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Ebolavirus is part of the Filoviridae family, which consists of three genera: Marbugvirus, Cuevavirus, and Ebolavirus. There are currently six known, genetically distinct, species of Ebolavirus—Ebola virus (EBOV), Sudan Ebolaviurs (SUDV), Tai Forest Ebolavirus (TAFV), Bundibugyo Ebolavirus (BDBV), Reston Ebolavirus (RESTV), and Bombali Ebolavirus (BOMV). No virus has triggered fear in the general population more than the filovirus Ebolavirus. EBOV is categorized among the deadliest viruses, with mortality rates up to 90%. The zoonotic origin of outbreaks are often the result of transmission from primates, although the suspected natural reservoir for EBOV, bats, is still being questioned. Since it was first identified in 1976 in Zaire (the actual Democratic Republic of Congo), 27 confirmed outbreaks, mainly in the central part of Africa, have occurred, and each outbreak was accompanied by high case fatality rates up to 88%, including the new declared outbreak ongoing in the North Kivu Province of the Democratic Republic of the Congo. The 2013–2016 Ebola outbreak is the largest (both by number of cases and geographical extension) ebolavirus outbreak ever reported, resulting in 28,610 cases and 11,308 deaths, with fatality rates of 70% in Guinea and Sierra Leone and 41% in Liberia. The number of cases in this single outbreak is far greater than the total number of all cases and deaths of the past outbreaks over the last 40 years. The reasons of such an extended outbreak are linked to societal factors (poverty, urbanization, population migration patterns, and changes of socio-economic conditions), together with the concomitant invasion of animal habitats, climate change, and deforestation. In fact, the emergence and re-emergence of such viruses in Africa or their potential introduction into new countries have usually been related to the mobility and international transport of infected animals or animal products, thus making ebolavirus and filoviruses a worldwide public health concern. Moreover, despite almost 40 years of research, filovirus transmission remains incompletely understood. In humans, EBOV has been found in a variety of body fluids, including blood, stool, breast milk, semen, urine, and saliva. There are multiple routes of transmission for EBOV. However, information about transmission in humans is incomplete, and defining the modes of transmission would greatly increase the ability of public health structures to limit the disease, as well as enable health care workers to avoid any unnecessary risk. So far, our understanding of EBOV transmission in humans mainly relies on epidemiological observations and contact with body fluids from EBOV-positive patients remain the most likely route of transmission. Notably, the number of past outbreaks and associated epidemiological studies carefully examining transmission patterns are small.
Ebola Virus Disease (EVD) is commonly associated with multiple organ systems, including the liver, renal organs, and lungs. So far, little is known about the involvement of the respiratory tract and EBOV pathogenesis in the lung. However, little evidence in filovirus animal outbreaks and animal studies highlights the involvement of the lungs and the respiratory tract in filovirus pathology. Over the years, there has been an increasing concern regarding the possible involvement of the lung in EBOV infection. This concern further increased during the 2013–2016 EBOV outbreak, which offered evidence of viral shedding in the lung, leading to a risk of aerosol transmission. The aim of this review is to highlight the pulmonary involvement in EVD, with a special focus on the new data emerging from the 2013–2016 Ebola outbreak.
About 70% of microbial agents causing outbreaks of emerging infectious diseases in humans originate directly from animals. Among respiratory virus infections, the influenza A viruses H5N1 and H7N9 from avian species, and the severe acute respiratory syndrome coronavirus from bats have caused large epidemics–. Atypical bacterial pathogens causing community-acquired pneumonia include Chlamydophila psittaci from psittacine birds and Coxiella burnetti from livestock and other animals. However, human outbreaks due to zoonotic bacteria associated with the emergence of a novel animal virus in the animal host were not previously documented.
In November 2012, an outbreak of human psittacosis affecting six staff members occurred at the New Territories North Animal Management Centre (NTNAMC) in Hong Kong. The human outbreak was preceded by an outbreak of avian chlamydiosis among the detained Mealy Parrots (Amazona farinose). Although birds in the tropical and sub-tropical areas are commonly infected with C. psittaci, most infected birds are asymptomatic,. Large human outbreaks are rare even among bird handlers. Although co-infection of C. psittaci and viruses has been reported in outbreaks of avian species–, no virus-bacterium co-infection of implicated avian species has ever been reported in outbreaks of human psittacosis. In this study, we sought to investigate viruses that cause avian co-infection, which may have led to this outbreak of psittacosis.
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).
A case was defined as a staff member working at the NTNAMC who was hospitalized for respiratory tract infection between November 1 and November 30, 2012, and confirmed to have C. psittaci infection by polymerase chain reaction (PCR) and/or a four-fold rise in serum microimmunofluorescent antibody titer against C. psittaci (Focus Diagnostics, Cypress, California, USA).
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.
Following on from the discovery of tobacco mosaic virus in 1892 and foot-and-mouth disease virus in 1898, the first ‘filterable agent’ to be discovered in humans was yellow fever virus in 1901. New species of human virus are still being identified, at a rate of three or four per year (see below), and viruses make up over two-thirds of all new human pathogens, a highly significant over-representation given that most human pathogen species are bacteria, fungi or helminths. These new viruses differ wildly in their importance, ranging from the rare and mild illness due to Menangle virus to the devastating public health impact of HIV-1.
In this paper, we take an ecological approach to studying the diversity of human viruses (defined as viruses for which there is evidence of natural infection of humans). First, we describe and analyse temporal, geographical and taxonomic patterns in the discovery of human viruses (§2). We then consider the processes by which new human viruses emerge (§3). There are a number of definitions of ‘emergence’; here, we are interested in all stages of the process by which a virus shifts from not infecting humans at all to becoming a major human pathogen. As experiences with HIV-1 and new variants of influenza A (and also with novel animal pathogens such as canine parvovirus) show, this shift can occur rapidly, over time scales of decades, years or even months.
Of course, not all newly identified human virus species are ‘new’ in the sense that they have only recently started to infect humans; many of them have been present in humans for a considerable time but have only recently been recognized (see for a more detailed discussion). Moreover, we recognize that ‘species’ itself is an imprecise designation, especially for viruses such as influenza A where different serotypes can have very different epidemiologies and health impacts. Indeed, the demarcation between genus, species complex, species and serotype (or other designations of sub-specific variation) can be somewhat arbitrary. Nonetheless, a study of currently recognized ‘species’ is a natural starting point for attempts to characterize and interpret patterns of virus diversity.
The evolution of emerging diseases is associated with factors embedded in the concept “host-agent-environment triangle” (1). To infect the host and cause disease, the pathogen needs to evade host defenses, which may occur through single point mutations, genome rearrangements, recombination and/or translocation (2). Genetic uniformity generated through genetic selection of the host (3) and the fact that demographic changes, intensification of farming, and international commerce have occurred markedly over the last decades, must be also considered as essential factors for the development of emerging diseases (4–6).
As well as in humans, emerging diseases drastically affect animal populations, especially food-producing animals. Livestock production in large communities (i.e., pig farms or poultry flocks) represents an excellent environment to facilitate the transmission and maintenance of huge viral populations, contributing to the pathogen evolution (through mutation, recombination and reassortment, followed by natural selection) (7–9). The intensification of livestock during the last four decades has probably been one of the main factors that contributed to the emergence of new pathogens and/or pathogen variants, leading to changes in the epidemiology and presentation of diseases (10).
The number of viral infectious diseases in swine has significantly increased in the last 30 years. Several important worldwide distributed viruses have been reported in this period, including Porcine reproductive and respiratory syndrome virus (PRRSV, family Arteriviridae), Porcine circovirus 2 (PCV-2, family Circoviridae) and Porcine epidemic diarrhea virus (PEDV, family Coronaviridae). In addition to those worldwide widespread viruses, an important number of novel swine pathogens causing different types of diseases has been described (11, 12). Although their economic impact might be variable, they are considered significant infection agents and their monitoring is nowadays performed in some parts of the world. Among others, relevant examples are Porcine deltacoronavirus (associated with diarrhea) (12), Senecavirus A (causing a vesicular disease and increased pre-weaning mortality) (11), Porcine sapelovirus (found in cases of polioencephalomyelitis) (13), Porcine orthoreovirus (assumed to cause diarrhea) (14), Atypical porcine pestivirus (cause of congenital tremors type II) (15) and HKU2-related coronavirus of bat origin (associated with a fatal swine acute diarrhea syndrome) (16).
Besides overt emerging diseases of swine, many other novel infectious agents have been detected in both healthy and diseased animals, and their importance is under discussion. This group of agents is mainly represented by Torque teno sus viruses, Porcine bocavirus, Porcine torovirus and Porcine kobuvirus, which are thought to cause subclinical infections with no defined impact on production (13, 17, 18). An exception may be represented by Hepatitis E virus (HEV); although it seems fairly innocuous for pigs, it is considered an important zoonotic agent (19, 20). Recently, a novel member of the Circoviridae family named Porcine circovirus 3 (PCV- 3), with unknown effects on pigs, has been discovered (21, 22).
Porcine circovirus 3 (PCV-3) was first described in 2015 in North Carolina (USA) in a farm that experienced increased mortality and a decrease in the conception rate (21). Sows presented clinical signs compatible with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. In order to identify the etiological pathogen, aborted fetuses and organs from the affected sows were collected for further analyses. Whilst histological results were consistent with PCV-2-systemic disease, both immunohistochemistry (IHC) and quantitative PCR (qPCR) methods to detect PCV-2 yielded negative results. Samples were also negative for PRRSV and Influenza A virus. Homogenized tissues from sows with PDNS-like lesions and three fetuses were tested through metagenomic analysis, revealing the presence of an uncharacterized virus (21). Further analyses using rolling circle amplification (RCA) followed by Sanger sequencing showed a circular genome of 2,000 nucleotides. Palinski et al. (21) also performed a brief retrospective study through qPCR on serum samples from animals clinically affected by PDNS-like lesions (but negative for PCV-2 by IHC) and pigs with porcine respiratory diseases. Results revealed PCV-3 qPCR positivity in 93.75 and 12.5% of the analyzed samples, respectively (21).
Interestingly, almost concomitantly, another research group from the USA reported a clinical picture pathologically characterized by multi-systemic and cardiac inflammation of unknown etiology in three pigs of different ages ranging between 3 and 9 week-old (22). Several tissues from these animals were tested by next-generation sequencing (NGS) methods and PCV-3 genome was found. Beyond NGS, in situ hybridization was performed in one out of these three pigs, confirming PCV-3 mRNA in the myocardium (cytoplasm of myocardiocytes and inflammatory cells mainly, although to a very low frequency).
Based on these two initial works, the name PCV-3 was proposed as the third species of circoviruses affecting pigs, since pairwise analysis demonstrated significant divergence with the existing PCVs. The novel sequences showed < 70% of identity in the predicted whole genome and capsid protein amino acid (aa) sequence compared to the other members of the Circovirus genus (22). Taking into account the economic importance and the well-known effects of PCV-2 on the swine industry, a new member of the same family like PCV-3 should not be neglected. Studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years, but the scientific community is still in its very beginning on the knowledge of this new infectious agent. Therefore, the objective of the present review is to update the current knowledge and forecast future trends on PCV-3.
Multisystemic inflammation and myocarditis were initially linked with the presence of PCV-3 (22). One single study described PCV-3 in weaned pigs that suffered from gastro-intestinal disorders (diarrhea), showing higher prevalence in pigs with clinical signs (17.14%, 6 out of 35) compared to those with non-diarrhea signs (2.86%; 1 out of 35) (87). In another report, animals with congenital tremors were analyzed and PCV-3 was the only pathogen found in the brain, with high amount of viral DNA (101).
More than 20 years after initial reports, porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a global swine industry problem with losses in the U.S. approaching $6 billion over the last decade. Belonging to the arteriviridae family in the order nidovirales, PRRSV is an enveloped RNA virus containing a single positive-strand RNA genome. The 15 kb viral RNA genome consists of seven open reading frames (ORF1-7). ORF1 comprises about 80% of the genome and encodes proteins with protease, replicase and regulatory functions. The smaller overlapping ORF2-7 encode five minor (GP2a, GP3, GP4, 5a and E proteins) and three major (GP5, M and N proteins) structural proteins. Several studies have shown that PRRSV possesses the capacity to subvert early innate immune responses in pigs by suppressing the production of antiviral cytokines, which also contributes to ineffective B- and T-cell responses. Superimposed on this suppressive activity is a high viral mutation rate, which has made the development of vaccines challenging. Modified live vaccines (MLV) used for control of PRRSV in the U.S. are based on only two virus isolates. Although MLV protect against some homologous field strains, their efficacy is not satisfactory due to failure to protect against infections of heterologous strains, as well as the potential risk for reversion to virulence. To develop successful vaccines against PRRSV infections, particularly those by heterologous strains, it is necessary to develop novel vector systems and to extensively categorize host factors critical in the virus-host interaction.
Several viral vectors, including those based on pseudorabies virus, poxvirus, adenovirus and transmissible gastroenteritis coronavirus (TGEV) have been used to express PRRSV structural proteins or host immune factors for developing anti-PRRSV immunity. For example, humoral immunity against PRRSV GP5 protein was detected in pigs immunized with fowlpoxvirus-coexpressing PRRSV GP5/GP3 and porcine IL-18, and in mice immunized with adenovirus-expressing GP5/GP3 fused with swine granulocyte-macrophage colony stimulating factor (GM-CSF). In this context, we and others have proposed to use PRRSV infectious cDNA clones or virus replicons as vectors for the expression of immune effectors that potentiate innate and adaptive immunity against a broad range of PRRSV isolates. Here we show that PRRSV infectious clones are effective vector systems to express exogenous antigens and host immune effectors. Specifically, we have constructed serial replication-competent viruses from a PRRSV infectious clone-based vector expressing indicator proteins and porcine type I interferons (IFNs). The indicator protein-expressing PRRS viruses efficiently produce progeny viruses and provide an efficient means for real-time monitoring of viral replication, thus allowing high-throughput elucidation of the role of host factors in PRRSV infection. In addition, the replication of some IFN-incorporated viruses is associated with the expression of active IFN peptides, which are capable of counteracting the subverted innate immune response and with potential to induce more effective adaptive immunity against PRRSV infection.
The order Nidovirales encompasses a diverse group of viruses that includes significant veterinary and human pathogens (1–6). These viruses cause a variety of diseases that range from mild enteric infection to severe respiratory disease or hemorrhagic fever (7, 8). Examples of disease-causing nidoviruses include the severe acute respiratory syndrome (SARS) coronavirus, a number of other coronaviruses that cause typically mild respiratory disease in humans, and agriculturally important animal pathogens, such as equine arteritis virus, porcine reproductive and respiratory syndrome virus, and yellow head virus. Nidoviruses are characterized by their overall genome architecture, distinct pattern of gene expression, and presence of a conserved set of functional domains in their nonstructural polyproteins. The nidoviruses cluster into five major groups, which have been taxonomically categorized into four families: Arteriviridae, Roniviridae, Mesoniviridae, and Coronaviridae. Viruses in the Coronaviridae family (subfamilies Torovirinae and Coronavirinae) have the largest known RNA genomes, an attribute thought possible because of a virally encoded proofreading exonuclease (ExoN) that increases replication fidelity (9–12). Although nidoviruses are known to infect mammals, birds, fish, and crustaceans, no nonavian reptile nidovirus has been previously described.
Ball pythons (Python regius) have become one of the most popular types of reptiles sold and kept as pets (13). Native to West Africa, these snakes make popular pets because of their relatively modest size (≤1.5 m), docile behavior, and ease of care. Selective captive breeding has resulted in a tremendous variety of colors and patterns (morphs), many of which command high prices. Since the late 1990s, veterinarians have been aware of respiratory tract disease as a common syndrome affecting ball pythons. This syndrome is often characterized by pharyngitis, sinusitis, stomatitis, tracheitis, and a proliferative interstitial pneumonia. The clinical and epidemiological characteristics suggested an infectious etiology.
In this study, we investigated the pathology and etiology of this disease. We obtained case samples from 7 collections around the United States, performed necropsies, and collected multiple tissues for light microscopy and samples of lung for transmission electron microscopy (TEM). Although TEM of the lung suggested a viral etiology, traditional molecular diagnostic methods did not identify an agent. Metagenomic sequencing was used to identify and assemble the genome of a novel virus in the order Nidovirales. Here we describe clinical and pathological manifestations of this disease, ultrastructural findings, tissue tropism, disease association, and subgenomic RNA expression and analyze the genome of this virus in the context of related viruses.
Next, all of the above findings were considered in conjunction with the literature data on the transmission ecology collated for each of the 36 viruses in Figure S1a. Pieced together on this basis was an outer- to inner-body line-up of viruses by organ system or combination of organ systems, guided by the one-to-four virus infiltration score, the corresponding virus organ system tropism, the matching virus transmission modes, length of the infection and shedding periods, infection severity level, and virus environmental survival rate, see Figure 3 and, also, Figure S1d.
The pathophysiological mechanism of pulmonary disease in patients with EVD is unknown. Notably, autopsies were performed on a limited number of humans (about 30 cases), primarily during the 1976 SUDV and 1995 EBOV EVD outbreaks and revealed interesting characteristics at microscopic level. During the first known SUDV outbreak, chest pain was almost universal (83% of patients), often accompanied by a dry cough. Autopsies were further performed on two patients and thickening of the alveolar walls due to proliferative accumulations of alveolar cells was found. Furthermore, a possible pathogenetic role of the virus in the respiratory tract was suggested by the fact that viral inclusions within alveolar macrophages and free viral particles within alveolar space were found in the lungs from fatal EVD cases who showed congestion, focal intra-alveolar edema, diffuse alveolar damage, and hemorrhaging..
One of the most common symptoms in EVD patients is a cough (up to 49%), especially during the progression of the disease, when viral loads in serum significantly increase, and the virus is copiously emitted in most body fluids, as well as in aerosol particles of various sizes. Among the reported EVD cases in the literature, respiratory symptoms were commonly reported with a wide range of symptoms, such as a cough (from 3% to 60%), dyspnoea or breathless (detected from 0% to 49%), and chest pain (from 7.5% to 98.6%). Moreover, a WHO study on the first 9 months of the epidemic in Western Africa found that nearly 30% (194 out of 665) of the patients experienced coughing and 2.4% (20 of 831) had a bloody cough. A study of 27 EBOV-positive patients of the 2013–2016 outbreak in Western Africa, treated in Europe and USA, reported that cough and dyspnoea were present at admission in seven (30%) and five (22%) EVD patients, respectively. At symptom onset, only a cough was reported in one patient. Furthermore, during hospitalization, 14 patients (52%) experienced hypoxemia while they were breathing ambient air, 12 patients (44%) had pulmonary oedema, seven patients (26%) had pneumonia, 39 patients (33%) had respiratory failure, and six patients (22%) had a diagnosis of acute respiratory distress syndrome (ARDS). Of these patients, four patients (15%) received non-invasive mechanical ventilation, and seven patients (26%) received invasive mechanical ventilation. Notably, the first EBOV-positive patient treated in Italy, mechanically ventilated for respiratory insufficiency for 5 days, had high levels of EBOV RNA in the lower respiratory tract secretions. The authors concluded that the absence of other identified respiratory pathogens in broncho-alveolar lavage fluids and aspirates supported the hypothesis of a direct contribution to the lung tissues damage by EBOV. Notably, EBOV RNA was detected in bronchial aspirate fluids when the EBOV RNA concentration in the concomitant blood samples was barely detectable. Furthermore, the blood EBOV RNA concentrations in the previous days were significantly lower than the concentrations detected in the bronchial aspirate samples. These findings suggest that this EBOV infection is unlikely a spillover from the blood compartment, eventually accompanied by delayed clearance. Instead, the most plausible explanation is that the virus actually replicated into the lower respiratory tract.
In the second EBOV-patient treated in Italy, our group investigated the presence of EBOV genetic material in the lungs and blood during the patient’s treatment and recovery. The patient showed a persistence of EBOV replication markers within the respiratory tract, with a prolonged detection of EBOV viral RNAs (negative and positive sense RNAs: neg-RNA and pos-RNA, respectively), known to be associated with EBOV replication, in the lower respiratory tract for up to five days after the EBOV viral load in blood was already undetectable. These results suggest that EBOV may replicate in the lungs, although it is possible that the lungs simply provided a protective environment that allowed RNA to linger longer than it did in the plasma. Nevertheless, the detection of pos-RNA together with neg-RNA in the sputum (until day 9 and 10 of the hospital stay, respectively) supports the concept of active viral replication within the respiratory tract, rather than plasma spill-over or prolonged RNA stability.
Overall, the pathophysiological mechanisms of pulmonary disease in patients with EVD are still uncertain, but there could be multiple contributing factors, including vascular leak from endothelial infection, cytokine dysregulation, or direct damage to EBOV-infected cells (Figure 2).
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.
For the epithelial, outer-body viruses it turned out that the length of the infection and shedding periods, as well as the virus environmental survival rate generally increased from respiratory tract to alimentary tract to skin. The respiratory viruses transmitted on the basis of aerosols, direct contact or fomites. Alimentary tract viruses were found to transmit on the basis of a fecal-oral cycle, through direct contact, contamination of feed and water, or involving fomites, persons and vehicles. Viruses infecting both respiratory and alimentary tract featured a mix of these transmission modes. Mostly, these viruses caused rather severe infections. Among the skin viruses, the more infiltrative viruses affecting all layers of the skin caused slowly healing lesions. The transmission of these deep-rooted skin viruses was found to rely on abrasion or biting flies rather than on direct touch or on indirect contact, more typical for superficial skin lesions. Some of the epithelial viruses are shed in feces over a prolonged time period, also in the absence of clinical signs, and these infections were considered to feature a systemic component. Next, the epithelial herpesviruses establishing latently in peripheral nerves and ganglia were found to cause a recurrence or persistence of the mucosal and/or skin infection, including of the distal urogenital tract and external genitalia.
PRRSV, a positive sense and single-stranded RNA virus, is a member of family Arteriviridae. Since it was emerged in the United States in 1987 and in Europe in 1990, PRRSV has rapidly spread in the swine producing regions and became one of the most important devastating diseases of swine worldwide. It can cause severe reproductive failure in sows and respiratory distress in young growing pigs. Infection with PRRSV also made pigs easy to secondary infection by other pathogens. Up to date, since there is no efficient method or drugs against PRRSV, it is very important and urgent to develop the effective therapeutic strategies to control PRRS.
The PRRSV genome has nine open reading frames (ORFs) composed of ORF1a, ORF1b, ORF2a, ORF2b, and ORF3-7. ORF1a and ORF1b could produce 16 nonstructural proteins (nsp1α, nsp1β, nsp2 etc.) [4–7]. Previous studies have shown that the nsp11 of equine arteritis virus(EAV), which is another member of family Arteriviridae, may play a key role in viral RNA synthesis and additional functions in the viral life cycle. Other and our previous work also demonstrated that PRRSV nsp11 inhibited the host innate immune responses such as the transcription of type I interferon, the RNAi innate immune response and the NLR family pyrin domain-containing 3 (NLRP3)-mediated production of IL-1β, which indicated that PRRSV nsp11 may play an important role in PRRSV infection. So the purpose of present study is to explore the effect of over-expression of nsp11 on PRRSV infection and whether the siRNAs targeting the PRRSV nsp11 could influence PRRSV infection.
Nidovirales is an order of enveloped, single-stranded positive genomic RNA viruses. They have the largest known viral RNA genomes and infect a broad range of hosts. The order of Nidovirales includes four virus families: Roniviridae, Arterividae, Mesoniviridae, and Coronaviridae (Figure 1). This classification is principally based on the organization of their viral genome, the closeness in genome sequences, the antigenic properties of the viral proteins, the replication strategy, the structure and physicochemical properties of the virions, the natural host range, the cell and tissue tropism, the pathogenicity, the cytopathology, and the mode of transmission. The name of Nidovirales, from the Latin word “nidus” for nest, refers to a nested set of viral subgenomic messenger RNAs that is produced during infection. Within the Coronaviridae family, the subfamily Coronavirinae is the one encompassing the larger number of viruses. Species in this subfamily, which include several human pathogens, can be grouped into four main subgroups on the basis of serological and genetic properties: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (Figure 1). Torovirinae is also a subfamily of Coronaviridae and four Torovirus species have been identified so far: the equine, bovine, porcine, and human Toroviruses (Figure 1). The Mesoniviridae subfamily has one genus, which contains one species, the Alphamesonivirus. Alphamesonivirus are mosquito-specific viruses with extensive geographic distribution and host range. Their virions are 60–80 nm in diameter, with club-shaped surface spikes and consist of eight major structural proteins, including a nucleocapsid protein, four differentially glycosylated forms of the membrane protein, and the spike S protein. Roniviridae contain the genus Okavirus and although still little is known about them, the yellow head virus (YHV) can cause significant economic losses to the shrimp industry and is listed as a notifiable disease by the World Organization for Animal Health. In recent years, veterinarians have also become very concerned about Arterividae, in particular the porcine reproductive and respiratory syndrome virus (PRRSV), which is causing economic losses to the USA swine industry that are estimated to US$560 million per year.
Nidoviruses rank among the most complex RNA viruses and their molecular genetics clearly discriminates them from other RNA virus orders. Still, our knowledge about their life cycle, mostly unveiled with studies on Coronaviruses (CoVs), is very limited. To enter cells, Nidoviruses bind to cell surface receptors, an event that precedes the fusion of the viral and cellular membranes (Figure 2, step 1), which is presumably mediated by one of the surface glycoproteins. The fusion takes place either at the plasma membrane or in the endosomes and releases the nucleocapsid into the host cell cytoplasm (Figure 2, step 1). After genomic RNA uncoating from the nucleocapsid, two large replicase open reading frames (ORFs), ORF1a and ORF1b, are translated by host ribosomes to yield two large polyprotein precursors that undergo autoproteolytic processing to eventually produce the non-structural (nsp) proteins. The nsp proteins interfere with the host defenses but also induce the formation of double-membrane vesicles (DMVs) and convoluted membranes, on which they collectively form the replication-transcription complexes (RTCs) (Figure 2, steps 2, 3, and 4). These complexes mediate the synthesis of the genomic RNA and a nested set of subgenomic RNAs that directs the translation of the structural proteins (the nucleocapsid N protein, the membrane M protein, the envelope E protein and the spike S protein) and some accessory proteins, like the hemagglutinin esterase in the case of Severe Acute Respiratory Syndrome (SARS)-CoV or Mouse Hepatitis Virus (MHV) (Figure 2, steps 5 and 6). Newly synthesized genomic RNAs associate with the cytoplasmic nucleocapsid proteins to generate the so-called ribonucleoprotein complexes. The viral structural envelope proteins are inserted into endoplasmic reticulum (ER) and targeted to the site of virus assembly, the ER, or the Golgi, where they interact with the ribonucleoprotein complex to initiate the budding of virus particles into the lumen of the membrane compartment (Figure 2, steps 7, 8 and 9). Newly formed virions then egress the host cell through secretion via the exocytic pathway (Figure 2, step 10).
Infectious viral diseases, both emerging, and re-emerging, pose a continuous health threat and disease burden to humans. In recent years, we have seen an increasing number of emerging virus disease outbreaks in human and animals alike with substantial health and economic impact. They are most likely related to accelerating environmental and anthropogenic changes, such as increased mobility and demographic changes, which alter the rate and nature of contact between animal and human populations. The influenza A viruses are probably the most notorious viruses which have shown their potential for repeated cross species transmission and pandemic potential (Claas et al., 1998; Koopmans, 2013; de Graaf and Fouchier, 2014; Bodewes et al., 2015). Schmallenberg virus caused an outbreak in ruminants with major impact on international trade of susceptible animals and animal products such as semen and embryos with more than 15 countries imposing restrictions on imports of live cattle from the European Union (EU) (Beer et al., 2013). Lately, Middle East Respiratory Syndrome (MERS) coronavirus was causing renewed concern as it spread from the Middle East to the Republic of Korea with 186 confirmed human cases, including 36 deaths in July 2015 (http://www.who.int/csr/don/21-july-2015-mers-korea/en/). Ebola virus has fruit bats (Pteripodidae) as natural hosts and the current epidemic is thought to be introduced into the human population by zoonotic transmission (Marí Saíz et al., 2014). Many of the most important human pathogens are either zoonotic or originated as zoonoses before adapting to humans (Taylor et al., 2001; Kuiken et al., 2005; Woolhouse and Gowtage-Sequeria, 2005; Cutler, 2010; Morse et al., 2012) and humanity is continuously being exposed to novel animal pathogens.
Breakthroughs in the field of metagenomics have had far-reaching effects on the identification and characterization of newly emerging viral pathogens (Fauci and Morens, 2012). Virus discovery metagenomics assays rely on sequence-independent amplification of nucleic acids from clinical samples, in combination with next-generation sequencing platforms and bioinformatics tools for sequence analysis. They are relatively simple and fast, and allow detection of hundreds of viruses simultaneously and unknown viruses even if they are highly divergent from those that are already described (Rosario and Breitbart, 2011; Miller et al., 2013; Smits and Osterhaus, 2013). If the new viral genome shows considerable similarity to previously characterized virus genomes present in public databases, the identification of a new virus and its genomic characterization can be finalized in a matter of days and a fraction of the costs compared to a few years ago. This is of utmost importance for timely disease outbreak management. Here, the guiding questions are: Is the group of diseased persons normal for the time of year and/or geographic area? If so, which pathogen(s) is causing the disease? Who gets infected? How do people get infected? What is the source of infection? What are transmission routes? How can infection be prevented, treated and/or contained? The fast discovery of a partial or full-length viral genome can also serve as basis for development of specific molecular diagnostic assays to confirm suspect cases and for development of vaccines and antivirals. This was exemplified after the discoveries of Schmallenberg virus and MERS Coronavirus, where molecular diagnostic protocols were made available within a matter of days after the discovery of the pathogen (Beer et al., 2013; Pollack et al., 2013).
Despite this promise, however, most new discoveries made through metagenomics in fact are viruses that belong to already known virus families as current data analysis strategies rely mostly on similarity searches against annotated sequences in public databases (Woyke et al., 2006; Chew and Holmes, 2009; Schmieder and Edwards, 2012; Garcia-Garcerà et al., 2013; Prachayangprecha et al., 2014; Schürch et al., 2014). Significant problems in characterization of full-length viral genomes from metagenomic datasets are encountered when dealing with a highly divergent new virus with no closely related genomes in public databases. Additional time-consuming experimental approaches are required to obtain full-length genome sequences (Van Leeuwen et al., 2010; Siegers et al., 2014). By optimally mining the metagenomic content, for example through effective assembly, k-mer frequency profiling, motif search, coverage profile binning, or other fragment linkage strategies, the likelihood, and speed of finding viral reads and the level of viral genome completeness can be increased. This also increases the number of reads for which a source can be assigned in metagenomes. An example was described by us recently (Smits et al., 2014) and showed that, using BLAST searches, 27.67, 5.82, and 0.11% of all reads were identified as being from the viral target genome (Figure 1), whereas after viral genome finishing, 69.52, 13.58, and 26.14% reads were tagged as belonging to the viral genome (Figure 1). At the same time, genome completeness increased from 7291, 7682, and 24,734 bases in the initial fragments, to full-length or nearly full-length genomes of 11, 15.5, and 33 kb (Figure 2). With the in silico methods described here, the need for laboratory follow-up can be minimized, thereby providing the necessary information in a timely, efficient, and more cost-effective manner.
Recovery of full-length genomes of novel viruses from metagenome data consists of four different steps: First, the assembly of the reads into long fragments, second, assignment of at least one contig (seed) as originating from the target virus, third, the linkage of other fragments to the seed contig to receive a draft genome and fourth, gap closing and finalizing of the draft genome to receive a full-length genome (Figure 3).
Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease for the swine industry worldwide. PRRS is characterized by reproductive failure in sows and respiratory disease in pigs of all ages. The annual loss associated with PRRS is approximately 664 million USD, as estimated by swine producers in the United States. PRRS virus (PRRSV) is a member of the Arteriviridae virus family in the order Nidovirales, along with equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). PRRSV is a small, enveloped virus with a single-stranded, nonsegmented, positive-sense RNA genome that is approximately 15 kb in length. The genome of PRRSV encodes at least ten open reading frames (ORFs) called ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5a, ORF5, ORF6, and ORF7. PRRSVs used to be grouped into European (type 1) and North American (type 2) genotypes, but currently classified as two species, betaarterivirus suid 1 and betaarterivirus suid 2, respectively. There is substantial genetic and antigenic variation among PRRSV strains, even within the same virus species. Since the first emergence of PRRS more than two decades ago, numerous efforts have been made to develop an effective control measure for this challenging disease. Currently, vaccination is the primary control method, and is widely used to control PRRS in pigs. However, the use of commercial vaccines with either modified live virus (MLV) or killed virus is inadequate to control PRRSV infection because of the highly divergent PRRSV strains in the field. Antiviral therapeutics could be an alternative or additional control measure to combat these viral infections when a successful vaccine that matches the circulating virus is not available. Therefore, pharmacological intervention might be a suitable and useful control strategy for PRRS. Many previous studies reported that a variety of natural compounds had been proven to exert antiviral activity against a number of viruses, such as equine herpesvirus 1 (EHV-1), hepatitis B virus, human cytomegalovirus (HCMV), and respiratory infections including the common cold and PRRSV. However, no such effective drugs are commercially available to prevent PRRSV infection. Chalcones (1,3-diaryl-2-propen-1-ones), which constitute a key class of natural products belonging to the Flavonoid family, were previously reported to have a wide range of biological activities, such as antiviral, antibacterial, antifungal, immunomodulatory, anti-inflammatory, antitumor, insect antifeedant, and antimutagenic activities. Thus, the present study aimed to evaluate the antiviral effects of DiNap [(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one], a synthetic analog of the chalcone flavokawain, on PRRSV replication in cell culture systems. A study was then carried out in a pig model to demonstrate the inhibitory effects of DiNap on PRRSV infection.
Lactate dehydrogenase-elevating virus (LDV) is a small, positive sense, single stranded RNA virus of the Arteriviridae family, related to coronaviruses such as the severe acute respiratory syndrome (SARS) virus,,,. It is a natural infectious agent of mice that causes very rapid lytic infections generally restricted to a minor subset of non-essential macrophages involved in scavenging extracellular lactate dehydrogenase,. The rapid loss of this subset results in the elevated lactate dehydrogenase levels for which the virus is named. Virus titers peak within the first day of infection as susceptible target cells are depleted, and then the infection is maintained at a much lower chronic level dependent on the replenishment of new macrophage targets. LDV establishes chronic infections regardless of mouse strain, age, sex or immune-status,,,. No clinical signs are typically associated with LDV infections, although co-infection with retroviruses can lead to CNS disease under certain circumstances,, and mice infected with LDV have suppressed immune responses,,,. We recently found that acute infection with LDV induced a state of partial and transient activation in the vast majority of splenic lymphocytes. Activation was characterized by high surface expression of the very early activation marker CD69. CD69 is the first surface marker known to be upregulated during the activation of lymphocytes and has recently been shown to interact with S1P1 to inhibit the egress of lymphocytes from lymphoid tissues. CD69 expression is upregulated by T cell receptor (TCR) ligation but is not dependent upon it and can be induced by inflammatory cytokines such as IFNα,.
Coronaviruses (CoVs) have a global distribution and infect a variety of human and animal hosts, causing illnesses that range from mostly upper respiratory tract infections in humans to gastrointestinal tract infections, encephalitis and demyelination in animals; and can be fatal. The International Committee for Taxonomy of Viruses (ICTV) reports four coronavirus genera, namely Alphacoronaviruses, Betacoronaviruses, Gammacoronaviruses and Deltacoronaviruses. CoVs are enveloped single-stranded, positive-sense RNA viruses with genomes ranging between 26.2–31.7 kb, the largest among known RNA viruses. This large, capped and polyadenylated genome contains seven common coronavirus genes in the following conserved order: 5'-ORF1a-ORF1b-S-ORF3-E-M-N-3'. ORF1a/b encompasses two-thirds of the genome and produces a genome-length mRNA (mRNA1) that encodes two overlapping viral replicase proteins in the form of polyproteins 1a (pp1a) and pp1ab.
These polyproteins are formed as a result of a -1 ribosomal frame shift that involves a complex pseudoknott RNA structure and are then proteolytically processed by virally encoded proteases into mature nonstructural proteins (nsp1 to nsp16), which assemble to form a membrane-associated viral replicase-transcriptase complex (RTC). The last third of the genome produces subgenomic (sg) mRNAs that encode the four structural proteins, spike (S), envelope (E), membrane (M), and nucleocapsid (N), as well as a number of accessory proteins.
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].
Coronaviruses (CoV) are enveloped viruses with plus-strand RNA genomes belonging to the family Coronaviridae, which together with the Roni- and the Arteriviridae, form the order Nidovirales. CoV infect a large range of birds and mammals, including humans, and their pathogenesis has been intensively studied since the 1960s. CoV generally cause respiratory and/or intestinal infections, although some may spread systemically. Human CoV (HCoV)-OC43 and HCoV-229E, for example, cause mild upper respiratory tract infections, although they are occasionally associated with severe pulmonary diseases in newborns and immuno-compromised people. In spring 2003, a new human CoV became infamously notorious due to an outbreak in South East Asia and Canada. The accused virus was rapidly identified as the SARS-CoV. Unlike HCoV-OC43 and -229E, the SARSCoV causes a severe respiratory disease, with nearly 10% mortality and it also spreads systemically. Since 2003, two additional new human CoV have been characterized, i.e., HCoV-NL63 and HCoV-HKU-1, which are also mainly associated with mild upper respiratory tract infections [8–10].
Several animal CoV are economically important pathogens. For example, transmissible gastroenteritis virus (TGEV) causes diarrhea in pigs [12–14], feline infectious peritonitis virus (FIPV) leads to a fatal systemic disease in cats [15–17], the bovine coronavirus (BCoV) causes respiratory tract diseases and diarrhea in cattle and the avian infectious bronchitis virus (IBV) is the etiological agent of severe respiratory tract and kidney diseases in chickens. The mouse hepatitis virus (MHV) has been extensively used to study the replication and assembly of CoV in cell culture models as well as in whole animals and is thus considered as a prototype CoV.
Fixed and frozen samples from affected snakes from collections in Wisconsin, Texas, Florida, Oklahoma, and Pennsylvania were collected between 2006 and 2013 (see Materials and Methods and Table 1). Necropsies were performed on 9 snakes with clinical signs of respiratory disease, and multiple tissues were collected for histopathology; samples of lung from two snakes were collected for TEM.
Lesions were evident in the upper and lower respiratory tracts of all diseased animals (Table 1 and Fig. 1). Four snakes had stomatitis/pharyngitis, with one having caseous material in the choanae (sinusitis) (Fig. 1A). Three snakes had pulmonary hemorrhage, three had either mucoid or caseous material in air passageways, and four snakes had moderately to severely thickened lungs (Fig. 1B). At a microscopic level, four snakes had mild to severe stomatitis (see Fig. S1A in the supplemental material). Eight of nine snakes had tracheitis, with multifocal mild to severe changes, including infiltrates of heterophils, macrophages, and lymphocytes in the lamina propria, and in four of these cases, there was hyperplasia in the tracheal epithelium (see Fig. S1B).
In comparison to the histology of normal snake lung, all nine snakes had a proliferative interstitial pneumonia of various severities (Table 1). In some cases, inflammatory cells were primarily around the terminal smooth muscle bundle of each septum that projected into the central lumen of the lung (Fig. 2A and B). Hyperplastic alveolar cells completely proliferated over capillary beds that are normally distributed at the surface of the primitive faveoli (Fig. 2C and D). Epithelial cells were tall and columnar with abundant apical mucus production (mucous metaplasia). Periodic acid-Schiff (PAS) and alcian blue staining were positive, consistent with the presence of mucopolysaccharides. Heterophils, lymphocytes, and in some cases macrophages infiltrated the interstitium of the pulmonary septa (Fig. 2D) and often were seen in air passageways along with sloughed epithelial cells. In deeper areas of the faveolar spaces, the lung was histologically normal. In more severe cases, the proliferative changes occurred diffusely throughout the lung and were seen from the proximal to distal surfaces lining faveolar structures.
Three snakes had mild to marked bronchial epithelial hyperplasia, with mild to moderate smooth muscle hypertrophy of the terminal muscle bundles. In these cases, the bronchus-associated lymphoid tissue was mildly to moderately hyperplastic. Sections of lungs of two cases were stained with Warthin-Starry stain and Brown-Hopps Gram stain; there was no positive staining for bacteria between cilia and microvilli of pneumocytes overlying muscle bundles. Three of five nasal cavities that were examined revealed mild subacute rhinitis/sinusitis. The vomeronasal organ in one snake was almost obliterated by foamy macrophages. Ziehl-Neelsen acid-fast staining was negative for acid-fast bacteria, and methenamine silver staining was negative for yeast and hyphae. Three cases had mild to moderate stomatitis/pharyngitis. The changes seen in the respiratory tract were consistent with a viral infection.
Of the 9 necropsied snakes, lesions were also observed in other areas of the body. These included mild nonsuppurative encephalitis characterized by periventricular perivascular lymphocytic cuffing (in 1 of 9 snakes examined), mild to moderate acute nephritis (1/9), mild nephrosis (1/9), mild to moderate multifocal subacute dermatitis (1/9), mild acute salpingitis (1/9), and hepatic lipidosis (5/9). In the eye of one snake, there was moderate necrotizing conjunctivitis, with diffuse corneal ulceration, mild superficial keratitis, and mild inflammation and necrosis of the inner layer of the spectacle. One snake had mild bacterial colitis. It is unclear whether these other lesions were related to the observed lung pathology.
The synthesis of the flavokawain analog DiNap is presented in Scheme 1, which began with the preparation 2-hydroxy-4,6-dimethoxyacetophenone (2). Compound 2 was quantitatively obtained from the reaction of 2,4,6-trihydroacetophenone (1) with dimethyl sulfate in the presence of potassium carbonate in acetone. The corresponding acetophenone 2 was then subjected to the Claisen–Schmidt condensation reaction with 1-naphthaldehyde (3) in the presence of potassium hydroxide in methanol to provide flavokawain analogs (DiNap) with a yield of 32%.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded enveloped RNA virus which belongs to the genus Arterivirus, family Arteriviridae and order Nidovirales. Recently, a new proposal has classified PRRSV isolates into two species in the genus Porartevirus, PRRSV-1 and PRRSV-2, that replace their previous designations of European-like and North American-like genotypes, respectively [2, 3].
The antibody response to PRRSV infection is highly complicated and is still not fully understood. However, multiple methods have been established to detect PRRSV specific antibody as a serological marker for PRRSV infection, such as enzyme-linked immunosorbent assay (ELISA), immunoperoxidase monolayer assay (IPMA) and immunofluorescence and immunochromatographic strip-based assays [4–9]. Generally, PRRSV-specific Neutralizing antibodies (NAbs) appear typically after 28 days post-inoculation (dpi) [1, 10], but non-protective antibodies produced within the first week post-infection may be more useful for early detection of PRRSV infection. These early antibodies include non-neutralizing antibodies specific for structural proteins such as PRRSV N protein or nsps [1, 11]; N protein and certain nonstructural proteins (nsp1, nsp2 and nsp7) have been demonstrated to be highly immunogenic [12, 13]. Indeed, most currently available commercial ELISA kits that detect PRRSV-specific antibodies (e.g., IDEXX HerdChek PRRS ELISA) employ anti-N antibody as a serological marker for PRRSV infection or Modified Live Virus (MLV) immunization status.
Although commercial tests such as ELISA are highly sensitive for determining the presence of PRRSV-specific antibodies in serum samples, ELISAs are not suitable for quantitative analysis of antibody levels. This shortcoming is due to the fact that OD values obtained from ELISAs usually vary within a narrow range (from 0.1 to 2). Moreover, most ELISA kits use prokaryotically expressed single recombinant PRRSV structural antigens (generally PRRSV-N protein) as coating antigens. Consequently, such systems cannot evaluate PRRSV-specific antibody responses against other PRRSV enveloped proteins or nonstructural proteins (nsps). It should be noted here that systems employing multiple PRRSV antigens will probably not be developed due to the tremendous effort required for expression and purification of multiple ELISA plate coating antigens. Moreover, due to the general nature of ELISA coating antigens expressed in E. coli, false positive or false negative results have been frequently reported [12, 14]. Therefore, development of an improved method for both accurate detection and quantification of PRRSV specific antibodies is urgently needed.
In this study, we developed a modified assay based on luciferase immunoprecipitation systems (LIPS) and hereafter referred to as the luciferase-linked antibody capture assay (LACA) for PRRSV specific antibody detection, [15–17]. Briefly, the LACA detects PRRSV-specific antibodies in pig serum samples using mammalian cell-expressed recombinant PRRSV protein antigens (N and nsp1α) fused with Rellina luciferase. Similar to LIPS, LACA utilizes the enzymatic activity from captured luciferase-fused antigen-antibody complexes to convert a substrate to luminescent form to quantify antibody levels indirectly [15, 16]. As an immunoprecipitation assay, LIPS was originally developed by Dr. Peter D. Burbelo from the National Institutes of Health (NIH). LIPS utilizes luciferase-fused antigen and has been used for detection of antigen-specific antibodies or autoantibodies resulting from infection or autoimmune disease, respectively [18–20].
Compared to other antibody detection assays, antigen used for LIPS is easily obtained from cell lysates of mammalian cells previously transfected with plasmids coding for luciferase-fused antigens. Although few reports detail the use of LIPS for swine pathogen, a recent study reported the application of LIPS to the characterization of swine acute diarrhea syndrome coronavirus (SADS-CoV) originating in bats in China.
In this study, a modified ELISA-like form of LIPS, the LACA, was adapted as a diagnostic method for detection of PRRSV-specific antibodies. Two antigen targets (PRRSV-N protein and nsp1α) were selected for evaluation of specificity and sensitivity of the LACA for anti-PRRSV antibody detection using serum samples that were either negative or positive for anti-PRRSV antibodies. Moreover, sequentially collected serum samples obtained from piglets infected with PRRSV were evaluated for anti-PRRSV antibodies to compare the sensitivity of LACA to ELISA for earlier detection of anti-PRRSV antibodies during the course of PRRSV infection.