terminal repeat (LTR)-type retroelements that account for approximately 10% of human or murine genomic DNA.

Among human retroviruses, HIV-1 is a lentivirus with an RNA genome formed by two copies of a single-stranded, positive-sense RNA. The HIV-1 RNA genome is associated to the nucleocapsid protein (NC) and to viral enzymes, thus it is “protected” within the viral capsid mainly formed by the p24 protein. Upon entry into the target cell, the viral RNA genome is reverse transcribed into double-stranded DNA by a virally encoded reverse transcriptase that is transported along with the viral genome into the virus particle. The viral DNA is imported into the cell nucleus and integrated into the cellular DNA by a virally encoded integrase and host co-factors. Once integrated, the virus may become latent, or may be transcribed, producing new RNA genomes and viral proteins that are packaged and released from the infected cell as new virus particles that will infect other cells to begin the new replication cycle. Many aspects of the life cycle of retroviruses are intimately linked to the functions of cellular proteins and RNAs. HIV-1 and Moloney Murine Leukemia Virus (MoMuLV) have been studied for the dimerization of two RNAs.

In traditional virology, most viruses found in humans are considered to be pathogenic to their hosts; however, recent studies have shown that there are some viruses that have symbiotic relationships with their hosts and do not cause disease. Infection with one virus may protect the host from a superinfection with another pathogen. Barton et al.16 demonstrated that latent infection with the herpesviruses murine gammaherpesvirus 68 or murine cytomegalovirus, which are genetically related to the human pathogens Epstein‐Barr virus and human cytomegalovirus, respectively, led to cross‐protection in mice. Infection with these viruses induced prolonged production of the antiviral cytokine interferon‐gamma and systemic activation of macrophages that protected the mice from subsequent bacterial infections with either Listeria monocytogenes or Yersinia pestis.16 Moreover, it has been reported that superinfection with hepatitis A virus suppressed hepatitis C virus replication in patients with chronic hepatitis C in at least two cases,17 and infection with human cytomegalovirus (HCMV) suppressed superinfection with HIV‐1 in vitro as a result of the downregulation of the expression of CCR5, a co‐receptor for HIV‐1, induced by the HCMV infection.18

Some viruses also have beneficial effects with respect to non‐infectious diseases. Epidemiologic studies suggest that virus infections in childhood might confer protection against some cancers later in life. For example, the risk of chronic lymphoid leukaemia in subjects who had measles in childhood is relatively low,19 and mumps infection in childhood might protect against the development of ovarian cancer in adults.20 However, infection with oncoviruses is known to increase the risk of development of some cancers (e.g. cervical cancer and liver cancer induced by the human papillomavirus and hepatitis B virus/hepatitis C virus infection, respectively).21 Such information is important when considering strategies for cancer immunotherapy and/or vaccination campaigns. In addition, the infection of non‐obese diabetic mice with lymphocytic choriomeningitis virus prevented the infected mice from developing autoimmune disease and subsequent type I diabetes (insulin‐dependent diabetes mellitus).22, 23 Chronic viral infection of mice with murine cytomegalovirus (CMV) increased epithelial turnover and wound repair via antiviral cytokine type I interferons (IFNs),24 but CMV infection can promote cancer malignancy; this phenomenon is known as ‘oncomodulation’.25, 26

Recent metagenomic studies have revealed that virus infection sometimes confers benefits including the regulation of microbiota in the gut. Bacteriophages are abundant in the gut and are thought to modulate the gut microbiota by infecting specific bacterial populations. Accordingly, potential therapeutic applications of bacteriophages in humans (e.g. control of antibiotic‐resistant bacteria, stabilisation of healthy gut microbes) have been considered.27, 28

Therefore, the elucidation of the symbiotic effects of viruses on the physiological functions and immune responses of their hosts, as well as clarification of the functional mechanisms involved, will lead to an understanding of the essential roles of viruses in regulating the biological processes of their hosts.

Mammalian orthoreoviruses (MRVs), prototypes of the genus Orthoreovirus (family Reoviridae), are non-enveloped viruses with a segmented double-stranded RNA (dsRNA) genome (∼23,500 bp). MRVs have four major serotypes (type 1 Lang, type 2 Jones, type 3 Dearing, and type 4 Ndelle) [1, 2]. Each MRV particle contains 10 genome segments divided into three size classes based upon their characteristic mobility during gel electrophoresis: three large (L1, L2 and L3) segments, three medium segments (M1, M2 and M3), and four small segments (S1, S2, S3 and S4) [1–3]. The virions have an average diameter of 70–80 nm with a typical icosahedral, double-layered protein capsid structure [1–3].

Although MRVs had been assumed to cause mild respiratory or gastrointestinal diseases, recent studies have shown that they can cause severe illnesses in humans and other mammals, including upper respiratory tract infections, encephalitis, and diarrhea [4, 5]. MRVs have been isolated from many mammalian species, including humans and bats [4, 5]; however, the natural reservoirs or direct progenitors remain unclear. The significance of bats as a source of emerging infectious diseases has been recognized. Bats also are being increasingly recognized as reservoir hosts for viruses which can cross species to infect humans and other domestic and wild mammals [6–8]. Indeed, many recent outbreaks of emerging viruses, such as the Hendra virus, Nipah virus, Ebola virus and severe acute respiratory syndrome coronavirus (SARS-like CoVs), have been associated with bat transmission events [9–13].

Here, we describe the first isolation of a novel natural reassortant MRV strain, named RpMRV-YN2012, from the least horseshoe bat (Rhinolophus pusillus) in China. The whole genome sequence of RpMRV2012 was determined. Its evolution and evidence of genetic reassortment were analyzed by sequence comparison and phylogenetic analysis.

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.

Hantaan virus (HTNV) is a worldwide pathogen that causes serious infectious disease featured with febrile, mucocutaneous hemorrhage, renal damage and shock. The disease is thus named hemorrhagic fever with renal syndrome (HFRS). The cases of HFRS in China account for nearly 90% of all HFRS cases worldwide. During 1950-2007, 1,557,622 HFRS cases and 46,427 deaths from HFRS (a death rate of 3%) were reported in China, and HFRS cases are reported in most provinces and cities (28/31) of mainland China annually, making HFRS a notable public health problem. Shaanxi province, which is located in northwest of China, is one of the most seriously afflicted areas. Over the past decade (2001-2010), the majority of HFRS cases (65%) occurred from October to December annually. The composition of the population of HFRS cases has changed with the rapid development of society and the environment in recent years; however, the infected population is primarily 16- to 59-year-old farmers.

HTNV (genus Hantavirus, family Bunyaviridae) is an enveloped virus with a single-stranded, negative-sense RNA genome. HTNV was the first hantavirus described and was isolated in 1978. The virion encloses three gene segments named L (large), M (medium), and S (small), which encode different proteins. The L segment encodes an RNA-dependent RNA polymerase, the M segment encodes two glycoproteins, Gn and Gc, and the S segment encodes a nucleocapsid protein (NP). Variation in the S and M segments may alter the virulence and antigenicity of hantaviruses. The transmission pathway of hantavirus within rodents and from rodents to humans includes inhalation of aerosolized excreta (i.e., saliva, urine and feces), the consumption of contaminated food, or rodent bites. Seven species of hantaviruses have been identified in China, but only HTNV, which is carried by Apodemus agrarius, and Seoul virus (SEOV), which is carried by Rattus norvegicus, are associated with HFRS [10–12]. However, the disease caused by HTNV is more clinically severe than that caused by SEOV. HTNV has been reported to be the predominant etiological agent for HFRS in the Xi’an district of Shaanxi province, and SEOV and other hantavirus species are seldom detected.

In clinical practice, the commonly used diagnostic methods for detecting of HTNV are primarily based on serological techniques, such as the enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). Other molecular biological methods, such as conventional RT-PCR and nested PCR, have also been developed but are mainly used for experimental research. Because it is time-consuming (usually requiring 5-7 days to perform the test), a plaque assay, which is the classical virus titration approach, is seldom used in laboratories. However, none of these detection methods provides information about the load of the infectious virions.

With the development of the quantitative RT-PCR (qRT-PCR) assay, an easier method to quantify viral load has become available, especially for the quantifying of HBV, HCV and HIV in patient peripheral blood [13–15]. The TaqMan-based real-time PCR assay has been described for the detection and quantification of hantavirus [16–18].

SYBR Green Ⅰ-based qRT-PCR is technically simpler and less expensive than the TaqMan probe or molecular beacon-based assays. This method uses fluorescent dye that directly binds to the amplified double-stranded DNA sequences; therefore, the fluorescence signal is proportional to the amount of PCR products accumulated in the reaction tubes. When coupled with melting curve analysis of the amplification products, SYBR Green Ⅰ-based qRT-PCR assay offers an alternative choice to TaqMan-based assays.

In this study, a SYBR Green Ⅰ-based one-step qRT-PCR assay was established to target the S segment of the HTNV genome for quantification of the HTNV RNA viral load, and the performance of the qRT-PCR assay was evaluated using serum samples from HFRS patients.

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.

RT-PCR and Next-Generation DNA Sequencing

Viruses that use RNA as their genetic material are a diverse group of obligate parasites associated with some of the most infectious and deadly human diseases. The genomes of RNA viruses are highly varied and can be either double-stranded or single-stranded, with the latter being of negative or positive polarity. RNA virus genomes can also be either segmented, whereby each segment typically encodes one viral protein, or unimolecular, in which a single polyprotein is translated and cleaved to form individual viral proteins. The genetic diversity of RNA viruses stems from a high rate of spontaneous mutation and has made development of antiviral therapies and vaccines extremely challenging. The increased mutation frequency is primarily due to a lack of proofreading by the RNA-dependent RNA polymerases (RdRp) required for replication of many RNA viruses. This elevated mutation rate is believed to be the reason that RNA virus genomes are usually restricted to lengths of less than 30 Kb and therefore tend to be smaller and encode fewer proteins than those of many DNA viruses.

RNA viruses have diverse modes of replication that largely depend on their genome configuration. In positive-sense single-stranded RNA viruses, such as coronaviruses and hepatitis C virus (HCV), the viral genome functions as mRNA and can be directly translated into a viral polyprotein by a host ribosome. Viral proteins can then form membrane-associated replication complexes (RC) in the cytoplasm that provide sites for viral RNA synthesis. Negative-strand RNA viruses, such as Rabies and Influenza, must initially use a viral RdRp to produce monocistronic mRNAs. These mRNAs are translated to produce viral proteins while production of new viral genomes first requires synthesis of positive-sense RNA which is then converted to negative-sense viral genomes. In this case, virus replication and assembly requires formation of ribonucleoprotein (RNP) complexes that contain viral polymerases and nucleoproteins.

Retroviruses are a unique class of RNA viruses whose lifecycles contain a DNA intermediate that requires integration into the host cell genome prior to viral gene expression. The retrovirus genome consists of two identical positive-strand RNA molecules of between 7 and 12 kb that are capped at the 5' end and polyadenylated at the 3' end. Following cell entry, viral reverse transcriptase (RT) enzymes are used to generate double-stranded DNA (dsDNA) molecules from the RNA templates. These are then transported to the nucleus and integrated into cellular DNA which requires another virus-encoded enzyme known as integrase (IN). The viral mRNA is exported to the cytoplasm where it is translated to viral proteins and the remainder is packaged as new viral genomes. Since integration of the viral cDNA requires breaks in cellular DNA, activation of host DNA repair pathways is a consequence of retroviral infection that is discussed in detail below.

For some RNA viruses, such as members of the families Orthomyxoviridae and Retroviridae, at least part of the replication cycle takes place in the nucleus. For the majority, however, replication occurs exclusively in the cytoplasm and therefore the impact of the viral lifecycle on the nucleus may be less severe than is the case for many DNA viruses. However, proteins encoded by RNA viruses are often transported to the nucleus where they can perturb cellular functions and inhibit the antiviral response. As is discussed below, these nuclear activities may involve the direct or indirect introduction of DNA damage and impairment of the subsequent cellular response.

Bats belong to the order Chiroptera, and are the second largest order of mammals after rodents. This order includes 19 families and 962 species distributed across the globe1. More than 130 kinds of viruses have been detected in bats, including more than 60 species of zoonotic viruses which are highly pathogenic in humans2, such as severe acute respiratory syndrome (SARS)-like coronavirus (SL-CoV), Ebola virus, Nipah virus, and Hendra virus3–6. In 2002, SARS outbreak in China infected more than 8,000 people worldwide and killed at least 800 people. Chinese horseshoe bats were proved to be the natural reservoirs of SARS-CoV and that intermediate hosts may not be necessary for direct human infection by a bat hosting SL-CoV7. Similarly, Middle East respiratory syndrome (MERS)-associated coronavirus were detected in bats, and required dipeptidyl peptidase 4 cell receptors for its invasion into host cells8. It seems likely that bats might act as natural hosts which critical roles in viral iner-host transmission.

In the last five years, the use of second-generation sequence technology allowed us to elucidate a flood of viruses, like SARS-CoV, hepatitis B virus, rotavirus and other important viruses9–11. In 2010, meta-genomic analysis was conducted for the first time, following the second generation sequencing of oral swab and faecal samples from 41 bats of three common North American bat species. The results showed that sample pools contained strong matches to at least three novel group of CoVs, and large numbers of insect and plant virus sequences were identified12. One bat virome analysis conducted by Ge et al. on fecal samples of bats from six locations in China13, and 97 contigs were found to be related to eukaryotic viruses, including coronavirus. Then, one bat virome analysis in Myanmar conducted by He et al. has identified many new mammalian viruses of Myanmar bats14, showing that the composition of bat viromes differs depending on geographical location and bat species. These studies show that the study of the bat viruses by the metagenomic analysis can be insightful.

The coastal wetlands southeast of China consist of growing ports, industrial districts, and port cities. Studies show the occurrence of natural focal diseases, such as Dengue fever and hemorrhagic fever with renal syndrome, in species-rich and densely populated southeast of China15, 16. It is important to understand the distribution of pathogens in different animals across different habitats. An understanding of the natural habitat of bat-associated viruses can prevent newly emerging and re-emerging zoonoses. To expand these studies to southest China, Here we combined second-generation sequencing technology with meta-genomics to understand the outbreak of new infectious diseases caused by animal-origin pathogens and explore the unknown viruses from the natural environment, humans, and animals. In this study, we collected 235 bats from six locations in the southeastern coastal area of China from July 2015 to August 2015. Using Illumina platform for sequencing gut and lung tissue from bats, we detected a total of 25 species of the virus family, including norovirus, which was detected first. Also, we sequenced Myotis formosus for the first time from the southeastern coastal area and found that astrovirus. This work extended our understanding the diversity of bats harboring virsues and provide new clues to monitor these transmittable zoonotic viruses.

Clinical (e.g., age, vaccination record, and clinical signs) and farm history should be provided to clinicians for determining the cause of diarrhea. Once the specimens are submitted to a veterinary diagnostic laboratory, the diagnostician sorts the samples to ensure proper delivery to testing laboratories based on the history and sample type. Generally, fecal sample are examined by microscopy (for C. parvum and Coccidia), bacterial culturing (for Salmonella spp., E. coli, and C. perfringens), and PCR (for BRV and BCoV). In contrast, intestinal tissues are subjected to immunohistochemistry or bacterial culturing. More recently, nucleic acid-based techniques such as PCR and an antigen-capturing enzyme-linked immunosorbent assay (Ag-ELISA) have been more commonly used for the rapid detection of various bacterial and viral pathogens in clinical specimens from diarrheic calves. When the laboratory test results are available, clinicians should consider the overall farm and clinical history in conjunction with lab results before identifying the causative pathogen.

Bocaparvoviruses (BoVs) belong to the genus Bocaparvovirus and are emerging pathogens of the Parvoviridae family. BoVs are nonenveloped, single-stranded DNA viruses with an icosahedral symmetry and were originally named according to their first identified members, bovine parvovirus (BPV) and minute virus of canine (MVC). In the past few years, novel BoVs have been identified in a variety of animals, including bats, camels, gorillas, marmots, pigs, and rodents. BoVs are comprised of 21 species, including carnivore BoV 1–6, chiropteran BoV 1–4, lagomorph BoV 1, pinniped BoV 1 and 2, primate BoV 1 and 2, and ungulate BoV (UBoV) 1–6. A few new UBoVs have been identified in dromedary camels (tentatively UBoV7 and UBoV8) but have yet to be classified by the International Committee on Taxonomy of Viruses (ICTV).

Initially, the classification of parvoviruses required the isolation of the virus; however, reporting of the viral sequence containing all the non-structural and structural coding regions is now acceptable provided the genomic, serological, or biological data supports infectious etiology. Most of the members of the Bocaparvovirus genus have been identified using molecular methods and lack isolation in cell culture. Human BoVs cause severe respiratory and gastrointestinal infections in young children. Bovine parvovirus (BPV) causes gastrointestinal and respiratory symptoms, reproductive failure, and conjunctivitis in cattle worldwide. Another important member of the BoV genus, canine minute virus (MVC), causes sub-clinical disease and fetal infections often leading to neonatal respiratory disease or abortions. However, Koch’s postulates have yet to be fulfilled to link newly emerging BoVs with the clinical disease in animals.

Alpaca (Vicugna pacos) are domesticated members of the new world camelids closely related to llama (Lama glama), guanaco (Lama guanicoe), and vicuna (Vicugna vicugna). Over the past couple of decades, alpacas have gained significant popularity as pets, show animals, and fiber animals in the United States, with a total of 264,587 alpacas registered in the US as of May 2019. A variety of viruses have been identified in alpacas, including bovine viral diarrhea virus, coronavirus, adenovirus, equine viral arteritis virus, rotavirus, rabies, bluetongue virus, foot-and-mouth disease virus, bovine respiratory syncytial virus, influenza A virus, bovine papillomavirus, vesicular stomatitis virus, parainfluenza-3 virus, West Nile virus, and equine herpesvirus. However, BoVs have yet to be reported in alpacas.

An alpaca farm in the mid-eastern United States reported recurrent diarrhea and respiratory failure in young alpacas, with a case fatality rate up to 100%. In 2017, an alpha coronavirus was identified as causing clinical disease in two animals, and vaccination was subsequently attempted. However, diarrhea and respiratory distress continued to occur in juvenile animals despite increased biosecurity measures and supportive herd management. In 2018, an intestinal sample from a deceased alpaca was submitted to Kansas State University Veterinary Diagnostic Laboratory for metagenomic next-generation sequencing (NGS) to further evaluate potential causes of disease. The intestinal sample was processed, extracted, and sequenced using previously described methods. The raw data was analyzed using a custom bioinformatic pipeline. Reads were trimmed, and the adapter/index sequences were removed using Trimmomatic, Sickle, and Scythe.

A total of 334,052 cleaned reads were classified as eukaryotes (41%), bacteria (28%), viral (7%), and other organisms (4%) by Kraken software, which applies a k-mer search strategy from a sequence database to taxonomically classify reads (Figure 1). Kraken revealed a majority of the viral reads (22,170) as BoV (77%); bacteriophages (14%); and miscellaneous viruses composed of retroviruses, bacteria viruses, and unclassified viruses (9%). Reads lacking classification (no hits, n = 67,604) and identified as viral reads (n = 22,170) were de novo assembled into contigs and BLAST (Basic Local Alignment Search Tool) against the National Center for Biotechnology Information (NCBI) database, identifying a contig with a 58.58% nucleotide percent identity to a camel BoV from Dubai (KY640435). A full-length genome (5155 nucleotides) of an alpaca BoV (AlBoV, GenBank number MK014742) had an average read coverage of 2440X. AlBoV was aligned with the 108 complete UBoV genomes from GenBank using Multiple Alignment using Fast Fourier Transform (MAFFT) in Geneious Prime. AlBoV shared a 57.77–58.58% whole genome nucleotide identity to the UBoV8 strains (Table 1). Recombination events were not detected in the UBoV alignment using RDP4 software, although single-stranded DNA viruses such as parvoviruses possess a mutation rate similar to single-stranded RNA viruses and a higher mutation rate than double-stranded DNA viruses.

AlBoV contained three open reading frames (ORFs), NS1, NP1, and VP1/VP2, which were 2154 bp (411 to 2564 bp), 507 bp (2701 to 3207 bp), and 1395bp (3194 to 4588 bp), respectively. ICTV indicates a new parvovirus species should have less than 85% amino acid identity of the NS1 protein with other parvovirus species. The AlBoV identified in the present study shared the highest NS1 amino acid percent identity (57.89–67.85%) with camel BoVs in UBoV8 (Table 1) and represents a tentative new BoV species, UBoV9. The ancillary protein NP1, which is a unique feature of BoVs, is known to influence RNA processing events by suppressing internal polyadenylation and splicing of an upstream intron. Unlike some of the other BoV sequences, the coding region of NP1 of AlBoV did not overlap with the C-terminal region of NS1. Interestingly, VP1/VP2 gene of AlBoV was the shortest among the identified UBoVs in the GenBank (Figure 2). Frameshift mutation were lacking in the AlBoV VP1/VP2 sequence, and the nucleotide sequence after the VP1/VP2 stop codon varied among the 108 complete UBoV sequences in the GenBank.

To study the phylogenetic relationship between AlBoV and other UBoVs, whole genome, NS1, NP1, and VP1 phylogenetic trees were created using a maximum likelihood method (phyML), using 500 bootstrap replicates in Geneious Prime. The trees were curated in FigTree (available from http://tree.bio.ed.ac.uk/software/figtree/) and Adobe Illustrator CS6 (Adobe Systems Inc, San Jose, CA, USA). Whole genome and NS1 phylogenetic trees illustrated that AlBoV shared a common ancestor with the UBoV8 species from camels (Figure 3). All eight species of UBoV (1–8) illustrated clear grouping in phylogenetic trees, which was observed in NP1 and VP1 phylogenetic trees as well.

To investigate and identify the presence of virulence attributes, AlBoV was screened for the ATP or GTP-binding Walker loop motif (GPASTGKT) and Phospholipase A2 (PLA2) motif with the calcium-binding loop and phospholipase catalytic residues; GPASTGKT and PLA2 were found in the NS1 and N-terminal of VP1 proteins, respectively (Figure 4). These protein motifs are conserved and are required for parvovirus infectivity. Phospholipase A2 activity, with the calcium-binding loop and phospholipase catalytic residues, is critical for efficient transfer of the viral genome from the late endosomes/lysosomes to the nucleus for the initiation of replication, and hence is considered essential for virus infectivity. Mutations of critical amino acid residues in the VP1 protein of human parvovirus B19 induces a strong reduction in phospholipase A2 activity and virus infectivity. Considering their vital role in parvovirus infectivity, PLA2 inhibitors are also targeted for antiviral drugs against parvovirus-associated diseases. The presence of the Walker loop and Phospholipase A2 motifs suggests that the newly identified alpaca BoV possesses the virulence determinants necessary to cause disease.

A new species of UBoV was identified in an alpaca intestinal sample. Bocaparvoviruses cause respiratory and gastrointestinal infections in humans, bovines, and other animals. However, the causation of clinical disease in this alpaca farm is unclear. Establishing an association between the presence of BoV and clinical disease would require comprehensive PCR testing of the alpaca farms. Moreover, Koch’s postulates is required to establish a virus–disease association. A causal association between the presence of BoV and clinical disease is often difficult due to prolonged viral shedding by the host after infection, high prevalence of BoV infection, and high rate of co-infections. Nevertheless, the discovery of a new BoV will help in developing new PCR diagnostics to determine the prevalence of BoV in alpaca herds and also to develop vaccines to prevent clinical disease. Given the high mutation rate of BoVs and increasing domestication of alpacas, identification of a new BoV in alpaca presents a true risk of cross-species transmission to other mammals.

Over time, for a pathogen with a truly predominant airborne transmission route, eventually sufficient numbers of published studies will demonstrate its true nature. If there are ongoing contradictory findings in multiple studies (as with influenza virus), it may be more likely that the various transmission routes (direct/indirect contact, short-range droplet, long-, and even short-range airborne droplet nuclei) may predominate in different settings [16, 20], making the airborne route for that particular pathogen more of an opportunistic pathway, rather than the norm. Several examples may make this clearer.

The selected pathogens and supporting literature summarized below are for illustrative purposes only, to demonstrate how specific studies have impacted the way we consider such infectious agents as potentially airborne and ‘aerosol-transmissible’. It is not intended to be a systematic review, but rather to show how our thinking may change with additional studies on each pathogen, and how the acceptance of “aerosol transmission” for different pathogens did not always followed a consistent approach.

The 15,012 viral tags were classified into 25 virus families (Fig. 2). About 94.7% (14222/15012) of tags were annotated for the mammalian families, including a total of 16 virus families: Herpesviridae, Coronaviridae, Poxviridae, Picornaviridae, Adenoviridae, Asfarviridae, Astroviridae, Caliciviridae, Circoviridae, Hepeviridae, Papillomaviridae, Reoviridae, Retroviridae, Flaviviridae, Parvoviridae,and Togaviridae. The proportion of viruses in this study was not the same compared with previous research13, 14, 17. The proportion of Parvoviridae accounted for a maximum of 88.16% (13234/15012) in vertebrate virus, which accounted for more than 97.43% (12894/13234) in the Parvoviridae was further annotated as Dependoparvovirus, followed by the 2.67% Coronaviridae (402/15012). The remaining Herpesviridae, Poxviridae, Picornaviridae, Retroviridae, and Flaviviridae existed in each group, indicating that viruses are a common presence in bats aspreviously reported. Although the viruses Adenoviridae, Circoviridae, Asfarviridae, and Caliciviridae annotated to a small number of sequences (<40 tags), but NCBI results show that known viruses exhibit low nucleotide (nt) or amino acid (aa) sequence identities.

Insect virus families contributed to 1.66% (249/15012) tags, including the following virus families: Baculoviridae, Iridoviridae, Rhabdoviridae, Polydnaviridae, and Iflaviridae. Among them, Iridoviridae accounted for the most, with 49.8% of insect virus sequences. Iridovirus was originally detected in insect body, after isolating from several other animals, especially aquatic animals. Also, Iflavirus tags had >78% nucleotide identity with the Perina nuda virus of genus Iflavirus.

Plant viruses accounted for 0.51% (77/15012) of annotated tags, which included the following four viral families: Luteoviridae, Totiviridae, Virgaviridae, and Phycodnaviridae. Phycodnaviridae accounts for the largest proportion of plant viruses (77%). Phycodnaviridae virus infection is widely present in fresh water algae or seaweed across the world.

It is worth mentioning that 35.27% virus sequences shared low similarity with known viral sequences. To verify the reliability of metagenomic sequencing results, six viruses (coronavirus, mastadenovirus, mamastrovirus, circovirus, norovirus, and bocavirus) closely related to human disease were selected, and nucleic acid from bat tissue samples of all 235 bats was amplified by PCR (Table 2). Among the five species of bats, Rhinolophus pusillus carried the broadest spectrum of viruses including coronavirus, adenovirus, norovirus, circovirus, and astrovirus. Of the remaining four bat species, bocavirus was detected in the bat species Myotis davidii for the first time (N = 2), coronavirus was detected in the bat species Rhinolophus ferrumequinum (N = 2), andastrovirus was detected in bat species Myotis formosus (N = 1). However other viruses were absent.

Gene therapy is the experimental use of genetic manipulation techniques to correct errors associated with genetic diseases or to modify undesirable Deoxyribonucleic acid (DNA) sequences. The ever extending list of genetic diseases opens the door wide to gene therapy as a new hope for targeting the aetiology rather than the symptoms of these diseases. Plenty of disciplines of gene therapy are currently discussed in the literature. However, there is a general agreement on few main issues to be thoroughly addressed before commencing a clinical trial for a novel gene therapy. Those include the precise diagnosis of the addressed genetic error, the relation between the causative gene defect and the resultant disease, the specific targeted tissue in the body, the dosage form design, and the choice of route of administration. Gene therapy approaches to corneal pathological disorders are being studied extensively to provide much needed progress against specific corneal malfunctions. Unlike protein based therapy, gene therapy has more research-attractive benefits being cheaper, better controlled, and more efficient in many occasions.

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen that causes life-long recurring disease. Two HSV serotypes exist, HSV-1 and HSV-2, with distinct tropism reported for each. The cold sore virus (HSV-1) is a leading cause of corneal blindness and rejection of corneal grafts in the developed world. The worldwide seroprevalence rates of HSV-1 ranges from 50 to 90%. Most of the population acquires the infection during early age and adolescence. Both serotypes are reported to infect the cornea and the neural cells too. However, the vast majority of HSV infections in the eye are caused by HSV-1 serotype. Soon after the HSV-1 infection, the virus develops lifelong latency in the sensory ganglion of the trigeminal nerve (trigeminal ganglion) as confirmed in several reports [4–7]. When active, HSV-1 travels from the trigeminal ganglion to different destinations, including the cornea. However, there is a growing body of evidence suggesting that the virus may be able to remain in its quiescent stage within the stroma [8–10]. Acyclovir and corticosteroids are normally prescribed either alone or in combination for common HSV keratitis. For example, prednisolone drops with prophylactic oral antiviral drug is common in stromal keratitis. The treatment of recurrent epithelial keratitis includes cycloplegia as well as antiviral eye drops such as trifluridine.

As of January 2012, the online record gene therapy clinical trials worldwide provided by the Journal of Gene Medicine (http://www.abedia.com) shows 28 currently active clinical trials addressing different ocular diseases, such as age-related macular degeneration, choroideraemia, and glaucoma. Interestingly, recent progress has been made targeting HSV-1 genome, as a new anti-viral class of medications [13, 14]. In this article, we review the current situation of ocular gene therapy against HSV-1 infections, including viral and non-viral vectors, routes of delivery of therapeutic genes and targets by screening and analyzing publications on ocular gene therapy from the published literature, and identify promising pathways, new techniques, and crucial research ideas. We also examine the advancements and prospects of ocular gene therapy, the progress and inadequacies, with potential solutions in this field of research. Targeting the genome of HSV-1 is a promising novel strategy in gene therapy field, and is the focus of this paper.