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Vaccinia virus (VV) is a member of the Poxviridae, which constitute a large family of enveloped DNA viruses and replicate entirely in the cytoplasm of the infected cells with a linear double-stranded DNA genome of 130–300 kilo base pairs. Poxviruses have a broad range of eukaryotic hosts including mammals, birds, reptiles and insects and can grow in many cell lines in vitro. Some poxviruses are causative agents of human diseases. Variola virus caused a deadly human disease smallpox until its global eradication in 1977, in which VV was used as a vaccine. Other poxviruses causing human diseases are molluscum contagiosum virus and the zoonotic monkeypox virus. Notably, variola and monkeypox viruses are transmitted to humans by respiratory route, whereas molluscum contagiosum virus is mainly transmitted through the skin. Variola and monkeypox viruses cause systemic infections with high levels of lethality, but the details of their pathogenesis are not well-understood.
Intranasal inoculation of different VV strains in mice shows different levels of virulence and only neurovirulent strains cause lethality. Western Reserve (WR) strain was generated by intracerebral mouse passages, and an intranasal inoculation results in an acute infection of the lung followed by dissemination of the virus to various organs. Intranasal infection with a low dose of WR strain induces an inflammatory infiltrate in the lung, and the virus was cleared 10 to 15 days after infection; however, infection with a high dose of WR strain caused lethality, which has been used as a challenge model to study the effect of antiviral drugs, immune IgG, soluble viral proteins and other vaccine strains. In one report intranasal infection with the WR strain caused pneumonia showing severe alveolar edema and acute necrotizing bronchiolitis and peribronchiolitis as well as neutrophilic infiltrates in the interstitium of the lung. The mechanisms of lethality in mice infected with the lethal dose of WR strain are, however, not well-understood.
In this study, we focused on the differences in virus replication and host immune responses between lethal and non-lethal respiratory infections with VV. We used two VV strains; neurotropic virulent WR strain and the less virulent Wyeth strain. Although BALB/c mice are frequently used for intranasal challenge of vaccinia virus, we used the C57BL6/J strain of mouse in these experiments for two reasons. One is that most knockout mice lacking genes involved in immune responses have been made with C57BL6/J genetic background. The other is that we and one other group have characterized cellular immune responses, especially CD8+ T cell responses, to vaccinia virus in C57BL6/J mice, when this study was planned. Infection of C57BL/6J mouse with a high dose (106 p.f.u. (plaque-forming units)) of the WR strain was lethal, whereas a high dose (106 p.f.u.) of Wyeth strain and a lower dose (104 p.f.u.) of WR strain were not lethal. The WR strain replicated and produced higher titers of virus in the lung and the brain compared to the Wyeth strain. There was, however, no difference between the virus titers in brains of mice infected with the high or low dose of WR strain. Lethal infection with WR strain resulted in fewer lymphocytes and an altered phenotype of T cells in the lung compared to non-lethal infection and uninfected controls, and induced severe thymus atrophy with a marked reduction of CD4 and CD8 double positive (DP) T cells.
Human microbiologic infections, known as zoonoses, are acquired directly from animals or via arthropods bites and are an increasing public health problem. More than two thirds of emerging human pathogens are of zoonotic origin, and of these, more than 70% originate from wildlife. In novel environments, viruses, particularly RNA viruses, can easily cross the species barrier by mutations, recombinations or reassortments of their genetic material, resulting in the capacity to infect novel hosts. Because of their adaptive abilities, RNA viruses represent more than 70% of the viruses that infect humans. When socio-economic and ecologic changes affect their environment, humans may encounter increased contact with emerging viruses that originate in wild or domestic animals.
Wolfe et al. in 2007 and Karesh et al. in 2012 described different stages in the switch from an animal-specific infectious agent into a human-specific pathogen. The key stage is the transition of a strictly animal-specific infectious agent (originating from wildlife or domestic animals) to exposed human populations, resulting in sporadic human infections (Figure 1). If the pathogen is able to adapt to its human host and acquire the means to accomplish an inter-human transmission, horizontal human-to-human transmission occurs and maintains the viral cycle. Sometimes, an intermediate host, such as a domestic animal, is the link between sylvatic viral circulation and human viral circulation. For example, some human infections originating from bats, such as Nipah, Hendra, SARS and Ebola viral infections, may involve intermediate amplification in hosts such as pigs, horses, civets and primates, respectively (Figure 1). Genetic, biologic, social, political or economic factors may explain a switch in viral host targets. For example, climate changes may influence the geographical repartition of vector arthropods, leading to new areas of the distribution of infectious diseases, like Aedes albopictus and Chikungunya infections in the Mediterranean. Morens et al. listed different key factors that may contribute to the emergence or re-emergence of infectious diseases, such as microbial adaptation to a new environment, biodiversity loss, ecosystem changes that lead to more frequent contact between wildlife and domestic animals or human populations, human demographics and behavior, economic development and land use, international travel and commerce, etc.. These patterns of transmission allow identifying different animals to follow in order to monitor the appearance of new or re-emerging infectious agents before its first detection in the human populations. Therefore, hematophagous arthropods, wildlife and domestic animals may serve as targets for zoonotic and arboviral disease surveillance, particularly because sampling procedures and long-term follow-up studies are more easily performed in these hosts than in humans.
Historically, classic viral detection techniques were based on the intracerebral inoculation of suckling mice or viral isolation in culture and the subsequent observation of cytopathic effects on cell lines. Later, immunologic methods, e.g., seroneutralization or hemaglutination, were used to detect viral antigens in various complex samples. These techniques were based on the isolation of viral agents. With the progresses of molecular biology, polymerase chain reaction (PCR)-based methods became the main techniques for virus discovery and allowed the detection of uncultivable viruses, but these techniques required prior knowledge of closely related viral genomes. Next-Generation Sequencing (NGS) techniques make it possible to sequence all viral genomes in a given sample without previous knowledge about their nature. These techniques, known as viral metagenomics, have allowed the discovery of completely new viral species. Because of their low cost, the use of NGS techniques is exponentially increasing.
The transmission of infections between humans occurs after a pathogen from a wild or domestic animal contacts with exposed human populations. The human exposures may or may not be mediated by the bite of bloodsucking arthropods. Surveillance programs may target wildlife, domestic animals or arthropods for emerging viruses before their adaptation to human hosts.
The porcine circovirus (PCV) belongs to the family Circoviridae and contains a single-stranded circular DNA genome. There are three types of PCV: porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus 3 (PCV3). During the past few decades, PCV2 has been widely studied and is considered to be the main pathogen responsible for porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), which are characterized as clinical or subclinical PCV2 infections among pigs. The most representative symptoms of the diseases include porcine dermatitis and nephropathy syndrome (PDNS), which mainly occurs during the growing or finishing stage of pigs; postweaning multisystemic wasting syndrome (PMWS), which affects nursery and growing pigs; and porcine respiratory disease complex (PRDC), which usually occurs in pigs 14–20 weeks of age.
To date, the exact mechanisms of PCVD/PCVAD are currently unknown. However, many studies have reported co-infection with other swine pathogens, such as porcine reproductive and respiratory syndrome virus, porcine parvovirus, swine influenza virus, Mycoplasma hyopneumoniae, and Salmonella spp., are important cofactors that may enhance PCV2 infection and the severity of PCVD/PDVAD. Furthermore, vaccination failure, stress or crowding together with PCV2-infected animals also cause PCVD/PCVAD. As co-infections with viruses are frequently detected in domestic pigs and wild boars, we discuss co-infections of pigs with PCV2 and other swine viruses in this review. Furthermore, co-infections of different PCV2 strains, which cause recombination and genomic shifts in recent years, are also reviewed.
Adult female C57BL6/J mice were inoculated with various doses of the WR and Wyeth strains intranasally. Weight change and survival of infected mice were recorded daily. Inoculation with higher doses (106 p.f.u. and 105 p.f.u.) of the WR strain induced rapid and severe weight loss, which became obvious at 3 days post-infection (Fig. 1A), and most of mice died at 7–10 days post-infection (with 106 p.f.u. all mice died by 8 days post-infection) (Fig. 1B). Lower doses (104 p.f.u. and 103 p.f.u.) of the WR strain caused mild weight loss, and all mice survived. These mice recovered their weight after 6–8 days post-infection (Fig. 1A). The 50% lethal dose (LD50) of WR strain was calculated as 4.2 × 104 p.f.u., which is similar to the LD50 reported for BALB/c mouse. Wyeth strain did not kill mice (Fig. 1B) or cause weight loss (Fig. 1A) even when 106 p.f.u. of the virus was inoculated.
Adult ferrets (weight, 500–1500 g) were housed at bioCSL under a Support Services Agreement with the Victorian Infectious Diseases Reference Laboratory. Ferrets were seronegative (hemagglutination inhibition [HI] titer, <10) to currently circulating influenza virus strains before use. Experiments were conducted with approval from the CSL Limited/Pfizer Animal Ethics Committee, in accordance with the National Health and Medical Research Council, Australia, code of practice for the care and use of animals for scientific purposes.
Astroviruses (AstV) are small, nonenveloped, RNA viruses that are a major cause of gastroenteritis in infants, immunocompromised people, and the elderly, and they also cause disease in mammals and birds. Despite the disease burden, little is known about the immune response to astrovirus infection. Human clinical studies have demonstrated that an antibody-mediated response may be responsible for limiting astrovirus infection and clinical disease. Recent work using small animal models and cell culture systems have revealed an important role in the innate immune response in restricting astrovirus replication and pathogenesis. This review will summarize the current knowledge of the innate and adaptive immune responses to astrovirus infection using studies of humans, small animal models, and cell culture systems and will discuss how astroviruses evade the immune system. This review will also highlight the increasing reports of astroviruses as possible causes of central nervous system disease, especially in immunocompromised individuals. Finally, we will conclude with unanswered questions, future studies, and how the use of a newly developed mouse model can enhance our understanding of the immune response to astrovirus infection, and how these responses play a role in astrovirus-induced disease.
Chlamydiae are implicated in a wide variety of diseases in both animals and humans. Although acute infections in animal chlamydioses are the most commonly reported, chronic chlamydial infections are also associated with a variety of diseases in humans and animals. These latter infections are characterized by inflammation and scarring resulting in significant damage of the host. A causative role in chronic diseases requires that chlamydiae persist within infected tissue for extended periods of time. Current theories, based primarily on in vitro data, suggest that chlamydial persistence, and the resulting chronic inflammation, is linked to morphological and metabolic conversion of the actively replicating and intracellular reticulate body (RB) into an alternative, non-replicative form known as an aberrant body (AB). In vitro, alterations of the normal developmental cycle of Chlamydia trachomatis and Chlamydia pneumoniae can be induced by Interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and penicillin G exposure as well as amino acid or iron deprivation and monocyte infection. To date, in vitro models for animal pathogens, Chlamydia abortus and Chlamydia pecorum have not been described although both organisms are associated with chronic disease in koalas and small ruminants.
In pigs, several chlamydial species, including Chlamydia abortus, Chlamydia psittaci, Chlamydia pecorum and Chlamydia suis, have been implicated in a variety of disease conditions including conjunctivitis, pneumonia, pericarditis, polyserositis, arthritis, abortion and infertility. In the gastrointestinal tract, chlamydiae appear to be highly prevalent but only occasionally cause enteritis. They have been found in the intestine of diarrheic and healthy pigs and could be demonstrated in mixed enteric infections. Pospischil and Wood first described an association between Chlamydiaceae and lesions in the intestinal tract of pigs and assumed a synergistic effect in co-existence with Salmonella typhimurium. Further, mixed infections with Eimeria scabra, cryptosporidia, and porcine epidemic diarrhea virus (PEDV) have been described in the past. PEDV, a member of the family Coronaviridae, is a well-known cause of diarrhea in pigs. After the identification of PEDV in 1978 by Pensaert and Debouck, more than a decade passed before the virus could be adapted for propagation in cell cultures. Examination of infected Vero cell cultures by direct immunofluorescence revealed single cells with granular cytoplasmic fluorescence as well as formation of syncytia with up to 50-100 nuclei or more. Typical features of syncytial cells were growth, fusion and detachment from cell layers after they had reached a certain size. Biomolecular studies revealed major genomic differences between cell culture-adapted (ca)-PEDV and wild type virus.
Cell culture model of co-infection with ca-PEDV and Chlamydia has been established recently to investigate the interaction of ca-PEDV and Chlamydiaceae in mixed infections and to detect possible synergistic or additive effects of possible significance in clinical enteric disease in pigs. In that study, abnormally large chlamydial forms were observed in dually infected cell layers by immunofluorescence suggesting that ca-PEDV co-infection might alter the chlamydial developmental cycle in a manner similar to that observed during persistent infections. To confirm these initial observations, we established a cell culture model of mixed infections with Chlamydia and a cell culture-adapted porcine epidemic diarrhea virus (ca-PEDV) and hypothesized that this would result in the generation of persistent chlamydial forms. This data demonstrates that ca-PEDV co-infection, indeed, alters the developmental cycle of Chlamydia pecorum and Chlamydia abortus in a similar manner to other inducers of chlamydial persistence.
It is estimated by the World Health Organization (WHO) that approximately 36.9 (34.3~41.9) million persons worldwide are infected with human immunodeficiency virus (HIV), which is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS), including 1.7 million in the United States. Influenza A virus can cause acute respiratory infection in humans and animals throughout the world, and has continued to be a significant public health threat, leading to substantial global morbidity and mortality and an average of approximately 23,600 deaths annually in the United States alone.
The impact of influenza virus on HIV infection has not been well investigated. Little is known about influenza virus infection in HIV-positive individual. HIV infection has been shown to be related to worse prognosis of influenza. HIV-infected patients in Canada who also had pandemic 2009 influenza A (H1N1) virus (pH1N1) infection had more severe illness than those who did not have the co-infection, and fatality was higher than for patients who were not co-infected in California (USA). Recent reports indicate that adults with AIDS experience substantially elevated influenza-associated mortality.
Influenza virus infection has been associated with viremia in human and animal models. Viral RNA has been detected in blood in severe human pH1N1 infection. Encapsidated pH1N1 RNA is stable in blood derived matrices and influenza viruses can be transmitted by blood transfusion in ferrets. Normally, influenza A virus infection is confined to the airways where the virus replicates in respiratory epithelial cells. Cumulated reports indicate that influenza viruses can infect and replicate in blood cells, such as dendritic cells, primary monocytes/macrophages, and T cells.
Infection with HIV-1 can result in apoptotic cell death through activation of both death receptor-mediated and Bax/mitochondrial-mediated apoptotic pathways, which cause a progressive depletion of a select group of immune cells namely the CD4+ T helper cells leading to immunodeficiency. While HIV directly and selectively infects CD4+ T cells, the low levels of infected cells in patients is discordant with the rate of CD4+ T cell decline and argues against the role of direct infection in CD4 loss. A viral protein, neuraminidase (NA), derived from the human influenza virus was reported to enhance the level of HIV-1-mediated syncytium formation and HIV-1 replication. However, it is not known whether influenza A virus in blood affects HIV-1 replication, or reactivates HIV-1 replication in HIV-1-infected cells. Here, we showed that pandemic influenza A (H1N1) virus infection increased apoptotic cell death and HIV-1 replication in HIV-1 infected Jurkat cells.
Infectious Bursal Disease (IBD) is an acute, highly contagious, and immunosuppressive disease of young chicken, caused by double-stranded RNA virus belonging to the genus Avibirnavirus of family Birnaviridae. It is characterized by the destruction of dividing lymphoid cells in the bursa of Fabricius causing cytolysis leading to immunosuppression in addition to severe economic losses due to impaired growth, death, and excessive condemnations of carcasses because of skeletal muscle hemorrhages. The virus is evolutionarily related to rotaviruses (Reoviridae) and picornaviruses (Picornaviridae) (Dalton and Rodriguez,). The virus can be adapted to grow and produce cytopathic effects in chicken embryo fibroblasts (CEF).
Nitric oxide (NO) has been shown to inhibit a number of viruses, including Herpes Simplex virus type 1, Ectromelia virus, Vaccinia virus, Vesicular Stomatitis virus, and murine Friend leukemia retrovirus. Lin et al. have reported the inhibitory effect of NO on Japanese encephalitis viral RNA synthesis, viral protein accumulation, and virus release from infected cells. NO also inhibited the replication cycle of Encephalomyocarditis virus, Coxsackie virus, Marek’s diseases virus, Respiratory Syncytial virus and Severe Acute Respiratory Syndrome virus. NO combines with superoxide radical to produce peroxynitrite radical (ONOO−) that reacts with capsid proteins on Coxsackie virus, leading to the inhibition of viral entry into cells. NO also inhibits a variety of transcription factors and viral proteinases that are required for viral replication. Takhampunya et al. reported the inhibitory effect of NO on Dengue virus infection, partly via the inhibition of the RdRp (RNA-dependent RNA polymerase enzyme) activity, which then down-regulates viral RNA synthesis. Jena could demonstrate the inhibition of IBD virus (IBDV) replication in CEF by NO.
Previously, we have shown profound inhibition of IBDV in CEF by root extract of the Indian ginseng, Withania somnifera (Linn.) Dunal (WS); however, the mechanism of inhibition was not clear. WS is a well-known inducer of NO. Iuvone et al. found that WS significantly increased NO production in vitro through concentration-dependent up-regulation of inducible nitric oxide synthetase (iNOS) expression. Hence, the present investigation was undertaken to ascertain the production of NO as an underlying mechanism of the inhibitory effect of WS against IBDV in CEF.
Streptococcus equi subspecies equi is the causative agent of strangles, a highly contagious upper respiratory tract infection of horses. Typical clinical signs of disease include fever, inappetance, lethargy, submandibular or retropharyngeal lymphadenopathy or purulent drainage, or purulent nasal discharge. Complications of S. equi infection can occur and include airway obstruction from lymphadenopathy, disseminated abscesses from hematogenous spread, or purpura hemorrhagica and various diseases caused by immune‐mediated processes.1, 2, 3, 4, 5
Streptococcus equi M protein (SeM) antibody titers are typically measured to determine if a horse has developed a complication of strangles, such as purpura hemorrhagica or metastatic abscess formation, or to determine if a horse is at risk of purpura hemorrhagica if they were to be vaccinated. Both the 2005 and 2018 American College of Veterinary Internal Medicine consensus statements on strangles state that a very high titer (≥1:12 800) is associated with metastatic abscess formation or purpura hemorrhagica and that high titers (1:3200‐1:6400) are detected 4‐12 weeks after infection.1, 2 Anecdotally, horses can have high titers (≥1:12 800) 4‐8 weeks after infection and no signs of complications (authors' personal observations, KMD, LAB, ACT). The objective of this study was to measure SeM antibody titers on horses after outbreak to determine if titers detect the presence of complications.2 An additional objective was to follow SeM antibody titers out to 7 months after infection to determine immunoglobulin decay and to monitor for development of additional complications. We hypothesized that the magnitude of SeM antibody titer after infection (SeM titer ≥1:12 800) will be useful to monitor for the presence of complications or for the risk of development of complications.
West Nile virus (WNV), a plus-sense, single-stranded neurotropic flavivirus, has been a public health concern in North America for more than a decade. The virus is maintained in an enzootic cycle that involves mosquitoes and birds, with humans and horses as incidental hosts. Infection in humans results from mosquito bites, blood transfusion, organ transplantation, breast feeding, and in utero or occupational exposure. WNV infection of the central nervous system (CNS, neuroinvasive disease) commonly presents as encephalitis, meningitis, or acute flaccid paralysis. The overall mortality rate in persons who develop WNV neuroinvasive disease is about 10%, although the mortality rate increases significantly in the elderly and immunocompromised. Recently, some WNV convalescent patients were reported to have significant long-term morbidity years after their acute illness; symptoms include muscle weakness and pain, fatigue, memory loss, and ataxia. At present, there is no specific therapeutic agent for treatment of the infection. No approved human vaccines are available for its prevention.
WNV has been studied in various animal models, including mice, hamsters, monkeys, and horses. The murine model is an effective in vivo experimental model to investigate viral pathogenesis and host immunity in humans. Following the initial subcutaneous or intraperitoneal inoculation in mice, WNV induces a systemic infection and eventually invades the CNS. Mice die rapidly when encephalitis develops, usually within one to two weeks. The severity and symptoms of lethal infection observed in the murine model mimic the symptoms caused by WNV infection in humans. Studies from experimental animal models, in vitro cell culture, and/or WNV patient samples have provided important insights into host immunity to WNV infection. Natural killer (NK) cells and γδ T cells are two innate lymphocytes that respond rapidly and non-specifically to viral infection. They are also known to form a unique link between innate and adaptive immunity. Moreover, the characteristics of these two cell types in adaptive immunity have been described in several disease models. In this review, we will discuss recent studies on these two unique cell types in both protective immunity and viral pathogenesis during WNV infection.
Vaccinia virus (VV), a member of the Poxviridae family, is an enveloped, DNA virus with a genome of 192 kb encoding about 200 proteins. Various cell lines can be infected by VV, including HeLa, CV-1, mouse L, and chicken CEF cells. VV causes major changes in host cell machinery shortly after infection, and cytopathic effects (CPE) are observed several hours after infection with VV. VV infection modulates host cell gene expression: several previous studies have shown that mRNA synthesis in the host cells was inhibited immediately after VV infection. Microarray analysis showed that around 90% of the host genes were down-regulated after VV infection, including genes involved in DNA replication, transcription, translation, apoptosis, and the proteasome-ubiquitin degradation pathway. Only a smaller fraction of host genes were up-regulated after VV infection, including WASP protein, and genes implicated in immune responses.
Several viral factors of VV utilize ATP and several steps in viral multiplication of VV require ATP. ATP is also required for DNA packaging and capsid maturation of herpes simplex virus, for capsid assembly and release of type D retrovirus, for capsid assembly of human immunodeficiency virus, and for budding of influenza virus. Therefore, it was expected that viral factors would modulate cellular energetics to benefit the virus, though this area is understudied.
In this study, the possible up-regulation of host cell genes after VV infection was analyzed by differential display-reverse transcriptase-polymerase chain reaction (ddRT-PCR), a simple technique with high sensitivity and specificity . Two mitochondrial genes involved in the electron transport chain (ND4 and COII) to generate ATP were found to be up-regulated after VV infection using this assay.
Intensive poultry farming leads to higher risk of infectious disease emergence causing great economical losses. Boundary spanning between clinical manifestations of different agents is peculiar to the course of many infections nowadays. More and more infectious diseases progress in association with different microorganisms and it effects significantly the clinical manifestation and differential diagnosis of the disease.
Currently, the viral infections such as avian influenza, Newcastle disease, infectious bronchitis, and infectious bursal disease, etc., are a potential threat to poultry farming in the Republic of Kazakhstan. Monitoring these economically significant avian diseases is the question of the day for poultry industry.
Avian influenza virus belongs to the Orthomyxoviridae family, Influenza A virus genus. From the beginning of year 2016 the disease outbreaks were recorded in 30 countries. Different AIV strains can cause 10 to 100% mortality among poultry.
The agent of the Newcastle disease is an RNA-containing virus, a member of the Paramyxoviridae family, Rubulavirus genus. In 2016 13 countries reported Newcastle disease cases to the OIE. In poultry industrial farms, all infected birds need to be sacrificed due to threat of dissemination of the infection across countries.
The agent of the infectious bursal disease is RNA-containing virus of Avibirnavirus genus in Birnaviridae family. In outbreaks of the infectious bursal disease practically the entire population is affected and the lethality rate can approach 90%, the reconvalescent birds become susceptible to the majority of infectious diseases of viral and bacterial etiology.
The causative agent of infectious bronchitis is an RNA-containing Coronavirus avia of Coronavirus genus in Coronaviridae family. Economical losses due to infectious bronchitis is composed of reduced egg and meat productivity, compulsory slaughter of sick birds, high death rate in young population. When the infection circulates in the farm for the first time the lethality rate can reach 70%.
Currently, standard immunological methods or methods based on polymerase chain reaction (PCR) [8, 9] are widely used to identify the above mentioned viruses. Unfortunately, they can detect only one agent in a specimen.
There are also multiplex RT-PCR assays that make possible simultaneous detection of more than one infectious agent by using multiple primer pairs. Advantage of the multiplex RT-PCR is in combination of sensitivity and quickness of PCR alongside with elimination of need to test clinical specimens for each agent separately [10, 11].
Avian viruses can cause diseases independently, in alliance with each other or in association with bacterial agents. Thereby, rapid and sensitive methods of detection are required that are able to differentiate viral infections for surveillance of newly emerging avian viruses as well as for disease control.
Application of DNA microarray technology that makes possible multivariate analysis of genetic material is a highly promising way for simultaneous detection of several agents (AIV, NDV, IBV and IBDV) in one specimen.
The paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescent-labelled viral cDNA on the microarray and its specificity tested with use of AIV, NDV, IBV, IBDV strains as well as biomaterials from poultry.
The objective of this study is to develop an oligonucleotide microarray for rapid diagnosis of avian influenza, Newcastle disease, infectious bronchitis, and infectious bursal disease that will be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections.
A/Tasmania/2004/2009 (A[H1N1]pdm09), A/Perth/16/2009 A(H3N2) and B/Brisbane/1/2007 (B/Florida/4/2006-like; B/Yamagata lineage) viruses were passaged in the allantoic cavity of embryonated hen eggs and stored at −80°C. Infectious virus was measured by 50% tissue culture infectious doses (TCID50) assays, using hemagglutination as the read-out.
Viruses can be identified by a wide range of techniques, which are mainly based on comparisons with known viruses. Historic methods include electron microscopy, cell culture, inoculation in suckling mice and serology, but these methods have limitations. For example, many viruses cannot be cultivated, excluding the use of cell line isolation and serologic techniques, and can only be characterized by molecular methods. In 2011, Bexfield summarized the different molecular techniques that identify new viruses such as microarray, subtractive hybridization-based and PCR-based methods. Although these techniques have allowed the discovery of many viruses, the prior knowledge of similar viruses is required. Recent advances in sequence-independent PCR-based methods have overcome this limitation, and Sequence-Independent Single Primer Amplification (SISPA), Degenerate Oligonucleotide Primed PCR (DOP-PCR), random PCR and Rolling Circle Amplification (RCA) methods have emerged. The end result of most of these PCR methods is amplified DNA that requires definitive identification by sequencing.
Novel DNA sequencing techniques, known as “Next-Generation Sequencing” (NGS) techniques, are new tools providing high-throughput sequence data with many possible applications in research and diagnostic settings. With the development of different NGS platforms, it is now possible to sequence all viral genomes in a given sample without previous knowledge about their nature with the use of sequence-independent amplification followed by high-throughput sequencing. This combination of techniques, known as viral metagenomics, allows the discovery of completely new viral species within a complex sample and, due to decreasing costs, are nowadays exponentially increasing.
NGS techniques are able to generate a huge number of sequences, ranging from thousands to millions of reads, in only one reaction. In order to fully benefit from this depth of sequencing to identify infectious agents present in a given environment, host DNA/RNA should previously be removed from samples. Preliminary treatments are therefore required prior to nucleic acid amplification and sequencing, mainly based on nucleases treatments and/or viral purification by ultracentrifugation on sucrose, cesium chloride or glycerol gradients. These strategies are known as “Particle-Associated nucleic acid amplification”, i.e., they try to isolate intact (i.e., infectious) viral particles from their environment, protected from the action of nucleases. Subsequent low amount of nucleic acids have required the use of Sequence-Independent Amplifications (SIA) such as SISPA, DOP-PCR, random PCR, RCA. Although these techniques allow generating enough nucleic acid material for sequencing, their main disadvantage remains that they distort quantitative analyzes by introducing bias of amplification in viral diversity studies. As a consequence, quantitative analyses of the composition of resulting viromes may not reflect the reality.
In diagnostic virology, in either human or veterinary medicine, viral metagenomics has allowed the discovery of causative viral agents of disease conditions. Virome analyses have also been conducted to describe the baseline viral diversity in healthy human conditions, as a prior knowledge before studying the viral flora of pathologic conditions.
In the same way, the use of viral metagenomics as a tool for arboviral and zoonotic disease surveillance requires prior knowledge of the viral diversity associated to hematophagous arthropods and animals in close contact with humans. This review thus summarizes our current knowledge of the diversity of viral communities associated with several arthropods, wildlife and domestic animals and present its potential applications for the surveillance of zoonotic and arboviral diseases.
PCV2 is divided into five genotypes according to the Cap gene sequence: PCV2a, 2b, 2c, 2d, and 2e. Moreover, the PCV2b genotype is classified into three clusters, 1A to 1C, and the PCV2a genotype is subdivided into five clusters, 2A to 2F. Recently, a retrospective study of PCV2 infection between 1996 and 1999 in China revealed a novel genotype PCV2f which shared lower sequence identity with the other known genotypes. Since the discovery of PCV2 in the late 1990s, the virus has continued to evolve, and two major genotype shifts have been observed. The first genotype shift in PCV2 was from PCV2a to PCV2b in 2004/2005. Since 2012, the predominant PCV2b has been gradually replaced by the PCV2d genotype in North America, China, South Korea and Uruguay. Besides, PCV2F becomes the predominant genotype in the PCV2a cluster in China.
It has been reported that concurrent infections with different PCV2 genotypes have been detected in the same pig, resulting in inter- and intra-genotype recombination. One hundred and eighteen PCV2-positive DNA samples isolated from diseased pigs were analyzed using a modified differential polymerase chain reaction (PCR) assay, and the results indicated that the coexistence rates of PCV2 genotypes were 32.2% (38/118) in sick pigs. The sequencing results of 38 co-infected samples showed that the coexisting genotypes were PCV2a-PCV2b (12/38), PCV2a-PCV2d (15/38) and PCV2e-PCV2d (11/38). One group reported that the recombination rate of the PCV2 isolates was 27.7% (17/54) in the samples collected from 2006 to 2016 in China, and the recombination mainly occurred in the ORF1 gene of PCV2. Furthermore, co-infections with different PCV2 genotypes may cause more serious disease. In cells infected with replicating viruses, both PCV2a and PCV2b genotypes were equally present. Further studies have demonstrated that pigs with dually heterologous inoculation or naturally infected with multiple PCV2 genotypes or strains displayed more severe lesions. These results suggest that the coexistence of different strains of PCV2 might contribute to the development of more severe clinical symptoms in pigs and more recombination events between strains in the field. Therefore, more studies need to focus on analyzing the recombination trends of PCV2 strains, which may provide a better strategy for vaccine development and vaccination strategy.
Given its continuous exposure to the outside environment, the respiratory mucosa is highly susceptible to viral infection. The human respiratory tract can be infected with a variety of pulmonary viruses, including respiratory syncytial virus (RSV), influenza virus, human metapneumovirus (HMPV), rhinovirus (RV), coronavirus (CoV), and parainfluenza virus (PIV) (1). The severity of disease associated with respiratory viral infection varies widely depending on the virus strain as well as the age and immune status of the infected individual. Symptoms can range from mild sinusitis or cold-like symptoms to more severe symptoms including bronchitis, pneumonia, and even death. RSV is the leading cause of severe lower respiratory tract infection in children under 5 years of age (2). RSV is commonly associated with severe lower respiratory tract symptoms including bronchiolitis, pneumonia, and bronchitis and is a significant cause of hospitalization and mortality in children, the elderly, and immunocompromised individuals (2–6). Similarly, PIV commonly infects children and is a major cause of croup, pneumonia, and bronchiolitis (7, 8). Seasonal influenza infections, most often of the influenza A virus (IAV) subtype, are responsible for 3–5 million cases of severe infection annually (9). Seasonal IAV infections also result in approximately 290,000–650,000 deaths per year, most commonly in either young children or elderly populations (9–11). However, infection with emerging pandemic IAV strains, such as the 2009 H1N1 pandemic strain, primarily induces severe disease and mortality in otherwise healthy adults younger than 65 years of age (12). In contrast, respiratory infection with HMPV, RV, and CoV are most commonly associated with symptoms of the common cold (13–15). Two notable exceptions are severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome (MERS) CoV, which cause acute respiratory distress and mortality in infected individuals (16–18).
Despite their profound impact on human health, most common respiratory viruses lack an approved vaccine. The strategy employed most often in vaccine development is the induction of robust neutralizing antibody responses. However, the hallmark of many respiratory viral infections, including RSV, HMPV, and RV, is the ability for reinfections to occur frequently throughout life (19–21). This suggests that the antibody response to these respiratory viruses may wane over time. Indeed, despite a correlation between pre-existing nasal IgA and protection from reinfection, the development of long-lasting RSV- and RV-specific mucosal IgA responses was poor in infected adults (22, 23). Although IAV-specific neutralizing antibodies are elicited efficiently through either infection or vaccination, IAV vaccine formulations must be redeveloped annually to account for the rapid mutations of HA and NA genes in seasonal strains (24). Therefore, vaccinations that solely promote the induction of neutralizing antibodies may not be optimal in providing protection against many respiratory virus infections. The induction of cellular immune responses has thus far received little attention in respiratory virus vaccine development. CD8 T cells play a critical role in mediating viral clearance following many respiratory virus infections including RSV, IAV, and HMPV (25–27). In addition, recent murine studies utilizing CD8 T cell epitope-specific immunization strategies observed significantly reduced lung viral titers following IAV, RSV, or SARS challenges (28–30). Therefore, the induction of virus-specific CD8 T cell responses has the potential to improve upon the efficacy of current vaccination strategies. Here, we review the current literature on CD8 T cell responses following respiratory virus infections and discuss how this knowledge may best be utilized in the development of future vaccines.
The Japanese encephalitis virus (JEV) is the main pathogen that causes severe encephalitis in humans. JEV belongs to the genus of Flavivirus, which also includes other arboviruses, such as the Dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV is a positive-sense single-stranded RNA virus. The genome of JEV is approximately 11 kb in length, containing a single open reading frame (ORF) flanked by the 5′- and 3′-untranslated regions (UTRs). The ORF encodes a long polyprotein that is cleaved into three structural proteins (capsid [C], pre-membrane [prM], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural proteins make up the infectious viral particle and the nonstructural proteins participate in multiple steps of viral life cycle including viral replication, virion assembly, and immune evasion.
Since the first record of the virus in the late 1800s, JEV has posed a significant threat to global health. It is reported that there are 69,000 cases of JEV infection per year. The average mortality rate caused by JEV can be as high as 30% in the past 30 years, and the proportion of survival with permanent neurological or psychiatric sequelae is approximately 44%. With its epidemic area expansion, JEV affects approximately 25 countries in Asia, and approximately 60% of the population lives with a risk of JEV infection. At present, vaccination is the most effective way to prevent JEV infection. The common vaccines include the inactivated mouse brain-derived vaccine (JE-VAX), inactivated BHK-21 cell-derived vaccine, live-attenuated vaccine (SA14-14-2), inactivated Vero cell-derived vaccine, and the chimeric attenuated vaccine. However, approximately 80% cases of the JEV infection occur in areas covered by the JEV vaccination program due to the failures of immunization strategies or the limitation of vaccines themselves. To date, no clinically approved antiviral agents have been available for the treatment of JEV infection. Furthermore, few randomized clinical trials have tested treatments for JEV. In the past 30 years, only six agents for the treatment of JEV infection have been tested by clinical trials, but none of them have been found effective. Therefore, it is essential and urgent to find a safe and effective treatment.
Drug repurposing has recently become a very popular method for drug discovery; drug repurposing provides old drugs (including approved drugs, under research drugs, and withdrawn drugs) with new indications by exploring new molecular pathways and targets. With this strategy, finding an alternative agent to treatment JEV infection will be fast and safe. During the past decades, the traditional method for drug repurposing depends on high-throughput screening of small-molecule libraries consisting of approved and developing drugs. However, the success rate of high-throughput screening for effective repurposed drugs has dropped dramatically. With the development of computational methods, the high-throughput omics data, virtual screening, and text mining have been used for drug repurposing. One of the computational methods for antiviral drug repurposing is to target pathogen to block its lifecycle. Using the crystal structure of the E protein and the strategy of structural-based virtual screening (SBVS), Leal et al. identified a compound exhibiting marked antiviral activity against DENV with its EC50 being 3.1 µM. The other methods for antiviral drug repurposing are targeting host genes to inhibit pathogen infection. Identifying the proteins participating in the pathogen infection process is the basis of host-targeted drug repurposing approaches. Quan et al. identified 170 Mycobacterium tuberculosis (Mtb) infection-associated genes by theoretical genetic analysis, and obtained high potential anti-Mtb drugs by targeting these genes. Therefore, it is possible to rapidly identify effective therapeutics for JEV infection using the method of drug repurposing through targeting JEV-susceptible genes.
Systems biology has been used to identify the pathogenic mechanisms of complex human diseases by integrating genetic variation, genomics, pathways, and molecular networks. The advent of systems biology provides a powerful method for facilitating drug development and drug repurposing. The representative algorithms used in the systems biology field include GeneRank and HotNet2. In this study, we applied the methods of HotNet2 and GeneRank to identify the genes essential in JEV infection (Figure 1). Additionally, we analyzed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) athway enrichment of these genes to validate our results. Using the information of the drug-target, we obtained the agents that have a potential treatment effect on JEV infection. We found that multiple targets of bortezomib play critical roles in the progress of JEV infection based on the analysis of the PheWAS data of encephalitis and of the gene expression data of human microglial cells after JEV infection. Furthermore, we investigated the effect of bortezomib using a JEV-infected mouse model. Overall, our research provided a novel agent for the treatment of JEV infection.
Feline immunodeficiency virus (FIV) is a lentivirus closely related to human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) that naturally infects numerous wild and domestic feline species. Individual feline species typically harbor genetically-distinct species-specific FIV strains [1–3]. Infection with the domestic cat (Felis catus) strain of FIV (FIVFca) results in CD4+ T-cell depletion and pathogenic disease which progresses to AIDS-like immune dysfunction and ultimately death. FIV infection and disease in domestic cats bears many similarities to HIV infection and AIDS in humans including similar routes of infection, cell and tissue tropism, clinical symptoms and course of disease. Thus, FIV infection of the domestic cat is a useful animal model for studying HIV infection and vaccine development. In contrast, FIV infection of nondomestic felid species has little measurable impact on survival of the natural host [2,10–13], but may be associated with long-term immune cell depletion and other disease sequelae [14–17]. Cross-species transmission events are thought to be limited by lack of contact between host species and the action of host-specific cellular restriction factors. Experimental infection of domestic cats with a lentivirus (puma lentivirus (PLV) or FIVPco) native to the cougar (Puma concolor) results in productive yet avirulent infection with no detectable T-cell depletion or clinical disease, providing a model to evaluate mechanisms for restriction of lentiviral cross-species infection. PLV viral load diminishes over time to virtually undetectable levels in circulation and lymphoid tissues with low level infection of the gastrointestinal tract. Intensive sequence analysis of PLV genomes integrated in cat blood cells showed a strong bias toward G-to-A hypermutation, a hallmark of cellular cytidine deaminase-mediated viral restriction [22–25], suggesting that cellular restriction likely plays a role in control of PLV infection in domestic cats. Thus PLV infection is a model for cross-species transmission of a virus which is not well-adapted to long-term replication in the new host and remains nonpathogenic.
Previously we have studied whether infection of domestic cats with PLV can impart resistance to subsequent infection with virulent FIV. Cats infected with PLV 28 days prior to FIV challenge were protected from the marked CD4+ T-cell depletion experienced by cats challenged with FIV alone. PLV/FIV co-infected cats had a unique immunologic profile distinct from FIV single infected cats—including elevated levels of CD8, CD25 and FAS expressing cells and elevated expression of the cytokines IL-4 and IFNγ. In contrast, we did not detect evidence of adaptive immunity to FIV such as neutralizing antibodies or virus-specific cytotoxic T-cells. These data suggest that innate rather than adaptive immune mechanisms are associated with PLV-induced protection from FIV disease. Additionally, examination of FIV population genetics demonstrated that during PLV/FIV co-infection FIV underwent a population bottleneck at approximately three weeks post-infection not observed in FIV single infection. The nature of this bottleneck remains to be determined, but the data are consistent with PLV-induced host restriction of normally virulent FIV. Collectively, these studies suggest that host innate immunity has a role in mediating PLV-induced modulation of FIV infection and disease. However, the exact nature of PLV-induced protection remains to be elucidated.
Our previous studies aimed at determining the mechanism(s) of PLV-induced protection from FIV disease have focused largely on timepoints early after FIV infection (<120 days post-PLV infection) using peripheral blood mononuclear cells (PBMC) for analysis. In this study we focused on PLV and FIV infection and innate immune response in multiple infected cat tissues at a later chronic infection timepoint (159 days post-PLV infection) for the purpose of detecting changes in infection and immunity which may be specific to important tissue reservoirs of infection and provide insight into PLV mediated protection. Further, evaluating distribution of both provirus and viral RNA transcription across a suite of tissues allows analysis of the hypothesis that PLV infection alters the distribution and tissue reservoirs of susbsequent FIV infection.
We found that PLV co-infection altered the magnitude and variability of FIV infection and innate immunity in tissues compared to FIV single infection—to a remarkable extent considering our analyses were conducted nearly five months following co-infection. While somewhat surprisingly, FIV proviral load tended to be higher in tissues from co-infected versus single-infected animals, viral transcripts tended to be lower in the co-infection state. Comparison of FIV proviral load and FIV mRNA load among single-infected versus co-infected tissues indicated that PLV co-infection limits FIV productive infection (viral mRNA expression) relative to FIV proviral load. Collectively, these data suggest that PLV-induced protection from FIV disease may be at least partially mediated by persistent alterations of innate immunity resulting in limitation of FIV productive infection. It is also possible that restriction of target cell populations via PLV-induced immune activation or alteration of susceptibility for other reasons results in an altered FIV infection landscape. This hypothesis is supported by the finding that viral and cytokine transcription rates were more variable during single FIV infection, and as reported previously, FIV replication in the face of previous PLV infection is highly constrained during acute infection. If during co-infection, FIV is restricted to a cell type with longer half-life that is less permissive for viral replication, we would predict an outcome similar to that observed in this study. These experiments pose a new paradigm for assessment of protective immunity against HIV/AIDS—namely that perturbation of early innate immune parameters and circulating cell phenotype can alter the outcome of a virulent lentiviral infection.
Four distinct papillomavirus sequences were identified in two bat species, E. helvum (Eidolon helvum papillomavirus 8; EhPv-8) and T. perforatus (T. perforatus papillomavirus 1, 2 and 3; TpPv-1, -2, -3). Complete coding sequences were recovered for EhPv-8 (acc. no. KX434763, 6985 bp), TpPv-1 (acc. no. KX434764, 7180 bp) and TpPv-3 (acc. no. KX434767, 7083bp). EpPv-8 shared 63% aa similarity to E. helvum papillomavirus type 1 across the DNA helicase protein (E1). TpPv-1, TpPv-2, and TpPv-3 (acc. no. KX434766, partial genome) shared 44%, 47% and 44% aa similarity across the E1 protein with human papillomavirus 63, Miniopterus schrebersii papillomavirus 1, and Castor canadensis papillomavirus 1, respectively (Fig 7).
Currently, most methods of AIV, NDV, IBV, IBDV and other avian viral agents detection are adapted to specific detection of one agent in a sample. Multiplex RT-PCR is successfully used for detection of AIV and its subtypes [19, 20] and for diagnosing double infections such as combination of NDV and AIV. Also methods with use of multiplex real-time RT-PCR for AIV, NDV and IBV subtypes differentiation have been developed [22–24]. At present development of a test based on microarray technology for simultaneous detection of AIV, NDV, IBV and IBDV in one sample is important for poultry industry in the Republic of Kazakhstan.
Use of microarray improves quality and shortens the analysis duration in molecular diagnosis of infectious diseases and therefore is employed as an independent method in screening for several genes of large numbers of pathology samples [25–27]. There are biochips for influenza diagnosis that allow screening not only for HA and NA, but for M and NP genes of influenza A virus [25, 28]. In identification of NDV molecular methods with use of oligonucleotides specific to conservative regions of NP-gene of NDV were used. Recently VP2 gene region of IBDV is successfully used in synthesis of oligonucleotide primers and probes from highly conservative regions for molecular diagnosis [30–33]. Molecular methods for IBV diagnosis are oriented at using more conservative sequences located in S1 and S2 genes of IBV [34, 35].
In the proposed microarray probes were developed on the basis of conservative regions of gene fragments encoding NP and M (AIV), NP (NDV), VP2 (IBDV), S1 (IBV) array proteins from Genbank Database. All viral gene fragments demonstrated high rate of conservatism and therefore the test is universal for detecting AIV, NDV, IBV and IBDV strains. So, high homology of nucleotide sequences of gene regions encoding AIV, NDV, IBV and IBDV array proteins compared to GenBank data confirms specificity of the developed microarray for rapid diagnosis of avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease.
Total analysis duration without time required for the viral RNA extraction is 5–6 h, and 16 specimens can be simultaneously assayed. Duration of the assay with use of the proposed microarray is not longer than in other molecular methods and simultaneous testing of samples for AIV, NDV, IBV and IBDV provides its advantage over other methods.
Various methods have been developed for the diagnosis of bird infection, such as virus isolation in cell culture, embryonated chicken eggs, or young specific-pathogen-free (SPF) chickens and localization of the virus in infected tissues by electron microscopy, fluorescence assay, agar immunodiffusion, antigene-capture enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry. All these methods have disadvantages, such as being time consuming, labor intensive, expensive, or nonspecific. These methods lack the ability to detect low levels of antigens in tissues [36–40].
In the present study field samples (122 in total) were used to test effectiveness and reliability of the microarray. Nevertheless, positive result of using molecular and biological methods, being very important in emergency cases, should always be confirmed by the method of virus isolation.
The results of the study show that diagnostic sensitivity (99.16%) and diagnostic specificity (100%) of the DNA microarray are comparable with the same of the real-time RT-PCR (99.15 and 100%, respectively).
Diagnostic effectiveness as percentage ratio of true results to the total number of obtained results for the developed DNA microarray and real-time RT-PCR was 99.18%.
Analysis of the obtained data shows that the microarray test for rapid diagnosis of avian infections demonstrates the effectiveness comparable to that of the molecular method real-time RT-PCR and is more rapid and less resource-consuming owing to its ability to detect simultaneously AIV, NDV, IBV IBDV positive samples in the course of one experiment. Universality of the test makes it suitable for wide use in veterinary laboratories for prompt detection of avian infections.
The successful replication of a viral agent in a host is a complex process which consists of a number of interactions, most of them related to the coevolution of pathogen and host. This coevolution often leads to a species specificity of the virus and can make interspecies transmission difficult. Therefore, natural host range switches by viruses are rare events. However, when they occur the results can become severe because the viruses may then spread widely through non previously adapted, and therefore immunologically naïve host populations.
Upon transmission to a new host species, viruses must usually adapt to a new genetic and immunologic environment in order to replicate and spread to other individuals within the species. The high rates of mutation and replication of RNA viruses, such as human immunodeficiency virus (HIV) and influenza, facilitate the occurrence and fixation of those mutations that become beneficial under certain conditions. Viral adaptations to new hosts primarily manifest as amino acid substitutions which can allow more efficient virus cell entry into the new host, block interactions with detrimental host proteins or promote escape from both the new and the old host's immune responses.
Influenza A is the paradigm of a virus capable of interspecies and interclass transmission. Those viruses are found in humans as well as in other animals, including swine, horses and birds, waterfowl being considered the natural reservoir. Subtypes of Influenza A are distinguished by the two surface glycoproteins: haemagglutinin (HA) and neuraminidase (NA). Periodically a subtype of influenza can make the shift from aquatic birds to humans, possibly through an intermediate host, resulting in a widespread pandemic in an immunologically naïve population. These antigenic shifts can occur either through the transfer of an entire virus from one host to another or through a reassortment process where genomic segments of the avian virus mix with genomic segments of a virus currently circulating in humans.
A number of proteins have been implicated in determining host specificity of the virus. Influenza haemagglutinin binds to sialic acid linked to galactose on the surface of the targeted cell, and the differing nature of the sialic acid-galactose linkages in birds and humans provides an important barrier to host shift events. In this sense, a number of amino acid substitutions have been produced in influenza haemagglutinin to adjust to the different receptors. Neuraminidase, the protein responsible for cleaving the haemagglutinin from the receptor surface, also seems to be adapted to the particular sialic acid linkages. Proteins in the viral replication complex (PA, PB1, PB2 and NP) have also been implicated in limiting host range by restricting replication and intra-host spread in mammals (for a review see). In particular, a specific substitution in the PB2 gene has been identified as crucial for replication and intra-host spread in mammals.
Severe acute respiratory syndrome coronavirus (SARS-CoV) is a recently identified human coronavirus. The extremely high homology of the viral genomic sequences between the viruses isolated from humans (huSARS-CoV) and those of palm civet origin (pcSARS-CoV) suggested possible palm civet-to-human transmission. Genetic analysis revealed that the spike (S) protein of pcSARS-CoV and huSARS-CoV was subjected to the strongest positive selection pressure during transmission, and there were six amino acid residues within the receptor binding domain of the S protein that were potentially important for SARS progression and tropism. It has been demonstrated that the double substitution of two amino acid residues of pcSARS-CoV for those of huSARS-CoV made pcSARS-CoV capable of infecting human cells, suggesting that these two residues are involved in the palm civet-human transmission.
Under certain circumstances, even a genetically stable DNA virus can gain the mutation required to adapt to a new host. That is the case of canine parvovirus (CPV) which emerged in 1978 as the cause of new enteric and myocardial diseases in dogs. The new virus spread globally in a pandemic and has since remained endemic in dogs throughout the world. Phylogenetic analysis showed that all CPV isolates obtained so far, termed CPV type 2, descended from a single ancestor closely related to the feline panleukopenia virus (FPV) which infects cats, mink and raccoons, but not dogs or cultured dog cells. FPV and CPV type 2 isolates differ by as little as 0.5% in DNA sequence and it is possible that changes of only two amino acid residues in the capsid protein could have introduced the canine host range. During 1979 a CPV variant (CPV type 2a) emerged, spread worldwide within 1 year and replaced the CPV type 2 strain. CPV type 2a contained five substitutions in the capsid sequence compared to CPV type 2 and also infected and caused disease in cats. Therefore, the emergence of CPV seems to have been a multistep process, where a small number of mutations in the capsid protein gene allowed the virus to efficiently infect and spread within a new host order.
Viruses of lower vertebrates include a large number of viral agents, belonging to different viral families and genera, with RNA and DNA genomes, and displaying different host specificities. In fact, some viruses have a very narrow host range, whereas others are known to be able to infect a wide range of species. The wide host range suggests that, in any moment along the viral evolution, those viruses may have been involved in different host shift events. In the present review we will focus on well documented or hypothesized cases of host shift as well as variations in host range for the genera Ranavirus, Novirhabdovirus, Betanodavirus, Isavirus and several herpesvirus. However, the suspicion for interspecies transmission in other fish viruses remains.
Worldwide, the use of vaccines is seen as critical for the prevention and control of many economically damaging outbreaks of poultry diseases. In Caribbean countries, the use of vaccination to prevent poultry diseases is variable and is mainly influenced by the size of the poultry industry in the various countries. Many of the larger poultry-producing CARICOM countries (for example, T & T, Jamaica, Belize, Barbados, and Guyana) with large poultry (broiler and layer) operations have structured and rigid regimes of vaccination in place, while smaller island states (for example, Grenada and St. Lucia), with smaller poultry and egg production operations, often do not vaccinate their poultry. The larger intensive broiler and layer production units in the Caribbean routinely vaccinate their birds against IBV, NDV, and IBDV, whereas some smaller semi-intensive and backyard operations often do not carry out vaccination against these three viral pathogens. Routine vaccination is not carried out in the region against other viral pathogens (AIV, ILTV, APV, FADV Gp1, and EDSV) included in this review, although occasional vaccination against FADV Gp1 has been carried out in the face of disease outbreaks. All reports/publications describing the detection of viruses and outbreaks of disease in both vaccinated and unvaccinated poultry were reviewed. When information pertaining to vaccination history was given in the relevant report/publication, this information was included.
EV-D68 preferentially causes severe respiratory symptoms in children and adults that have a prior history of asthma. Thus, in addition to naïve mice, HDM-sensitized and -challenged mice also been studied. In mice with allergic airways disease, EV-D68 enhances allergen-induced type 2 inflammation with increased expression of lung IL-5, IL-13 and Muc5ac and augmentation of bronchoalveolar lavage fluid eosinophils and airway responsiveness.
Streptococcus equi subspecies equi M protein antibody titers
Streptococcus equi subspecies equi M antibody titers on all 48 horses at 8 weeks after infection are shown in Figure 1. One (2%) had a titer of 1:400, 7 (15%) had a titer of 1:800, 9 (19%) had a titer of 1:1600, 7 (15%) had a titer of 1:3200, 8 (17%) had a titer of 1:6400, 12 (25%) had a titer of 1:12 800, and 4 (8%) had a titer of ≥1:25 600. The mean age was similar for each SeM antibody titer. Figure 1 shows the distribution of SeM antibody titers of horses with and without any clinical signs of S. equi infection. The median SeM antibody titers for horses with and without clinical signs were 1:12 800 and 1:1600, respectively. A correlation for horses with clinical signs to have SeM antibody titers ≥1:6400 (Pearson's R = 0.76, P < .05) was noted. S. equi M protein antibody titers of affected horses were compared to their specific clinical syndromes (Figure 2). The median SeM antibody titer was 1:6400 for uncomplicated cases and was 1:12 800 for both persistent GP infection and complicated cases. A correlation for horses with persistent GP infection or complicated cases to have SeM antibody titers ≥1:6400 (Pearson R = 0.58, P < .05; Pearson R = 0.45, P < .05, respectively) was also found. Six out of 16 (38%) horses with very high SeM antibody titers ≥1:12 800 had evidence of complicated cases (Figure 2). S. equi M protein antibody titers of nonsurvivors (n = 4) were all ≥1:6400. For this outbreak, sensitivity and specificity for a SeM antibody titer ≥1:12 800 detecting complications were 75% (95% CI 45‐105) and 43% (95% CI 23‐64), respectively. Using a cutoff of ≥1:6400 instead, the sensitivity and specificity for detecting complications were 100% (95% CI 100‐100) and 22% (95% CI 5‐39), respectively. All horses with persistent GP infection had SeM antibody titers ≥1:6400, and 8 of 12 had a SeM antibody titer ≥1:12 800. Sensitivity and specificity for SeM antibody titer ≥1:12 800 detecting persistent GP infection were 67% (95% CI 40‐93) and 42% (95% CI 20‐64), respectively. Using a cutoff of ≥1:6400 instead, the sensitivity and specificity for detecting persistent GP infection were 100% (95% CI 100‐100) and 26% (95% CI 7‐46), respectively.
Of the 8 vaccinated horses, 2 had a titer of 1:800, 1 had a titer of 1:1600, 2 had a titer of 1:3200, 1 had a titer of 1:6400, and 2 had a titer of 1:12 800. The median titer of vaccinated horses (1:3200) compared to unvaccinated horses (1:6400) was not significantly different (P = .19).
At 12 weeks after infection, SeM antibody titers were measured on 18 horses. Previous titers on these horses were 1:800 (n = 1), 1:3200 (n = 1), 1:6400 (n = 3), 1:12 800 (n = 11), and > 1:25 600 (n = 1). At the 12 week time point, 1 had a titer of 1:400, 5 had a titer of 1:3200, 9 had a titer of 1:6400, and 3 had a titer of 1:12 800 (Figure 3). One horse with a titer of 1:12 800 was being treated for metastatic abscess formation, the second horse with a titer of 1:12 800 had a persistent GP infection with no evidence of other complications, and the third horse with a titer of 1:12 800 had no clinical evidence of disease or complications. Two other horses at 12 weeks after infection had a persistent GP infection and had a SeM antibody titers ≤1:6400. Fifteen out of 18 horses at this time point had a decrease in SeM antibody titer. At 28 weeks after infection, SeM antibody titers were measured on 36 horses. This included all horses that were still on the property or in the area. Eight horses were lost to follow‐up and 4 had been euthanized secondary to S. equi infection. At the 28 week time point, 3 of 36 (8%) had a titer of 1:400, 9 (25%) had a titer of 1:800, 18 (50%) had a titer of 1:1600, 4 (11%) had a titer of 1:3200, 2 (6%) had a titer of 1:6400, and 0 (0%) had titers ≥1:12 800 (Figure 3). No horse at this time point had evidence of clinical disease. Thirty out of 36 horses demonstrated a decline in SeM antibody titer. There was a decline (r = −0.44, P = .001) in mean SeM reciprocal antibody titer at each time point: 7292 (range 400‐25 600) at 8 weeks after infection, 6244 (range 400‐12 800) at 12 weeks after infection, and 1744 (range 400‐6400) at 28 weeks after infection (Figure 3).