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.

PCRs testing were repeated on the 50 fruit bats original samples including the Kidney, heart, lung, liver, spleen, intestine, rectal swab sample, and brain samples. Two bat’s QPCRs results were positive. One bat’s QPCRs result was positive in the lung, intestine sample (Cangyuan virus isolated) and rectal swab sample, and the Ct (Threshold Cycle) of QPCR were 19.86 ± 0.056, 19.52 ± 0.041, 19.64 ± 0.061 respectively. The Ct of another bat’s PCR were 23.07 ± 0.253, 22.53 ± 0.171 in the intestine sample and rectal swab sample, respectively.

To establish the evolutionary relationship between Cangyuan virus and other known orthoreoviruses, Homology were compared (Table 2, Table 3 and Additional file 1: Table S1, Additional file 2: Table S2 and Additional file 3: Table S3) and phylogenetic trees were constructed based on the nucleotide sequences of the L genome segments (Figure 2), the M genome segments (Figure 3) and the S genome segments (Figure 4). The Cangyuan virus L1-L3, M1-M3 segments sequence identity were 81.6% –94.2%, 83.8%–97.9%, 85.9%–97.6% ( Additional file 1: Table S1), 82.2%–94.1%, 78. 1%–95.0%, and 83.0%–93.9% (Table 2, Additional file 2: Table 2), respectively, by alignment with Pteropine orthoreovirus (PRV) species group. The phylogenetic trees for L2, L3, M1 and M2 segments demonstrated that Cangyuan virus was most closely related to Melaka and Kampar viruses, and was placed in Pteropine orthoreovirus (PRV) species group which covers all known bat-borne orthoreoviruses together with Nelson Bay orthoreovirus.

To better understand the genetic relatedness of Cangyuan virus to other known bat-borne orthoreoviruses, the published sequences for the S genome segment of bat-borne orthoreoviruses known for causing acute respiratory disease in humans were retrieved from GenBank and used to compare homology (Table 3 and Additional file 2: Table S2) and construct phylogenetic trees (Figure 4). The Cangyuan virus S1-S4 segments sequence identity were 55.3%–94.7%, 86.2%–95.5%, 86.5%–97.9%%, and 83.5%–98.2%, respectively (Table 3 and Additional file 2: Table S2). The S1 segment demonstrated a greater heterogeneity than other S segments in Pteropine orthoreovirus (PRV) species group.

Members of the Paramyxoviridae family are pleomorphic enveloped viruses divided into two subfamilies, Paramyxovirinae and Pneumovirinae. Paramyxovirinae has recently been subdivided into seven genera: Aquaparamyxovirus, Avulavirus, Ferlavirus, Henipavirus, Morbillivirus, Respirovirus, and Rubulavirus (http://ictvonline.org/virusTaxonomy.asp?version=2012). Viruses of this family affect a wide range of animals, including primates, birds, carnivores, ungulates, snakes, cetaceans and humans, and cause a wide variety of infections, such as measles, mumps, pneumonia and encephalitis in humans, and distemper, peste des petits ruminants, Newcastle disease and respiratory tract infections in animals. However, several paramyxoviruses (PVs) have not been classified into any of these seven genera, including Nariva virus (NarPV), Mossman virus (MosPV), Beilong virus (BeiPV), J virus (JPV),, Tupaia paramyxovirus (TupPV) and Tailam virus

[8], all of which belong to a group of novel paramyxoviruses isolated from wild animals, as well as Salem virus isolated from horses. Among them, only JPV has been shown to be pathogenic, causing extensive haemorrhagic lesions in rodents. Horizontal transmission is the principal mode of intraspecies PV infection, suggesting that contaminated faeces, urine or saliva may be responsible for spillover to other species.

Bats have a close evolutionary relationship with several genera of mammalian paramyxoviruses. Otherwise, bat-borne paramyxoviruses are in close relationship to known paramyxoviruses of mammalian. These small mammals are known to harbour a broad diversity of PVs, including emergent henipaviruses (Nipah virus and Hendra virus) and rubulaviruses [Menangle virus, Tioman virus, Mapuera virus, and Tuhoko virus 1, 2 and 3 (ThkPV-1, ThkPV-2 and ThkPV-3)]. A very broad diversity of paramyxoviruses, including Henipa-, Rubula-, Pneumo- and Morbilli-related viruses, have been detected in six of ten tested bat families. Whereas most of the viruses identified in bats do not seem to cause clinical disease in these animals, there have been reports of rabid bats, and of unusually large numbers of animals succumbing to infection by rabies virus.

As part of a large-scale investigation of viral diversity in bats and of associated zoonotic risks, we have previously detected a bat paramyxovirus in one insectivorous African sheath-tailed bat (Coleura afra), exhibiting several hemorrhagic lesions at necropsy. We therefore examined occurrence of this bat paraymxovirus in other bats.

Bats are considered a reservoir of severe emerging infectious diseases. Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), Nipah virus, Hendra virus, and Ebola virus are all thought to be bat-borne viruses1,2.

Notably, bats also host major mammalian paramyxoviruses from the family Paramyxoviridae, order Monone-gavirales3,4. While Henipaviruses (Nipah and Hendra viruses) in South East Asia and Australia are associated with fruit bats5, other paramyxoviruses have been detected not only in fruit bats but in insectivorous bats worldwide6–9. A potential pathway for Nipah virus transmission from bats to humans was found to be associated with a human-bat interface, specifically date palm sap shared by bats and humans10. In addition, serological evidence of possible human infection with a bat-originated paramyxovirus, Tioman virus11, reinforces the epidemiological role of bats in the emergence of pathogens such as paramyxoviruses in humans.

In addition to these bat paramyxoviruses with zoonotic potential, other new paramyxoviruses have been reported. These include several new mammalian paramyxoviruses such as Beilong virus and J virus, which remain unassigned under the family Paramyxoviridae12. Recent bat-associated paramyxoviruses were proposed to be grouped in a separate phylogenetic clade within a potentially separate genus such as Shaanvirus13 which was distantly related to Jeilongvirus14. In addition, novel strains of bat paramyxoviruses in diverse genera have been reported continuously15–17. Based on the recent papers, bat paramyxoviruses found worldwide to date have belonged to the genera Rubulavirus, Morbillivirus, Henipavirus and the unclassified proposed genera Shaanvirus. Expanded classifications for grouping newly identified viruses in bats can be accomplished by further studying the biological characteristics of novel paramyxoviruses as well as genome characterization18.

In this study, active surveillance was performed to reveal paramyxoviruses circulating in Korean bats. A total of 232 bat samples were collected at 48 sites in natural bat habitats and tested for the possible existence of paramyxoviruses.

This research was approved by the Institutional Ethical Committee of the University of Santander and VECOL, Colombia.

Many emerging infectious diseases are caused by zoonotic transmission, and the consequence is often unpredictable. Zoonoses have been well represented with the 2003 outbreak of severe acute respiratory syndrome (SARS) due to a novel coronavirus. Bats are associated with an increasing number of emerging and reemerging viruses, many of which pose major threats to public health, in part because they are mammals which roost together in large populations and can fly over vast geographical distances. Many distinct viruses have been isolated or detected (molecular) from bats including representatives from families Rhabdoviridae, Paramyxoviridae, Coronaviridae, Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, Arenaviridae, Herpesviridae, Picornaviridae, Filoviridae, Hepadnaviridae and Orthomyxoviridae.

The Reoviridae (respiratory enteric orphan viruses) comprise a large and diverse group of nonenveloped viruses containing a genome of segmented double-stranded RNA, and are taxonomically classified into 10 genera. Orthoreoviruses are divided into two subgroups, fusogenic and nonfusogenic, depending on their ability to cause syncytium formation in cell culture, and have been isolated from a broad range of mammalian, avian, and reptilian hosts. Members of the genus Orthoreovirus contain a genome with 10 segments of dsRNA; 3 large (L1-L3), 3 medium (M1-M3), and 4 small (S1 to S4).

The discovery of Melaka and Kampar viruses, two novel fusogenic reoviruses of bat origin, marked the emergence of orthoreoviruses capable of causing acute respiratory disease in humans. Subsequently, other related strains of bat-associated orthoreoviruses have also been reported, including Xi River virus from China. Wong et al. isolated and characterized 3 fusogenic orthoreoviruses from three travelers who had returned from Indonesia to Hong Kong during 2007–2010.

In the present study we isolated a novel reovirus from intestinal contents taken from one fruit bat ( Rousettus leschenaultia) in Yunnan province, China. In the absence of targeted sequencing protocols for a novel virus, we applied the VIDISCR (Virus-Discovery-cDNA RAPD) virus discovery strategy to confirm and identify a novel Melaka-like reovirus, the “Cangyuan virus”. To track virus evolution and to provide evidence of genetic reassortment PCR sequencing was conducted on each of the 10 genome segments, and phylogenetic analysis performed to determine genetic relatedness with other bat-borne fusogenic orthoreoviruses.

Hendra virus (HeV) causes serious systemic infection with pneumonia and encephalitis in humans, horses and various laboratory animals,,,. It is a single-stranded, negative-sense RNA virus belonging to the family Paramyxoviridae and is classified within the genus Henipavirus which it shares with one other virus, Nipah virus (NiV). HeV first emerged in the Brisbane suburb of Hendra in 1994, where it caused the deaths of one human and fourteen horses. Since then a further thirty four HeV outbreaks have been identified along the mid to north-eastern coast of Australia with infection of five more humans (of whom three died) and numerous horses,,,. Pteropid bats have been identified as the reservoir host, however epidemiological evidence does not support direct bat to human transmission. Horses have been an intermediate host in the transmission of disease to humans in all cases. There are as yet no readily available effective therapies or prophylaxis for HeV infection, either for use in humans or other susceptible animals.

Of necessity, HeV pathogenesis studies and evaluation of vaccine and therapeutic candidates must be carried out in animal infection models under Biosafety Level 4 (BSL4 conditions). Several species have been used for this purpose including: ferrets, hamsters, guinea pigs, pigs, cats, horses, and African green monkeys,,,,,,. With bats, the list comprises species from six orders including; Rodentia, Primates, Chiroptera, Cetartiodactyla, Perrisodactyla and Carnivora. The broad species susceptibility is unusual for a member of the family Paramyxoviridae and is attributed largely to the highly conserved nature of the host receptors for the virus, Ephrin B2 and B3,. Despite the possession of relevant receptors, the laboratory mouse, a most useful host on account of their small size, ease of handling, and vast library of available reagents, is reported to be resistant to HeV infection and disease.

Westbury et al in 1995 reported resistance of mice to HeV infection in a study that was designed to identify a suitable laboratory animal model of HeV disease. Juvenile BALB/c mice were inoculated with 5000 median tissue culture infective doses (TCID50) of virus by a parenteral route and observed for clinical signs of infection. Mice remained clinically well throughout the 21 day study period and, after euthanasia, there was no evidence of infection by gross or histological examination, virus isolation or serology. Similar results were reported by Wong et al in 2003, who investigated the susceptibility of mice to the closely related Nipah virus by inoculating juvenile Swiss brown mice by either parenteral or intranasal routes.

An understanding of the mechanisms of resistance of mice to HeV may provide novel targets for therapeutic and preventative intervention of human infections. Furthermore, circumvention of such mechanisms may induce a useful mouse model of HeV disease. Therefore, in view of the limited previous work, we decided to re-evaluate the apparent resistance of mice to HeV infection by investigating the outcome of HeV exposure by various routes to inbred mice of different ages and strains. Additionally, quantitative real-time polymerase chain reaction (qPCR), a technique not available at the time of the initial studies, would be used for detecting evidence of viral replication.

We found that mice are susceptible to HeV infection when exposed via the intranasal route, but resist infection when challenged by a parenteral route. Infection manifested as acute, transient, and asymptomatic virus replication in the upper and lower respiratory tracts, together with clinically significant encephalitis that has a longer incubation period than is reported for other models of fulminating HeV disease. The pattern of central nervous system involvement (CNS) supports neuroinvasion by the anterograde route (spread from the neuron cell body toward the axon terminus) and, importantly, transneuronal spread within the CNS. Over all, the study demonstrated that mice are susceptible to HeV infection and has provided a new and important model for HeV induced encephalitis.

Over one-half of all known human pathogens originated from animals, and over 75% of emerging infectious diseases identified in the last three decades were zoonotic.1 The threat of veterinary pathogens to human health continues to grow because of increasing population density and urbanization, global movement of people and animals, and deforestation accompanied by increased proximity of human and wildlife habitats. Recent emerging infectious diseases have been concentrated in tropical Africa, Latin America, and Asia, with outbreaks usually occurring within populations living near wild animals.1 Identification of animal reservoirs from which zoonosis may emerge and detection and characterization of pathogens in these reservoirs will facilitate timely implementation of control strategies for new zoonotic infections.2 Therefore, pathogen discovery studies in animal reservoirs represent an integral part of public health surveillance.

Bats have long been known as natural hosts for lyssaviruses, and more recently, they have been recognized as potential reservoirs for emerging human pathogens, including henipaviruses, filoviruses, and severe acute respiratory syndrome (SARS) related coronaviruses.3,4 Novel viruses are documented in bats every year, which has drawn increasing attention to these mammalian reservoirs that are uniquely associated with a variety of known and potential zoonotic pathogens. In this study, we report the detection of nucleic acids of adenoviruses, rhabdoviruses, and paramyxoviruses in bats from Kenya.

The above results suggested that aged mice were more susceptible to clinical disease following intranasal HeV exposure than juvenile animals. Using logistic regression analysis to determine if this trend was significant we found that 80% of aged animals developed clinical disease (s.e. = 12.7%) and only 10% of juvenile animals (s.e. = 9.5%) suggesting a real difference (70%, s.e. = 15.8%) in susceptibility to the development of disease between the aged and juvenile animals (p<0.01).

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.

Medical procedures that have the potential to create aerosols in addition to those that patients regularly form from breathing, coughing, sneezing, or talking are called AGMPs. While there are many suspected AGMPs, few AGMPs were confirmed to generate aerosols. In order to determine which AGMPs could be important for nosocomial virus transmission, we first need to characterize what aerosols are and how they are created.

Aerosols are particles suspended in air that can contain a variety of pathogens, including viruses, and there is ongoing debate about how to classify them. Many divide aerosols into the categories of small droplets (which some exclusively call aerosols) and large droplets, with small droplets having the potential to desiccate and form droplet nuclei that travel long distances, while large droplets do not evaporate before settling on surfaces. Classifying aerosols by their initial size is relevant in relation to their dispersal patterns, but it is also important to classify aerosols according to where they deposit in the respiratory tract because pathogenesis can be influenced by whether a virus deposits in the upper respiratory tract (URT) or lower respiratory tract (LRT). Dispersal and deposition depend on a variety of factors, and there is no exact cutoff for small and large droplets. Some authors use ≤5 µm in diameter as a cutoff for small droplets, while another possible cutoff between aerosol types is 20 µm, since aerosols ≤20 µm in diameter can desiccate to form droplet nuclei, and aerosols ≥20 µm do not deposit substantially in the LRT.

Often the term airborne transmission is used to describe infection by small droplet aerosols and droplet nuclei, while droplet transmission refers to the route of large droplet aerosols. Since aerosols can be of multiple sizes, we use the term aerosol transmission to generally describe transmission through the generation of infectious small and large droplet aerosols. In addition to these modes of transmission, AGMPs may also create opportunities for direct contact and fomite transmission, which may be difficult to distinguish.

HCWs are considered to be at risk for nosocomial virus transmission from both small and large droplet aerosols, for both seem to play a role in human-to-human virus transmission. Small droplets can be inhaled into the LRT, while large droplets can splash into the eyes or mouth and deposit in the URT. Certain respiratory viruses, like influenza A virus, are believed to transmit between people by both small and large droplets, whereas other nonrespiratory viruses, like EBOV, could theoretically be spread by large droplets because small droplets containing these viruses are not known to form in the human respiratory tract. It is unknown whether certain AGMPs generate either small or large droplets, or both. Therefore, depending on what aerosols are formed, AGMPs could potentially amplify a normal route of transmission for respiratory viruses or open up a new route of transmission for other viruses.

We can group possible AGMPs into two categories: procedures that mechanically create and disperse aerosols and procedures that induce the patient to produce aerosols (Figure 1 and Table 1). Procedures that irritate the airway, such as bronchoscopy or tracheal intubation, can cause a patient to cough forcefully, potentially emitting virus-laden aerosols, and both of these procedures are associated with the possibility of increasing the risk of SARS-CoV transmission among HCWs. The pressure on a patient’s chest during cardiopulmonary resuscitation can also induce a “cough-like force”, which was another possible source of SARS-CoV nosocomial transmission. Sputum is also routinely collected from patients for diagnostic purposes by cough induction, but it is not associated with nosocomial virus transmission.

In contrast to causing a patient to produce aerosols, AGMPs can also mechanically create and disperse respiratory aerosols through procedures such as ventilation, suctioning of the airway, or nebulizer treatment. Both manual ventilation, using a bag-valve-mask, and other forms of noninvasive ventilation (NIV), such as continuous positive airway pressure (CPAP), bilevel positive airway pressure (BiPAP), and high-frequency oscillatory ventilation (HFOV) are associated with SARS-CoV nosocomial transmission. Although the exact mechanisms of how these procedures create virus-laden aerosols in the respiratory tract remain unknown, it is possible that forcing or removing air from the respiratory tract could generate aerosols.

While AGMPs are traditionally thought of in regard to the generation of respiratory aerosols, AGMPs can also aerosolize infected fluids in other regions of the human body. Surgical techniques can aerosolize blood and possibly viruses. For example, infectious HIV-1 was found in the aerosols generated by surgical power tools, and a tracheotomy was associated with SARS-CoV transmission. Lasers can create plumes of debris that contain infectious aerosolized virus, as well. It is important to recognize the range of AGMPs and the circumstances under which they might be performed on infected patients. In order to associate certain AGMPs with nosocomial virus transmission, researchers need to test whether certain procedures generate aerosols with infectious virus, either through hospital sampling or laboratory procedures.

Influenza virus is a (–)ssRNA virus and a member of the Orthomyxoviridae family.5 There are four influenza genera within this family, called A, B, C, or D. Influenza A and B contain hemagglutinin and neuraminidase envelope glycoproteins. Influenza C and D have a single surface glycoprotein called the hemagglutinin-esterase fusion protein.6,7 Antigenic variation in these glycoproteins results in limited vaccine protection. Influenza, or the flu, presents with symptoms such as headache, cough, fever, sore throat, malaise, and chills.8 Generally, the flu lasts from 5 days to 2 weeks and the severity of infection is determined by the host. The highest incidence of influenza infection occurs in younger patients (<25 years old) where a shorter infection is typical, while those at risk for longer and more severe illness and complications associated with infection are the pediatric (<2 years old) and geriatric populations (>65 years old), pregnant women, and immunocompromised individuals.9,10 It is estimated that 3–5 million cases of the flu occur annually around the globe, with a quarter to half million deaths resulting from these illnesses.11

Until recently, PIV, HMPV, and RSV were all categorized in the Paramyxoviridae family due to their phylogenetic proximity in the order Mononegavirales, the non-segmented negative-strand RNA viruses. More recently, RSV and HMPV have been assigned as members of the newly formed Pneumoviridae family.12 While influenza outbreaks are most prevalent in the winter, some viruses such as PIV persist year-round. Human PIV has four types (1 to 4) and was known historically to induce respiratory complications mainly in children and the immunocompromised; however, more recently, it has been identified as a concern in the adult population as well.13 Symptoms of PIV include upper and lower respiratory tract infection, middle ear inflammation, bronchitis, pneumonia, and croup, the last of which results in the most hospitalizations in the pediatric patients infected by this virus.14,15 Up to one-third of the nearly 5 million annual cases of lower respiratory tract infection in children is at least partially due to the presence of PIVs.16

RSV and PIV infections are among the most common reason for hospitalization of young children.17,18 The two strains of RSV, A and B, are distinguished by genetic variations in the G surface glycoprotein.19 Dissimilar to PIV, RSV occurs mostly in the winter months in its target pediatric population. Symptoms include runny nose, nasal inflammation, cough, sore throat, low-grade fever, wheezing, bronchiolitis, and pneumonia.20 Current estimates in developing and industrialized countries suggest as many as 33 million cases of RSV worldwide in the pediatric population less than 5 years old, 10% of which require hospitalization, and 2% to 18% of hospitalized cases result in mortality. This amounts to between 66,000 and 600,000 deaths in young children annually.18,21

HMPV, like RSV and influenza, tends to have greatest prevalence in the winter and studies have shown that by the age of 5 years, nearly all children have been infected with this virus.22 The clinical manifestations of infection with this virus are upper and lower respiratory tract infections, bronchiolitis, middle ear inflammation, fever, chills, pneumonitis, and wheezing.23 Of note, HMPV tends to occur in populations with seasonal inconsistency as studies done on Italian populations shortly after its discovery from 2000–2002, showed a range of infection from 7% to 40% depending on the year. Patterns of seasonal irregularity like this have been noted with other respiratory viruses, particularly RSV and influenza.24

Intensive poultry farming leads to higher risk of infectious disease emergence causing great economical losses. Boundary spanning between clinical manifestations of different agents is peculiar to the course of many infections nowadays. More and more infectious diseases progress in association with different microorganisms and it effects significantly the clinical manifestation and differential diagnosis of the disease.

Currently, the viral infections such as avian influenza, Newcastle disease, infectious bronchitis, and infectious bursal disease, etc., are a potential threat to poultry farming in the Republic of Kazakhstan. Monitoring these economically significant avian diseases is the question of the day for poultry industry.

Avian influenza virus belongs to the Orthomyxoviridae family, Influenza A virus genus. From the beginning of year 2016 the disease outbreaks were recorded in 30 countries. Different AIV strains can cause 10 to 100% mortality among poultry.

The agent of the Newcastle disease is an RNA-containing virus, a member of the Paramyxoviridae family, Rubulavirus genus. In 2016 13 countries reported Newcastle disease cases to the OIE. In poultry industrial farms, all infected birds need to be sacrificed due to threat of dissemination of the infection across countries.

The agent of the infectious bursal disease is RNA-containing virus of Avibirnavirus genus in Birnaviridae family. In outbreaks of the infectious bursal disease practically the entire population is affected and the lethality rate can approach 90%, the reconvalescent birds become susceptible to the majority of infectious diseases of viral and bacterial etiology.

The causative agent of infectious bronchitis is an RNA-containing Coronavirus avia of Coronavirus genus in Coronaviridae family. Economical losses due to infectious bronchitis is composed of reduced egg and meat productivity, compulsory slaughter of sick birds, high death rate in young population. When the infection circulates in the farm for the first time the lethality rate can reach 70%.

Currently, standard immunological methods or methods based on polymerase chain reaction (PCR) [8, 9] are widely used to identify the above mentioned viruses. Unfortunately, they can detect only one agent in a specimen.

There are also multiplex RT-PCR assays that make possible simultaneous detection of more than one infectious agent by using multiple primer pairs. Advantage of the multiplex RT-PCR is in combination of sensitivity and quickness of PCR alongside with elimination of need to test clinical specimens for each agent separately [10, 11].

Avian viruses can cause diseases independently, in alliance with each other or in association with bacterial agents. Thereby, rapid and sensitive methods of detection are required that are able to differentiate viral infections for surveillance of newly emerging avian viruses as well as for disease control.

Application of DNA microarray technology that makes possible multivariate analysis of genetic material is a highly promising way for simultaneous detection of several agents (AIV, NDV, IBV and IBDV) in one specimen.

The paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescent-labelled viral cDNA on the microarray and its specificity tested with use of AIV, NDV, IBV, IBDV strains as well as biomaterials from poultry.

The objective of this study is to develop an oligonucleotide microarray for rapid diagnosis of avian influenza, Newcastle disease, infectious bronchitis, and infectious bursal disease that will be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections.

Aerosol-generating medical procedures (AGMPs) are increasingly being recognized as important sources for nosocomial transmission of emerging viruses. Intubation was investigated as a possible cause of Ebola virus (EBOV) transmission among health-care workers (HCWs) in the United States. Additionally, the high rate of nosocomial transmission of Middle East respiratory syndrome coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) caused speculation about the role of AGMPs. Crimean–Congo hemorrhagic fever orthonairovirus (CCHFV), was also associated with nosocomial infection secondary to AGMPs. While guidelines were developed for performing AGMPs on patients with certain viral infections, assessing and understanding the risk that specific viruses and AGMPs pose for nosocomial transmission could improve infection control practices, as well as reveal relationships in virus transmission.

Despite the perceived importance of AGMPs in nosocomial transmission of viruses and other infectious agents, scarce empirical or quantitative evidence exists. In order to assess the risk that certain viruses and AGMPs create for nosocomial transmission, we first need to identify potential AGMPs and viruses. The second step is then to determine the risk associated with these viruses and procedures, either through retrospective analysis, investigating the circumstances of nosocomial transmission, or through experiments, such as using air sampling during AGMPs to determine the risk of generating infectious virus-laden aerosols. Lastly, we can use this knowledge to re-evaluate current guidelines and communicate which viruses and AGMPs pose the highest risk for nosocomial transmission.

RNA was extracted from virus-containing material with TRizol (“Invitrogen”, USA) according to the manufacturer's instruction.

CoVs are enveloped, positive sense, single stranded RNA viruses belonging to the family Coronaviridae. There are six coronaviruses divided in two genera that can infect humans. CoV 229E and NL63 belong to the genus alphacoronavirus and OC43, HKU1, severe acute respiratory syndrome (SARS)-CoV, and Middle East respiratory syndrome (MERS)-CoV are members of the genus betacoronavirus. The genome codes for two non-structural proteins and four structural proteins (van der Hoek, 2007; Greenberg, 2011).

While CoVs are an important cause of the common cold in the general population, there is limited information on the clinical manifestations in immunocompromised hosts. Milano et al. described the epidemiology and risk factors of CoVs infection among HCT recipients. The cumulative incidence estimated at day 100 was 11.1%. The median time of first detection was 53 days (range, 2–93 days). Seasonal outbreaks were common in the winter with 13 of 22 cases first detected in December through March. The median time of shedding was over 3 weeks (Milano et al., 2010).

Ogimi et al. described several risks factors related to prolonged viral shedding, such as higher viral load, use of steroids, and myeloablative conditioning (Ogimi et al., 2017a). Univariate analysis of potential risk factors showed no significant association of acquisition with patient age, gender, underlying disease risk, stem cell source, CMV serostatus, donor type, acute GVHD, conditioning regimen, or engraftment status. Infection with coronaviruses was not associated with mortality in this cohort (Milano et al., 2010). However, another group reported similar mortality rates in HCT recipients to those observed with other viruses such as RSV, influenza virus, and PIV (Ogimi et al., 2017b).

A recent article from Seattle Children's Hospital looked at 85 immunocompromised and 1152 non-immunocompromised children with HCoV infection. Other than median age which was significantly higher for immunocompromised patients, demographic characteristics were similar. Viral co-infection was most commonly seen in non-immunocompromised patients, mostly due to detection of RSV. CoVs strains did not differed between the two groups. The most common clinical presentation was URTI. Younger age, male sex, presence of an underlying pulmonary disorder, and detection of a respiratory co-pathogen, particularly RSV, were associated with an increased likelihood of LRTD or severe LRTD. However, lymphopenia was not associated with more severe disease in this cohort (Ogimi et al., 2018a).

There is currently no treatment available for coronaviruses. However, current in vitro evaluation of HCoV therapy includes investigation of antiviral, as well as human monoclonal antibodies (Pyrc et al., 2007; Adedeji et al., 2013) (Table 4).

The RhVs belong to the genus Enterovirus within the family Picornaviridae. They include three genetically distinct groups: A, B, and C. They are single-stranded, positive sense, RNA viruses. The genome encodes one polypeptide that is cleaved in four structural proteins (VP1-4) and 7 non-structural proteins. RhVs are antigenically diverse with over 100 serotypes identified to date (Greenberg, 2011; Jacobs et al., 2013).

Rhinovirus has been described as the most common respiratory pathogen isolated in immunocompromised children undergoing HCT (Loria et al., 2015; Fisher et al., 2017), with an incidence as high as 22.3% by day 100 (Milano et al., 2010).

The median time for a first positive tests has been reported at 44 days following HCT but can be as early as 2 days and as late as 93 days. There is no apparent seasonality for RhV detection. Prolonged viral shedding is common with a median of 3 weeks (Milano et al., 2010). Higher viral load at initial presentation has been significantly associated with prolonged viral shedding (Ogimi et al., 2018b). In the former study, thirteen percent of patients with RhV infection had no respiratory symptoms at the time of diagnosis. The remaining patients had URT symptoms. Four patients underwent a BAL for concerns of progression to LRTI, of these two died of respiratory failure (Milano et al., 2010). These results agreed with a study from Seo et al looking at 697 transplant recipients with rhinovirus infection. In this cohort, eighty one percent of patients presented with URTI. Patients with low monocyte count, oxygen use and steroid dose > 1 mg/kg/day had significantly increased risk of LRTI. Mortality was higher in those with LRTI (41 vs. 6%) (Seo et al., 2017). A multicenter study of respiratory viral infections in pediatric HCT recipient reported an all cause fatality rate of 10% in those patients with RhV infection (Fisher et al., 2017).

Pre-transplant rhinovirus detection has been significantly associated with increased mortality at day 100 (Campbell et al., 2015). However, Abandhe et al. did not find any differences in ICU admission, length of hospitalization or mortality (Abandeh et al., 2013). Currently, treatment of RhV infection consists of supportive care. Antiviral medications for RhV are under investigation (Table 4).

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).

The BelPV nucleotide sequence obtained showed similarity with the JPV and BeiPV sequences. BelPV has previously been reported to hold a phylogenetic position between the genera Henipavirus and Morbillivirus. The same phylogenetic position had been observed with MosPV and J-V.

In this study, organs with high BelPV concentrations are different from those found with high paramyxovirus concentrations in pteropodids and microchiroptera bats. In microchiroptera bats from Brazil, spleen has been found more positive than the others organs with highest viral load, as in Eidolon helvum (megachiroptera bat) in Africa. However, in our study majority of spleen were not available.

The within-host BelPV distribution tended to be organ-specific. BelPV seemed to be restricted to the heart and liver. In contrast, JPV has been isolated from blood, lung, liver, kidney and spleen of experimentally infected laboratory mice but not in heart. The BelPV distribution for the heart and liver, together with the high viral load in heart tissue, could suggest that this virus is likely to be present in the bloodstream and might thus be transmitted during aggressive contacts between bats, or by blood-sucking vectors. Nethertheless, viremia was not proven. BelPV RNA was not searched from blood because in these small species of bats blood was difficult to collect in the field.

We detected BelPV only in Coleura afra and not in other bat species sharing the same roosting sites and living in very close proximity in the two caves sampled. However, it has been shown that bats of different species occupying the same roosting sites can share the same viruses. Marburg virus had been detected in Rousettus aegyptiacus and Hipposideros sp. bats living in Kitaka cave in Uganda and Miniopterus inflatus and Rousettus aegyptiacus bats caught in Goroumbwa mine in the Democratic Republic of the Congo. These bat species are known to live in close proximity. Thus, virus transmission between different bat species is possible. Thus, we can speculate that the failure to detect BelPV in other bat species sharing the same caves would suggest that this virus has strong host specificity for C. afra, as well as restricted intraspecies transmission. Henipaviruses occur naturally in fruit bats belonging to the genus Pteropus

[26], and this also appears to be true of severe acute respiratory syndrome-like coronaviruses in Rhinolophus bats,.

In view of our data we can assume that BelPV might have pathogenic potential for its host C. afra. Indeed, high viral load was detected in the heart of the diseased bat, and the lesions were consistent with those reported in wild rodents and mice experimentally infected by JPV.

Although BelPV RNA was also detected in asymptomatic bats, pathogenicity may appear in long term under some immunological and/or ecological conditions. Indeed, virus must not induce pathology to persist or adapt within its reservoir host. Many authors suggested that persistence in the absence of pathology or disease appears to be a common characteristic of bat viruses in their natural host population,. However, a severe immunodepression for instance, may increase the risk of infection with opportunistic pathogens. Under some environmental conditions (cool environments for example), some avirulent pathogens, such as Geomyces destructans, causative agent of white-nose syndrome, may become pathogenic in hibernating bats in North America,. Nevertheless, infection by BelPV may be mild for bats and thus the pathology observed not directly related. Otherwise, it may also be that this animal had an underlying disease or infection with a different pathogen. Even in this case, we might not draw any conclusions neither establish a link with lesions seen. Therefore, the pathogenicity of the BelPV should be demonstrated by experimental animal infection. Otherwise, viral antigens or RNAs should be detected histologically in the lesions of naturally-infected bats. However, the unavailability of biological tissues from the diseased bat failed to perform these analyzes. Consequently, other captures of Coleura afra species are considered in order to find BelPV again for further studies (pathogenicity to its host, isolation and complete genome characterization). However, Coleura afra is a migratory species living in colonies of several hundred individuals. In Gabon, this species, which has been recently described, is not present all year round in the caves of the north-east of the country, making the studies on this species difficult and thereof partly explaining the lack of virological studies.

Some viruses appear to cause clinical disease in wild-living bats; these include lyssaviruses and an ebola-like filovirus named Lloviu virus,,.

Bats are the natural reservoirs for many viruses, including emerging zoonotic viruses such as SARS-CoV, Hendra and Nipah viruses,, Ebola virus, Marburg virus,, rabies virus and other Lyssaviruses. In general, humans are infected through an intermediate amplifying host such as palm civets for SARS-CoV, horses for Hendra virus and pigs for Nipah virus. However, in humans Nipah virus outbreaks linked to bats exposure have been reported. It remains to be shown whether the BelPV reported here presents a zoonotic risk. Nonetheless, like most RNA viruses, for example coronaviruses, characterized by high mutation and/or recombination rates, PVs may adapt to novel hosts, including humans. A serological test capable of detecting antibodies to this virus in human populations living in the vicinity of these animals is needed to assess zoonotic potential.

All the blood-sucking arthropods collected from bats, as well as mosquitoes collected in the caves where bat sampling took place, were negative for BelPV, in keeping with the lack of known PV vectors. However, BelPV transmission by blood-sucking vectors within the Gabonese population of C. afra cannot be ruled out. Indeed, a haemosporidian parasite (Polychromophilus) was found in a blood parasite vector (Penicillidia fulvida) in Faucon cave in Gabon in 1977 and also in its host M. inflatus (greater long-fingered bat) from the same cave in 2010 and 2011. In addition, the methodology used to collect flying hematophagous insects (based on light traps) possibly introduced a bias by selecting only those attracted by light. Therefore, we can not exclude that additional sampling techniques could increase the number of mosquitoes species or groups known to colonize caves such as sandflies or biting midges. Hence, the natural mode of transmission of this unclassified paramyxovirus in bat populations, through bat-bat aggression for example, remains to be determined.

This association between C. afra and BelPV could serve as an interesting model, (i) to evaluate modes of transmission within host populations, (ii) to study host-virus interactions (pathogenesis and host specificity), and (iii) to evaluate the zoonotic risk of a newly identified virus.

Further studies of C. afra populations and a broader diversity of arthropod vectors, spanning larger areas and time scales, are needed to confirm this apparent host-virus specificity, and to determine the modes of BelPV transmission. Further studies are needed to characterize complete BelPV genome and demonstrate the pathogenicity of this virus for its host Coleura afra.

Bovine respiratory disease complex (BRDC) is a major problem for cattle breeders worldwide, causing serious economic losses. BRDC is associated with infection by certain viruses, bacteria, and parasites (33). In addition to these infectious agents, stress factors such as transport, gestation, and poor management conditions play an important role in the onset of the disease (30). Bovine herpes virus 1 (BHV-1), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza virus-3 (BPIV3) are the most common viral agents of the respiratory system. Some opportunistic agents (Mannheimia haemolytica, Pasteurella multocida, Haemophilus somnus, and Mycoplasma spp.) contribute to the appearance of clinical signs and thus increase mortality and cause losses in the herds (18). Suppressed immunity also has an important role in the prognosis. Diseases such as bovine leucosis and bovine viral diarrhoea suppress immunity and lead to more animal loss by worsening clinical symptoms.

BPIV3 (the new name of which is bovine respirovirus 3) is an RNA virus assigned to the Paramyxoviridae family under the Respirovirus genus. BRSV (the new name of which is bovine orthopneumovirus) is in the Pneumoviridae family under the Orthopneumovirus genus. To date, three genotypes of BPIV3 have been described. These genotypes, termed A, B, and C, were differentiated based on phylogenetic analysis. Genotype A strains have been isolated in North America, China, and Japan. Genotype B was originally found in Australia. Isolations of genotype C were in China, South Korea, and Japan. In addition, all three genotypes have been reported in Argentina (23).

Initially BRSV subgroups were identified (A, B, and AB or intermediary) based on monoclonal antibody and polyclonal sera analyses against F and G proteins (31). Additionally, Valarcher et al. (36) proposed that six genetic subgroups may be found in BRSV strains, when F, G, and nucleoprotein sequences are phylogenetically analysed by maximum-likelihood algorithms. Therefore, six subgroups were detected in BRSV. These subgroups termed I (the subgroup B prior to the recommendation of Valarcher et al. (36)), III (subgroup A), and II, IV, V, and VI (subgroup AB) were differentiated based on phylogenetic analysis. Subgroup I consists of European strains (UK and Switzerland). Subgroup III includes viruses exclusively from the USA. Subgroup II aggregates strains from the Netherlands, Belgium, France, Denmark, Sweden, and Japan. Subgroup IV is of European and USA strains while subgroups V and VI are found only in French and Belgian isolates (29, 36). Subgroup VII was detected in later years (9) and some strains are known which are still not classified (these are regarded as untyped) (10).

BPIV3 and BRSV can cause mild symptoms or subclinical disease when present alone. However, when there is a co-infection, they may cause bronchopneumonia, severe cough, high fever, and nasal discharge and contribute to a more serious clinical course of infection (33). Regardless of the infecting agent in BRDC, clinical symptoms may be similar and the process of detecting the underlying primal agent may be hindered due to mixed bacterial infections. This situation makes viral diagnosis difficult and decreases the specificity and sensitivity of the molecular methods (when compared to immunofluorescence antibody tests) (15).

Data on virological detection of these agents in Turkey is limited (2, 6), but there are more studies on seroprevalence of these viruses among cattle herds. The studies reported lowest and highest seropositivity of 11% (1) and 92.8% (13) for BPIV3 and 28% (1) and 94% (13) for BRSV. Serological studies on BRSV and BPIV3 were previously conducted in different geographic regions of our country. In these studies the following percentage values for BRSV and BPIV3 prevalence were determined respectively: Alpay et al. (5) 26.6% and 44.6%, Alkan et al. (3) 62.0% and 44.6%, Avci et al. (7) 78.2% and 85.6%, Çabalar and Can Sahna (11) 67.3% and 18%, Yavru et al. (40) 46% and 53.9%, and Yesilbag and Gungor (41) 73.0% and 43%. These studies were conducted either countrywide (3) or in selected regions (40, 41).

The aim of this study was the detection and molecular characterisation of BPIV3 and BRSV strains retrieved from nasal swabs and lung samples of cows in the eastern region of Turkey. The determination of BRSV and BPIV3 types and associated co-infections for respiratory system infections was conducted.

We next observed preliminary cross-reactivity in indirect immunofluorescence assay between several human viruses belonging to the family Paramyxoviridae and the bat paramyxovirus B16-40. 6-week-old female BALB/cA mice maintained under SPF conditions were intramuscularly inoculated twice, with 2 weeks in between, with a 1:1 ratio mixture of AddaVax adjuvant and live virus stock (bat paramyxovirus B16-40, human respiratory syncytial virus A (KBPV-VR-41), human mumps virus (KBPV-VR-51), and human parainfluenza virus 1 (KBPV-VR-44), respectively, from the Korea Bank for Pathogenic Viruses. Two weeks after the last immunization, antisera against the virus were obtained by cardiac puncture for further studies related to cross-reactivity in indirect immunofluorescence assay. Mouse antisera against the bat paramyxovirus B16-40 (105.75 TCID50/mL) and human parainfluenza virus 1 (KBPV-VR-44, 105.75 TCID50/mL) were further compared through serum titration using the indirect immunofluorescence assay and serum neutralization test.

EV-A71 belongs to the genus Enterovirus within the family of Picornaviridae. It was first characterized in 1969 in California, USA and is one of the main etiological agents of hand, foot and mouth disease (HFMD). Some cases of EV-A71 infections have been associated with neurological complications such as aseptic meningitis, brainstem encephalitis and acute flaccid paralysis. In China, enteroviruses such as EV-A71 and CV-A16 have caused 7,200,092 cases of HFMD between 2008 to 2012. The mortality rate was highest among children below the age of five. It was further reported that 82,486 patients developed neurological complications and 1617 deaths were confirmed by the laboratory to be caused by EV-A71.

The course of evolution through which EV-A71 evolves to escape the central nervous system (CNS) was investigated by complete sequencing and haplotype analysis of the strains isolated from the digestive system and the CNS. A novel bottleneck selection was revealed in various environments such as the respiratory system and the central nervous system throughout the dissemination of EV-A71 in the host. Consequently, a dominant haplotype resulting from the bottleneck effect caused a change from viruses harboring VP1–3D to VP1–31G where the amino acid 31 was a favorable site of selection among the circulating EV-A71 sub-genotype C2. VP1–31G was present at elevated levels amongst the population of mutants of EV-A71 in the throat swabs of subjects with severe EV-A71 infections. Furthermore, in vitro studies showed that VP1–31D virus isolates had higher infectivity, fitness and virion stability, which sustained the virus infections in the digestive system. Speculations were that such factors benefitted the virus in gaining added viral adaptation and subsequently enabled viral spread to more tissues. These beneficial abilities could also justify the reduced number of VP1–31D viruses located in the brain following positive selection. The VP1–31G viruses presenting the major haplotype in the central nervous system displayed increased viral fitness and growth rates in neuronal cells. This implied that the VP1–31G mutations aided the spread of the mutant virus in the brain which resulted in serious neurological complications in patients. It was speculated that the fluctuating degree of tissue tropism of EV-A71 at diverse inoculation sites resulted in the bottleneck effect of the viral population having a mutant spectrum. Hence, the adaptive VP1–31G haplotype became dominant in neuronal tissues and once the infection was achieved, VP1–31G viruses expedited bottleneck selection and propagation into the skin and CNS. Among the three minor haplotypes (C to E) which co-existed in various tissues, the minor haplotype C was isolated in the intestinal mucosa and throat swab specimens. The minor haplotype D was isolated from specimens obtained from the respiratory and digestive systems. However, the minor haplotype E appeared in throat swabs and the basal ganglia but not the intestinal mucosa, hence, suggesting that the intestinal mucosa is the initial replication site of the EV-A71. Collectively, these data showed that the EV-A71 quasispecies utilized the dynamic proportion of varying haplotype populations to co-exist, sustained the ability of the population to adapt and enabled the propagation in different tissues. Lastly, the study concluded that the selection of haplotype(s) might be a driving factor in viral dissemination and severity of infections in humans as well as the virulence in EV-A71 infected patients.

There are various diseases affecting young and old cattle worldwide. These diseases can impair the respiratory, digestive, and genital systems (4, 25, 34, 35). However, it is viral diseases associated with the respiratory system of cattle which are regarded as one of the main problems in cattle breeding, affecting both adults and young animals. This holds especially true when mortality rates increase in case of mixed infections. In this study, BRSV and BPIV3 virus strains were detected in pool of 155 lung tissue and nasal swab samples collected from cattle from Erzurum and neighbouring provinces.

BPIV3 and BRSV were detected by other researchers with serological and virological methods in cases of lower respiratory tract infections in cattle in Turkey (2, 6, 41). However, these studies were not performed by molecular methods and did not include genetic characterisation as we have done. Although it was proved that in cases of lower respiratory tract infections lungs could contain a high number of virus particles (8), we could detect only BPIV3, even though samples were collected in late winter. This may be because the animals were in a convalescent phase of infection or a majority of tissue samples suspected of being from pneumonic animals did not contain virus particles.

Enveloped viruses, paramyxoviruses in particular, are generally susceptible when exposed to the outside environment. Pirtle and Beran (27) reported that when diluted to 105.5 CCID50/mL and used to contaminate different kinds of media, the viability time was 1.5 h on rubber gloves, 20 min on skin, and a maximum of 6 h on an indoor surface. This is why samples must be admitted to the laboratory in a short time and tested in the shortest time possible. When low positivity rates are considered, both in this study and worldwide, there is always the possibility of false negativity because of virus sensitivity. PCRs specific for the gene-encoding fusion region for BRSV and the matrix gene for BPIV3 were used. The fusion gene of BRSV is a region that determines antigenic variations (26), and the matrix gene for BPIV3 is responsible for viral assembly, pathogenesis, and antigenicity of strains (20, 21). Phylogenetic analysis of BRSV based on the F protein sequence classified the isolates into seven different subgroups. The topology of the phylogeny was retained when an analysis of the N and G protein gene sequences was conducted (36). In our study, the determined strain was found to belong to subgroup III. Knowing the genotypes helps to determine the type-specific vaccine selection in the geographic region. Valarcher et al. (36) identified vaccine failure among animals infected with BRSV groups V and VI, indicating that commercial vaccines act poorly against infections caused by such viral groups. Therefore, selection of vaccines is important and should be specific to the type of strains present in the region. For example in the 2000s in Europe, vaccines including Rispoval RS (strain RB-94, subgroup II), Bayovac (strain Lehmkuhl 375, subgroup III), and Vacores (strain 220/69, subgroup II) were used. If these vaccines were currently in use, Bayovac could be suitable for our country. However, in vivo trials would be required for other vaccines. Therefore, genotyping studies of viruses are necessary for molecular epidemiology and vaccine studies.

It is well established that respiratory tract infections are mostly mixed. These types of infections may contain viruses, bacteria, and other agents or may be caused by virus-virus or bacteria-bacteria interaction (18, 33). We investigated the samples with single or mixed (BRSV + BPIV3) infections and no other mixed infections were detected. Therefore, when viruses are the sole cause of infection, it is highly likely that they will cause subclinical infections; this may explain the low rate of detection in this study.

Aetiology in respiratory infection is quite important (14, 38). A fast and reliable method must be selected to identify the underlying agent. Genotyping studies may provide information about strains that cause serious clinical onsets related to vaccine strains and preventive status. Phylogenetic analysis of the BRSV strain was performed (Fig. 2). Analysis of the partial sequence of BRSV shows that our strains were more closely related to American and Brazilian (USA: KU159366; and Brazil: FJ543092) strains instead of European ones. Although the strains identified in the study are expected to be close to the European strains (because of the geographical location of Turkey), the increase in meat and live animal imports into our country over the last 5 years may be a factor in this situation, because our country imports meat from USA, Canada, Brazil, and Uruguay. Many infectious diseases may be spread among cities, countries, and continents as a result of animal or animal product transportation (12, 16). In our study, BRSV sequences showed high genetic similarity with the American and Brazilian strains. This genetic similarity among BRSV strains suggests that it could have come from Brazil or America, as a result of live animal exports.

Infection with BPIV3 is common in cattle worldwide. The virus is divided into three genotypes: A, B, and C (19, 24, 39, 42). Genotype A is most common in America, genotype B in Australia and genotype C is seen mostly in Asia (24, 28, 42) (Fig. 1). Our samples clustered in genotype C in the phylogenetic tree. This is the first study to report BPIV3 of this type (genotype C) in Turkey. This also may be regarded as the first report in Europe since no published data is available as far as we are aware.

In conclusion, this paper is the first report of BRSV and BPIV3 infections in cattle in the east Anatolian region of Turkey and specifically Erzurum and its neighbouring provinces by molecular methods. There is a need for additional epidemiological studies to better understand the prevalence and control of the infections. Regional and local infections should be investigated and properly addressed through random sampling. For implementing control and eradication studies at a national level, full genome sequence data should be provided in further studies.

The avian paramyxoviruses (APMVs) are isolated from wild and domestic birds all over the world. The APMVs have been divided into nine serotypes (APMV 1 to 9) based on hemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays. More recently, viruses representing potential APMV serotypes 10 and 11 were isolated from Rockhopper Penguins and common teal, respectively. APMV-1, which includes all strains of Newcastle disease virus (NDV), has been extensively characterized because virulent NDV strains cause severe disease in chickens. NDV strains are divided into three pathotypes based on their virulence in chickens: highly virulent (velogenic) strains cause severe respiratory and neurologic diseases; moderately virulent (mesogenic) strains cause milder disease, and nonpathogenic (lentogenic) strains cause inapparent infection. Mesogenic and lentogenic strains of NDV are highly restricted for replication and highly attenuated in non-avian species including primates, and are being developed as vaccine vectors for animal and human pathogens. Previous studies have shown that APMV serotypes 2-9 replicate not only in avian species, but also in mice and hamsters,. However, their ability to replicate and possibly cause disease in primates, and their potential as human vaccine vectors, were unknown.

The APMVs belong to family Paramyxoviridae, a large and diverse family that includes viruses from a wide variety of mammalian, avian, reptilian, and fish species around the world. Some members of the family are responsible for major human and animal diseases, while others cause inapparent infections. Paramyxoviruses are pleomorphic and enveloped and contain a non-segmented, negative-sense, single-stranded RNA genome of 13–19 kb. On the basis of virus structure, genome organization and sequence relatedness, the family Paramyxoviridae is divided in to two subfamilies: Paramyxovirinae and Pneumovirinae

[6]. The subfamily Paramyxovirinae is divided into five genera: Respirovirus (including Sendai virus and human parainfluenza virus types 1 and 3), Rubulavirus (including parainfluenza virus 5 [previously known as simian virus type 5], mumps virus, and human parainfluenza virus types 2 and 4), Morbillivirus (including measles and canine distemper viruses), Henipavirus (comprising Hendra and Nipah viruses), and Avulavirus (comprising the APMVs). Subfamily Pneumovirinae contains two genera, Pneumovirus (including human and bovine respiratory syncytial viruses) and Metapneumovirus (comprising human metapneumovirus and the avian metapneumoviruses),

Although a lot of information is available for APMV-1, much less is known about the molecular biology, pathogenicity and host range of the other APMV serotypes. As an initial step towards their characterization, we have recently determined the complete genome sequences of APMV-2 to -9–[15] and we have developed reverse genetics systems for APMV-2, -3, -4 and -7–[19]. However, the biological characteristics and pathogenicity of APMV-2 to -9 remain poorly understood. APMV-2 has been associated with severe respiratory disease, reduced egg production and infertility in turkeys,. APMV-3 has been associated with encephalitis and high mortality in caged birds, respiratory disease in turkeys and stunted growth in young chickens,. APMV-4 strains have been isolated from chickens, ducks and geese. APMV-5 causes disease in budgerigars that is characterized by depression, dyspnoea, diarrhea and high mortality. APMV-6 and -7 cause mild respiratory disease in turkeys and are associated with a drop in egg production,. APMV-8 and -9, isolated from ducks, waterfowl, and other wild birds, did not produce any clinical signs of viral infection in chickens,

In the last 10 years, reverse genetic techniques have made it possible to engineer NDV as a potential vaccine vector for both human and animal uses–[39]. NDV vectors expressing a number of foreign antigens have been evaluated not only in avian hosts, but also in murine and nonhuman primate models–[33],–[42]. Several strains of NDV have been shown to be highly restricted for replication in these mammalian models, indicating that they are highly attenuated due to a strong host range restriction and represent promising vaccine vectors. However, NDV strains are highly related antigenically, and therefore the use of NDV vectors for multiple purposes would be compromised by the induction of vector-specific immunity. This limitation might be overcome by using other APMV serotypes that are antigenically distinct from NDV as alternative vaccine vectors. In addition, it is possible that one or more of the other APMV serotypes might have other advantageous properties that cannot be predicted, such as increased immunogenicity compared to NDV. Furthermore, the possibility exists that one or more of the other APMV serotypes might be pathogenic in certain non-avian hosts including primates. For example, an APMV-2-like virus was previously recovered from a cynomolgus monkey with respiratory disease, and an APMV-3-like virus was recovered from pigs. Therefore, evaluation of APMV-2 to -9 in non-avian species was warranted.

We recently showed that APMV-2 to -9 are competent to infect and replicate to low-to-moderate titers in mice and hamsters,. However, rodents are uncertain predictors of performance in other species such as humans because it is possible, and indeed likely, that there will be differences in the level of host range restriction between rodents and other species including primates. In the present study, we sought to evaluate the replication and pathogenicity of APMV-2, -3, -4, -5, -7 and -9 in rhesus macaques as a surrogate for humans. The viruses included biologically- and recombinantly-derived wt viruses as well as several recombinant viruses in which the F protein cleavage site had been modified to be multi-basic and to contain the optimal furin protease cleavage site motif RX(R/K)R↓ (signature R and K residues underlined). This was done because the presence of a furin motif in the F protein cleavage site typically facilitates cleavage and is a major determinant of virulence for NDV strains,, although this paradigm is uncertain for the other APMV serotypes,,. The present study showed that, except for APMV-5, all of the APMVs under evaluation replicated at varying levels in rhesus macaques without inducing any apparent clinical disease signs. Thus, APMV serotypes 2, 3, 4, 7, and 9 are infectious, replication-competent and attenuated in non-human primates. In future work, the reverse genetics system for APMV-2, -4, and -7 will be used for the development of APMV vectored vaccines.