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No data is available regarding the pathogenesis of PCV-3 infection. The lack of virus isolation has impeded the establishment of an infection model to date. It is known that PCV-3 can be found in different tissues of domestic pig and wild boar (86, 87, 95), indicating the systemic nature of the infection. However, the point of viral entry, primary replication, organic distribution and persistence are still unsolved issues. PCV-3 has been found in feces, nasal swabs, oral fluids, and trucks transporting pigs (82, 85, 95), which allows speculating that horizontal transmission through direct contact is probably an important route. Detection of viral genome in fetuses and stillborn from farms with history of reproductive failure (21, 70, 75), as well as in semen and colostrum, points out also to vertical transmission as another likely route. Definitively, more studies are needed to ascertain the potential excretion routes of this virus.
Although PCV-3 genome has been detected at higher prevalence in weaned pigs (48, 77, 82), only one study has monitored PCV-3 infection longitudinally (83). In this study, PCV-3 was found in pigs at all ages with a similar frequency. This infection dynamics contrasts with that of PCV-2, which infects pigs mainly between five and 12 weeks of age, and rarely in animals at the lactation phase (116–118). This is explained by the fact that colostrum antibodies are protective against infection and then decline during the lactation and weaning phases. Once maternally derived antibodies waned, an infection is followed by active seroconversion (117–119). This seroconversion usually occurs between 9 and 15 weeks of age and the antibodies may last until 28 weeks of age at least (117, 120–122). Regrettably, information about infection in sows, maternally derived immunity and how protective the immunity might be against PCV-3 is completely lacking at this moment. It is known that PCV-3 can be found in colostrum (84), implying the possibility of vertical transmission (sow to piglet) and emphasizing the potential importance of early infections. Again, available information regarding these issues on PCV-3 is still to be generated.
A larger number of AstVs were detected in both rodent and shrew samples (Additional file 1: Table S4). Fifty-five AstVs were selected for sequencing. Most of the rodent AstVs sequenced belonged to four main genetic lineages 1 to 4 within the genus Mamastrovirus and had less sequence similarity with AstVs in other hosts (Fig. 5c). One rodent AstV, RtRn-AstV-1/GD2015, was closely related to AstVs of cattle, deer, and pigs with > 90% nt identity. Two shrew AstVs, Shrew-AstV/SAX2015 and Shrew-AstV/GX2016, were related to mouse AstV with ~ 70% nt identity in the genus Mamastrovirus. Lineage 5 contained one shrew AstV and one mouse AstV, with 79% nt identity with each other. Lineage 5 branched out of the genus Mamastrovirus and showed a closer relationship with the genus Avastrovius.
Sixty rodent samples were identified as PicoV positive, and 23 strains underwent genome sequencing (Additional file 1: Table S4). Rodent viruses from the genera Enterovirus, Hunnivirus, Mosavirus, Cardiovirus, Rosavirus, Kobuvirus, and Parechovirus were found in this study and showed 48.3–56.4%, 80.4–80.8%, 47%, 46.8–60.3%, 60.9%, 63–76.9%, and 43.7–87.3% RdRp aa identities with known members in each genus, respectively (Fig. 5b and Additional file 1: Table S11). Eight viruses formed lineages 1 and 2 close to the bat PicoV clade with 38.1–43.6%, 33.5–38.8%, and 48.2–56.7% aa identities with bat PicoVs in the P1, P2, and P3 regions, respectively. Two novel lineages 3 and 4 were identified with < 10.2–28.9% aa identities in the P1 region, 17.3–23.6% in the P2 region, and 21.8–28.4% in the P3 region compared with other PicoVs (Additional file 1: Table S10). Viruses closely related to known PicoVs of other hosts were found (e.g., rodent viruses related to human aichivirus, human rosavirus, and bovine hunnivirus).
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.
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.
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.
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 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].
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.
After its first discovery in 1989 in cynomolgus macaques imported to Reston, Virginia, RESTV was detected in domestic swine in the Philippines in a co-infection with the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV, family Arteriviridae, genus Arterivirus) and Porcine Circovirus type 2 (PCV-2; family Circoviridae). Later on, RESTV was identified to cause asymptomatic infections with mild respiratory symptoms, which may result in severe mortality in cases of co-infections with other viral pathogens like viruses in the families Arteriviridae and Circoviridae. The virus was first isolated in lung and lymphoid tissues in the original disease investigation. However, the massive presence of the virus in the lungs may be due to the fact that RESTV infection in pigs has been mostly associated with other infections of the respiratory tract, which may contribute to the specific localization of the virus and the respiratory symptoms of the disease. Marsh et al. conducted an experimental study to rule out the effect of other pathogens affecting pigs, using a 2008 Philippines swine isolate of RESTV. Specifically, five-week-old pigs were exposed (via the oro-nasal or subcutaneous route) to the virus, and the subsequent viral replication in internal organs and shedding of the virus from the nasopharynx was observed. The researchers detected the highest levels of virus replication in lung and lymphoid tissues, confirming previous results.
The detection of RESTV in domestic swine raised important biosecurity concerns about the potential for the disease’s emergence in humans and other livestock, mainly in animals for food consumption. The evidence of RESTV seropositive individuals further increased the concern for human infections and the worries of researchers, farm owners, and the public at large (World Health Organization. WHO, 2009, Available online: https://www.who.int/csr/resources/publications/HSE_EPR_2009_2.pdf). Interestingly, so far RESTV has not been seen to result in any human disease, even if there is concern that its passage through swine may allow RESTV to diverge and shift its potential for pathogenicity.
On the other hand, several studies investigated if other Ebola viruses may be transmitted through the aerosol route and may result in primary pulmonary infection. Researchers reviewed the different animal models and offered an overview regarding the possibilities of Ebola viruses causing aerosol infections in non-human primates (NHPs) and other animals. Experimental studies analyzed the respiratory tract involvement in filovirus infections when the animals were exposed to the virus through different aerosol routes (artificially aerosolized virus or natural aerosol transmission). In these experimental studies conducted on NHPs and pigs, EBOV was inoculated via the aerosol route, and, following mucosal exposure, EBOV replicated, reaching high concentrations, mainly in the respiratory tract, with the development of severe lung pathology. Interestingly, Weingartl et al. demonstrated that piglets inoculated oro-nasally with EBOV and then transferred to a different room housing macaques in an open inaccessible cage system resulted in EBOV infection of all macaques, suggesting a need to revise prevention and control measures during outbreaks. Viral replication was observed within alveolar spaces, in type I pneumocytes and macrophages, and in type II pneumocytes, bronchiolar epithelial cells, and endothelial cells, supporting the respiratory involvement. The upper and lower respiratory tract, the lymphoid tissues, and the mediastinal lymph nodes showed infection signs, as well. Similarly, in experiments on cynomolgus macaques placed separately in cages with experimentally infected piglets, and on guinea pigs exposed via aerosols to a guinea pig-adapted EBOV strain, viral antigens were detected within alveolar and septal macrophages, pneumocytes, epithelial cells, endothelial cells, fibroblasts, and other interstitial cells of the respiratory tree. Considering the pathology of the respiratory system, the expression of disease in the lungs and the patterns of lesions seem to be influenced by the exposure routes (aerogenous or hematogenous). Broncho–interstitial pneumonia, characterized by injury to both the bronchiolar and the alveolar epithelium, is commonly caused by aerogenous viral infections. Moreover, such pathological features were generally not evidenced following the inoculation of EBOV by other routes in NHPs and laboratory animals. As shown in animal studies, primary pulmonary infections could occur and cause active viral shedding from the respiratory tract, thus potentially setting up a cycle of ongoing respiratory transmission in humans.
Overall, experimental works conducted so far have shown that EBOV infection induces respiratory complications, that the virus can be shed via the respiratory secretions, and that it can cause similar pulmonary lesions both in animals exposed to aerosols and in those kept nearby in separate cages with no close contact.
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 data set supporting the results of this article is included within the article.
The discovery curve for virus families is shown in figure 1b. Here, a family is included on the date of the first published report of human infection by a virus species from that family. There is too little data (n = 23) for detailed statistical analysis, but the figure does suggest a possible decrease in the rate of discovery, implying that the pool of undiscovered families may be relatively modest (see).
Strikingly, no new families have been added to the list since 1988, the longest such interval on record. However, several viruses (specifically Torque Teno (TT) virus, TT mini virus and TT midi virus) newly reported since 1988 remain unassigned to a family.
It should also be noted that there are three virus families that, although they do not contain any known human virus species, do contain species that infect other mammals: Arteriviridae (several species including simian haemorrhagic fever virus); Asfarviridae (African swine fever virus); Circoviridae (including mammal infecting circoviruses as well as gyrovirus which infects chickens). This suggests that the list of families containing human viruses may not yet be complete.
Immunosuppression refers to suppression of the immune system and its ability to fight infection. HIV and infectious bursal disease virus are examples of viral infections that destroy entire lymphoid cell populations that ablate or disable adaptive immune responses. Lymphoproliferative cancers block cellular differentiation and deprive the body of mature, effector lymphocytes, thus causing immunosuppression in a different manner. PRRSV does neither; infection does not lead to severe lymphoid depletion or ablation, and it does not interfere profoundly with lymphocyte differentiation or maturation. Leukocyte perturbations in lymphoid tissues are associated with PRRSV infection, suggesting that adaptive immunity might be weakened, though not destroyed.
The immune system also maintains peripheral tolerance to self and commensal bacteria through immunosuppressive mechanisms that include regulatory T cells (Tregs), characterized as CD4+CD25+Forkhead box p3 (Foxp3)+ T lymphocytes. Treg suppressive properties were discovered when thymectomized or Treg-depleted mice succumbed to autoimmune reactions. Tregs suppress effector and effector memory T cell proliferation by cytokine deprivation leading to polyclonal apoptosis, and by suppression of antigen presenting cells by cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and other mechanisms. Studies in PRRSV infections give an ambiguous picture about the role of Tregs. PRRSV-2 strains are reported to induce a strong Treg response which included transforming growth factor (TGF)β-1 secretion in vitro as well as in vivo. Other studies did not show Treg responses to infection with either PRRSV-1 or PRRSV-2. Interleukin-10 (IL-10), an immunosuppressive cytokine expressed by various cell types including Tregs, was induced by PRRSV-2 vaccination in weaned pigs in one study, but was not induced in weaned or adult pigs in another study. Additional in vitro and in vivo studies reported IL-10 mRNA transcription and cytokine production after PRRSV infection. However, kinetic analysis in serum of viremic pigs of various ages showed that elevated IL-10 levels were primarily a function of age and were not associated with infection status. The only exception was in weaned pigs infected with a virulent virus, in which a transient increase was associated with viral pathogenesis.
On balance, the immunological evidence for PRRSV inducing a state of immunosuppression does not appear to be compelling. Secondary infections following PRRS disease outbreak in swine herds, suggesting a reduced ability to fight infection, is an alternative indicator of immunosuppression. An early study showed concurrent pulmonary bacterial infections in 58% of 221 PRRS cases. However, the study did not determine if bacterial infections were present before the PRRS outbreaks. The immunosuppression question also was addressed in more controlled settings using dual infection models with PRRSV and various bacterial species. A summary of published literature in 2003 showed no predisposition to bacterial disease in 8 of 15 coinfection models, three ambiguous outcomes, and four cases in which severity of disease was increased. More recent studies found a positive association between PRRSV infection and replication of porcine circovirus 2 (PCV2) or swine influenza virus.
It is possible that bacterial infections in swine herds increase following PRRS outbreaks due an increased burden of viral infection on host resilience to pathogen burden. Subclinical viral and bacterial infections are common, with PCV2, Salmonella enterica, Haemophilus parasuis, various Mycoplasma species, Leptospira, and Escherichia coli being examples. Control of infection is maintained by a combination of immune resistance to microbial replication and tissue tolerance to damage. In a coinfection model of influenza virus and Legionella pneumophila, it was clearly demonstrated that L. pneumophila infection was subclinical in healthy mice, but was lethal in the presence of influenza virus. Overwhelming disease was due to loss of tissue resilience, since the bacterial load was unchanged. This model might account for mortalities observed in experimental swine following PRRSV exposure. Given the variable results of PRRSV coinfection models in swine and an alternative mechanism for increased disease in PRRSV-infected herds, generalized immunosuppression does not appear to be a key feature of PRRSV pathogenesis.
PRRSV, like many viruses, has developed countermeasures to host immune responses that enable it to survive and replicate for extended periods of time before the infection is resolved. PRRSV modulation of intracellular antiviral defense mechanisms has been reviewed extensively. The effects of PRRSV infection on adaptive immune response, i.e., antigen-specific T cell, B cell, and antibody responses, are less well characterized. The antiviral response of T cells to PRRSV, examined primarily by the IFNγ enzyme-linked immunospot (ELISPOT), appears to develop slowly over a period of weeks, and is not associated with changes in viral loads in blood or in infected lung and lymphoid tissues. Peripheral blood mononuclear cells (PBMC) from young, weaned pigs show limited IFNγ responses even when stimulated by phytohemagluttinin, which might account for the low anti-PRRSV responsiveness after re-stimulation in vitro. However, PBMC from growing pigs and mature sows, which showed higher levels of IFNγ sensitivity, still showed limited responsiveness. These findings indicate that PRRSV may interfere with specific cell-mediated immunity, but more direct evidence is needed for a fuller understanding.
By contrast, the interaction of PRRSV with pigs does not appear to retard or attenuate the development of humoral immunity or B cell differentiation. Induction of antibody responses to PRRSV proteins, both structural and non-structural, occurred in the same time frame as antibody responses to keyhole limpet hemocyanin (KLH), an irrelevant protein antigen. The antibody response to KLH was also the same in the presence or absence of PRRSV infection. Similarly, PRRSV infection did not inhibit cellular or humoral immune protection in response to pseudorabies virus vaccination. Thus, the adaptive B cell response is not delayed or suppressed by PRRSV.
An extended viremia and prolonged survival in lymphoid tissues is characteristic of PRRSV infection. These features show that PRRSV has mechanisms of immune avoidance that are not present in viruses such as influenza virus and foot and mouth disease virus, in which sterilizing immunity is achieved within 10–14 days. It appears from the findings of field observations and experimental investigations that some type of PRRSV-specific T cell interference is present, whereas specific B cell inhibition or a generalized state of immunosuppression are not immunological hallmarks of PRRSV infection.
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.
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.
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).
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.
The effect of DiNap on the growth rate of the pigs was evaluated over the course of PRRSV infection. The average daily weight gain (ADWG) was measured for all pigs in each group up to 28 dpc. DiNap significantly enhanced the ADWG for both treatment groups (groups 2 and 3) compared with that of the pigs in group 1 (NC) (Figure 4).
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).
Nidovirales is a large order of positive sense, single-stranded RNA (ssRNA) viruses that consists of the many genera and species in the families Coronaviridae, Arteriviridae, Roniviridae and Mesoniviridae. Although the genomes of nidoviruses vary in length, ranging from 13 to 32 kilobases (kb), the organization of the genomes are similar across the entire order [1–4]. The 5′ end of the genome encodes two replicase polyproteins (pp1a and pp1ab), structural proteins and accessory proteins. Genes downstream of the replicase polyprotein gene are expressed from a nested set of 3′-coterminal subgenomic mRNAs, a replication strategy unique to the Nidovirales
Nidoviruses infect a broad range of hosts including humans and other mammals, birds, fish, insects and crustaceans [8–11]. Although reptiles are susceptible to infection by a wide variety of viruses (as reviewed in), nidovirus infections have not previously been described. Viruses affecting the reptile respiratory tract include herpesviruses, iridoviruses, adenoviruses, flaviviruses, and, of particular importance in snakes, paramyxoviruses and reoviruses.
Here we report the discovery of a novel nidovirus in a collection of ball pythons (Python regius) in upstate New York with pneumonia, tracheitis and esophagitis. The snakes were found dead between July 2011 and September 2013.
Gross postmortem examination was performed on 4 snakes. Snake 1, a 6-year-old, female piebald color morph ball python, was submitted in July of 2011. Snake 2, a 6-year-old, male lesser platinum color morph ball python, was submitted in December of 2011. Snake 3, a 3-year-old female paradox color morph ball python, and snake 4, a 7-year-old female pastel color morph ball python, were submitted in September 2013. After gross evaluation, samples of tissues were collected and saved in 10% neutral buffered formalin, routinely processed and mounted in paraffin. Five μm paraffin ribbons were cut and stained with either hematoxylin and eosin (H&E) or Gram’s stain for histologic examination. Eleven tissues, including lung (n = 4), liver (n = 1), spleen (n = 3) and esophagus (n = 3) from the 4 snakes were collected and stored at −20°C.
Gross and histologic findings in all four snakes were primarily restricted to the respiratory and upper gastrointestinal tracts (Table 1). Hematoxylin and eosin stained sections (Figure 1A through F) revealed marked hyperplasia of epithelial cells lining air exchange areas (pneumocytes) with significant mononuclear (lymphocytes and plasma cells) and granulocytic (heterophils) interstitial inflammation and epithelial necrosis (Figure 1B). Similar inflammatory and hyperplastic changes were also present in the trachea (Figure 1D), esophagus (Figure 1F) and oral cavity. Gram-negative stained bacteria are shown in lung tissue from a snake with bacterial bronchopneumonia (Figure 1H).
Total nucleic acids were extracted from snake samples (lung and spleen for snake 1, lung, spleen and esophagus for snake 2, spleen and liver for snake 3, lung and trachea for snake 4) using the EasyMag (bioMérieux, Inc.) platform; Samples from snakes 1 and 2 were depleted of ribosomal RNA (Ribo-Zero™ rRNA Removal, Epibio) and treated with DNAse I (TURBO DNA-free™, Ambion). cDNA synthesis was performed using SuperScript II first-strand synthesis supermix (Invitrogen). Viral discovery was performed using broadly reactive consensus PCR assays targeting common respiratory viruses of animals, including paramyxoviruses [19–21], reoviruses and caliciviruses. When PCR analysis failed to yield a causative agent, high-throughput sequencing was performed on all samples originating from snakes 1 and 2 (Ion PGM, Life Sciences). On average, 850,000 reads were obtained from each sample. All reads were processed by trimming primers and adaptors, length filtering, and masking of low-complexity regions (WU-BLAST 2.0). To remove host sequences, the remaining reads were subjected to a homology search using BLASTn against a database consisting of ribosomal and genomic metazoan sequences. Following the processing, an average of 250,000 reads per sample remained for further analysis.
Nucleotide sequence analysis (BLASTn) of processed reads was uninformative; however, amino acid analysis (BLASTx) revealed multiple reads with amino acid homology of <50% to the polyprotein region of the Nidovirales subfamily Torovirinae, including Breda virus, White bream virus, and Fathead minnow virus. Assembly of all Torovirinae-like reads generated a 3,408 nt contig with 33% amino acid homology to the replicase polyprotein 1ab of Fathead minnow virus. The presence of this 3,408 nt sequence in both snakes was confirmed by PCR using primers shown in Table 2. Cycling conditions are described in a footnote to Table 2. Samples from multiple tissues of snakes 3 and 4 were also screened and tested positive for this virus.
For phylogenetic analysis, the 3,408 nt sequence was translated with Se-Al v2.0a11, and a 1,136 amino acid fragment was aligned against all Nidovirales sequences from GenBank using ClustalW. The best-fit model of amino acid substitution, the Whelan and Goldman (WAG) matrix, was selected using the maximum likelihood method implemented in MEGA version 5.2. A Bootstrap-supported (1000 replicates) maximum likelihood phylogenetic tree was constructed using MEGA version 5.2. The ball python-associated virus clustered within the Torovirinae subfamily (Figure 2). A neighbor-joining phylogenetic method was also implemented with congruent results. Based on the phylogenetic position and the genetic distances between species, this virus, tentatively called ball python nidovirus (BPNV, GenBank accession number KM267236) may represent a new species within the subfamily Torovirinae.In situ hybridization to a 934 nt fragment of the genomic polyprotein 1ab region was used to assess viral infection and distribution in the lung tissue. Positive cytoplasmic staining, consistent with the presence of viral nucleic acid, was confirmed in the cytoplasm of pulmonary cells, presumably epithelial cells (Figure 3A). The specificity of probes for in situ hybridization was confirmed by the absence of signal when the same probe was used on control pulmonary tissue from an uninfected, 6-year-old, female ball python maintained at College of Veterinary Medicine, Cornell University (Figure 3B).
Respiratory disease can be an important cause of morbidity and mortality in both wild and captive reptiles. In captivity, reptiles, and particularly snakes, are frequently maintained in collections with a high population density in relatively small spaces. As such, disease transmission within collections can occur rapidly, and early detection and diagnosis is critical in controlling disease spread.
Although 8 of 12 snakes with disease in the collection showed gram-negative rods by Gram stain and follow up culture in 4 revealed the presence of Aeromonas sp., Pseudomonas sp., Serratia sp., no evidence of bacterial infection was found in 4 snakes. In contrast, all snakes with epithelial hyperplasia in the trachea, lung and esophagus and mononuclear inflammatory infiltrates had viral signal by PCR and ISH. In concert, these data suggest a role for this nidovirus in the pathogenesis of respiratory disease. However, unequivocal implication will require experimental infection studies.
The identification of this novel nidovirus expands our understanding of nidoviral diversity and provides insight into the pathogenesis of respiratory disease in snakes. Phylogenetic analysis indicated that the virus belongs to a novel genus within the Torovirinae subfamily distinct from the Torovirus and recently characterized Banifivirus genera. Due to overlapping clinical signs and pathologic lesions of the newly discovered nidovirus with the best characterized viral respiratory pathogens of snakes, paramyxoviruses and reoviruses [17, 18], it is possible that nidoviral infections were previously misdiagnosed or overlooked. PCR-based detection methods to rapidly determine infection status and etiology of respiratory disease in snakes are recommended to guide decisions for managing husbandry and veterinary care.
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.
To evaluate the analytical sensitivity of LACA for diagnosis of early PRRSV infection, LACA and IDEXX PRRS ×3 ELISA were performed side-by-side to measure specific antibody detection in 38 serum samples collected at a series of time points from 6 HuN4-inoculated pigs. Among the 6 pigs, sequential samples were collected at 0, 3, 7, 10 14, 21 and 28 dpi and for 4 pigs sequential samples were collected at 0, 3, 7, 10 and 14 dpi (Fig. 4a). Based on our results of the N-Rluc LACA, serum samples from two infected pigs (33.33%) were identified as anti-PRRSV antibody-positive as early as 3 dpi and serum samples from five pigs (83.33%) were positive at 7 dpi. All sera collected at 10 dpi were positive using N-Rluc LACA (Fig. 4a). For the nsp1α-Rluc LACA, PRRSV-specific antibodies were not detected before 7 dpi. However, all serum samples tested positive at 10 dpi and thereafter (Fig. 4b). Meanwhile, all serum samples analyzed by IDEXX ELISA were anti-PRRSV antibody-negative at 3 dpi but tested antibody-positive at 7 dpi (Fig. 4c). Taken together, it appears that the N-Rluc LACA demonstrated superior sensitivity for early PRRSV detection (3 dpi) when compared to nsp1-Rluc LACA and IDEXX ELISA.