Two new polyomaviruses were identified in 2007 in respiratory tract samples following large scale molecular screening using high throughput DNA sequencing of random clones and have been named after the institutes where they were found: KI (Karolinska Institute) polyomavirus (KIPyV) and WU (Washington University) polyomavirus (WUPyV). Data on seroprevalence indicate that infection is widespread ranging from 54.1 and 67% for KI and from 66.4% and 89% for WU in North American and German blood donors. Since their first identification, KI and WU viral sequences have been confirmed worldwide in respiratory samples from children with respiratory tract disease ranging from 0.2% to 2.7% and from 1.1 to 7%, respectively. However WUPyV and KIPyV were found at similar frequencies in control groups without respiratory diseases so the link between these polyomaviruses and acute respiratory diseases remains speculative.

Careful analysis is complicated by high co-infection rates with other well-characterized viral respiratory pathogens. A co-detection rate of 74% has been observed for KIPyV and rates ranging from 68 to 79% for WUPyV. Therefore, in a recent study in Southern China, hospitalized children with WUPyV infection displayed predominantly cough, moderate fever, and wheezing, but were also diagnosed with pneumonia, bronchiolitis, upper respiratory tract infections and bronchitis. As in most of infected children a single WUPyV infection was detected, it was suggested that the newly described polyomavirus can cause acute respiratory tract infection with atypical symptoms, including severe complications. Nevertheless these data have to be confirmed in further studies.

The presence of WUPyV and KIPyV in samples from children but not from immunocompetent adults suffering from LRTIs suggests that these viruses primarily infect the young population. A correlation between immunosuppression and reactivation of the two novel polyomaviruses has been suggested in immunocompromised patients and in AIDS patients at the molecular level, but no evidence of a role of these viruses as opportunistic pathogens has been given.

Overall, these data support the hypothesis that, in analogy with BK and JC polyomaviruses, KIPyV and WUPyV can establish persistent infection, and that virus replication may increase in immunocompromised hosts. However, in a recent study on immunocompetent and immunocompromised adult patients, real-time PCR detected KIPyV and WUPyV in 2.6% and 4.6% of HIV-1–infected patients respectively and in 3.1% and 0.8% of blood donors respectively, while no association was found between CD4+ cell counts in HIV-1 positive patients and infection with KIPyV or WUPyV.

KIPyV and WUPyV are also incidentally detected in adults with community acquired pneumonia, in immunocompromised hosts, and in patients with lung cancer; they are more often found in patients suffering an underlying medical condition and coinfections with KIPyV and WUPyV with other respiratory viruses are common. A recent study evaluating the prevalence and viral load of WUPyV and KIPyV in respiratory samples from immunocompromised and immunocompetent children showed that the prevalence of WUPyV and KIPyV is similar in hematology/oncology patients compared with that of the general pediatric population. High co-detection rates with other respiratory viruses, mainly RSV and enterovirus or rhinovirus, were found for WUPyV and KIPyV in both groups, in analogy with previous reports. However, higher viral loads for KIPyV in the immunocompromised group were detected, suggesting that there may be an increased replication of this virus in this population.

A similar association was not observed for WUPyV. Furthermore, in the immunocompromised group, infection with either virus occurred in older children compared with controls, which may indicate viral-reactivation Table 1.

HRVs are currently classified in the Picornaviridae family, genus Enterovirus, that includes 3 species: HRV-A, HRV-B, and HRV-C. Within each species there are multiple HRVs designated as “serotypes”, “types”, or “strains”. Several recent epidemiological studies suggest that HRV-A and HRV-C are the predominant species associated with acute respiratory illnesses in hospitalized children and adults, compared to HRV-B which are rarely detected.

The new HRV lineage designated HRV-C has been identified using molecular methods and associated with severe clinical presentations in infants and immunocompromised adults. Symptoms of patients infected with this new strain were mainly bronchiolitis, wheezing, and asthmatic exacerbation in cases from Australia and Hong Kong, which peaked in fall and winter whereas in New York the new rhinovirus genotype was detected in cases of influenza like illness (ILI) that were clustered within an 8-week period from October to December. A recent study describes a clinical case of severe respiratory and pericardial disease in an infant infected by HRV-C suggesting tha viral tropism is not strictly restricted to the respiratory tract. A study focusing on the global distribution of novel rhinovirus indicates its association with community outbreaks and pediatric respiratory disease also in Africa and in symptomatic subjects living in remote locations having limited contacts with other human populations. Moreover evidence for a role of HRV-C in lower respiratory tract disease and febrile wheeze in infants and asthma exacerbations in older children was reported. Recent studies making comparisons between HRVs species, found the HRV Cs more so than As or Bs as the major contributors to febrile wheeze and asthma exacerbation in infants and children, respectively . However, the severity of clinical manifestations for HRV-C is comparable to that for HRV-A in children with community-acquired pneumonia. In HRV C studies so far, no clear clinical difference has been noted between patients with single or mixed HRV-C infection. In a study, monoinfection was observed in more than half of cases and was more common than RSV monoinfection in patients with upper RTD, however the duration of hospitalizations was not significantly different between the HRV-C monoinfection group, HRV-A or HRV-B monoinfection group and RSV group suggesting that HRV-C is an important etiological factor in children with RTI. Most HRV-C co-detections are with RSV, however in a large study HRVs were statistically the least likely virus of 17 examined to be associated with co-infections Table 1.

BPI-3V sometimes cause severe disease as a single agent and can predispose the animal to bacterial infections of the lung. Our results revealed high BPI-3V seroprevalence (47.1%) in the three explored municipalities that indicate most adult cattle have been exposed to this pathogen. These results agree with those by Carbonero et al., who found high seroprevalence values in cattle of Yucatan, Mexico. However, the results obtained in this study differ with those published by Betancur et al., who reported lower seroprevalence values (13.5%) in cattle from Monteria, Colombia. The high seroprevalence of BPI-3V found in this research is in agreement with the ubiquitous nature of the virus and with its worldwide distribution. In this research, the seroprevalence was higher in the age group of >24 months of age (Table-4). This age group was a significant risk factor for BPI-3V transmission (OR=3.5). Possibly, due to the presence of some stress factor in these animals that favors reinfections with or without respiratory signs. In adults, especially BPI-3V, it is subclinical unless it is part of concomitant infections with other viruses and bacteria such as Pasteurella multocida, Mannheimia haemolytica, Mycoplasma spp., and immunosuppressive factors. With regard to the clinical signs, conjunctivitis had a statistical association with the BPI-3V seroprevalence values, and regarding sex, female was a significant risk factor for BPI-3V infection (OR=3.6). This result differs with those published by Betancurt et al., who found no statistical association between BPI-3V infection and sex.

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.

The prospective cross-sectional study was conducted from March 2013 to April 2016. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant institutional guides on the care and use of laboratory animals (Chulalongkorn University Animal Care and Use Committee Approval, No. 1431005).

BRSV infection is widely spread around the world, most likely as a direct result of the movement of cattle. Regardless of the geographical location, infectivity rates are usually rather high, suggesting that viral transmission is a common event among herds. Cattle are the principal reservoir of infection; however, sheep can also become infected. Intra-herd transmission usually occurs by aerosols, allowing the virus to enter susceptible cattle via the respiratory tract. However, local spread and airborne transmission between herds are not of great importance for inter-herd transmission despite the circulation of BRSV in a given geographical region. On the other hand, direct transmission between herds is frequently a consequence of the introduction of new infected animals, while indirect transmission occurs by individuals visiting farms. Some of the main risk factors for BRSV transmission include large herd size and common farm practices such as not providing boots to visitors and dual-purpose farms. Additionally, it has also been proposed that good management and better hygienic routines have a direct impact on overall health status.

BRSV outbreaks commonly occur during winter. Thus, clinical disease is commonly diagnosed during autumn and winter in temperate regions. Nevertheless, infection can also be observed during summer. The sero-prevalence of BRSV infection varies greatly across different geographical regions. The distribution of BRSV is most likely affected by the movement of cattle, as insect vectors are not believed to play a role in viral transmission. The morbidity of the disease is quite high, and in some instances, it has been responsible for up to 60% of the clinical respiratory diseases among dairy herds. In general, the frequency of BRSV is strongly associated with cattle population density in the region and with the age of the host. Interestingly, BRSV infection is also associated with a high morbidity of up to 80% and with mortality that can reach up to 20% in some outbreaks.

BRSV outbreaks can become epidemics affecting animals in all age groups. However, the age distribution of BRSV infection seems to be a function of exposure. In other words, herds that have been previously exposed to the virus tend to experience infections that are limited to younger, more susceptible animals. In consequence, morbidity is commonly high during the occurrence of outbreaks. Importantly, natural infection affects both beef and dairy cattle, although management practices can significantly impact the infectivity rates. Climate also favors the dissemination of the virus during winter, after the sudden drop in temperature, although infection can occur throughout the year.

The mechanisms that are responsible for the survival of the virus within a given population are not fully understood. Controversial information has been reported about viral persistence. Nonetheless, chronicity has been proposed as a mechanism that might play role in disease spread. BRSV can be isolated from asymptomatic animals and can persist for several months. Thus, one possibility is the existence of persistently infected calves, which might start shedding the virus under specific conditions. Therefore, latent infection among herds might occur, providing a possible explanation for the occurrence of outbreaks among relatively isolated calves. However, some reports have suggested that subclinical infection in cattle is not a plausible mechanism for the persistence of BRSV in dairy herds.On the other hand, clinically ill animals are believed to be the most likely sources of infection, and therefore, the most likely explanation for recurrent infections is the reintroduction of the virus into the herd before the occurrence of a new outbreak. This controversial issue requires more detailed studies aiming to assess the role of persistence in virus spread to fully understand the mechanism exploited by the BRSV to warrant transmission.

The prevalence of the disease varies greatly in North America. For instance, in the United States, original studies conducted during the early 1970s showed a frequency of infection of 67% among adult cattle. However, in a few instances, 100% of the animals within herds showed the presence of specific antibodies. Subsequently published reports showed that up to 81% of herds had specific antibodies against BRSV. Additionally, the incidence of sero-conversion soon after the occurrence of an outbreak has been reported to be as high as 45%. Furthermore, the sero-positivity rate among asymptomatic cattle has been shown to reach up to 95%. Importantly, the sero-positivity rate seems to be closely associated with the age of the host, showing a higher prevalence among older animals. Moreover, southern regions of the U.S. commonly exhibit a higher sero-prevalence than the northern parts of the country. These discrepancies could be explained by differences in the vaccination practices in those regions, as well as by management practices or by sampling errors. Thus, the frequency of BRSV infection in a particular region is subject to a number of factors that can drastically change the prevalence of the disease. Interestingly, the mortality among herds experiencing respiratory disease in the U.S. can reach up to 13%. While other viruses, such as the bovine viral diarrhea and parainfluenza viruses, could also account for the elevated mortality rates seen in the U.S., BRSV remains as a very important ethological agent and a probable cause of death due to respiratory disease in cattle.

In Canada, initial reports have shown that the overall frequency of BRSV infection might be nearly 36%. However, during outbreaks, the percentage of sero-positive individuals within a herd can range between 22% and 53%. Subsequent studies confirmed the high frequency of the disease (40%) among feedlot calves. Similar to what has been observed in the U.S., BRSV is still an important cause of mortality among cattle in Canada. In Mexico, the circulation of BRSV was recently reported in two different regions of the country. Both studies reported rather high overall frequencies of BRSV infection (52% and 90.8%). The reasons for the significant differences in the distribution of BRSV infection between the two regions are not known; however, age played a major role in the distribution of infection. Additionally, BRSV has also been shown to circulate in other regions of the Americas.

In Europe, shortly after the virus’s discovery, it was reported to have widely circulated in different parts of the continent. In Sweden, different studies have shown high frequencies of antibodies in milk, ranging between 41% and 89% depending on the geographical region of the country. In general, higher antibody frequencies in milk samples were observed in samples from the southern regions of Sweden, while lower frequencies were detected in samples from the northern part of the country. The authors attributed the discrepancies observed among differences herds to cattle population density, as highly populated regions were more common in the southern part of Sweden. More recently, the geographical distribution of BRSV in the country has been studied. The aforementioned study showed that infection with BRSV occurs predominantly in the central-western and southern parts of Sweden. In particular, two regions of the country, Skaraborg and Skåne, displayed a high prevalence of BRSV infection. Similarly, Danish reports have also shown rather high frequencies of BRSV infection (54%). Likewise, a high prevalence of BRSV infection has been observed in Belgium. Circulation of the virus in Scotland has also been reported, although conclusive figures regarding BRSV prevalence are not available. Furthermore, the presence of antibodies in bulk tank milk samples has been reported in herds from England. Additionally, a high sero-prevalence of BRSV has also been reported in Northern Europe.

In Africa, Ethiopia and South Africa have also been shown to have high incidences of BRSV infection. Other countries, in different regions, such as Turkey, have also been shown to a have high sero-prevalence, which can reach up to 43%. Unsurprisingly, high sero-prevalence has also been associated with large-capacity facilities, rather than with small farms. Interestingly, organic farms have been shown to exhibit lower antibody prevalence when compared to conventional farms. These findings highlight the importance of management for the effective control of viral transmission and disease spread, which are closely associated with different farming methods

Thus, more information is required to understand the mechanisms that allow for viral survival in a given geographical region. Monitoring of outbreaks among herds is likely to provide valuable information that might help us to understand in greater detail the epidemiology of BRSV infection.

Focussing on upper respiratory tract samples (nasal and tonsillar swabs), viral nucleic acids were detected in 31 of 214 diseased dogs (14.5%). Sixteen dogs tested positive for CRCoV (7.5%), 14 dogs for CPiV (6.5%) and one of these dogs additionally for CAV‐2‐specific nucleic acid (0.5%). One single dog tested positive for CDV‐specific nucleic acid (0.5%). In none of the obtained samples from the upper respiratory tract was CIV‐specific nucleic acid detected. Of those 31 positive dogs, 21 were privately owned (group A), and 10 kept in shelters (group B). They consisted of five puppies, 12 adolescent dogs and 14 adult dogs. Twenty‐seven of the 31 positive dogs (87.1%) showed acute onset of signs, three suffered from chronic disease (9.7%) and for one diseased dog this information was not available (Table 2).

Furthermore, upper respiratory tract samples from two dogs (4.0%) of the clinically healthy control group C tested positive for CRCoV‐specific nucleic acid (Table 2).

Nine dogs from group A (5.2%) and seven dogs out of group B (17.0%) tested positive for CRCoV in either nasal, tonsillar or both samples at one time. One of these dogs belonged to the subgroup of puppies; nine dogs were from the adolescent subgroup and six animals from the subgroup of adult dogs. With one exception, all these animals showed acute onset of CIRD (93.7%).

Fourteen diseased dogs (6.5%) tested positive for CPiV. From those, 11 belonged to group A and three to group B. They all harboured CPiV‐specific nucleic acid in sample material from the nose and one dog concurrently from the tonsils. Four of these dogs were classified as puppies; three dogs were from the adolescent subgroup and seven dogs were adults. Twelve out of these 14 animals showed acute onset of clinical signs (85.7%), one dog was chronically ill, and for another dog this information was not available. Seven dogs (50.0%) were regularly vaccinated‐including against CPiV.

In one of these 14 CPiV‐positive dogs, CAV‐specific nucleic acid was detected concurrently. This dog was privately owned (group A) and tested positive for CAV in both nasal and tonsillar swabs and CAV‐2 strain (Toronto) was confirmed by DNA sequencing. Belonging to the subgroup of adults this dog had been irregularly vaccinated and received its latest vaccine 45 days before sample collection. It presented with a several week history of clinical signs including severe coughing, nasal and ocular discharge, dyspnoea and fever. Apart from that case, in no other dog was viral nucleic acid of two or more different viruses detected. In addition, no other proband of the study tested positive for CAV.

One dog from group A tested positive for CDV‐specific nucleic acid in a sample retrieved from the tonsils. RNA sequencing enabled the identification of a CDV vaccine strain (Onderstepoort). The dog was an adult and presented with chronic respiratory disease but no other signs consistent with CDV infection. The vaccination status of this dog was unknown.

Additional information regarding all PCR‐positive dogs is summarised in Table 3.

All BALF samples collected from 31 chronically ill dogs revealed negative PCR results.

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.

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.

Exercise has anti-inflammatory effects and in the long term can protect the development of chronic diseases and obesity. Regular exercise of moderate-intensity is associated with a reduced incidence of upper respiratory tract infection. However, long hours of intensive training appear to make children more susceptible to upper respiratory tract infections. The recommended means of aerobic exercise is walking, with an optimal frequency of three to five days a week and an optimal duration of 20 to 30 minutes of continuous activity. In a recent study, the IgA secretion rate was negatively correlated with the incidence of infections. A recent randomized trial comparing meditation and exercise with wait-list control among adults aged 50 years and older found significant reductions in ARI illness.

Immune function and anti-viral defenses have a number of components, both specific and non-specific. Asthmatic children can improve their immune function by following some simple advice including a healthy life style with regular exercise, a balanced diet, adequate sleep and avoiding environmental tobacco smoke, stress and unnecessary antibiotics.

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–[3]. 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–[12], 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.

No conflicts of interest have been declared.

In the present study, we demonstrated the possibility to detect dual infection which caused by RSV and hMPV. Due to the lack of sensitivity of conventional methods detection of co-infections with different respiratory viruses had been underestimated (13). Severe RSV bronchiolitis in small patient series had been recognized because of co-infection with other viral pathogens (14). Some previous studies have lighted up increased clinical severity in case of dual infection caused by RSV and hMPV (8, 9). Greensill et al. observed a 70% co-infection rate with HMPV and RSV and a 90% co-infection rate among intubated infants with hMPV and RSV admitted to their PICU (8). Although Greensill et al. did not include an appropriate control group in their study, these findings suggest that co-infection with both hMPV and RSV is common and that together the two viruses may contribute to increase the severity of disease (9, 14). Whereas in other reports hMPV did not contribute to the severity of RSV infection (15, 16). The basis of the pathogenesis of severe RSV disease is multifactorial. Since severe RSV disease may develop in apparently healthy children, known host risk factors cannot completely account for instances of severe illness. Preexisting or maternally acquired immunity, innate immunity, viral factors and genotypes and environment all likely contribute to disease pathogenesis. We detected co-infection of hMPV and RSV in Kawasaki patient without special respiratory sign and symptoms, we could not find the patient to follow up her and study more about the immunity status and other possibilities of this scenario. More investigation is suggested specially on Kawasaki patients to detect the viral pathogens.

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).

Our study characterized the virome in fecal samples of pediatric HFMD patients during a 2012 widespread outbreak in Thailand. We used high-throughput next-generation sequencing to better understand the viruses present in HFMD patients who tested negative for enterovirus species A EV-71 and CV-A16/A6, predominant causes of HFMD and the main focus of many existing diagnostic assays [26, 27]. Analysis of the sequences either assembled into contigs or as singletons revealed a complexity of the virus population. Although previous examination of these samples did not initially detect enteroviruses using PCR-based assays, the present study utilizing deep sequencing enabled the detection of enterovirus sequences as well as other enteric virus at concentration as low as a few genome copies, providing a more systematic analysis of the prevalence of the viral genome.

The majority of viral reads identified here belonged to the family Picornaviridae, and were dominated by several members of the genus Enterovirus, namely HRV-C, CV-A21, and CV-A10. In addition to the commonly identified EV71 and CV-A16, other enteroviruses have been reported to cause HFMD outbreaks in different countries including CV-A10 and CV-A6 in Finland and France in 2010 and in China during 2008–2012. CV-A21 was least reported to cause HFMD with only 42 detections throughout the 36 years of surveillance in the United States. However, a study in China cited a high incidence rate of CV-A21 infection in adults with RTI. Another rarely detected virus found was EV68, originally isolated in the USA in 1962 from patients with RTI and has been detected sporadically thereafter. EV68 is unusual among EV in that it shares phenotypic properties of both enterovirus and rhinovirus. Reports suggested that this virus was associated with RTI including pneumonia and bronchiolitis with greater severity in infant and school-aged children. Between 2008 and 2010, an increasing number of EV68 clusters in cases of RTI have been reported worldwide [85–88]. Outbreaks have also been reported in the USA in 2014 when EV68 infection affected more than a thousand people, mostly young children and resulting in at least 12 deaths. The association of EV68 infection and diseases other than RTI remains uncertain. Studies have linked the virus with infection of the central nervous system including acute flaccid paralysis and fatal meningomyeloencephalitis. In addition, by using un-biased metagenomic analysis, our results not only expanded the number of identified types of enterovirus, both common and rare, but also revealed possible circulation of multiple enterovirus types during the outbreak.

Nearly completed genome of HRV species C was detected in the virome. Although the ability for HRV to replicate in the gastrointestinal tract is unknown, the detection of HRV in fecal samples has been reported [42, 43]. The amount of HRV RNA detected in feces could be as abundant as enteroviruses and feces-derived HRV can retain infectivity in cell culture [91, 92]. No role for HRV has been postulated for HFMD and the virus is typically present in respiratory secretion and associated with asthma exacerbation. Nucleic acids from HRV and other typical respiratory viruses such as RSV in feces may reflect inactivated particles in swallowed respiratory secretions or actual replication in the digestive tract possibly aided by adaptive mutations.

The second most abundant viral sequences identified belonged to astroviruses. The classic HAstV species consisting of eight serotypes has been associated with diarrhea particularly in immunodeficient patients. Astroviruses detected here belonged to recently described species MLB1 and MLB2. MLB1 was initially sequenced from the fecal samples of a child with diarrhea and serological survey revealed it to be a common childhood infection. A study of children in India failed to associate MLB1 with diarrhea. Meanwhile, MLB2 was also initially detected in fecal samples from Indian children with diarrhea and in the plasma of a child with undifferentiated fever, suggesting replication beyond the digestive tract. Other astroviruses in human and animal tissues have also indicated likely neurological involvement [99–101], therefore a wide range of diseases may be associated with astrovirus infections.

Viral metagenomic studies of human fecal samples have been reported [102–105] and had suggest evidence of frequent co-infections with known enteric viruses from the family Picornaviridae, Astroviridae, and Parvoviridae [106, 107]. Our study found enterovirus, cardiovirus, astrovirus, rotavirus, norovirus, adenovirus, bocavirus, and picobirnavirus in stools from pediatric HFMD patients. Several of these viruses are known to cause gastrointestinal diseases. Although co-infections with different viruses and correlates of disease severity are ongoing, it is conceivable that viral co-infections may aggravate clinical manifestation of an otherwise mild virus infection, which could result in compromised gut function and upregulated immune response leading to increased risk of morbidity. For example, during an HFMD outbreak in Sarawak, Malaysia in 1997, a subgroup of adenovirus and enterovirus were isolated from three fatal cases. It is therefore conceivable that co-infection could precipitate or aggravate HFMD symptoms. Future studies to examine viral profile in individual clinical samples are warranted to clarify the possible role of co-infection with disease severity.

Since a significant fraction of HFMD cases remains negative for the viral enterovirus pathogens and a direct or aggravating role for other viruses is conceivable, our study illustrates an overview of the virus community in stools from pediatric HFMD patients using unbiased sequencing approach. Our finding shows that such EV71, CV-A16/A6 PCR-negative children shed multiple types of picornaviruses and other enteric viruses in their feces including enterovirus, cardiovirus, astrovirus, rotavirus, norovirus, adenovirus, bocavirus, and picobirnavirus. Such overview information may help clinicians figure out the contribution of different types of picornaviruses as well as other pathogens to HFMD, with potential public health implications on the disease control.

The analysis of pooled samples and the deep sequencing method used presented limitations in the interpretation of the results. First, it was not possible to equate viral read numbers with the number of children infected. Second, comparison of the viral loads of viruses with different genome types (e.g. ssRNA, dsRNA, dsDNA) was not possible since the relative efficiency of converting their genomes into next-generation sequencing-compatible DNA may vary. Although stool samples used in this study were convenient samples collected for our previous study, inclusion of other clinical specimens such as throat swab, vesicular fluid, and skin lesions would be ideal. Stool samples may harbor unknown inhibitors of PCR and present nucleic acids of non-viral origin, thus complicating analysis. Nevertheless, our findings highlight the potential advantage of next generation sequencing to detect viruses from clinical specimens, which may be present below the limit of detection by conventional PCR assay. Assessing the role, if any, of these viruses in HFMD will require the study of larger populations, including epidemiologically matched healthy controls, and the analysis of individual, rather than pooled, clinical samples.

Our results revealed high BRSV seroprevalence (98.6%) in the three explored municipalities that indicate most adult cattle have been exposed to this pathogen. The herd seroprevalence of BRSV (100%) found in this research is consistent with published data of Solis-Calderon et al., Saa et al., and Carbonero et al., who reported a herd prevalence of 90.8% (Mexico), 91.3% (Ecuador), and 95.8% (Argentina), respectively. However, these results differ from those reported by Obando et al. Contreras and Parra, who found lower seroprevalence values in other studies. The individual seroprevalence of BRSV (98.6%) agrees with the findings of Saa et al. who reported 80.4% of seroprevalence in herds of Ecuador. This result also agrees with those of Betancur et al. and Betancur et al. who found 13% and 31% of seroprevalence in dairy cows and calves, respectively, in herds of Montería state, Colombia.

Nevertheless, these results differ from those published by Carbonero et al., who reported 46.6% of seroprevalence in Argentina. The results obtained demonstrate that BRSV is widespread among animals and dual-purpose cattle herds in Cesar department. Probably, after the initial infection occurs in some animals, the virus is rapidly spread throughout the animals, probably by aerosols, particularly in herds without prior exposure to the virus, increasing seropositivity. The several herds in Colombia are not being vaccinated against BRSV and result from this research demonstrates that this virus circulates among the animals and herds from the three municipalities. It would be important to include BRSV in vaccination programs with the aim of controlling infection in this region.

Regarding the age, BRSV infection was observed in both age groups in this research. Although the analysis was not done in younger animals, as reported in the literature, the clinical disease is more frequent in calves. This seroreactivity in adult animals suggests possible reinfections during the course of their life. The result obtained in this research agrees with those reported by Betancur et al., who found no statistical association between infection and age group. Nevertheless, the results obtained differ from those published by Bidokhti et al., who found statistical differences with respect to the age of the infected animals. They demonstrate that after infection with BRSV, the animals will remain seropositive for several years. The older cows were seropositive while the younger cows were seronegative, i.e., there had been no virus circulating for several years. In this study, municipality, sex, and herd size were not a significant risk factor (Table-4). Regarding the clinical signs, animals with respiratory problems (34.9%) and conjunctivitis (38.5%) were found (Table-3). However, there was no statistical association (p>0.05) between seroprevalence values and respiratory signs in tested animals. These results are due to BRSV, which is observed in any age group, but infections that result in severe clinical disease are typically observed in calves. Nevertheless, there was no sampling in calves in this research.

All ethical issues including plagiarism, Informed Consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc have been completely observed by the authors.

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–[8], 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

[11]. 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–[14]. 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.

The results of the indirect ELISA revealed that the overall prevalence of BRSV in the Nineveh Governorate was 83.11%, with the highest prevalence in cattle that were aged >7 months-1.5 years (relative risk (RR)=2.12) (Table-1). BRSV prevalence was higher in imported animals, compared to animals of a local origin (RR=1.17) (Table-1), and in animals originating from large herds (100 animals), compared to those from small herds (40 animals) (RR=1.48) (Table-1). There was no significant difference (p<0.05) between the prevalence of the disease in male and female animals (Table-1). BRSV prevalence varied significantly (p<0.05) across the different geographical areas of the Nineveh Governorate with the samples collected from the northern region displaying the highest prevalence (RR=1.33) (Figure-1). In addition, samples collected in the winter displayed the highest prevalence of BRSV (RR=1.38) compared to those collected in the spring, summer, or fall (85.09%, 83.18%, and 75.18%, respectively) (Table-2).

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

[7]. 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.

Table 2 presents the results from the individual investigation of lactating cows in the 11 selected herds where paired BTM samples ­indicated new infection. Herd size, the number of lactating cows ­contributing to the BTM, the association between levels of antibodies detected in BTM and individual milk results from the lactating cows are presented. Indications of time since presence of BRSV based on the results of the antibody testing of serum of animals at different ages are also shown. This includes the samples of young stock. In eight of the herds, all the tested young stock was positive. In three of the herds, all the young stock up to a certain age was negative, and all animals above that age were positive. The time of exposure was therefore presumed to be between the age of the oldest negative and the youngest positive animal. The results indicated that 9 of the 11 herds had a recent infection (<17 months ago). For two of the herds (herds 3 and 4), the results indicated that BRSV had not been in the herd for the last five to seven years. The level of antibodies detected was also lowest for these herds. The percentage of positive animals contributing to the BTM was also lowest for herds 3 and 4, but the mean PP of the positive animals was high (114 and 86, respectively).

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.

Statistical analysis was performed using IBM SPSS Statistics for Windows, version 19 (IBM Corp., Armonk, N.Y., USA). The two-sided Chi-square test and Fisher’s exact test were used to assess the difference in BRSV prevalence and various risk factors in the different cattle groups. The rate of relative ratio (RR) between BRSV risk factors was calculated at 95% significance using Epi-Info TM 7, version 7 (CDC, Atlanta, GA, USA).