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The orthopoxviruses (family Poxviridae, genus Orthopoxvirus) are a diverse group of large, enveloped viruses that contain a covalently closed, double-stranded DNA genome of approximately 200 kbp. The genus is comprised of at least 10 recognized species. Several viruses within this group are significant human pathogens, including Variola virus (the causative agent of smallpox), monkeypox virus, cowpox virus, and Vaccinia virus. Other members, including raccoonpox, camelpox, ectromelia (mousepox), taterapox, and volepox viruses have only been isolated from their respective mammalian hosts. Although many orthopoxviruses specifically infect certain animal hosts, others (e.g., monkeypox and cowpox viruses) can also infect humans and are considered zoonotic pathogens. In humans, symptoms of orthopoxvirus infections range from mild skin lesions to fatal systemic disease. For example, smallpox produced a generalized rash that progressed from the papular to vesicular to pustular stages and resulted in a greater than 30% mortality rate in unvaccinated persons. Although naturally-occurring smallpox was eradicated nearly three decades ago, official stocks of the virus still remain in two locations, one at the U.S. Centers for Disease Control and Prevention in Atlanta, GA and the other at the State Research Center of Virology and Biotechnology, Novosibirsk, Russia. This, in addition to waning immunity against smallpox within the human population, has led to concerns that Variola virus might be used as a bioweapon.
Monkeypox virus causes a disease similar to smallpox in humans, but results in a lower fatality rate. Monkeypox virus is primarily transmitted to humans through direct contact with infected animals, generally various species of rodents or squirrels in the rain forests of central Africa. However, additional attention was brought to bear on this virus when, in the spring of 2003, it emerged for the first time in the Western Hemisphere and caused a cluster of cases in the U.S. Midwest.
Vaccinia virus is famous for being the vaccine that was used to eradicate smallpox. It was also the first animal virus to be purified and chemically analyzed and was the first to be genetically engineered. Despite its notoriety, however, its origin and natural history remain obscure. Recent evidence suggests that Vaccinia virus and horsepox virus are very similar phylogenetically and share a relatively recent common ancestor. Vaccinia virus is often confused with cowpox virus; although it is now well established that they are distinct virus species. In fact, cowpox virus is considered a zoonotic pathogen and is seen in a broad range of host species, most notable wild rodents, but rarely in cattle, as its name would imply. Interestingly, wild and domestic cats and elephants appear to be highly susceptible to infection with cowpox virus. Numerous recent studies have uncovered several novel vaccinia-like viruses that have caused zoonotic outbreaks in Brazil,,,,,,. It is likely that as various ecological niches are examined further, even more species of Orthopoxvirus will be identified.
Several genus-specific assays have been described for the detecting and discriminating various Orthopoxvirus members that require conventional PCR followed by restriction endonuclease digestion and subsequent gel electrophoresis,. More recently, several real-time LightCycler-based PCR assays have been developed for pan-Orthopoxvirus or specific orthopoxvirus species detection,,. For these assays, differentiation of the various orthopoxvirus species requires the use of different TaqMan probes in separate reactions or melt-curve analysis of hybridization probes. Here, we describe a rapid, high-throughput, multi-locus method for identifying orthopoxviruses based on PCR amplification followed by electrospray ionization mass spectrometry (PCR/ESI-MS) performed on the Ibis T5000 instrument,,,. This technology has been applied to detection and identification of other viral pathogens, including alphaviruses, influenza viruses, adenovirus, and coronaviruses. The assay described here is extremely sensitive and able to detect and identify each species from a diverse collection of orthopoxviruses.
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.
Influenza, also known as the flu, is a respiratory illness caused by viruses belonging to the family Orthomyxoviridae. This family consists of four influenza virus genera (influenza virus A, influenza virus B, influenza virus C, and influenza virus D) that are classified based on differences in their internal glycoproteins nucleoprotein (NP) and matrix (M). Influenza type A viruses can infect humans, birds, pigs, horses, and other animals, while influenza B and C viruses are found only in humans. Influenza viruses contain a single stranded negative sense RNA genome that encodes 11 proteins. Based on the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), influenza A viruses are divided into various subtypes. There are 18 HA (H1–H18) and 11 NA (N1–N11) subtypes of influenza A viruses, that potentially form 144 HA and NA combinations. Aquatic birds including ducks, geese, and swans, are considered to be the natural reservoir of these subtypes.
Each year influenza viruses, both influenza A and influenza B are responsible for seasonal epidemics accounting for over 200,000 hospitalizations and 30,000–50,000 deaths. As per World Health Organization (WHO) estimates, influenza viruses infect between 5%–15% of the global population, annually resulting in 250,000 to 500,000 deaths, making it the leading cause of mortality after acquired immune deficiency syndrome (AIDS). In addition to annual seasonal epidemics H1N1 and H3N2 viruses have also resulted in four major influenza pandemics: The “Spanish flu” in 1918, the “Asian flu” in 1958, the “Hong Kong flu” in 1968, and the more recent 2009 H1N1 pandemic. Since 1997, human infections with a novel H5N1 subtype of highly pathogenic avian influenza (HPAI) have been reported. The first cases of human infection with H5N1 influenza were reported in 1997, when HPAI outbreaks in poultry farms and markets in Hong Kong resulted in eighteen cases and six deaths. Since then, this virus has spread to many countries in Asia, Africa, and Europe, resulting in over 424 human cases with a mortality rate greater than 60%. In addition, H9N2 and H7N7 avian influenza subtypes have also been reported to cause human infections. The most recent strain infecting humans was H7N9 in China, detected in 2011.
A number of diagnostic techniques, including virus isolation, nucleic acid amplification test (NAAT), immunochromatography-based rapid diagnostic test (RDT), etc., have been used for detection of influenza viruses in humans. Here, we review various approaches currently available or under development for diagnosis of influenza infections in humans.
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).
Human bocavirus (HBoV) was discovered in 2005 by Allander et al. in respiratory samples from children with suspected acute respiratory tract infection (ARTI) using a novel technique. This molecular virus screening is based on a random PCR-cloning-sequencing approach and was employed on two chronologically distinct pools of nasopharyngal aspirates (NPAs). It revealed a parvovirus-like sequence, with close relation to the members of the bocavirus genus.
A retrospective study revealed 17 (3.1 %) out of 540 NPAs positive for HBoV, with 14 specimens tested negative for other viruses, giving the suggestion that HBoV is a causative agent of respiratory tract infections.
Recovery of influenza viruses in clinical samples by propagation in mammalian cells or embryonated eggs is the most traditional method for influenza diagnosis. Introduced in the 1940s, the viral culture approach is considered one of the gold standards for the diagnosis of viral infections. This approach involves inoculation of permissive cell lines or embryonated eggs with infectious samples, propagation for 7–10 days to monitor development of cytopathic effect, and final confirmation of influenza virus infection by specific antibody staining, hemadsorption using erythrocytes, or immunofluorescence microscopy. Influenza virus isolation using this approach is usually performed on established cell lines, such as Madin Darby canine kidney (MDCK), A549, mink lung epithelial cell line (Mv1Lu), rhesus monkey kidney (LLC MK2), and buffalo green monkey kidney (BGMK), or primary cell lines, such as rhesus monkey kidney (RhMK) or African green monkey kidney (AGMK). In some labs, viral culture is used in parallel with NAAT assays.
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).
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.
Rapid laboratory diagnosis is critical for infection control, so several diagnostic tests have been developed and are available for the detection of influenza viruses. Rapid influenza diagnostic tests are less sensitive and specific than fluorescent antibody assays and RT-PCR. RT-PCR is the preferred diagnostic assay for influenza virus. These tests are the most sensitive and specific and can differentiate between influenza types (A or B) and subtypes. The main problem of this technique is that it could not be available in all laboratories, so there is a need of other tests in these settings.
Real-time PCR is much more sensitive than other methods of detection and is available for detecting influenza virus but is more expensive.
With respect to parainfluenza viruses, types 1 to 4 have been associated with bronchiolitis, croup and pneumonia in children, but also in elderly and immunocompromised patients. PCR is an adequate technique for the detection of parainfluenza virus, above all in immunocompromised patients. PCR has a sensitivity of 100% and specificity that range 95-98% if compared with culture method. Multiplex PCR assays can differentiate between a wide variety of respiratory pathogens. Also, a rapid and sensitive multiplex real-time PCR assay for detection of four serotypes of parainfluenza viruses has been developed.
For more information, it can see the article entitled “Laboratory detection of respiratory viruses by automated techniques”.
HBoV is a putative member of the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. Before identification of HBoV, parvovirus B19 of the genus Erythrovirus was the only known human pathogen in the family of parvoviruses. Parvovirus B19 is widespread and manifestations of infection vary with the immunologic and hematologic status of the host. In immunocompetent children, parvovirus B19 is the cause for erythema infectiosum. In adults it has been associated with spontaneous abortion, non-immune hydrops fetalis, acute symmetric polyarthropathy, as well as several auto-immune diseases.
Based on its genomic structure and amino acid sequence similarity shared with the namesake members of the genus, bovine parvovirus (BPV) and canine minute virus (MVC), HBoV was classified as a bocavirus and therefore provisionally named human bocavirus.
Other subfamily Parvovirinae members known to infect humans are the apathogenic adeno-associated viruses of the genus Dependovirus and parvovirus 4. Parvovirus 4 has not yet been assigned to a genus, but it was proposed to allocate it to the genus Hokovirus as it shares more similarities to the novel porcine and bovine hokoviruses than with other parvoviruses. Recently a second human bocavirus has been identified, HBoV2, with 75.6 % nucleotide similarity to HBoV. HBoV2 was found in stool samples from Pakistani children as well as in samples from Edinburgh (1 of the 3 positive samples was derived from a patient >65 years old), indicating that it is not restricted to one region or to young children.
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.
Viruses are infective obligate parasites that can replicate only in the living cells of animals, plants, fungi, or bacteria. Although extremely small in size and simple in structure, viruses cause numerous diseases such as cancer, autoimmune disease, and immunodeficiency as well as organ-specific infectious diseases including the common cold, influenza, diarrhea, hepatitis, etc.,,,.
Recent progress in the formulation of antiviral therapies and vaccines has helped to prevent, shorten the duration, or decrease the severity of viral infection,,. Most antiviral agents are designed to target viral components, but mutations in the viral genome often result in drug resistance and immune evasion, creating a major hurdle for antiviral therapies and vaccine development. In addition, the continuous emergence of new infectious agents such as the Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) necessitate the advancement of novel therapeutic approaches. Accordingly, great attention has recently been drawn to the development of antivirals with broad-spectrum efficacy and immunomodulators which improve host resilience by increasing host resistance to the viral infection.
Korean ginseng (the root of Panax ginseng Meyer) is one of the most popular medicinal plants used in traditional medicine in East Asian countries including Korea. Ginseng contains various pharmacologically active substances such as ginsenosides, polysaccharides, polyacetylenes, phytosterols, and essential oils, and among those, ginsenosides are considered the major bioactive compounds. Korean Red Ginseng (KRG) is a heat-processed ginseng which is prepared by the repeated process of steaming and air-drying fresh ginseng. KRG has been shown to possess enhanced pharmacological activities and stability compared with fresh ginseng because of changes in its chemical constituents such as ginsenosides Rg2, Rg3 Rh1, and Rh2, which occur during the steaming process.
Currently, numerous studies have reported the beneficial effects of KRG on diverse diseases such as cancer, immune system disorder, neuronal disease, and cardiovascular disease,,,. In addition, KRG and its purified components have also been shown to possess protective activities against microbial infections. In this review, we summarize the current knowledge on the effects of KRG and its components on infections with human pathogenic viruses and discuss the therapeutic potential of KRG as an antiviral and vaccine adjuvant.
Both viruses were recovered in cell cultures in 1971. BK virus is associated with nephropathy, above all in renal transplant patients as well as in patients with ureteral stenosis and hematuria. On the other hand, it is well knowing the association of JC virus infection with progressive multifocal leukoencephalopathy in immunocompromised patients (such as those with AIDS).
Conventional PCR for detection of JC virus in the cerebrospinal fluid has replaced the brain biopsy for the diagnosis of presence of this virus in patients with leukoencephalopathy. This technique had sensitivity that range 70-90% and a specificity that range 90-100% before the highly active antiretroviral therapy. However, at the moment, the application of this therapy recovery the immune system, so the viral replication becomes to be decreased, being JC virus PCR negative in CSF. Currently, it has been developed a qualitative real-time PCR for JC virus detection in CSF.
BK virus can be detected in urine samples by conventional PCR; also, a quantitative real-time PCR technique has been developed to monitoring BK virus DNA in renal transplant recipients. Active BK virus nephropathy is associated with high quantitative levels of BK virus DNA, and resolution of nephropathy was correlated with decreased DNA virus level in urine. However, although it is a sensitive test, the presence of BK virus DNA in this kind of samples does not necessarily means a true infection because there is asymptomatic reactived infection in 10-45% of renal transplant patients. Therefore, the result should be confirmed using blood samples. In this sense, research has demonstrated that renal transplant recipients with higher urine DNA levels are more likely to show detectable DNA in blood. On the other hand, a negative result does mean no association of BK virus with nephritis.
Finally, PCR from kidney biopsy specimens is not an appropriate test for primary diagnosis of BK nephropathy, because persistent but low-level target DNA in biopsy specimens can be detected in asymptomatic patients.
As shown in Figure 2, all of the four primer pairs produce amplicon that can distinguish Variola virus from any other orthopoxvirus. Primer pair VIR979 could resolve Variola major virus from Variola minor virus and camelpox strain CMS from strain M-96. Primer pairs VIR982, VIR985 and VIR988 could resolve the two strains of monkeypox tested: VR-267 and Zaire-96-1-16. All of the vaccinia isolates tested including rabbitpox and horsepox, which are sub-species of vaccinia, have a common base count signature for primer pair VIR9888 ( 34A, 16G, 19C, 30T). Vaccinia, Copenhagen strain and horsepox (gi|111184167) can be distinguished from each other and other vaccinia isolates based upon their unique base-count signatures for primers pairs VIR985 and VIR979.
In addition to providing sub-species resolution, the use of four PCR primers in the assay enabled the detection and identification of an orthopoxvirus at the stochastic limit of PCR: 4–8 copies/PCR reaction. For example if only 4–8 genomes/PCR are used in the assay there is a very high probability that at least one of the four primers would detect the virus and provide information sufficient for its identification.
The assay is specific to Orthopoxvirus. Nucleic acid extracts from the blood of non-infected rabbits (N = 4) and humans (20 ng and 500 ng/PCR reaction) failed to produce an amplicon other than the internal positive controls. As expected, swinepox, a suipoxvirus (ATCC VR-363), in the family Poxviridae failed produce an amplicon other than the internal positive controls using the assay. We further tested a panel of DNA viruses in the assay to further define specificity including HSV1, adenovirus types 1, 5, 8, 4, 7A, varicella zoster virus (VZV), HPV16 & 18, human parvo virus B19, BK virus and JC virus. None of these viruses cross-reacted using the assay.
More than 70% of the emerging infectious disease agents are caused by microbes jumping from animals into human. This has been well exemplified by the highly fatal human infection due to avian influenza A H5N1 in 1997. The outbreak of severe acute respiratory syndrome (SARS) caused by a novel coronavirus in 2003, confirmed again that microbes can jump species from animals to humans with unpredictable consequence. The human SARS coronavirus was traced to caged civets in the market, and later Chinese horseshoe bat, Rhinolophus sinicus, was suggested to be a likely reservoir of SARS coronavirus. Bats are ideal incubators for new emerging infectious agents as they are mammals which roosted together and can fly over vast geographical distance. This has reignited the interest in seeking for new bat viruses including many bat coronaviruses and the recent discovery of bat influenza virus. Besides the SARS coronavirus, viruses in bats often infect human through intermediate hosts such as horses for Hendra virus, pigs for Nipah virus, and chimpanzees for Ebola virus. It is therefore important to catalogue as comprehensively as possible the animal viruses present in wild life especially the bats and birds, the food animals such as pigs and cattles, the pet animals such as cats and dogs, and monkeys which are phylogenetically close to humans. Using consensus primer polymerase chain reaction (PCR) screening, we have been able to discover relatively closely related species of virus in many different animals,–. However more distant or novel families of virus can only be found by metagnenomics using deep sequencing with the newer generation sequencers,. We report in this paper the discovery and characterization of a novel bat papillomavirus (PV) from rectal swab samples randomly collected from asymptomatic wild, food and pet animals using a metagenomic approach.
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].
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.
Currently, there are about 15 known zoonotic marine mammal pathogens (reviewed in). For instance, Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, was introduced to the Americas via pinnipeds. In addition, Influenza A virus, which poses a global human threat, is present in cetacean and pinniped populations and has been shown to be transmitted from seals to humans [8–10]. Since aquatic mammals are phylogenetically our closest sea relatives they serve as sentinel species for both human and ocean-related health. Thus identifying pathogens in marine mammals may help assuage disease outbreaks and prevent zoonotic transmission.
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.
Rotavirus is the leading cause of acute gastroenteritis in young children age ≤ 5 years. Two live oral rotavirus vaccines (Rotarix by GlaxoSmithKline, Unitied Kingdom, and RotaTeq by Merck, United States) are available, and the implementation of rotavirus vaccines in childhood immunization programs has significantly reduced the morbidity and mortality associated with Rotavirus infection. Nevertheless, there is no antiviral drug to treat rotavirus infection, and mostly, therapeutics involve the prevention of dehydration,.
In traditional medicine, ginseng has been known to improve gastrointestinal function and prevent gastrointestinal problems such as diarrhea. A recent study researched the active constituent in ginseng and reported that two pectic polysaccharides isolated from hot water extract of ginseng prevented cell death from viral infection. The polysaccharides, named GP50-dHR and GP50-her, did not have virucidal effects but inhibited viral attachment to the host cells thereby protecting them from virus-induced cell death. Given these results and an additional report that other pectin-type polysaccharides in ginseng inhibited the adherence of Helicobacter pylori to gastric epithelial cells and the ability of Porphyromonas gingivalis to agglutinate erythrocytes, further evaluation of the antimicrobial effects of acidic polysaccharides with the structure of pectin is merited.
Xenotransplantation is being developed to overcome the shortage of human tissues and organs needed to treat organ failure by allotransplantation. Pigs are the preferred species to be used as donor animals for a number of reasons including the size of the animals, their similar physiology, and the ease with which they can be genetically modified and cloned.
Although there are still some hurdles that have to be overcome, such as immunological rejection, physiological incompatibility and risk of transmission of porcine microorganisms, recent achievements in breeding genetically modified pigs, in the development of new immunosuppressive regimens and in approaches to safety suggest that xenotransplantation may soon be introduced in the clinic (for review see [1–3]).
In the past, only a few porcine viruses with known or suspected ability to infect human cells or to accelerate transplant rejection have been analysed within the context of xenotransplantation. Next generation sequencing or deep sequencing techniques and metagenomic analyses now give the opportunity to analyse the entire virome of pigs and its impact on xenotransplantation. These studies on the pig virome are, like investigations into the virome of humans and other species, only at their very early stages. The development of metagenomics has revolutionised virus discovery, leading to the identification of many new viruses. These studies will generate an enormous amount of data concerning the prevalence of porcine viruses although these will be difficult to interpret, especially with regard to the virus safety of xenotransplantation.
In this study, we examined the virus diversity in serum samples from Nicaraguan children with unknown acute febrile illness. We performed Virochip microarray and deep sequencing analyses on 7 positive control and 123 undiagnosed samples. Both of these methods succeeded in detecting the expected virus in the positive control samples. Virochip analysis produced putative viral hits in 10/123 (8%) of the previously negative samples, whereas deep sequencing revealed virus or virus-like sequences in 45/123 (37%). This study demonstrates the utility of these metagenomic strategies to detect virus sequence in multiple human serum samples and is the first to utilize second-generation sequencing to simultaneously investigate many cases of acute unknown tropical illness.
Monitoring the emergence and spread of novel human pathogens in tropical regions is a central public health concern. Metagenomic analysis enables more systemic viral detection of both known and novel viral pathogens and can be employed as diagnostic supplements to pathogen detection as part of public health monitoring systems and epidemiologic surveys–,–,,,. Despite the headway, metagenomic virus detection studies will have to confront several remaining difficulties concerning diagnostic accuracy. Foremost concerns include enhancing the sensitivity and specificity of deep sequencing-based diagnostic methods and re-evaluating the evidence for disease causality in light of increasingly sensitive nucleic acid detection and pathogen discovery methods. The former will require improved strategies to biochemically enrich and computationally identify viral sequences while reducing host background sequences. The latter will require a cautious reconsideration of criteria used to establish causal links between microbes and disease, as well as extensive case-by-case follow-up studies employing classical laboratory methods, such as serological analysis and cell culture amplification. It is important to highlight that observing viral sequence in sequencing data is insufficient to establish the role of a virus in disease causality. Like other detection strategies, deep sequencing will serve to inform secondary tests, including seroconversion assays, further nucleic acid testing, cell culture amplification, and additional investigations into plausible disease mechanisms.
We detected virus sequence at concentrations as low as ∼2 in 106 reads. Virus sequence detected in a clinical sample at vanishingly low copy numbers may reflect several possible host-microbe scenarios. The sequence detected may be that of a pathogenic virus capable of causing illness at low copy number or through indirect effects, a ubiquitous non-disease causing microbe, a virus outside of its primary replication site, low-level contamination, an artifact of sample collection timing/processing, or remains of incomplete immune clearance. Additional evidence must be considered in each case to define the host-microbe relationship.
In this study, we compared the performance of the Virochip and deep sequencing for detecting virus sequence in human serum. The limit of detection of the Virochip was approximately one part in 105 for the poliovirus controls, for which there are microarray probes with perfect sequence complementarity (Figure S1). The sensitivity of deep sequencing is limited by the number of reads generated per sample, or read depth. In this study, we detected virus sequences down to two parts per million. Nearly every virus that was detected on the microarray was also detected by deep sequencing; additionally, in numerous samples (n = 44), sequencing revealed viruses not detected by the Virochip (Table 1). There were two instances where Virochip analysis identified a virus (TTV) that was not detected by deep sequencing (Table 1). Deep sequencing, therefore, is a superior method for novel virus discovery, because it is more sensitive and provides more conclusive genotypic information than the Virochip. Nevertheless, the Virochip is a relatively fast and inexpensive method that is best applied to samples with expected virus copy numbers present at levels greater than 1 in 105 host sequences.
We were unable to detect a virus in two thirds of the 123 dengue-like illness samples. These results could reflect true negative status, which would result from a non-viral infection, illness due to non-infectious agent, or complete immunologic clearance. Alternatively, the negative results could reflect failures in our diagnostic approaches due to imperfect sensitivity, unsatisfactory sample preparation, improper sample type, or failure to recognize highly divergent viral sequences. The presence of sequences that lack even remote similarities to known species also highlights the need for further development of de novo assembly methods for metagenomic data. Assembled data, increased depth, and enhanced sequenced comparison methods should enable more sensitive detection of divergent viruses in metagenomic samples.
Determining the etiology of human diseases with symptoms that overlap with dengue-like illness is important for understanding the full spectrum of emerging or previously uncharacterized pathogens in tropical populations. In this study, 10% of acute serum samples negative for dengue virus from cases of pediatric dengue-like illness were positive for HHV-6. Primary HHV-6 infection causes undifferentiated febrile illness and exanthem subitum (roseola infantum or sixth disease), an acute illness with high fever and rash that typically resolves in three to seven days. Exanthem subitum is a common disease of infants worldwide, and HHV-6 infection most frequently occurs between 6 and 12 months of age, with seropositivity estimates of >95% in adult populations in developed countries. The HHV-6 positive patients in this study were between 7–12 months old, and presented with fever and rash (Table S3). We detected multiple kilobases of HHV-6 sequence in each positive sample, with sequence deriving from multiple viral genomic regions (Figure 2).
After acute infection, HHV-6 can latently persist in the host quiescently, with no production of infectious virions or with low levels of viral replication. Latency is believed to endure in several cell types, including monocytes and bone marrow progenitor cells,, and may undergo chromosomal integration that can be vertically transmitted. The confounding effects of chromosomal integration make differentiating between active and latent HHV-6 infections difficult when detecting HHV-6 sequence in serum DNA,. A previous study detected integrated HHV-6 genomic sequence in ∼1% of healthy blood samples. Since detection of HHV-6 nucleic acid in serum alone does not prove active viral infection, we cannot definitively confirm that the HHV-6 sequences in these samples were not derived from the vertical transmission of chromosomally integrated virus. However, the clinical, epidemiological, and virus sequence data suggest HHV-6 may be the etiologic agent in these febrile illness cases.
Primary HHV-6 infection is a major cause (∼20%) of infant hospitalizations in the United States, a clinical burden likely shared throughout the tropical world given similar seroprevalence rates. The results of this study illustrate the importance of administering HHV-6 diagnostic tests to cases of suspected dengue-like illness in infants from dengue-endemic regions to differentiate between cases of exanthem subitum, a ubiquitous self-limiting childhood illness, and dengue fever, which carries a greater risk of severe clinical complications and death.
Similarly, the one sample positive for Parvovirus B19 sequence may be a case of acute infection with a commonly acquired childhood virus. Parvovirus B19 can manifest as erythema infectiosum (fifth disease), a condition associated with characteristic “slapped cheek” rash. Infection can also be subclinical or result in mild nonspecific symptoms. It is possible that Parvovirus B19 infection caused the symptoms in this case (Table S3), though as with HHV-6, the identification of viral sequences does not definitively demonstrate causality.
Epstein Barr Virus (HHV-4) sequences were found in the serum of one patient who presented with relatively severe symptoms, and died during hospitalization (Table S3). HHV-4 infection is a nearly universal occurrence in the first two decades of life,. Primary infection in adolescents or adults can manifest as infectious mononucleosis, and chronic infection is associated with various malignancies later in life. Primary infection during childhood, however, is usually asymptomatic or produces only mild symptoms. It is not clear that HHV-4 infection or HHV-4 alone caused the illness in this case.
In addition to the viruses for which a plausible disease association exists, many samples contained sequences from viruses with no well-established link to human disease. These included the two samples positive for GBV-C and those containing ASFV-like, TTV-like, and circovirus-like sequences.
The Circoviridae family is an extraordinarily diverse group of small, single-stranded circular DNA viruses that includes cycloviruses (genus Cyclovirus) and circoviruses (genus Circovirus), which are commonly detected in human stool and blood, and also in environmental samples–. Some circovirus species, such as beak and feather disease virus and porcine circovirus 2, have been associated with disease in bird and pig hosts, respectively, but the pathogenic potential of circoviruses in humans remains unconfirmed,. The circovirus-like sequences reported here were detected in nucleic acid libraries prepared from acute human serum and were most closely related to circovirus-like viruses (Figure 3), which were first reported in environmental samples and in bats,. We were unsuccessful in recovering a full genome sequence corresponding to any of the circovirus-like sequences, and it has not yet been possible to prove that these sequences were not an environmental artifact introduced during sample preparation. It is also possible that these sequences derive from other organisms, such as Giardia intestinalis or Entamoeba dispar, whose genomes encode proteins that share amino acid similarity with circovirus replicase proteins (Figure 3). Furthermore, it has yet to be established whether circoviruses are capable of replicating in humans. Pending additional screening and serologic studies, the detection of circovirus-like sequences from human serum should be interpreted with caution.
Metagenomic approaches provide an effective high-throughput method to detect uncharacterized virus diversity in a tropical setting from many samples simultaneously. The findings presented in this study further our knowledge of well-characterized and previously unknown viruses present in serum collected from pediatric dengue-like illness patients and advance our understanding of the application of metagenomic approaches to human pathogen detection. Deep sequencing analysis of clinical samples holds tremendous promise as a diagnostic tool by permitting the detection of many different viruses simultaneously, including those present at low-copy numbers and of divergent origin. Major remaining barriers to high-throughput sequencing strategies becoming standard diagnostic practice include prohibitive cost, lengthy sample preparation time, and computationally intensive data analysis requirements. These challenges are magnified in resource-limited settings, such as Nicaragua, but are gradually being addressed. Industry hardware and technical advancements have steadily decreased the per-base cost of deep sequencing, and the results presented here strengthen our expectations of multiplexed sample preparation and bioinformatic data filtering within the framework of current second-generation sequencing platforms. Long-term bi-directional partnerships with developing country collaborators facilitate easier access to techniques not currently available on-site, such as deep sequencing, and are also important in providing training opportunities for local scientists and developing relevant pathogen tests and diagnostic policies.
This study expands our understanding of the virus diversity in pediatric dengue-like illness in Nicaragua and the application of genomic detection techniques in a tropical setting, findings that are particularly valuable given the pressing need for improved global emerging pathogen surveillance.
Food safety and its related issues are attracting interest worldwide because they are closely related to human lives and health conditions. Pathogens from the environment may contaminate food and food products, thus foodborne pathogens and their detection are directly related to human life and safety. Foodborne viruses, among all other pathogens, are relatively new, gaining more attention due to their emerging contamination events and the small scale of outbreaks.
Global public food safety issues have been increasing in recent years. Foodborne disease outbreaks related to fresh produce have been increasing in North America and European Union 1. In the United States, norovirus is the main pathogen, responsible for 59%, followed by Salmonella, which is responsible for 18% of foodborne diseases related to fresh produce. In European Union, norovirus is responsible for 53%, followed by Salmonella, which is responsible for 20% of foodborne diseases related to fresh produce. In Canada, Salmonella is the main pathogen, responsible for 50%, and hepatitis A virus is responsible for 0.1% of foodborne diseases from fresh produce 2. Although viruses are not the major pathogen in Canadian fresh produce, they are prevalent in farm-level infection such as hepatitis E virus (34%), porcine enteric calicivirus (20%), and rotavirus (7%) in finisher pigs 3. These viruses are hypothesised to infect humans zoonotically through swine and pork exposure.