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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.
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).
The bovine respiratory syncytial virus (BRSV) has been recognized as a pathogen in cattle responsible of an acute respiratory disease syndrome in beef and dairy calves since the early 1970s. The impact of BRSV infection on the cattle industry results in economic losses due to the morbidity, mortality, treatment and prevention costs that eventually lead to loss of production and reduced carcass value.
BRSV is an enveloped, non-segmented, negative-stranded RNA virus belonging to the Pneumovirus genus within the subfamily Pneumovirinae, family Paramyxoviridae. The BRSV virion consists of a lipid envelope containing three surface glycoproteins (glycoprotein [G], the fusion protein [F] and the small hydrophobic protein [SH]) (Figure 1). The envelope encloses a helical nucleocapsid composed by the nucleoprotein (N), the phosphoprotein (P), the viral RNA-dependent polymerase protein (L) the M protein and a transcriptional anti-termination factor known as M2-1. The genomic RNA (~15,000 nucleotides in length) also encodes an RNA regulatory protein M2-2 and two non-structural proteins, NS1 and NS2.
BRSV is closely related to human RSV (HRSV), and the epidemiology and pathogenesis of infection between these two viruses share some similarities and also many differences. The similarities between the two viruses have facilitated the unveiling of some of the mechanisms by which BSRV can cause disease. However, the means used by the virus to warrant transmission among individuals within and between herds have remained elusive.
Understanding of the global epidemiology and molecular epidemiology of BRSV has significantly improved over recent years. In this review, we discuss various aspects of the epidemiology and molecular epidemiology of BRSV as well as their relationship with viral evolution.
Metapneumovirus was first recognized in 2001 in the Netherlands from nasopharyngeal aspirates collected during a 20-year period in 28 hospitalized children and infants with acute respiratory tract infection (RTI) having signs and symptoms similar to that of RSV infection. The virus genomic sequence was identified by using a randomly primed PCR protocol and revealed to be closely related to the avian pneumovirus, a member of the Metapneumovirus genus, in the Paramixoviridae family, Initial studies following the first hMPV identification indicate that it causes upper and lower RTIs in patients of all ages, but mostly in children aged below 5 years. A large epidemiological retrospective study examined nasal washes collected over a 20-year period during acute respiratory illnesses in an outpatient cohort of children. Over the entire study period, hMPV was detected in 1%-5% of pediatric upper RTIs (UTRIs), with variation from year to year. Several reports indicate that hMPV is a commonly identified cause of pediatric lower RTIs, and is second only to RSV as cause of bronchiolitis in early childhood. While bronchiolitis, is the most common presentation of hMPV illness, other reported syndromes have included asthma exacerbation, otitis media, flulike illness, and community-acquired pneumonia. Several studies have found hMPV –RSV co-infection rates of approximately 5-14%. Nevertheless, in a study conducted in the Netherlands in children admitted to hospital for lower RTIs (LRTIs), no virus co-infection between RSV and hMPV was detected. Different controversial reports suggest an association between RSV-hMPV coinfection and an increase in the disease severity or the absence of an association between dual infection and disease severity. Greensill and colleagues reported a 70% rate of co-infection with hMPV in a cohort of infants with critical RSV bronchiolitis who required intensive care in the United Kingdom, suggesting that dual infection with RSV and hMPV may predispose for a more severe disease. In another study from the United Kingdom, hMPV and RSV co-infection was associated with increased disease severity and higher risk of admission to the pediatric intensive care unit. Similar findings are supported by other studies suggesting that in young children, coinfections with RSV and hMPV are more severe than infections with either RSV or hMPV alone, requiring a longer hospitalization and supplemental oxygenation. However, such synergistic association has not been found in other population-based and case–control studies of hospitalized children. In particular, two studies evaluated the epidemiology of hMPV coinfection in children with LRTI caused by RSV and demonstrated no hMPV and RSV co-infection in mechanically ventilated children suggesting that co-infection with hMPV is not associated with a more severe course of RSV-LRTI. In addition, in a prospective 2-year study in hospitalized infants with acute respiratory diseases, the role of RSV as a major respiratory pathogen was not influenced by the co-circulation of other emerging viral agents with similar seasonal distribution. In particular, RSV-hMPVs co-infections were significantly observed in less severe respiratory disease when compared to unique RSV infections. The possible synergistic interaction between hMPV and the severe acute respiratory syndrome (SARS) coronavirus was also suggested during the 2003 SARS outbreak in Hong Kong and Canada. In one case report, in an infant with SARS CoV infection, fatal encephalitis was correlated with hMPV infection as hMPV RNA was detected post-mortem in brain and lung tissue. Nevertheless, in experimental studies performed in macaques, a synergy between hMPV and SARS was not confirmed. In addition, infections of hMPV with respiratory viruses different from RSV, have also been occasionally reported but no sufficient data are available to discuss epidemiology or association with clinical disease presentation (Table 1).
Human Bocavirus (HBoV) was discovered in 2005, in Sweden by Allander and colleagues by using a large-scale molecular viral screening technique including DNase sequence-independent single-primer amplification. Since initial observations, several studies have reported the prevalence of human Bocavirus infection all over the world ranging from 2 to 21.5%, mainly in children younger than 3 years of age where it has been associated with upper and with lower RTIs. In a study from Norway, HBoV was detected in 12% of children with RTI and it was the fourth most common virus after RSV, HRV and hMPV. Recently, in children with radiographically confirmed community acquired pneumonia in which 17 respiratory viruses were tested during the acute phase of the disease, HBoV was the most frequently detected virus after RSV and HRV. Since the discovery of the first HBoV (HBoV1), three other related bocaviruses (HBoV2, 3 and 4) have been identified in stool samples and associated with gastrointestinal diseases. Serological studies on HBoV1 are in line with molecular data.
Serological studies have shown that the mere presence of HBoV DNA in the respiratory tract is not proof of an acute primary infection. These data are also supported by studies on consecutive respiratory samples showing that HBoV DNA can persist for several months in the respiratory tract. Prolonged viral shedding could explain both data reported in some papers in which HBoV DNA was found more often in asymptomatic than symptomatic cases and the high percentage of co-infections. In fact, HBoV infections tend to be associated with high rates of coinfections with other viral pathogens such as HRV, adenoviruses, RSV, as well as with bacteria such as Streptococcus spp and Mycoplasma pneumoniae[46,54,68-70,77,81-83]. Characteristics of persistence and high frequency of coinfections have led to a debate over its role as a true pathogen. Our current knowledge of HBoV infection suggests that the virus is sometimes a passenger and sometimes a pathogen in acute respiratory tract disease and that diagnosis should not be solely based on qualitative PCR in respiratory samples. Indeed, in many studies a positive correlation was seen between respiratory illness and high copy numbers of HBoV1 DNA or the presence of HBoV1 monoinfection. A study performed by Allander and colleagues suggests that acute HBoV infections are associated with the presence of viral DNA in the blood of patients. In fact, HBoV DNA was reported more frequently in patient blood during the acute symptoms than after recovery. In addition, high load and viremic HBoV infection were associated with respiratory tract symptoms, while detection of a low viral load in the nasopharinx alone resulted to have no clinical relevance. Other studies confirmed that HBoV is the most probable cause of respiratory tract disease if the patient has a single infection and high viral load in NPA and viremia. However, despite these diagnostic challenges it is becoming increasingly evident that HBoV1 is an important respiratory pathogen. Severe and life-threatening disease has been recently well documented in a 8-month-old child with acute respiratory distress attending an emergency department in Germany. Don et colleagues found serological evidence of an acute HBoV infection in 12% of children with pneumonia and in more than half of these cases with single HBoV infection. In most cases a significant rise in IgG antibodies between paired sera was found in children admitted to hospital for radiologically confirmed pneumonia. IgM antibodies were also detected in all but one patient. This study suggests that HBoV may be a fairly common cause of pneumonia in children Table 1.
Due to worldwide occurrence, substantial morbidity and mortality rates, respiratory viral infection which swiftly and easily spread, pose a serious public health problem (1). Human metapneumovirus (hMPV) is one of the etiological agents of acute respiratory tract infections (ARTIs) which can infect people in all age groups (2). It induces clinical symptoms ranging from upper to lower respiratory tract illnesses such as bronchiolitis, bronchitis and pneumonia (3–5). In 2001, HMPV was identified in samples from children with respiratory tract disease for the first time (6). In addition, it is a new member of the family paramyxoviridae, subfamily pneumovirus, genus metapneumovirus (7). RSV is one of the most important respiratory pathogens of childhood, with detection rates reaching 70–85% in hospitalized infants during seasonal winter epidemics worldwide (1, 8). RSV causes severe lower respiratory infections like bronchiolitis or pneumonia in infants and young children (9). Coinfection of hMPV with RSV in infants has been suggested to be a factor that influences the severity of bronchiolitis (9). Phylogenetically, RSV is the closest human virus related to hMPV, and the clinical manifestations of hMPV may share an overlapping spectrum with RSV, so that these two viruses cannot be distinguished by clinical manifestations (4, 7, 9, 10). RSV and hMPV might have similar seasonal patterns, so co-infection is possible (6, 9).
This report describes a case who did not have the respiratory sign and symptoms, while was actually infected with RSV and hMPV simultaneously.
The Paramyxoviridae family within the order of Mononegavirales includes a large number of human and animal viruses that are responsible for a wide spectrum of diseases. Measles virus (MV) is one of the most infectious human viruses known, and has been targeted by the World Health Organization for eradication through the use of vaccines. The paramyxovirus family includes several other viruses with high prevalence and public health impact in humans, like respiratory syncytial virus (RSV), human metapneumovirus (HMPV), mumps virus (MuV), and the parainfluenza viruses (PIV). In addition, newly emerging members of the Paramyxoviridae family – hendra and nipah virus – have caused fatal infections in humans upon zoonoses from animal reservoirs,,. In animals, Newcastle disease virus (NDV) is and Rinderpest virus (RPV) was among the viruses with the most devastating impact on animal husbandry. Members of the Paramyxoviridae family switch hosts at a higher rate than most other virus families and infect a wide range of host species, including humans, non-human primates, horses, dogs, sheep, pigs, cats, mice, rats, dolphins, porpoises, fish, seals, whales, birds, bats, and cattle. Thus, the impact of paramyxoviruses to general human and animal welfare is immense.
The Paramyxoviridae family consists of two subfamilies, the Paramyxovirinae and the Pneumovirinae. The subfamily Paramyxovirinae includes five genera: Rubulavirus, Avulavirus, Respirovirus, Henipavirus and Morbillivirus. The subfamily Pneumovirinae includes two genera: Pneumovirus and Metapneumovirus
. Classification of the Paramyxoviridae family is based on differences in the organization of the virus genome, the sequence relationship of the encoded proteins, the biological activity of the proteins, and morphological characteristics,. Virions from this family are enveloped, pleomorphic, and have a single-stranded, non-segmented, negative-sense RNA genome. Complete genomic RNA sequences for known members of the family range from 13–19 kilobases in length. The RNA consists of six to ten tandemly linked genes, of which three form the minimal polymerase complex; nucleoprotein (N or NP), phosphoprotein (P) and large polymerase protein (L). Paramyxoviruses further uniformly encode the matrix (M) and fusion (F) proteins, and – depending on virus genus – encode additional surface glycoproteins such as the attachment protein (G), hemagglutinin or hemagglutinin-neuraminidase (H, HN), short-hydrophic protein (SH) and regulatory proteins such as non-structural proteins 1 and 2 (NS1, NS2), matrix protein 2 (M2.1, M2.2), and C and V proteins,.
Routine diagnosis of paramyxovirus infections in humans and animals is generally performed by virus isolation in cell culture, molecular diagnostic tests such as reverse transcriptase polymerase chain reaction (RT-PCR) assays, and serological tests. Such tests are generally designed to be highly sensitive and specific for particular paramyxovirus species. However, to detect zoonotic, unknown, and newly emerging pathogens within the Paramyxoviridae family, these tests may be less suitable. Development of virus family-wide PCR assays has greatly facilitated the detection of previously unknown and emerging viruses. Examples of such PCR assays are available for the flaviviruses, coronaviruses, and adenoviruses. For the Paramyxoviridae, Tong et al. described semi-nested or nested PCR assays to detect members of the Paramyxovirinae or Pneumovirinae subfamily or groups of genera within the Paramyxovirinae subfamily. Although these tests are valuable for specific purposes, nesting of PCR assays and requirement for multiple primer-sets are sub-optimal for high-throughput diagnostic approaches, due to the higher risk of cross-contamination, higher cost, and being more laborious.” Here, a PCR assay is described that detects all genera of the Paramyxoviridae with a single set of primers without the requirement of nesting. This assay was shown to detect all known viruses within the Paramyxoviridae family tested. As the assay is implemented in a high-throughput format of fragment analysis, the test will be useful for the rapid identification of zoonotic and newly emerging paramyxoviruses.
Bovine respiratory disease complex (BRDC) is a major cause of economic losses in the cattle industry worldwide. The most important viral agent include bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza-3 virus (BPI-3V). BRSV, belonging to the genus Pneumovirus within the family Paramyxoviridae, and is one of the most important causes of lower respiratory tract infections in calves; however, adult animals with subclinical infection are the main source of infection, since reinfections are common in the herds. It is highly prevalent in cattle, with a significant economic impact as the most important viral cause of BRDC worldwide. BVDV is a Pestivirus from the family Flaviviridae, which affects the digestive, respiratory, and reproductive systems in different production animals. Clinical signs include pyrexia, diarrhea, reduced production, and highly morbid disease but cause low mortality of infected animals. Infectious bovine rhinotracheitis (IBR) is an important infectious disease of domestic and wild cattle caused by BHV-1. This virus is a member of genus Varicellovirus, which belongs to the Herpesviridae family. Clinical signs infection includes symptoms of inflammatory reactions in respiratory, genital tracts, abortion, and neurological disorders. Betancur et al. found a statistical association between seropositive animals for BHV-1 with respect sex and age in Colombia, while Ochoa et al. reported higher infection in cows older than 5 years of age. BPI-3V is in the genus Respirovirus of the family Paramyxoviridae, which cause serious economic losses in small and large ruminants. Clinical disease is usually mild, with symptoms of fever, nasal discharge, and cough. Betancur et al. reported a statistical association between seroprevalence values for BPI-3V and age groups.
Aguachica, Rio de Oro, and La Gloria municipalities are located in Cesar department, which, in turn, is located in the Northeast of Colombia, and is very important agricultural and fish raising region, being the dual-purpose cattle husbandry one of the most important agricultural components of the regional economy, with a participation of 8% in the cattle national inventory. According to the National Agricultural Institute, the state has a population of 1,305,984 heads of cattle, being 30% located in the three municipalities.
Information about the prevalence of these viral pathogens is available from several countries in which these diseases have been reported. Nevertheless, there is very little epidemiological information on viral pathogens in cattle, mainly in the Northeast region of Colombia. Therefore, the present study was conducted to estimate the seroprevalence of respiratory viral pathogens in dual-purpose cattle and evaluate risk factors in the municipalities of Aguachica, Rio de Oro, and La Gloria in the department of Cesar.
Canine infectious respiratory disease complex (CIRDC) is a major cause of respiratory illness and morbidity. It is a complex condition for disease occurrence, involving multifactorial etiologies. Overcrowding, stress, age and other underlying factors result in interference with the dog's immune response and play a role in disease predisposition [2, 3, 4, 5, 6]. Infectious agents serve as a key factor in promoting the severity of a clinical symptom. The CIRD viruses (CIRDVs) that are commonly associated with CIRDC are canine parainfluenza (CPIV), canine distemper (CDV), canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CaHV-1), canine respiratory coronavirus (CRCoV), and canine influenza virus (CIV) [1, 7, 8, 9].
During the last decade, novel viruses, such as canine pneumovirus (CnPnV) [10, 11] and pantropic canine coronavirus, which induce respiratory problems in dogs, have also been discovered. In addition, canine bocavirus [13, 14], canine hepacivirus and canine circovirus have been detected in respiratory tract samples of dogs showing respiratory illness, but the pathogenic roles of those viruses are poorly determined. Although new viruses have emerged, the common CIRDVs are still important contributors to respiratory disease [17, 18]. Additionally, current core vaccines that are routinely used in dogs prevent some CIRDVs, such as CPIV, CDV, and CAdV-2, but not CaHV-1, CRCoV and CIV. This lack of a vaccine for CaHV-1, CRCoV, and CIV is likely to allow the spread of infection.
Hospital-associated infection (HAI) has recently been described as an “ever-present risk”. The current literature includes reported outbreaks of CPIV and CaHV-1 in animal healthcare facilities, where these nosocomial infections worsened any ongoing disease and enhanced the morbidity and mortality rates. In addition, infections among communities are documented and serve as a source for disease dissemination. Community-acquired infections (CAIs) are those that are acquired without exposure to hospitals or with limited regular exposure with a health care center or both. Currently, CAIs with CIRDVs are believed to have a concomitant effect in respiratory disease [1, 21, 22, 23, 24].
The identification of multiple-viral-induced CIRDC involves the simultaneous rapid nucleic acid-based detection, typically by the reverse transcription polymerase chain reaction (RT-PCR) for RNA viruses and PCR for DNA viruses. These detection methods have led to an increased and better realization of the prevalence of CIRDC worldwide [3, 25, 26]. However, whether the severity of the respiratory disease depends on the number of viral infections is equivocal. Although epidemiological evidence of CIRDC has been reported periodically, the classification among HAIs and CAIs has largely not been evaluated [7, 24].
A comprehensive understanding of the source of infection (HA or CA) and other associated risk factors may help guide the successful implementation of preventive strategies. To emphasize this point, this study focused on the associations between the incidence of all six common CIRDVs, source of infection and the possible related risk factors of the dog's age, sex and vaccine status. Moreover, the relationship between clinical severity and number of viral infections was also evaluated in respiratory-ill dogs during 2013–2016 in Thailand.
Canine infectious respiratory disease (CIRD), synonymous for infectious tracheobronchitis or “kennel cough,” is a disease caused by single or multiple infectious agents with a high worldwide prevalence. Apart from several viral and bacterial agents, the individual health and constitution, vaccination status and environmental influences including husbandry conditions (e.g. crowding of animals) may have an impact on the manifestation of clinical signs. Non‐complicated forms of typically self‐limiting character may be distinguished from complicated forms associated with, possibly fatal, pneumonia. A severe course of disease typically develops as a consequence of coinfections (Chalker et al. 2003, Chvala et al. 2007, Schulz et al. 2014a). However, even isolated viral infections [e.g. canine influenza virus (CIV)] may lead to clinically relevant and sometimes lethal respiratory disease (Crawford et al. 2005). Commonly recognised viral causes of CIRD are canine parainfluenza virus (CPiV), canine adenovirus type 2 (CAV‐2) and canine distemper virus (CDV) (Ford 2012).
However, according to more recent studies, the understanding of this disease complex has changed. New viral pathogens have been detected within the past two decades. In 2003, canine respiratory coronavirus (CRCoV) emerged as a cause of CIRD in a rehoming centre in the UK (Erles et al. 2003). Further studies from several countries detected CRCoV‐specific nucleic acid in dogs suffering from respiratory disease (Yachi & Mochizuki 2006, Decaro et al. 2007, Spiss et al. 2012, Schulz et al. 2014a, Viitanen et al. 2015).
In association with an outbreak of respiratory disease in racing greyhounds in Florida, CIV types closely related to influenza subtype H3N8, originally detected in horses were isolated (Crawford et al. 2005). Subsequently, several studies from different countries detected CIV isolates in respiratory samples and concurrent anti‐CIV antibodies in dogs with mild respiratory signs as well as cases of fatal respiratory disease (Yoon et al. 2005, Daly et al. 2008, Payungporn et al. 2008, Song et al. 2008, Kirkland et al. 2010, Li et al. 2010, Song et al. 2013). Furthermore, isolation of human‐related influenza strains from dogs was successful (Lin et al. 2012). To date, at least seven influenza virus subtypes showing different ability of interspecies and intraspecies transmission have been isolated from dogs. These subtypes are mainly prevalent in the USA (H3N8), Eastern China and South Korea (e.g. H3N2), but some of them have also been reported from European countries (Sun et al. 2013, Xie et al. 2016) supporting the hypothesis that dogs may play a role in transmission and spread of influenza virus among animal species and even humans.
Detection of further viral pathogens (e.g. canine herpesvirus, canine reovirus, canine pneumovirus (CnPnV), pantropic canine coronavirus, canine hepacivirus and canine bocavirus) has been associated with respiratory disease in dogs (Buonavoglia & Martella 2007, Decaro & Buonavoglia 2008, Kawakami et al. 2010, Renshaw et al. 2010, Decaro & Buonavoglia 2011, Kapoor et al. 2011, Kapoor et al. 2012, Mitchell et al. 2013b, Priestnall et al. 2014). However, these viruses are uncommonly detected in dogs with CIRD or their possible role as causative agents is not yet completely determined.
The aim of this study was to assess the prevalence of common CIRD‐associated viruses (CPiV, CAV‐2, CDV) in dogs in and around Vienna, Austria. Although there may be environmental factors specific to this location, our findings are likely to generalise to other locations within Western Europe and possibly further afield. It was further investigated whether emerging viruses (CRCoV and CIV) have a significantly higher prevalence in dogs with CIRD compared to dogs without respiratory disease.
Canine infectious respiratory disease (CIRD) is a multifactorial disease affecting dogs of all ages, which is typically induced by simultaneous viral and bacterial infections. Apart from well-known canine respiratory pathogens, such as canine adenovirus type 2, canine herpesvirus, canine distemper virus, and canine parainfluenza virus, novel viruses are being continuously associated with CIRD occurrence in dogs. These include canine influenza virus, canine respiratory coronavirus, canine pantropic coronavirus–, canine bocaviruses, and canine hepacivirus.
Pneumoviruses (family Paramyxoviridae, subfamily Pneumovirinae, genus Pneumovirus) are enveloped, single-strand negative-sense RNA viruses that are associated with respiratory disease in mammals and birds. Apart from the prototype species human respiratory syncytial virus (HRSV) and its ruminant relative bovine respiratory syncytial virus (BRSV), a murine pneumovirus (MPV), also known as pneumonia virus of mice, is included in the genus Pneumovirus
. This virus, which is only distantly related to human and ruminant RSVs, is a natural rodent pathogen circulating among research and commercial rodent colonies.
Recently, a pneumovirus was associated to respiratory disease in canine breeding colonies in the United States–. The virus, designated as canine pneumovirus (CnPnV), was found to be very closely related to MPV, displaying 95% nucleotide identity with the MPV prototype isolate J3666. Experimental infection of mice with the canine isolate demonstrated that CnPnV is able to replicate in the mouse lung tissue inducing pneumonia. Although the virus was discovered more than 4 years ago, to date there is no complete genomic sequence, which prevents a comprehensive comparative study with other members of the Pneumovirinae subfamily.
The aim of the present manuscript is to report the detection and molecular characterisation of this emerging virus in dogs with respiratory disease in Italy. The full-length genome of a prototype strain was determined and analysed in comparison with American strains and other pneumoviruses.
Canine infectious respiratory disease (CIRD), also known as “Kennel cough”, is an endemic syndrome with multiple viral and bacterial pathogens being involved in disease causation. CIRD is most common when dogs are kept in large groups with continuous intake of new animals, particularly in kennels, but also occurs in singly housed pets. Clusters of infection have also been documented in veterinary hospitals. Common clinical signs include nasal discharge, coughing, respiratory distress, fever, lethargy and lower respiratory tract infections [1, 3–5]. The clinical signs caused by the different pathogens associated with this syndrome are similar, which makes differential diagnosis challenging. Vaccination plays an important role in managing CIRD, and as such, several mono and multivalent vaccines are available; however, despite the widespread use of vaccines to prevent CIRD, clinical disease is still common in vaccinated dogs [2, 6]. Vaccines are commercially available for some, but not all pathogens, which may explain the occasional lack of protection.
The complex multifactorial etiology of this disease involves the traditional CIRD viral and bacterial agents, canine parainfluenza virus (CPIV), canine adenovirus (CAV), canine distemper virus (CDV), canine herpesvirus (CHV), and Bordetella bronchiseptica. New or emerging microorganisms associated with CIRD include canine influenza virus (CIV), canine respiratory coronavirus (CRCov), Mycoplasma cynos and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus). Other novel canine respiratory agents include canine pneumovirus, canine bocavirus, canine hepacivirus [17, 18] and canine picornavirus. There is debate on whether these are truly new emerging pathogens or pre-existing pathogens that are now easier to detect due to the advent of sophisticated molecular diagnostic tools and more frequent diagnostic testing. In recent years, the role of other bacterial agents such as Mycoplasma canis has been questioned [13, 20]. It is unknown whether certain Mycoplasma species such as M. canis act as a commensal, primary or secondary agent.
The detection of co-infections of CIRD pathogens in a single dog has been previously documented [2, 12, 20]. It is most likely that a single pathogen alters the protective defense mechanisms of the respiratory tract, thereby allowing additional pathogens to infect the respiratory tissues. The presence of co-infections may increase disease severity compared with single pathogen infections [2, 5, 20]; however, the prevalence and role of co-infections in CIRD causation remain unclear.
Previous epidemiologic studies of CIRD pathogens in the United States have focused on asymptomatic dogs or on specific pathogens implicated in clinical cases [11, 22, 23]; therefore, a comprehensive etiologic and epidemiologic study involving multiple CIRD agents in a diverse population of dogs has not yet been reported. Understanding disease prevalence facilitates the improvement or establishment of new vaccination programs and alternative treatments. To aid in addressing this question, we conducted a disease surveillance study using molecular methods to detect nine pathogens currently known to be involved in CIRD using samples from symptomatic and asymptomatic dogs that were received at a veterinary diagnostic laboratory. The aim was to attain information regarding pathogen occurrence according to age, seasonality, sex, clinical signs, and vaccination history. This study also aimed to evaluate the role of co-infections in disease severity, and to develop a novel probe-based multiplex real-time PCR assay to simultaneously detect and differentiate M. cynos and M. canis.
Paramyxoviridae is a large and diverse family whose members have been isolated from many species of avian, terrestrial, and aquatic animal species around the world. Paramyxoviruses are pleomorphic, enveloped, cytoplasmic viruses that have a non-segmented, negative-sense RNA genome. The family is divided into two subfamilies, Paramyxovirinae and Pneumovirinae, based on their structure, genome organization, and sequence relatedness. The subfamily Paramyxovirinae contains five genera: Respirovirus, Rubulavirus, Morbillivirus, Henipavirus, and Avulavirus, while the subfamily Pneumovirinae contains two genera, Pneumovirus and Metapneumovirus. All paramyxoviruses that have been isolated to date from avian species can be segregated into two genera based on the taxonomic criteria mentioned above: genus Avulavirus, whose members are called the avian paramyxoviruses (APMV), and genus Metapneumovirus, whose members are called avian metapneumoviruses. The APMV of genus Avulavirus are separated into nine serotypes (APMV-1 through -9) based on Hemagglutination Inhibition (HI) and Neuraminidase Inhibition (NI) assays. Various strains of APMV-1, which is also called Newcastle disease virus (NDV), have been analyzed in detail by biochemical analysis, genome sequencing, and pathogenesis studies, and important molecular determinants of virulence have been identified. As a first step in characterizing the other APMV serotypes, complete genome sequences of one or more representative strains of APMV serotypes 2 to 9 were recently determined, expanding our knowledge about these viruses.
APMV-1 comprises all strains of NDV and is the best characterized serotype because of the severity of disease caused by virulent NDV strains in chickens. NDV strains vary greatly in their pathogenicity to chickens and are grouped into three pathotypes: highly virulent (velogenic) strains, which cause severe respiratory and neurological disease in chickens; moderately virulent (mesogenic) strains, which cause mild disease; and non-pathogenic (lentogenic) strains, which cause inapparent infections. In contrast, very little is known about the comparative disease potential of APMV-2 to APMV-9 in domestic and wild birds. APMV-2 strains have been isolated from chickens, turkeys and wild birds across the globe. APMV-2 infections in turkeys have been found to cause mild respiratory disease, decreases in egg production, and infertility. APMV-3 strains have been isolated from wild and domestic birds. APMV-3 infections have been associated with encephalitis and high mortality in caged birds. APMV-4 strains have been isolated from chickens, ducks and geese. Experimental infection of chickens with APMV-4 resulted in mild interstitial pneumonia and catarrhal tracheitis. APMV-5 strains have only been isolated from budgerigars (Melopsittacus undulatus) and cause depression, dyspnoea, diarrhea, torticollis, and acute fatal enteritis in immature budgerigars, leading to very high mortality. APMV-6 was first isolated from a domestic duck and was found to cause mild respiratory disease and drop in egg production in turkeys, but was avirulent in chickens. APMV-7 was first isolated from a hunter-killed dove and has also been isolated from a natural outbreak of respiratory disease in turkeys. APMV-7 infection in turkeys caused respiratory disease, mild multifocal nodular lymphocytic airsacculitis, and decreased egg production. APMV-8 was isolated from a goose and a feral pintail duck. APMV-9 strains have been isolated from ducks around the world. APMV types -2, -3, and -7 have been associated with mild respiratory disease and egg production problems in domestic chickens. There are no reports of isolation of APMV-5, -8 and -9 from poultry. But recent serosurveillance of commercial poultry farms in USA indicated the possible prevalence of all APMV serotypes excluding APMV-5 in chickens.
APMV-1 (NDV) is known to replicate in non-avian species including humans, although its only natural hosts are birds. APMV-1 infections in non-avian species are usually asymptomatic or mild. Clinical signs in human infections commonly involve conjunctivitis, which usually is transient and self-limiting. Presently, APMV-1 is being evaluated as a vaccine vector against human pathogens. When administered to the respiratory tract of non-human primates, NDV is highly restricted in replication, but foreign antigens expressed by recombinant NDV vectors are moderately to highly immunogenic. One of the major advantages of this approach is that most humans do not have pre-existing immunity to APMV-1. Pre-existing immunity is a potential drawback to using vectors derived from common human pathogens, and also can be a concern for any vector if two or more doses are necessary to elicit protective immunity. Therefore, we are investigating APMV types 2 to 9, which are antigenically distinct from APMV-1, as alternative human vaccine vectors. Also, some of these additional APMV types likely will have differences in replication, attenuation, and immunogenicity compared to APMV-1 that may be advantageous. However, the replication and pathogenicity of APMV-2 to -9 in non-avian species has not been studied. As a first step, we have evaluated the replication and pathogenicity of APMV-2 to -9 in hamsters. In this study, groups of hamsters were infected with a prototype strain of each APMV serotype by the intranasal route and monitored for virus replication, clinical symptoms, histopathology, and seroconversion. Our results showed that each of the APMV serotypes replicated in hamsters without causing adverse clinical signs of illness, although histopathologic evidence of disease was observed in some cases, and also induced high neutralizing antibody titers.
Infectious causes of respiratory disease are common in dogs; canine distemper virus, adenovirus 2, parainfluenza, influenza, herpesvirus, pneumovirus, respiratory coronavirus, Bordetella bronchiseptica, various Mycoplasma spp., and Streptococcus equi var. zooepidemicus are documented causes.1 Molecular diagnostic assays to detect viral and bacterial pathogens are available for these agents. In the United States, modified live vaccines (MLVx) for intranasal (IN) administration are currently available for adenovirus 2, B. bronchiseptica, and parainfluenza. These vaccines do not induce sterilizing immunity, and vaccinated dogs can still develop clinical signs of disease if exposed to virulent strains of the organisms.2 It is currently unknown if IN administration of MLVx against these agents results in positive molecular diagnostic assay results in dogs without previous vaccination. If transient positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays to predict disease caused by these agents in dogs would be decreased.
The purpose of this study was to determine the impact of administration of a single IN dose of a commercially available MLVx adenovirus 2, B. bronchiseptica, and parainfluenza containing vaccine,1 included as part of a facility standard initial vaccination series with a parenteral administration of MLVx containing adenovirus 2, canine distemper virus, and parvovirus, on the results of a commercially available polymerase chain reaction (PCR) panel that amplifies the RNA or DNA of the agents.2
The study was completed with Institutional Animal Care and Use approval. Beagle puppies housed at a commercial breeding facility were used.3 The puppies were housed in a closed facility without contact with other dogs and staff members followed facility barrier precautions over the course of the study. A sterile cotton swab was gently rubbed at the entrance to the external nares, and a second swab gently rubbed against the mucosa of the oropharynx in nonsedated puppies. The swabs were stored separately at 4°C in sterile plastic tubes and stored until shipped by overnight express on cold packs for performance of the molecular assays.2
A total of 12 puppies were screened twice as described, 1 week apart, and all were negative for nucleic acids of the target organisms. Eight puppies were randomly selected for the study and housed in a separate room at the breeding facility for the duration of the study. The puppies were approximately 9 weeks of age when samples were collected on Day 0 before the SQ administration of a MLVx containing adenovirus 2, canine distemper virus, and parvovirus4 and the IN administration of a MLVx1 containing adenovirus 2, B. bronchiseptica, and parainfluenza following manufacturer's instructions (approximately ½ mL per nares). Nasal and pharyngeal swabs were then collected on days 1, 2, 3, 4, 5, 6, 7, 10, 14, 17, 21, 24, and 28 for molecular analysis.2
Sneezing or coughing which have been associated with IN MLVx administration was not noted over the course of the study. Adverse effects associated with the collection of the nasal and oropharyngeal swabs were not noted. At the time the study was performed, the PCR panel utilized also included primers for canine distemper virus RNA; and none of the samples collected over the course of the study were positive. In contrast, nucleic acids of adenovirus 2, B. bronchiseptica, and parainfluenza were amplified from both sampling sites, from all 8 puppies, on multiple days after vaccine administration (Table 1). Because adenovirus 2 was administered in both vaccine types, source of that virus cannot be determined. Increasing numbers of positive samples after vaccination suggest local replication of the vaccinal strains. Decreasing numbers of positive samples over time suggest immune responses inhibiting organism replication. However, quantitative PCR assays normalized to total DNA/RNA on the swab would be needed to confirm or deny these hypotheses. The PCR laboratory adheres to standard operating procedures including use of positive and negative controls thus erroneous results are unlikely.
Agents considered most common for kennel cough syndrome include canine distemper virus, adenovirus 2, parainfluenza, and B. bronchiseptica. However, emerging pathogens include influenza, herpesvirus, respiratory coronavirus, pantropic coronavirus, pneumovirus, and others.1 All of these agents, as well as S. equi var. zooepidemicus and Mycoplasma spp., have been identified as causes of canine infectious respiratory disease. Determination of the agent is important for targeting treatment, particularly for dogs who fail to respond to standard treatment recommendations.2 In animal shelter environments, agent identification is critical for outbreak control and individual case management.3
Bacterial and viral shedding postvaccine administration complicates diagnostic testing and treatment. This is especially problematic in shelter environments as dogs are routinely vaccinated on intake. Viral shedding after vaccination has been detected in cats,4 people,5 cattle,6 pigs,7 and dogs.8 A vaccine strain of B. bronchiseptica was detected via nasal culture up to 4 weeks after IN vaccination of 2–week‐old puppies.9
Commercially available respiratory PCR panels are a relatively cost and time effective diagnostic method for identifying multiple respiratory pathogens. However, amplification of nucleic acids may inherently lead to inaccurate clinical diagnosis because small amounts can be amplified from some animals even though the agent may not be present in sufficient quantity to cause disease. In this study, nucleic acids of all 3 organisms contained in the IN vaccine were amplified from both sites on multiple days via PCR, although no clinical signs of respiratory disease were observed. Thus, interpretation of PCR panel results for diagnoses should include consideration of recent vaccination status and clinical signs of disease. Use of quantitative PCR and wild‐type sequence differences may be able to differentiate between vaccine and pathogenic agent shedding and may be used diagnostically in the future.
Real‐time reverse transcriptase PCR has been used to amplify canine distemper virus RNA in blood, urine, and conjunctival swabs after administration of SQ MLVx.10 In this study, the PCR panel did not amplify distemper virus RNA from nasal or pharyngeal swabs. Further studies are needed to determine whether the negative result is because this strain of vaccine virus does not reach the nasal or pharyngeal tissues or was present at levels below the detectable limit of the assay used.
Bovine respiratory syncytial virus (BRSV) is a pneumovirus of the paramyxoviridae family (Valarcher and Taylor 2007). The infection is prevalent worldwide (Elvander 1996, Paton and others 1998, Uttenthal and others 2000, Klem and others 2013), and BRSV is considered one of the major pathogens of the bovine respiratory disease complex (Griffin 1997, Snowder and others 2006, Brodersen 2010). To diagnose the infection, different strategies can be chosen according to the given situation and the purpose. For larger epidemiological studies, antibody analyses are used (Hägglund and others 2006, Beaudeau and others 2010, Ohlson and others 2010, Klem and others 2013). Infected animals seroconvert and IgG can be measured in serum and milk long after the virus is no longer present. Studies on the longevity of antibodies against BRSV are scarce, but Elvander (1996) found that such antibodies could be detected in the serum of adult cattle for at least two years postinfection.
The virus is reported to spread effectively in a herd during outbreaks, resulting in high within-herd prevalence of antibody positive animals (Rossi and Kiesel 1974, Stott and others 1980, Verhoeff and van Nieuwstadt 1984, Bidokhti and others 2009). Antibodies in bulk tank milk (BTM), pooled milk samples from a selected number of cows or serum from several animals at a selected age have been used to classify a herd or given population (Paton and others 1998, Uttenthal and others 2000, Klem and others 2013, Ohlson and others 2013). Since animals are known to be seropositive for a long time, classification of herds based on serology in young animals (Klem and others 2013), or pooled milk samples from primiparous cows (Ohlson and others 2013), will give a more up-to-date picture than BTM testing. Recent studies show that testing of antibodies in BTM is a reliable tool for identification of BRSV-negative herds (Klem and others 2013, Ohlson and others 2013).
For several important infections in dairy cattle, testing of antibodies in BTM is used as an effective and inexpensive method to determine a herd's exposure to infectious agents (Niskanen 1993, Booth and others 2013). Cost-effective methods to classify the infection status of herds are of interest for several reasons. Such knowledge can be used to reduce the risk of virus transmission when animals are traded and as a diagnostic tool in the investigation of herd health problems. It may also be used in large-scale studies, such as screenings for surveillance or control purposes.
Several methods are available for detection of antibodies against BRSV, including virus neutralisation tests and different ELISAs, of which the indirect ELISA is most commonly used. In a neutralisation test, the level of antibodies in a sample is measured quantitatively. The indirect ELISA is not developed as a quantitative assay; its function is primarily to differentiate between negative and positive samples, and to be semiquantitative at antibody levels where seroconversion in individual animals is usually seen. Outside this range, there may be a weaker correlation between the ELISA optical density (OD) values and the actual level of antibodies in the sample.
The aim of the present study was to see how the level of antibodies against BRSV in BTM can be interpreted with respect to the time at which herds are infected with BRSV. Subsidiary aims were to investigate
A 24 mo old girl, who was originally from Tehran, Capital of Iran, was admitted to a pediatric hospital in Tehran on 29 Jan 2007 with a 2 wk history of fever and erythema of cheeks, lips, throat, and mouth since 4 d ago. In spite of treating with antipyretics, fever still was persistent. On physical examination, the vital signs were as follow: respiration, 22 breath/min; pulse, 104 beats/min and temperature, 38° C. Cardiac, abdominal, neurological and respiratory findings were normal. Laboratory findings were as follows: WBC count, 9.9×103/μl; RBC, 3.77×106/μl; Hb, 10.6 g/dl; Hct, 34.5%; PLT, 443×103/μl; ESR, 90; CRP, +1; Wright/Widal and salmonella Para A/B, Typhi D were negative. Stool exam was normal. In the abdominal sonography, there was no problem and chest X ray was also normal. According to the findings, possible diagnosis was Kawasaki syndrome and treatment with IVIG, aspirin and acetaminophen was started. After one day, fever was resolved but dry cough started at the day four of the admission. Because of the history of contact with chickens and possibility of H5N1, throat swab specimen of the patient was collected and sent to the National Influenza Center in Tehran university of Medical Sciences for influenza surveillance. The specimen was tested for Influenza virus types and subtypes by real time PCR assay using CDC procedure, CDC Real-Time RT-PCR (r.RT-PCR) protocol for detection and characterization of influenza virus (version.2007), but the result was negative. Subsequently, as a part of a project, the specimen was tested for RSV and hMPV by hemi-nested multiplex PCR and parainfluenza viruses type 1–4 by hemi-nested multiplex PCR (11), and adenovirus by hemi-nested PCR (12). In our surprise positive results for RSV and hMPV were observed without any special respiratory sign and symptoms. The test was repeated and the results were confirmed again. The nucleotide sequence of the PCR product of the detected hMPV (the M gene fragment) was submitted to GenBank (Accession no. GQ219792).
Bovine respiratory syncytial virus (BRSV) is one of the main causes of severe pneumonia, interstitial edema, and emphysema in cattle. BRSV is an RNA virus with a non-segmented, single-stranded, negative-sense 15.2 kb genome that belongs to the genus Pneumovirus and family Paramyxoviridae. BRSV infection leads to sudden fever, rhinitis, cough, respiratory distress, abdominal breathing, and decreased appetite.
The previous epidemiological studies reported that the prevalence of BRSV in cattle ranged from 28% to 70%, depending on animal age and environmental conditions. The disease occurs in most countries worldwide and affects cattle of all ages, with younger animals being at the greatest risk of severe BRSV disease.
Diagnosis of BRSV can be confirmed through different laboratory tests including virus isolation and antibody detection in serum and milk. Serological investigations such as serum neutralization test, complement fixation test, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) are commonly used methods for BRSV diagnosis. BRSV can also be accurately diagnosed using reverse transcriptase polymerase chain reaction.
A seroprevalence survey for BRSV in cattle has never been carried out in the Nineveh Governorate, Iraq. Therefore, this study aimed to ascertain the seroprevalence of BRSV in this region and to investigate the risk factors associated with the disease.
Enzyme-linked immunosorbent assay (ELISA) is the enzyme facilitated colorimetric detection of specific protein-antibody complexes. ELISA is extensively used for detection of various proteins at very low concentration in different sample types, thus making it clinically significant in routine diagnosis of pathogens. ELISA for RSV detection is mainly based on targeting RSV F protein (antigen). Recently, several modifications of the classical ELISA technique have been efficiently developed and employed for the detection of RSV. Sensitivity of ELISA was increased by using the high affinity anti-RSV F antibody peptides derived from the motavizumab. Motavizumab is a high affinity antibody based therapeutic against RSV which binds to RSV F protein; however, it was disapproved by the FDA due to higher hypersensitivity in patients receiving motavizumab as compared to palivizumab. Motavizumab also caused urticaria. It is presumed to be a better binding target than the conventional F protein ELISA. This method could prove more effective than the F protein ELISA due to its higher sensitivity to the degradation of motavizumab. In a simple thin layer amperometric enzyme immunoassay, RSV was detected as early as 25 minutes, at low cost with comparative sensitivity to that of real-time PCR and immunofluorescence assays. The assay is based on the development of a double layer sandwich method similar to ELISA. It involves a polystyrene microarray slide coated with monoclonal antibody which captures the antigen (RSV). The antigen-antibody complex is detected by horse radish peroxidase conjugated secondary antibody on a screen printed electrochemical cell coated with the substrate.
Worldwide, there are reportedly about 12 million severe and 3 million very severe cases of lower respiratory tract infection (LRTI) in children. Respiratory syncytial virus (RSV) is a common contributor of respiratory infections causing bronchiolitis, pneumonia, and chronic obstructive pulmonary infections in people of all ages but affects mainly children and elderly along with other viral infections leading to high mortality and morbidity [2–4]. A recent global survey suggests that RSV is not prevalent throughout the year in the tropical regions of the globe, but the incidence peaks in winter with a wide ranging persistence depending on the geographical topology. RSV has been reported to be a prevalent lower respiratory tract pathogen distributed worldwide including countries from both, the developed and developing world. The major countries with RSV seasonal outbreaks include USA, Canada, Cambodia, Mexico, Uruguay, Brazil, Peru, France, Finland, Norway, Sweden, Latvia, Denmark, Germany, Netherlands, Ireland, Italy, Turkey, Iran, Saudi Arabia, Australia, New Zealand, China, Korea, Hong Kong, Japan, India, Pakistan, Bangladesh, Nepal, Taiwan, Vietnam, Myanmar, Thailand, Madagascar, Kenya, Zambia, Nigeria, and Columbia. The data about human RSV described in literature over the years seem to have been unchanged significantly, indicating the severity of RSV and the urgent concern to address this issue. An estimate of more than 2.4 billion US dollars per year is the economic cost of viral lower respiratory tract infection in children.
RSV is a Paramyxovirus belonging to the genus Pneumovirus. RSV is an enveloped, nonsegmented, negative, single stranded linear RNA genome virus (Figure 1). RSV genome (~15 kb) has 10 genes encoding 11 proteins with two open reading frames of gene M2 [7, 8]. Other genes include nonstructural proteins NS1 and NS2 (type I interferon inhibitors), L (RNA polymerase), N (nucleoprotein), P (Phosphoprotein cofactor for L), M (Matrix protein), M2.1 and M2.2 (required for transcription) SH (small hydrophobic protein) G (glycoprotein), and F (fusion protein). Being a negative strand RNA genome virus, RSV packages its own polymerase into the nucleocapsid. Of these proteins, fusion protein (F) is indispensable for viral attachment to the host and entry into the host cell. Although the G protein is responsible for the preliminary attachment, the F protein is necessary for the fusion, budding, and spread of the virus [9, 10]. After attachment to the host cell, RSV fuses with the host cell membrane using the F protein through the 6 helix coiled-coil bundle of the F protein, a mechanism characteristically found in paramyxoviridae members. Although the detailed mechanism of RSV infection is not fully understood, the most accepted mechanism is the entry of the nucleocapsid into the host cell mediated by the F protein through clathrin mediated endocytosis. The RNA is first converted into a plus strand, which serves as the template for replication; whereas for transcription, the RNA genome itself transcribes mRNA for protein synthesis without any intermediate.
Almost all children of 2 years of age will have had an RSV infection and leading to 160,000–600,000 deaths per year. Approximately, 25% to 40% of infants and children at the first exposure to RSV have signs or symptoms of bronchiolitis or pneumonia. These symptoms include rhinorrhea, low-grade fever, cough, and wheezing. The symptoms in adults may include common cold, with rhinorrhea, sore throat, cough, malaise, headache, and fever. It can also lead to exacerbated symptoms such as severe pneumonia in the elderly, especially residing in nursing homes. Usually, children show symptoms within 4 to 6 days of infection and most of them recover in 1 to 2 weeks while serving as carriers of the virus for 1 to 3 weeks. RSV infection in children of nosocomial origin is associated with higher mortality than community-acquired illness because of the pre-existing morbidity [14, 15]. Severe RSV disease risk hovers for the elderly and adults with chronic heart or lung disease or with weakened immune system. RSV infection does not provoke lasting immunity therefore, reinfection is very common. Recently, RSV infection was reported to account for hospitalizations and mortality in elderly people. RSV accounted for severe lower respiratory tract infections including chronic lung disease, systemic comorbidities, and even death. At present, there is no specific treatment for RSV infection ever since its first discovery in 1956. Currently, Food and Drug Administration (FDA) approved prophylactic drug for RSV that includes palivizumab and ribavirin; administered along with symptomatic treatment drugs and supportive care. Currently, techniques used for diagnosis of RSV include ELISA, direct immunofluorescence, western blot, PCR, and real-time PCR. The diagnosis and treatment scenario has significantly changed with the advent of advanced techniques and in-depth understanding of RSV biology, but the execution of these clinical developments in practice requires extensive study and time. This review presents the recent advances in the diagnosis, prevention, and treatment of RSV (Figure 2).
The family Paramyxoviridae consists of enveloped viruses with a nonsegmented, single-stranded, negative-sense RNA genome. These viruses have been isolated from a great variety of mammalian and avian species around the world. Many members of the family cause important human and animal diseases, while the disease potential of many other members is not known. The family is divided into two subfamilies: Paramyxovirinae and Pneumovirinae. The subfamily Paramyxovirinae comprises five genera: Rubulavirus, Respirovirus, Morbillivirus, Henipavirus, and Avulavirus. Subfamily Pneumovirinae is divided into two genera: Pneumovirus and Metapneumovirus. All paramyxoviruses isolated from avian species are classified into the genus Avulavirus, except avian metapneumoviruses, which are classified in the genus Metapneumovirus. Avian paramyxoviruses (APMVs) have been divided into nine different serotypes (APMV 1 to 9) based on Hemagglutination Inhibition (HI) and Neuraminidase Inhibition (NI) assays. APMV-1 comprises all strains of Newcastle disease virus (NDV) and has been well characterized because of its economic importance in poultry industry. As an initial step towards characterizing other APMV serotypes, complete genome sequences of one or more representative strains of APMV serotypes 2 to 9 have been determined–.
The genomes of all paramyxoviruses range between 15 and 19 kb in length and contain 6–10 genes that encode up to 12 different proteins. For members of subfamily Paramyxovirinae, efficient RNA replication requires that the nucleotide (nt) length of the genome is an even multiple of six, known as ‘rule of six’, reflecting the precise packaging of the polynucleotide in the nucleocapsid. The genomes of APMVs are very similar in organization. All the members contain six genes in the order of 3′-N-P-M-F-HN-L-5′ with the exception of APMV-6, which contains an additional small hydrophobic protein (SH) gene in its genome. APMVs encode a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutinin-neuraminidase protein (HN), and a large polymerase protein (L). Two additional proteins called, V and W, are produced by RNA editing of the P gene. The F and HN proteins form spike-like projections on the outer surface of the viral envelope and are the neutralizing and protective antigens of NDV. Significant sequence divergence in these two proteins exists among APMV serotypes. The HN protein possesses receptor-binding and neuraminidase activity; whereas, the F protein is directly involved in membrane fusion which is necessary for the entry of the virus. Homotypic interactions between the HN and F proteins are hypothesized to control initiation of the fusion process for most paramyxoviruses,. The M protein forms the inner layer of the envelope and plays a key role in assembly by interacting with the HN and F proteins as well as ribonucleocapsid,.
APMVs have been isolated from many different avian hosts. APMV-1 is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. NDV isolates vary greatly in their pathogenicity for chickens, ranging from no apparent disease to severe respiratory and neurological disease causing 100% mortality. NDV strains are categorized into three main pathotypes: lentogenic (avirulent), mesogenic (moderately virulent), and velogenic (virulent), based on their pathogenicity in chickens. In contrast, the disease potential of APMV-2 to -9 is not well known because many of these viruses were isolated from birds dying in quarantine, hunter killed, trapped wild birds, apparently healthy poultry or exotic birds. APMV-2 and -3 have been reported to cause significant disease in poultry, whereas the pathogenic potential of APMV-4 to -9 is generally unknown. In general, APMV-2 strains have been isolated from chickens, turkeys and wild birds across the globe and have been found to cause mild respiratory disease, decreases in egg production, and infertility–. APMV-3 strains have been isolated from wild and domestic birds and their infections have been associated with encephalitis and high mortality in caged birds. APMV-5 strains have only been isolated from budgerigars (Melopsittacus undulatus) and cause depression, dyspnoea, diarrhea, torticollis, and acute fatal enteritis in immature budgerigars, leading to very high mortality. Infections from APMV-4, -8, and -9 appear to be restricted to ducks and geese. APMV-6 and -7 infections in turkeys cause drops in egg production and induce respiratory disease. There are no reports of isolation of APMV-5, -8 and -9 from poultry. But recent serosurveillance of commercial poultry farms in the U.S. indicated the possible prevalence of all APMV serotypes excluding APMV-5 in chickens. The pathogenicity of APMV-2 and APMV-3 has been studied in experimentally infected chickens,. However, replication, pathogenicity, and neurovirulence of APMV serotypes 2 through 9 have not been comprehensively studied. Therefore, we characterized in vitro replication of APMVs (growth kinetics and cytopatic effect in chicken fibroblast cells) and their in vivo replication and tropisms by infecting prototype strains of each serotype in two different ages of chickens (1-day-old and 2-week-old chickens) and 3-week-old ducks. Specifically, neurotropism of APMVs was evaluated in primary chicken neuronal cells and brain tissue of 1-day-old chicks.
The NS samples were collected by gently inserting a sterile rayon tipped applicator (Puritan®, Puritan Medical Product, ME, U.S.A.) into the nostril to a one-third depth of the nasal passage, while the OS samples were collected by rolling the swab onto the soft palate. The swabs were immersed in 0.5 mL of 1% sterile phosphate buffered saline (PBS) and stored at -80 °C until assayed.
Respiratory viruses first infect nasal epithelial cells which triggers an antiviral response. This response is driven by type I (α/β) and III (λ) interferons (IFN) that are induced following recognition of viral ribonucleic acid (RNA) by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are cell surface and endosomal PRRs, whilst the RNA helicase receptors (RIG-I and MDA-5) and NOD-like receptors (NOD2), detect viral RNA in the cytoplasm. Signalling via the PRRs activates transcription factors (IRF-3, IRF-7, NF-κB), which lead to the production and secretion of type I and III IFN. The IFNs then bind to cell surface receptors to activate a separate pathway leading to the production of interferon stimulated genes (ISGs) which encode antiviral proteins that combat infection, as well as PRRs and transcriptional factors which further amplify IFN production. The respiratory syncytial virus (RSV), human meta-pneumovirus (hMPV) and human rhinovirus (HRV) are all single stranded RNA viruses but engage differently with cell signalling pathways. In airway epithelial cells RSV and hMPV RNA are primarily detected by RIG-I in the cytoplasm. RSV can also be detected by NOD2. HRV is endocytosed by epithelial cells, and is therefore primarily detected by TLR3 in the endosome early in the infection process and by RIG-I and MDA-5 later in infection following upregulation of these PRRs. The fusion (F) protein of RSV is recognised by TLR4 at the epithelial cell surface.
A successful antiviral response would see the infection limited to the upper airway, as is the case clinically with the majority of viral infections in healthy individuals. Should such a response be deficient, then predominantly upper-airway viral infections, such as HRV, may spread to the lower airways, causing lower respiratory symptoms and an exacerbation of asthma in predisposed individuals.
While definitive data are yet to be produced, experimental HRV infections in adult volunteers initially suggested that asthmatics were more likely to develop lower respiratory infections (LRI) than healthy adults, i.e. less likely to be able to limit viral replication to the upper airways. Subsequent in vitro infection of primary airway epithelial cells from asthmatic and healthy adults with HRV have demonstrated that asthmatic cells produce less IFN-β and IFN-λ making them potentially more susceptible to infection, slower to clear infection, and more susceptible to virus-induced cell cytotoxicity. Deficiencies in the IFN-α response of peripheral blood mononuclear cells and plasmacytoid dendritic cells from asthmatic adults and children has also been observed, in these particular studies, in response to RSV, HRV and Influenza A. It is likely that the overall impaired innate immune response of the asthmatic airway epithelium is a result of deficiencies in the antiviral response of both epithelial cells and immune cells. Childhood, especially infancy, is characterized by developmentally-regulated deficiencies in innate and adaptive immunity. Such deficiencies are likely to increase the risk of viral LRI in children, especially in those at high risk for asthma and allergies.
There is currently no immunoprophylactic strategy to generate broad spectrum protection against diverse respiratory viral pathogens. Conventional strategies to protection against respiratory pathogens have relied on vaccination against individual virus strains or the use of drug based therapies. However, the efficacy of these immunoprophylactic strategies is often significantly limited by either difficulty in engineering effective vaccines to some respiratory viruses such as respiratory syncytial virus, or because of evasion of acquired immunity as a result of antigenic variation of circulating respiratory viral pathogens (H1N1 swine influenza pandemic, 2009). In addition, conventional strategies are generally not applicable to newly emerging viruses (SARS outbreak, 2003) since their sudden appearance does not allow for time to develop a vaccine.
Since many pathogens either directly infect the mucosal epithelium or enter a host by penetration of mucosal surfaces, the induction of effective pathogen-specific immunity at local mucosal sites is becoming a desirable objective of novel vaccination strategies. Mucosal, as opposed to systemic vaccination is often more efficacious against mucosal pathogens due to their ability to induce secretory IgA,. Despite such an advantage, vaccination of mucosal epithelial surfaces, especially those of the lung, pose significant safety concerns due to the fragility of the tissue and the likelihood of triggering unwanted pulmonary inflammation. Consequently, it is critical that the development of novel methods for pulmonary immunization focus not only on the generation of effective mucosal immunity but also on the amelioration of concomitant inflammatory responses. Thus, new immunoprophylactic strategies that facilitate the local generation of protective immunity against a broad spectrum of lung pathogens and are able to diminish any detrimental inflammatory sequelae associated with the onset of primary adaptive and/or innate immune responses at the site of infection would be highly advantageous.
Therapies that cause the formation of inducible bronchus-associated lymphoid tissue (iBALT) may provide an alternative immunoprophylactic approach for broad spectrum viral protection in the lung. Although iBALT development is known to occur in association with pulmonary immune responses,, the significance of the role of iBALT in the resolution of infections has only recently been investigated, most notably in its role in adaptive immunity to influenza virus infections,. Remarkably, iBALT can replace the function of the spleen and other secondary lymphoid tissues in primary adaptive immune responses and also support the maintenance and re-expansion of adaptive immune memory responses following influenza virus infections. The association of iBALT with lung pathologies in autoimmune diseases, or the necessity of infection for its induction, has diverted attention from the immunoprophylactic potential of iBALT. Here, we demonstrate that iBALT can be induced asymptomatically without infection or ensuing pathology, and that pre-existing iBALT enhances and accelerates the primary adaptive and likely innate immune responses to respiratory viruses in the naïve host.
Avian respiratory tract infections are associated with massive economic losses particularly under poor intensive rearing conditions in winter seasons. Several pathogens, adverse environmental conditions and poor managemental factors are involved [1–3]. Pathogens include avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), avian pneumovirus (APV), Mycoplasma gallisepticum (MG), and avian pathogenic E. coli (APEC). Avian respiratory tract infections can be caused by single pathogens or in combination with each other in form of preceding, concurrent or secondary infections [3–5]. Change in the ecosystem of the respiratory microbiome, mechanical damage of the epithelium, loss of ciliated cells and the impairment of innate immune responses are a consequence of primary respiratory infections and associated with enhanced disease and economical losses during superinfections.
In Egypt, respiratory diseases outbreaks caused significant economic losses in commercial poultry production during recent years. Laboratory investigations revealed that IBV represents the most common pathogen detected during these outbreaks [8–10]. IB is a highly contagious disease affecting the respiratory, renal and reproductive tract of chickens. The disease caused by single-stranded, enveloped RNA virus belongs to the genus of Gamma coronavirus. The spike (S) glycoprotein represents the major structural and immunogenic domain, and plays a pivotal role in tissue tropism and carry the receptor-binding site. The S glycoprotein is cleaved post transitionary into S1 (globular head) and S2 (stalk) domain. The S1 domain possesses three hypervariable regions (HVR 1, 2 and 3) responsible for the high genetic and antigenic variability of IBV. Despite massive vaccination strategies used in Egypt, IBV extensively circulates in all over Egypt in vaccinated and non-vaccinated chickens with continuous viral evolution. Complications between APEC and IBV infections are widely observed under field conditions and are reproduced under experimental conditions resulting in enhanced severe colibacillosis and enhanced pathogenicity post IBV infection.
Under field conditions, commercial broiler farms with inadequate biosecurity measures and with the presence of colibacillosis in early age bird are associated with more severe IBV infections in late age birds (>20 days old). Despite the wide spread use of antibiotics and vaccination against IBV, complications as a consequence of mixed infections of avian bacterial pathogens including APEC and IBV became a general phenomenon associated with significant economic losses.
In the present study samples were collected from broiler farms with birds with respiratory manifestations. All samples were tested for the presence of IBV and APEC. Recovered IBV isolates were genetically characterized by partial spike protein sequencing. Recovered APEC were characterized by serotyping and antibiotic profiling. Bacteriophages were isolated from water samples collected from the same farms and enriched against APEC O78. In vivo study was conducted to evaluate the efficacy of bacteriophages in reducing the pathogenicity of single and mixed infections with APEC and IBV.