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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)
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
75. The clinical diagnoses most commonly associated with HMPV are bronchiolitis and pneumonia
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.
Zika virus (ZIKV) is a recently emerged mosquito-borne virus, which in 2016 was declared as an international public health emergency by the World Health Organization (WHO, http://www.who.int/csr/en/). ZIKV is a member of the Flavivirus genus that belongs to the Flaviviridae family and is closely related to other mosquitoes-transmitted flaviviruses of public health relevance such as Dengue virus (DENV), Yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV). ZIKV was first isolated in 1947 of a sentinel rhesus monkey in the Zika forest of Uganda and has been associated with sporadic human cases detected across Africa and Asia, resembling a mild version of DENV or Chikungunya virus (CHIKV). These similarities with DENV and CHIKV has interfered with ZIKV diagnosis and most probably underestimated the number of cases for ZIKV infections. Symptomatic disease generally is present with a mild febrile illness characterized by fever, rash, muscle pain, headache and conjunctivitis, although as up to 80% of the ZIKV cases are asymptomatic. However, the outbreak in the island of Yap in 2007, French Polynesia in 2013–2014 and the massive epidemic that emerge in Brazil in 2015 have caused major concerns due to the association of ZIKV infection with severe congenital abnormalities, including microcephaly in infants and an increased risk of Guillain-Barré syndrome in adults. ZIKV is mainly transmitted to people through the bite of an infected Aedes spp. mosquito (Ae. Aegypti and Ae. Albopictus), which carries a high risk for pregnant woman due to the ability to cross the placenta and infected fetal nervous tissues. In addition to maternal-fetal transmission, ZIKV can also be transmitted from mother to child during pregnancy or spread through sexual contact, breastfeeding, blood transfusion and non-human primate bites.
Approximately two thirds of emerging infectious diseases (EIDs) that affect humans originate from bats, rodents, birds, and other wildlife [1–3]. In many of these reservoir host species, emerging viruses appear to be well adapted, with little or no evidence of clinical disease. However, when these viruses spill over into humans, the effects can sometimes be devastating [4–6]. Previously, our limited knowledge of the viral population and ecological diversity harbored by wildlife have complicated the study of EIDs. Thus, comprehensive understanding of the viral community present in wildlife, as well as the prevalence, genetic diversity, and geographical distribution of these viruses, could be valuable for prevention and control of wildlife-origin EIDs.
The order Rodentia is the largest mammalian order, with 33 families and 2,277 species (~ 43% of all mammal species). They live in close contact with humans and their domestic animals and act as a bond between humans, domestic animals, arthropod vectors (ticks, mites, fleas), and other wildlife [8–10]. This interface with humans has led to the rodent origin of important zoonotic viruses including members of the family Arenaviridae, Hantaviridae, Reoviridae, Togaviridae, Picornaviridae, and Flaviviridae [11–18]. Many of these viruses cause severe disease in humans (e.g., Lassa virus; tick-borne encephalitis virus, TBEV; lymphocytic choriomeningitis virus, LCMV; Sin Nombre virus; Hantaan virus, HTNV; Seoul virus, SEOV; and Puumala virus); have only recently been discovered (e.g., Whitewater Arroyo virus and Lujo virus); or appear to have a wider geographical range than originally thought (e.g., Junin virus, Guanarito virus, Machupo virus, and Sabia virus), suggesting that further viral discovery studies in wild rodent populations may be valuable for public health [8, 11–13, 15, 19–25]. Recent reports of rodent viruses have enabled new hypotheses regarding the evolution of hepaciviruses and the origin of coronaviruses (CoVs) and picornaviruses (PicoVs) such as hepatitis A virus [26–29].
China is a megadiversity country and harbors ~ 200 rodent species from 12 families. To develop baseline data on the origin of existing viral EIDs and identify other potential zoonotic viral reservoir hosts, we have conducted a series of viral surveys from rodents, bats, and other small animals and have simultaneously constructed online viral databases of these animals (DBatVir and DRodVir, http://www.mgc.ac.cn/) since 2010 [31–34]. In the current study, 3,055 small mammal individuals of 55 species from the orders Rodentia, Lagomorpha, and Soricomorpha across China were sampled by pharyngeal and anal swabbing. Virome analysis was then conducted to outline the viral spectrum within these samples. On the basis of virome data, we describe the community, genetics, evolution, and ecological distribution characteristics of viruses and determined whether these features change with their host species and locations. The identification of novel mammal viruses provides new clues in the search for the origin or evolution pattern of human or animal pathogens such as hantaviruses (HVs), arenavirus (AreVs), CoVs, and arteriviruses (ArteVs).
host response to viral infection
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).
On infection of a new host, the genomes of many viruses undergo rapid adaptive evolution, which may result in escape from host immune responses [1–8] and increases in viral growth rates. Although these genetic changes make viruses superior competitors within their current host, they do not necessarily favour improved transmission between hosts. A logical consequence of this process is ‘short-sighted’ evolution (see Glossary), by which adaptation at the within-host level occurs at the expense of the spread of the virus through the host population.
Susceptibility to short-sighted evolution will be influenced by two factors: the rate of viral adaptive evolution and the time between transmission events, which we refer to here as the ‘transmission interval’. For instance, acute viral infections, such as influenza and norovirus, are typically short-lived with little time for within-host adaptation before transmission to a new host. Their strategy is one of ‘smash and grab’: infect a new host, reproduce, and get out before the adaptive immune system removes the infection. Such viruses have short transmission intervals and will exhibit little short-sighted evolution, irrespective of their rate of adaptive evolution. Alternatively, persistent viral infections that use proof-reading polymerases, such as Herpes and Papilloma viruses, are unlikely to suffer short-sighted evolution for a different reason; their low mutation rates constrain the rate of host-specific viral adaptation, regardless of their transmission interval (Figure 1). It is common for these viruses to have larger genomes with many genes, which enable the virus to manipulate or hide from host immune responses, for example by persisting in a nonproliferative latent state.
In contrast, short-sighted evolution could be problematic for persistent chronic viral infections that use low-fidelity polymerases and which undergo active replication throughout infection, such as human immunodeficiency virus (HIV-1) and hepatitis C virus (HCV). High rates of mutation during replication, large viral population sizes, and long durations of infection combine to create considerable potential for within-host adaptation, enabling these viruses to outpace natural and induced immune responses. However, long transmission intervals mean that this adaptation may come at a cost of reduced transmissibility later in infection (Figure 1).
Chronic viral infections are clearly successful within their natural hosts, so how do those with long transmission intervals avoid the detrimental impacts of short-sighted evolution? Here we suggest that such viruses exhibit life histories that either (i) significantly reduce rates of within-host adaptation, or (ii) lead to the retention of a genetic archive of viruses that are similar to the founder strains that initiated the infection. This archive is analogous to the germline in multicellular animals, which does not carry somatic mutations that accumulate during the lifetime of an individual. We further speculate that mechanisms that limit the effects of short-sighted evolution in chronic viruses could themselves be under viral control and therefore subject to selection (Box 1).
To begin, we outline the evidence that within-host adaption can reduce viral transmissibility. We then discuss mechanisms by which viruses may avoid short-sighted evolution, before examining the evidence that, in rapidly evolving viruses, germline lineages are preferentially transmitted. Because they have been much more widely studied, most of the examples we use come from viruses that infect humans, but the general principles apply to all viruses.
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.
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).
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.
Mammalian genomes transcribe many short and long non-protein-coding RNAs (ncRNAs), but whether these RNAs play a role in the host response to virus infection remains an enigma. It is known that some small RNAs, such as microRNAs (miRNAs) (1), are involved in virus-host interactions. For example, in vitro studies have shown that the liver-specific miR-122 is required for hepatitis C virus (HCV) RNA replication (2). Distinct expression profiles of cellular miRNAs enabled researchers to differentiate infection by the lethal 1918 pandemic influenza virus from nonlethal seasonal influenza virus A/Texas/36/91 infection (3). It was also reported that HIV-1 virus is able to suppress expression of the polycistronic miRNA cluster miR-17/92 to enable efficient viral replication (4). However, changes in expression of other small RNAs during virus infection have not been systematically studied.
Using next-generation deep-sequencing technology, we recently discovered widespread differential expression of host long ncRNAs in response to virus infection (5), but the experimental protocol used was not designed to capture small RNAs (6). In this study, we used deep-sequencing technology to perform a complementary small RNA transcriptome analysis of the same severe acute respiratory syndrome coronavirus (SARS-CoV)-infected lung samples collected from four mouse strains as previously reported. In addition, lung samples collected from the same four influenza virus-infected mouse strains were included in the new analysis. Our results show that many known miRNAs responded differently to the two virus infections and that many of them were also differentially regulated during lethal influenza virus infection, as shown in a previous study (3). We also discovered many non-miRNA small RNAs and unannotated small RNAs that were differentially expressed during infection. The integration of transcriptome sequencing analysis of long transcripts and small RNAs showed the intricate interactions of short and long RNAs during virus infection. The changes in miRNAs positively correlated with the changes in long transcripts cotranscribing from the same locus, indicating that the miRNAs were transcriptionally regulated during virus infection. We predicted that differentially expressed miRNAs could target the majority of differentially expressed long transcripts during virus infection.
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.
Viruses are obligate intracellular pathogens that require host cells in order to replicate and produce infectious progeny. Virus entry into host cells is followed by capsid uncoating, genome transcription and replication, synthesis of viral proteins, assembly of progeny virions, and egress. For most viruses, genome replication and assembly take place in specialized intracellular compartments known as viral factories or inclusions, which are often composed of membranous scaffolds, viral and cellular factors, and mitochondria. Viral inclusions (VIs) serve multiple purposes during infection, including the concentration of viral and host factors to ensure the high efficiency of replication, sequestration of viral nucleic acids and proteins from innate immune responses, and the spatial coordination of consecutive replication cycle steps. Most double-stranded RNA (dsRNA) viruses form cytoplasmic inclusions with a characteristic morphology. These neoorganelles constitute sites of genome replication and virion assembly, and contain abundant viral RNA and proteins.
The combination of ultrastructural and functional studies has enhanced our knowledge about VI biogenesis. However, for many viruses, it is still not known how these structures form and mediate functions in viral replication. Here, we describe the current understanding of the morphogenesis and function of reovirus inclusions and compare these neoorganelles with the replication factories formed by other members of the Reoviridae family.
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.
Pharyngeal and anal swabs were collected from 3,055 individual small mammals captured from July 2013 to July 2016 in 20 provinces across China (Fig. 1a and Additional file 1: Table S1). These comprised 50 rodent species of the families Muridae, Cricetidae, Sciuridae, Dipodidae, Chinchillidae, and Gliridae; two lagomorphs of the family Ochotonidae; and three soricomorphs of the family Soricidae that reside in urban, rural, and wild areas throughout China. The most common species sampled were Apodemus agrarius, Niviventer confucianus, Rattus norvegicus, Rattus tanezumi, Rattus losea, and Sorex araneus. Due to repeated sampling of some species in the same location, swabs were combined into 110 pools for analysis.
Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic life cycle. It is the prototypical member of the orthopoxviridae genus of the Poxviridae family which includes Variola virus (the causative agent of smallpox), Monkeypox virus and Ectromelia virus. VACV was used as a vaccine in the successful global eradication of smallpox in the 20th century and closely related attenuated strains such as Modified Vaccinia virus Ankara (MVA) are now some of the most frequently used recombinant vaccine vectors against a variety of human and animal diseases including HIV, malaria and tuberculosis. Understanding the VACV life cycle is therefore important since it provides the base for the development of efficient and safe novel vaccines.
VACV, like all other viruses, harnesses the cell to enable its replication. It turns off or subverts multiple crucial anti-viral pathways including cytokine production, Toll-like receptor pathways, NF-κB activation and the dsRNA PKR response–. In addition VACV suppresses both intrinsic and extrinsic pro-apoptotic pathways and activates numerous anti-apoptotic, pro-survival pathways including the PI3K/Akt pathway,, the MEK/ERK pathway,, the p38 MAPK pathway and the MAPK/JNK pathway,. Modulation of so many different signalling pathways prevents viral-induced premature cell death and contributes to the ability of poxviruses to replicate in a wide range of cell types.
To investigate this complex pathogen-host relationship further, a RNAi screen of druggable host targets was carried out to analyse the effect of cellular protein depletion on VACV replication, using a multi-cycle VACV infection assay that monitors all stages of virus replication including virus spread. The screen identified a range of previously identified HFs, but also novel HFs and pathways influencing VACV infection that may facilitate the development of broadly effective anti-viral strategies and the optimisation of poxviral-based vaccine vectors.
Bats, comprising the second largest mammalian population in the world and distributed globally with the exception of the two polar areas, belong to the order Chiroptera with 17 families and 925 species. Bats are important virus reservoir animals and more than 60 viruses have been identified in them with many highly pathogenic to humans, including henipaviruses, Ebola virus, Marburg virus, dengue virus, lyssaviruses and SARS-like coronavirus–. Most recently, Bokeloh and Shimoni bat viruses, circovirus, bocavirus, retrovirus, astrovirus, and Cedar virus have been identified as new bat viruses with some never having been reported in other animals, suggesting that bats could be a large virus bank and breeding ground for viruses,–. In China, viruses are increasingly being detected in, or isolated from bats, such as coronavirus, circovirus, astrovirus, Xi River virus, Japanese encephalitis virus (JEV), Chikungunya virus, Tuhoko virus, picornavirus, adeno-associated virus and adenovirus,,,–. Notably, SARS coronavirus, which has infected more than 8,000 people and killed almost 800 worldwide, has been identified as likely originating from horseshoe bats in China,,. However, all available studies so far fail to provide a complete understanding of the pathogen ecology of bat populations.
To control the outbreaks of emerging or re-emerging viral diseases and prevent the transmission of viruses from wildlife, particularly bats, to humans, monitoring the virus infection situation in natural hosts and vector animals is important. Availability of next generation sequencing-based viral metagenomics in recent years has provided a powerful tool for large-scale detection of known and unknown viruses existing in host animals,. This new technology has been employed to explore the constitution of viral communities in such environments as oceans, lakes, various tissues, guts and feces of animals including bats has undoubtedly opened a new window to an understanding of the virus diversity in these environments and has provided a successful paradigm for future rapid discovery of new viruses in nature,–.
The region covering Southeast Asia and Southern China is a main epicenter of emerging or re-emerging viral diseases due to its high human population, inadequately developed public health systems, and abundant and diverse wild animal resources with their illegal trading. Yunnan province in China is a main and busy trading route between Southeast Asia and China and shares a long border with Myanmar, Laos and Vietnam. Studies have shown that many viruses, including Nipah virus, JEV, Chikungunya virus, and circovirus, are present in bats in Cambodia, Thailand and Yunnan,,,,–. To expand these studies to Myanmar, we applied viral metagenomics to determine the virome of bats collected from areas of Myanmar adjoining Yunnan. Results revealed 24 virus families in these bats, including phages and viruses of plants and insects as well as vertebrates. Several viruses of the genera Orthohepadnavirus, Mastadenovirus, Mamastrovirus, and Bocavirus, have been characterized as new viruses. This work has expanded our knowledge of bat viruses and their geodistribution in Southeast Asia and could be helpful in establishing effective surveillance of wildlife-associate zoonoses.
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).
Zika virus (ZIKV), a member of the Flaviviridae family, became a global public concern because of the correlation of Zika virus epidemic with fetal developmental defects, including highly publicized cases of microcephaly1. The viral genome is made of a positive sense, single-stranded RNA molecule (~10.8 kb) that contains a single open reading frame flanked by 5′ and 3′ untranslated regions2–4. The viral RNA is translated as a single polyprotein that is co- and post-translationally processed by viral and cellular proteases into three major structural proteins (capsid, pre-membrane and envelope) involved in viral entry, fusion and assembly5, and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) that are involved in viral RNA replication and transcription, assembly and evasion of the host antiviral responses6–9.
Although the majority of ZIKV infections are asymptomatic or associated with a mild febrile illness, infection during pregnancy has been associated with miscarriage and severe congenital malformations, including fetal microcephaly10,11, and in adults with the Guillain-Barré syndrome10,12,13. ZIKV is primary transmitted to humans through the bite of infected mosquitoes11. However, non-vector ZIKV transmission can occur through sexual contact14,15 or vertical transmission from infected mothers16,17. Currently, there are no approved vaccines or antivirals to prevent or treat ZIKV infection.
The establishment of plasmid-based reverse genetic systems for RNA viruses entails the rescue of recombinant viruses from cDNA clones containing the entire viral genome on a plasmid from transfected culture cells18. In 1981, Racaniello and Baltimore provided the basis of this approach with poliovirus19. Since then, numerous reverse genetics systems have been developed for positive- and negative-stranded RNA viruses4,18,20–24. Plasmid-based reverse genetic approaches allow the rescue of infectious recombinant viruses by the transfection of one or more plasmids encoding the components necessary for the de novo generation of infectious virus particles into cultured cells. Importantly, reverse genetic techniques are powerful platforms to modify the viral genome and they have provided critical insights into replication and pathogenesis of multiple viruses18. Importantly, viral reverse genetic approaches have also been used as platforms to develop novel and more effective live-attenuated vaccines (LAVs) against viral infections and have significantly contributed to the development of antiviral treatments18,20. However, there are intrinsic limitations in the in vitro recovery of recombinant viruses using reverse genetic approaches, mostly associated with cell culture-adaptive mutations that might restrict and/or change the phenotype of the virus in vivo25,26. In the case of LAVs, these mutations could lead to the reversion to a virulent phenotype27,28. Likewise, the current need of first generating recombinant viruses in cultured cells rather than directly in validated animal models of infection delays studies aimed to understand and assess viral infections in vivo. Therefore, it is of high significance to develop approaches that allow the recovery in vivo of infectious recombinant virus from cDNA infectious clones without the current need of a tissue culture step. In vivo reverse genetics or recover of virus directly from validated animal models could overcome the concern of virus adaptation to cell cultures and facilitate and simplify the study of the virus and the development of LAVs based on attenuated forms of these viruses.
Recently, we have described a reverse genetic approach for ZIKV based on the use of a bacterial artificial chromosome (BAC), by assembling the full-length genome of ZIKV Rio Grande do Norte Natal (RGN)27,29 or Paraiba/2015 strains30 under the control of the cytomegalovirus (CMV) immediate-early promoter. Importantly, we demonstrated the feasibility of using this infectious clone to generate a virulent rZIKV with similar in vitro and in vivo characteristics to the natural Paraiba/2015 isolate30. Using the BAC infectious cDNA clone of Paraiba/2015 strain (pBAC-ZIKV), we have explored the possibility to recover infectious rZIKV directly in vivo in the type-I interferon (IFN) receptor deficient (IFNAR−/−) A129 mouse model of ZIKV infection31–33. The pBAC-ZIKV cDNA clone was complexed with Lipofectamine 2000 (pBAC-ZIKV/LPF) and inoculated in IFNAR−/− A129 mice using different inoculation routes, which are typically used for ZIKV infections. Recovery of rZIKV in vivo with high efficiency was observed in mice. In addition, infectious rZIKV was also recovered by direct inoculation of the infectious cDNA clone in the absence of transfection reagent. More Importantly, we demonstrate the feasibility of using this in vivo rescue approach with a BAC cDNA infectious clone encoding an attenuated rZIKV (rZIKVatt)30 that resulted in sterilizing immunity against aggressive ZIKV challenge. In vivo recovery of fully infectious and/or attenuated RNA viruses expands the potential use of reverse genetic systems and open the possibility of developing similar approaches for other viruses, which could be an innovative technology for their study or the future development of LAVs.
Most human‐infective viruses are RNA viruses, 94% of which harbour a single‐stranded RNA (ssRNA) genome.1 These include established pathogens such as HIV and dengue virus (DenV), most high‐profile emerging pathogens this decade [e.g. Zika virus (ZikV), SARS‐coronavirus (SARS‐CoV) and avian influenza], re‐emerging pathogens including measles virus (MV) and every pathogen prioritised in the recent WHO R&D Blueprint.2 Furthermore, climate change‐related factors are likely to drive changes in future dispersion or transmission of viruses including mosquito‐borne viruses such as DenV and ZikV.3 The disease burden associated with many of the 214 human‐infective RNA virus species is large and growing, yet only five have US Food and Drug Administration (FDA)‐approved antivirals available and nearly all target virus proteins (Table 1).
While virus‐oriented approaches are efficacious, the genetic diversity of viruses often restricts such treatments to particular species or serotypes (Table 1). Furthermore, these antivirals are often costly and are ultimately susceptible to escape mutant selection. Simple point substitutions are often responsible for treatment failure,4, 5 while fitness costs associated with harbouring these substitutions may be trivially absorbed by the escaped strain upon accumulating compensatory adaptations.6 Tenofovir is an example of a highly effective single‐regimen treatment for chronic hepatitis B infection, a retro‐transcribing virus characterised by considerable genetic heterogeneity, by simultaneously imposing potent viral suppression, a high barrier for escape and reduced replicative fitness of escape strains. Despite these synergising effects, complex escape mutants harbouring multiple point substitutions in the viral reverse transcriptase have recently emerged.7 One way of enhancing treatment efficacy while minimising viral escape is to deploy existing antivirals as combination therapies, a strategy used extensively in current HIV (e.g. tenofovir/emtricitabine) and hepatitis C virus (HCV) treatment regimens.4, 5 While increasing the number of combinations increases the height of the escape barrier, proportional increases in treatment costs, adverse effects and counterindications make this strategy one of ever compounding challenges that ultimately remains exposed to the core problem of viral resistance. Treatment failure and the continuous need for the development of additional therapies are the realised costs of playing into such ‘strengths’ of virus evolution.
As obligate intracellular parasites, all viruses must subvert key resources of permissive hosts in order to replicate.8 Subverting multifunctional host proteins can confer significant fitness advantages by enabling RNA viruses to efficiently execute multiple steps in their replication strategy. Over time, these features are likely to be conserved within lineages and serve as foci of evolutionary convergence for viruses with a similar host range, while purifying selection eliminates steps rendered less efficient. Nevertheless, ideal targets of pathogenic viruses include those that are also vital to the host, thereby limiting its options for antiviral adaptation and driving more costly evolutionary innovation on its part. Similarly, the potential for adverse effects limits options for targeting such host proteins therapeutically.
Therapeutic drug availability, together with recent advances in areas including immunotherapy and precision medicine, is beginning to alleviate such constraints on host‐oriented approaches. Significantly, many of these technologies arose through examining evolutionarily diverse host–virus and immune interactions, which are being increasingly uncovered with the advent of mass next‐generation genome sequencing and machine learning‐assisted metagenomic analysis technologies. Furthermore, such interactions are increasingly found to perform crucial roles throughout our biosphere.9, 10, 11 As was once the case for the CRISPR/Cas bacterial immune system proteins now used in genome editing,12 these host–virus interactions often employ unique proteins of unknown function.10, 13, 14 This review examines how host–virus evolution may be leveraged towards solving disease and sustainability issues of our time. Multitasking or multifunctional host proteins as antiviral therapeutic targets, methods for targeting such proteins, vaccine design and neo‐virology as an emerging source of biotechnological innovation, will be discussed.
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.
Viruses are divided into two similar-sized groups depending on whether the virus particle contains DNA or RNA, and, as causes of human fatality, RNA viruses are by far the more important (1). New viral diseases continue to appear as a result of several changes in human activity: travel, population growth, interaction with wild habitats etc. Well-known novel, or emergent, RNA diseases include severe acute respiratory syndrome (SARS) (2), human immunodeficiency virus 1 (HIV-1) (3), and may come to include Avian influenza H5N1 virus (4). These emergent diseases are an important factor behind the increase in the number of genome sequences that NCBI treats as representing new species (Figure 1). In 2005, more than 200 new virus species were submitted to GenBank (more recent dates are less reliable because there is typically a delay between submission and public availability). As more emergent viruses appear, it is important to have a site that allows their genomes to be compared to those of known viruses. The origin of most major infectious diseases is unknown because of our ignorance of the diversity of pathogens in wild animals. This restricts our ability to both predict risks and develop treatments (5).
Despite some advances (6,7), the evolutionary history of RNA viruses is in general poorly known, especially the deep phylogenetic relationships between virus families (8,9). We believe that one of the reasons for this is a lack of easily available translated genes and genomes for all species, and the lack of aligned genome sequences representing different isolates of the same species.
In addition to the need to facilitate greater comparative analysis of RNA viruses is the need to link together the existing virus Web sites and their underlying databases. There are many Web sites that provide genomic data, tools for genetic analysis and/or biological information for some viruses (see ‘Links’ on our site home page). The RNA Virus Database is intended to complement these other sites by providing basic genomic information and tools for all RNA viruses and linking the user to more specialist sites, where they exist, e.g. for HIV-1 and hepatitis C virus (HCV), we provide links to sites such as the Los Alamos Laboratory on the main page for each of these viruses (find by typing HIV-1 or HCV into the search window in the top toolbar). For such viruses, we do not duplicate the work of other groups by attempting to display the available diversity of genomes. We intend that the RNA Virus Database should develop further as a hub for other sites and we therefore encourage other workers to contact us with details of their sites that they wish linked to ours. Also, we encourage workers to ‘adopt a virus’ and improve and/or expand the information that we provide for individual species. This can be done by emailing us or getting involved directly in developing the database, which is an Open Source project available at our GoogleCode site (http://code.google.com/p/rnavirusdb).
Some of the data and tools on the RNA Virus Database can be found elsewhere, but not all of them can, e.g. NCBI's Genome site provides genomic overviews of virus species and pairwise alignments of other isolates to the reference sequence, but it does not provide multiple alignments or complete translated genomes as we do. Similarly, its general Entrez site provides pair-wise alignments of the query sequence and similar sequences in the database, plus a phylogenetic tree calculated from those distances; however, no multiple alignment is built. We also corrected the (few) errors in the GenBank entries, and our database records features such as RNA editing (10) that make genome translation problematic.
We have, therefore, created the RNA Virus Database as a user-friendly site devoted to RNA viruses, providing essential genomic data and tools (discussed in more detail below) and links to the other virus Web sites. The three main features are as follows.
Provide multiple whole-genome alignments, gene and whole-genome translations for all RNA virus speciesIdentification and taxonomic searching facilityGuidance to other web resources.
Influenza A (IAV) and B (IBV) viruses belong to the Orthomyxoviridae family of enveloped viruses. IAV is able to infect several species and mostly exists in the wild aquatic fowl reservoir. On the other hand, IBV is mainly restricted and adapted to humans, although sporadic infections of seals have been documented.
IAV and IBV genomes contain eight negative sense, single-stranded viral (v)RNA segments (Figure 1). IAV and IBV vRNAs contain a central coding region that is flanked at both terminal ends by non-coding regions (NCRs), which serve as promoters to initiate genome replication and gene transcription by the viral polymerase complex. Influenza vRNAs in the virion are found as viral ribonucleoprotein (vRNP) complexes encapsidated by the viral nucleoprotein (NP) and a single copy of the viral polymerase complex. Influenza virus-encoded RNA-dependent RNA polymerase (RdRp) is a trimeric complex consisting of the polymerase basic 1 (PB1) and 2 (PB2) and acidic (PA) proteins and, together with the viral NP, are the minimal components involved in viral replication and transcription.
IAV and IBV share many features, but they differ in their host range, virion structure, genomic organization and glycan binding specificities. Despite having similar genomes encoding homologous proteins, IAV and IBV are distinguished by the different lengths of proteins and non-coding regions (NCRs) that serve as promoters for genome replication and gene transcription (Figure 1). Likewise, they can also be distinguished by the presence of accessory proteins encoded from overlapping open reading frames (ORFs) and by the antigenic differences of internal proteins (Figure 1A,B). For instance, IAV and IBV both encode ion channel proteins from the gene M segment 7, M2 and BM2, respectively. The M2 and BM2 proteins of IAV or IBV are encoded together with the matrix protein 1 (M1) and both are incorporated into virions and expressed on the surface of virus-infected cells. However, the M2 protein of IAV is translated from a spliced mRNA, while the IBV BM2 protein is translated using a different strategy, where the initiation codon of BM2 protein overlaps the termination codon of M1 protein (UAAUG, a stop-start pentanucleotide). In addition, IBV expresses the NB ion channel, which is absent in type A influenza virus (Figure 1B). However, both influenza viruses encode two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (Figure 1A,B). IAV and IBV HA proteins are involved in binding to cellular receptors and responsible for the fusion of the viral and endosomal membranes. Infection with IAV or IBV induces a protective immunity mediated, at least partially, by antibodies directed against the viral HA, which is the main immunogenic target in both natural infections and vaccine approaches. Influenza NA glycoprotein is responsible for the cleavage of sialic acid moieties from sialyloligosaccharides and facilitates the release of newly produced virions from infected cells. IAVs are classified on the basis of the antigenic properties of HA and NA into 18 HA (H1–H18) and 11 NA (N1–N11) subtypes. However, only IAV H1N1 and H3N2 subtypes are currently circulating in humans. On the other hand, two major lineages of IBV are circulating in humans, the Victoria-like and Yamagata-like subtypes that are divergent from the ancestral IBV (B/Lee/1940) and have been co-circulating in humans since the 1980s. These two subtypes are the predominant circulating virus strains about once every three years.