Influenza viruses are known to constantly evolve and cross species barriers. The genetic diversity of influenza viruses is ever increasing with more novel influenza subtypes being discovered periodically. The purpose of this review is to provide an up-to-date overview of ecology and evolution of influenza viruses including the novel influenza viruses in bats and cattle. In addition, we discussed the growing complexity of influenza virus–host interactions and highlighted the key research questions that need to be answered for a better understanding of the emergence of pandemic influenza viruses.

Influenza is among the major infectious disease problems affecting animal and human health globally. Several human influenza pandemics have been recorded since 1590 AD, with the most significant of those being the “Spanish flu” of 1918, often referred to as the “mother of all pandemics”. Spanish flu pandemic is believed to have affected approximately 25–30 percent of the world’s population and caused more than 50–60 million human deaths globally. Influenza infections in humans occur either as epidemic (seasonal or interpandemic) influenza caused by influenza A and B viruses, or as sporadic pandemic influenza caused by influenza A viruses. Study of influenza pandemics has been of great interest to epidemiologists. Influenza epidemics and pandemics have been repeatedly occurring for centuries, but to date the ability to predict a pandemic has not been achieved.

Lower respiratory tract infection (LRTI) remains one of the major causes of mortality and morbidity in children under five years globally. Viruses have already been recognized as important etiologies of respiratory infections with influenza virus which is considered as the main contributor. The epidemiology and public health impact of influenza infections are relatively well described as many studies and surveillance have been conducted in part of pandemic preparedness [2–5]. Most of the countries in the world, including Indonesia, have developed influenza surveillance, influenza-like illness (ILI) surveillance, and severe acute respiratory illness (SARI), which form the network under WHO through Global Influenza Surveillance and Response Systems (GISRS) [4–8]. This network improves influenza disease control by providing support on influenza vaccine recommendation, laboratory diagnostic tools, antiviral, and public health risk assessment. As influenza virus contributed only less than 30 percent of viral respiratory infections, there is an urge to investigate the contribution of other respiratory viruses for improving respiratory disease control program.

Recent advancement of molecular technology supports the investigation and characterization of several respiratory viruses. The molecular technology improves the capability to study respiratory viruses, which are previously identified: rhinovirus, adenovirus, respiratory syncytial viruses, parainfluenza virus, and also the new emerging viruses/strain viruses: MERS coronavirus, human metapneumovirus, and human rhinovirus strain C. Multiple detection platforms, which recently have been developed, allows relatively inexpensive and timely detection of several viruses [9–11]. The detection of multiple respiratory viruses will accommodate the efforts to determine the epidemiology of noninfluenza respiratory viruses in the community, which will further help the respiratory disease control program including the use of antimicrobial agents.

Previous results of the investigation on acute respiratory infection patients in several countries showed the difference in the prevalence of respiratory viruses among studies [3, 13–15]. Study design including the case definition, study population, time of the study, and diagnostic tools being used have been considered as factors that influenced the variation. Each virus has different seasonality circulation and an age-related prevalence that can lead to a specific pattern of virus cocirculation in many studies [17–19]. Moreover, the occurrence of virus coinfection in which two or more viruses are detected in a single patient has been described in recent studies using multiple pathogen detection platforms [20–22].

There are limited studies on viral pathogens of respiratory tract infection in low-middle income countries including Indonesia. Previous studies have been conducted mostly focused on specific viral pathogens, especially influenza [6, 23, 24]. The prevalence of noninfluenza respiratory infections is relatively unknown. Therefore, this study has an objective to investigate the prevalence of viral etiologies from ILI cases in Indonesia.

In 2005, Allander et al., reported the discovery of a previously undescribed human parvovirus in respiratory secretions from children with respiratory tract disease in Sweden. Phylogenetic analysis showed that this virus belonged to the genus Bocavirus (subfamily, Parvovirinae; family, Parvoviridae) and was most closely related to bovine parvovirus (BP) and minute virus of canines (MVC). The virus was thus named "human bocavirus" (HBoV).

Human bocaviruses (HBoV) contain 3 open reading frames (ORFs) encoding a nonstructural protein (NS1, NP1) and two capsid proteins VP1 and VP2, respectively. The genomic organization of HBoV closely resembles that of bovine parvovirus type 1.

HBoV has been reported in respiratory samples from children with acute respiratory tract disease in various parts of the world (including Australia, North America, Europe, Asia, and Africa), suggesting that the virus is circulating worldwide. Pneumonia, acute bronchitis, bronchiolitis, are the main manifestations of HBoV infection.

HBoV seems to be a new member of the community-acquired respiratory viruses such as respiratory syncytial virus, adenovirus, influenza virus, parainfluenza virus, and rhinovirus, which cause common respiratory tract infections in the community. Because of its very high copy numbers in respiratory tract secretions, aerosol and contact transmission are likely effective, as they are for other respiratory viruses.

HBoV has been detected also in nasopharyngeal, serum, fecal and urine samples obtained mainly from young children around the age of 2 years predominantly during the winter season. HBoV was detected in two pediatric patients after organ transplantation, in human immunodeficiency virus-infected pediatric patients, and immunosuppressed adult patients.

Diagnosis of HBoV infection is based on the PCR amplification of viral genome fragments present in human respiratory, serum, stool and urine samples. A great number of different PCR techniques employing varying sets of primers specific for the viral genes NP1[10], NS1, and VP1, VP2 have been described. In addition to the detection of viral genomes by PCR, recent reports describe the detection of HBoV-specific IgG and IgM-antibodies against HBoV VP2 in serum samples using western blot or immunofluorescence assays. Furthermore, a standardized ELISA for the quantitative determination of HBoV-specific antibodies has been established by. The aim of the work was determination of HBoV in respiratory specimens (NPA) of infants by qualitative PCR and determination of acute HBoV infection by estimation of IgM antibodies in serum by ELISA.

Human bocavirus (HBoV) was discovered in 2005 by Allander et al. in respiratory samples from children with suspected acute respiratory tract infection (ARTI) using a novel technique. This molecular virus screening is based on a random PCR-cloning-sequencing approach and was employed on two chronologically distinct pools of nasopharyngal aspirates (NPAs). It revealed a parvovirus-like sequence, with close relation to the members of the bocavirus genus.

A retrospective study revealed 17 (3.1 %) out of 540 NPAs positive for HBoV, with 14 specimens tested negative for other viruses, giving the suggestion that HBoV is a causative agent of respiratory tract infections.

Alpaca (Vicugna pacos, also known as Lama guanicoe pacos) are domesticated members of the New World camelid species (Lamini), which also include guanaco (Lama guanicoe), vicuna (Vicugna vicugna), and llama (Lama glama). The natural habitat for alpaca is at high altitude (3500–5000 m) in South America (Peru, Ecuador, Bolivia, and Chile) where they are kept as livestock in herds and their fiber is used much like wool. Approximately 300,000 animals are in the U.S. Compared to other livestock, e.g., about 96 million cattle, their number is still relatively small.

Previously reported viral infections in domestic alpaca include adenovirus, equine viral arteritis virus, rabies, bluetongue virus, foot-and-mouth disease virus, bovine respiratory syncytial virus, influenza A virus, rotavirus, orf virus, bovine papillomavirus, vesicular stomatitis virus, coronavirus, bovine parainfluenza-3 virus, West Nile virus, equine herpesvirus-1,, and bovine viral diarrhea virus–[13]. Bovine enteroviruses (BEV) have not previously been reported to infect alpaca. The bovine enterovirus species previously contained two types, BEV-A and BEV-B, although a new classification structure was ratified recently, redesignating these as species Enterovirus E (EV-E) and Enterovirus F (EV-F), respectively,. Each of the new BEV species includes multiple serotypes, with EV-E comprising four described serotypes (previously A1–4, renamed E1–E4), and EV-F containing six reported serotypes (previously B1–6, renamed F1–F6).

Recently developed approaches to virus detection have the potential to further expand understanding of viral disease in animals, including alpaca. Many of these approaches are based on non-specific PCR amplification used in conjunction with standard or high-throughput sequencing to identify PCR products.

We utilized such a method–[19] to investigate an outbreak of a respiratory infection in alpaca, identifying a bovine enterovirus (EV-F), named Enterovirus F, strain IL/Alpaca, after other techniques had failed to detect any pathogen.

Human bocavirus (HBoV) was newly discovered by Allander et al. and was stated to be the possible causative agent of respiratory illness. Phylogenetic analysis of the complete genome of HBoV revealed that the virus is most closely related to canine minute virus and bovine parvovirus, which are members of the Bocavirus genus of the Parvoviridae family.

HBoV was found in 1.5-11.3% of respiratory samples investigated worldwide. HBoV may be a causative agent of respiratory tract infections. However, some investigators argue that the association between HBoV and respiratory tract diseases remains unproven because of the high rate of codetection of HBoV with other respiratory pathogens. Its clinical epidemiology and role in respiratory infection have not yet been fully elucidated.

Although recently it was found that HBoV could be cultured in differentiated human airway epithelial cells, routine viral culturing of HBoV remains difficult. Real-time PCR has been used to estimate viral load and its usefulness has been proved as an indicator of the degree of active viral infection, interactions between the virus and the host, and the role of viral reactivation or persistence in the progression of disease. In this study, we investigated the epidemiology of HBoV and the clinical features of respiratory infection associated with HBoV, especially in terms of HBoV load.

Cultures of nasopharyngeal aspirates from 100 patients were positive in 67% of patients. Cultures yielded growth of one or more pathogen among upper respiratory tract normal flora. Gram positive cocci were isolated from 57/90 (63.3%) of specimens including: Streptococcus pneumoniae 25/90 (27.7%), Staphylococcus aureus 32/90 (35.5%). Gram negative bacilli were detected in 33/90(36.6%) of specimens including, Klebsiella pneumoniae 15/90 (16.6%), Escherichia coli 10/90 (11.1%), Pseudomonas aeruginosa 8/90 (8.8%), while Cultures of the control group showed growth of upper respiratory tract normal flora (table 1).

Bocavirus is a single-stranded DNA virus belonging to the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. Bocaviruses are unique among parvoviruses because they contain a third ORF between the non-structural and structural coding regions–[2]. The genus bocavirus includes viruses that infect bovine, canine, feline, porcine and some simian species as well as sea lions–[8]. Human bocavirus (HBoV) was first found in children with acute respiratory tract infections in 2005. It was then detected in children with respiratory tract infections in addition to gastroenteritis worldwide–[12]. The virus exists in four different serotypes HBoV1-4–[2],–[14]. Although HBoV 1 and 2 were reported in respiratory samples, all the 4 genotypes of HBoV have been identified in children with acute gastroenteritis.

HBoV has been reported in various countries, indicating its worldwide endemic nature. The virus has been identified in Europe–[17], America–[19], Asia,, Australia–[22], Africa, and the Middle East. The prevalence of HBoV ranges between 1.5 to 19.3%,. Primary infection with HBoV seems to occur early in life and children between the ages of 6–24 months seem to be mostly affected–[10], but older children can also be infected. Newborn children may become protected by maternally derived antibodies. HBoV infections are rarely found in adults–[27]. Lindner et al. detected anti-HBoV antibodies in 94% of healthy blood donors >19 years of age.

HBoV detection has been mostly performed on nasopharyngeal aspirates and swabs and relies mostly on classical,,,,, and real-time PCR,,. Real-time PCR possesses many advantages over conventional PCR, as it offers greater sensitivity, specificity, and reduced expenditure of time.

The current study aims to screen the epidemiological status and molecular phylogeny of HBoV isolates prevailing in pediatric patients with respiratory infection in Saudi Arabia.

Acute respiratory tract infection (RTI) is estimated to be the second important cause of death throughout the world among children less than 5 years old. Both respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important viral pathogens inducing RTI [2–4]. RSV and hMPV are non-segmented, negative-strand, enveloped RNA viruses. Both of them are classified within the Pneumovirinae subfamily of the Paramyxoviridae family. RSV is implicated in the majority of respiratory tract infection, which accounts for 60–80% of the bronchiolitis cases in children below 12 months of age. Recently, it has been demonstrated that hMPV also causes acute respiratory tract infections, similar to RSV [1, 6]. hMPV was first identified in 2001 from a pediatric patient with respiratory diseases in the Netherlands. hMPV causes a wide spectrum of disease ranging from mild upper RTI to more severe lower RTI such as bronchiolitis or pneumonia. Second to RSV, hMPV causes bronchiolitis and accounts for 5–15% of child hospitalizations for RTI [8, 9].

Both RSV and hMPV infections induce disruption of respiratory epithelial architecture, sloughing of epithelial cells, loss of cilation and acute pulmonary inflammation characterized by alveolitis, interstitial inflammation and peribronchiolitis [7, 9]. Following, acute otitis media is the most common complication of RTI due to RSV or hMPV. At present, the initial clinical manifestations of RSV are indistinguishable from those of hMPV [10–12]. One possible reference to distinguish RSV and hMPV infections is based on the epidemiological data. The median age of hMPV-positive hospitalized patients is 6-12 months, which is significantly higher than that of RSV (2-3 months).

RSV and hMPV do not appear to be significantly associated with asymptomatic carriage in the respiratory tract of healthy persons. However, the diagnosis efficiency for these 2 virus infection is low using the classical virus culture methods. Both viruses are labile in the environment and are susceptible to drying. For viral culture, RSV can growth in the HEp-2, A549, or Vero cells and usually takes 3–5 days to revealing characteristic syncytial cytopathic pattern [7, 15–17]. In contrast, hMPV can growth in the LLC-MK2 cells. The growth of hMPV is slow and often requires several blind passages before any cytopathic effect (CPE) is apparent, particularly following primary isolation, which often takes more than 10 days. These lengthy viral culture processes, in part, limited the opportunity for optimal initial diagnosis and the following therapeutic adjustment for RTI patient. Therefore, a reliable and rapid diagnostic tool is needed. Rapid and accurate laboratory diagnosis of viral RTI is crucial for optimal clinical management, diminishes unnecessary use of antibiotics, and allows for use of antivirals when appropriate.

The descriptive statistics were used to analyse demographics, clinical, and laboratory data using Microsoft Excel (Microsoft Corporation, Washington, US). To compare between single and multiple infection, two paired tests were used.

Bovine respiratory syncytial virus (BRSV) is one of the main causes of severe pneumonia, interstitial edema, and emphysema in cattle. BRSV is an RNA virus with a non-segmented, single-stranded, negative-sense 15.2 kb genome that belongs to the genus Pneumovirus and family Paramyxoviridae. BRSV infection leads to sudden fever, rhinitis, cough, respiratory distress, abdominal breathing, and decreased appetite.

The previous epidemiological studies reported that the prevalence of BRSV in cattle ranged from 28% to 70%, depending on animal age and environmental conditions. The disease occurs in most countries worldwide and affects cattle of all ages, with younger animals being at the greatest risk of severe BRSV disease.

Diagnosis of BRSV can be confirmed through different laboratory tests including virus isolation and antibody detection in serum and milk. Serological investigations such as serum neutralization test, complement fixation test, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) are commonly used methods for BRSV diagnosis. BRSV can also be accurately diagnosed using reverse transcriptase polymerase chain reaction.

A seroprevalence survey for BRSV in cattle has never been carried out in the Nineveh Governorate, Iraq. Therefore, this study aimed to ascertain the seroprevalence of BRSV in this region and to investigate the risk factors associated with the disease.

Saffold virus (SAFV) is an emerging pathogen identified in the United States in 2007. The virus´ name traces back to the name of the first author of the original description. The virus is classified in the species Cardiovirus B (formerly named Theilovirus) within the genus Cardiovirus (genus supergroup 1) of the family Picornaviridae [1–4].

SAFV is closely related to the other members of the Cardiovirus B species, i.e., Theiler's murine encephalomyelitis virus (TMEV), Theiler-like rat virus (Thera virus, TRV), and Vilyuisk human encephalomyelitis virus (VHEV). SAFV is a small non-enveloped virus with a single-stranded RNA of about 8,050 nucleotides. Up to now, eleven different SAFV genotypes have been identified (http://www.picornaviridae.com/).

SAFV-1 has initially been isolated from a fecal specimen of an 8-month-old girl with fever of unknown origin. Following this observation, SAFV was detected frequently in nasal specimens of children with respiratory infections (0.2%-24%,) and stool samples from children suffering from acute gastroenteritis (0.2%-3%,) [1, 4–13]. Due to these findings, SAFV was supposed to be a relevant new human pathogen, especially in children. According to antibody seroprevalence studies, SAFV infection is highly common and occurs early in life, with approximately 80% of seropositive children at the age of 2 years [14–16]. SAFV is spread worldwide [1, 3–4, 6, 9–10, 12–17].

The association of SAFV with various diseases is currently under research. Besides respiratory and gastrointestinal illnesses, the disease spectrum may involve type 1 diabetes and neurological disorders [7, 17]. Furthermore, the virus was detected in nasopharyngeal swabs from children with exudative tonsillitis (9/37,) and in autopsy samples from myocardium, lung and blood from a child with myocarditis. Additionally, the virus was found in feces from Asian children with non-polio acute flaccid paralysis and in cerebrospinal fluid specimens of patients with aseptic meningitis [12, 20–21]. This is of particular importance, because other members of the Cardiovirus B species are neurotropic. E. g., TMEV is known to cause a multiple sclerosis (MS)-like syndrome in mice. However, there are also reports i) describing SAFV detection with similar percentages in healthy and diseased individuals, e.g., in stool samples [9, 22–23] and, ii) reports failing to detect SAFV in samples from diseased patients, e.g., in cerebrospinal fluid from individuals with aseptic meningitis, encephalitis, and MS [24–25], and, finally, iii) reports co-detecting SAFV with common gastroenteritis pathogens in stool samples in case of diarrhea. The true clinical significance and pathophysiology of SAFV thus has to be elucidated.

In order to extend our knowledge on SAFV presence in healthy children, we analyzed adenoid tissue and throat swab samples from children who did not display symptoms of a respiratory tract infection for SAFV RNA. Samples were collected in the course of elective adenoidectomy.

Bovine respiratory syncytial virus (BRSV) is an important respiratory pathogen in cattle, detrimentally affecting the economy and animal welfare. The virus is distributed worldwide and is a major pathogen of the bovine respiratory disease complex [1, 2]. Viral respiratory infections are also of concern with regards to antibiotic resistance, as they predispose cattle to secondary bacterial infections that are commonly treated with antibiotics. Bovine respiratory disease is traditionally handled with management measures, vaccination and metaphylactic antibiotic treatment. Another possible strategy is to prevent inter-herd transmission of the main pathogens by increasing biosecurity measures at herd level. Because live animal transport is considered one of the main modes of BRSV transmission between herds [5, 6], proper mitigation must ensure that live animal transport be performed without compromising biosecurity. This requires knowledge on transmission risk associated with animal contact at different stages of infection. Knowledge of BRSV shedding related to clinical features would also be useful in order to assess the transmission risk of an infected herd without the use of viral diagnostic assays. For both of these areas, several knowledge gaps exists. Although way of infection may affect both viral shedding and clinical signs compared to naturally exposed animals, challenge studies are superior in the sense that aetiology and time of exposure is known and clinical features and virus excretion can be followed closely. Challenge studies, many of them aiming to evaluate the efficacy of vaccines [7–11] seldom last longer than one to two weeks. Grissett et al. and Gershwin concluded that shedding of BRSV begins on day three or four post-infection (p.i.) and usually lasts until day nine or ten. Grissett et al. summarized that the median time to appearance, peak and resolution of clinical signs was 3, 6 and 12 days, respectively, based on information from 22 inoculation studies [7–11, 14–22]. As studies outlasting the acute phase of infection are lacking, it is not known how long an animal can transmit infectious viruses to other animals. Appearance of clinical signs is usually the only information available in the field, and finding a clinical parameter that indicates shedding of infectious BRSV would be valuable. The existence of chronic or persistent infections in individuals is likewise still unclear [23–26].

During the acute phase of a BRSV infection, immunological protection develops, but it is assumed to be short-lived. This might enable early reinfection and new shedding of the infective virus, which complicates the risk assessment. A few BRSV studies have been performed to shed light on this. In a study by Kimman et al. they reported a strong local IgA response in the respiratory tract, but no virus shedding, when calves were re-exposed 3–4 months after primary BRSV infection. Stott et al. indicated, referring to their own unpublished results, that reinfection in calves and heifers may occur as early as three weeks post-infection. However, early reinfection with BRSV is not well-documented, and more precise knowledge of the occurrence is needed.

The existing literature on BRSV shedding and transmission is based on various laboratory methods, such as detection of viral RNA and culturing of the virus. Although resource-demanding, virus transmission studies are preferably performed using live animals in sentinel trials.

The aim of the present study was, therefore, to study basic features of BRSV infection in calves infected by exposure to BRSV-shedding calves. This was performed by:Investigating the shedding of viral RNA and infective virions:related to clinical outcome during the experimental period, lasting for two monthsin calves rechallenged by inoculation seven weeks p.i.Investigating whether the calves and their environment are not infectious to naïve in-contact calves four to nine weeks post-infection despite rechallenge with BRSV and mild stress induction.

Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production, as it is one of the most important causes of lower respiratory tract infections in calves. In dairy cattle, BRSV infection usually occurs in young calves aged between 2 weeks and 9 months. Adult animals with subclinical infection are the main source of infection, since reinfections are common in the herds [1, 4, 5].

BRSV, bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus (BVDV) and bovine parainfluenza type-3 (PI-3) are considered primary agents involved in the bovine respiratory complex. Additionally, secondary infection by Pasteurella multocida, Histophilus somni and mycoplasmas contribute to the aggravation of the disease. Clinical signs are characterized by respiratory symptoms, initially with moderated intensity, such as nasal and ocular discharges which can be aggravated leading to pneumonia. However, mainly in calves, an acute and severe onset is also observed, due to maternal antibodies not effectively protect against BRSV infection.

Considering the high prevalence of the disease, several studies determined risk factors involved in the epidemiology of BRSV. In Europe, risk factors were mainly attributed to herd size, herd density, purchasing of new animals, geographic location of the farms, herd type and concomitant BVDV infection [7–11]. Similar studies have also been performed in some Latin American countries and they showed that most of the animals probably have already been exposed to the virus with consequent high BRSV prevalence in cattle herds. In these countries, herd size, age group, presence of bordering farms, herd type and geographic location of the farms were the main risk factors associated with BRSV infection [12–16].

In Brazil, BRSV was first diagnosed in calves in the state of Rio Grande do Sul and some studies have shown that BRSV infection is widespread in Southern and Southeastern Brazil, with high serological prevalence rates [18–20]. Nevertheless, research has not been conducted in order to verify possible risk factors involved in BRSV epidemiology. Due to this, the current study aimed to determine antibody prevalence against BRSV and investigate some risk factors associated with BRSV seroprevalence in herds of an important milk producing region in São Paulo State, Brazil.

Bovine respiratory disease complex (BRDC) is a major cause of economic losses in the cattle industry worldwide. The most important viral agent include bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza-3 virus (BPI-3V). BRSV, belonging to the genus Pneumovirus within the family Paramyxoviridae, and is one of the most important causes of lower respiratory tract infections in calves; however, adult animals with subclinical infection are the main source of infection, since reinfections are common in the herds. It is highly prevalent in cattle, with a significant economic impact as the most important viral cause of BRDC worldwide. BVDV is a Pestivirus from the family Flaviviridae, which affects the digestive, respiratory, and reproductive systems in different production animals. Clinical signs include pyrexia, diarrhea, reduced production, and highly morbid disease but cause low mortality of infected animals. Infectious bovine rhinotracheitis (IBR) is an important infectious disease of domestic and wild cattle caused by BHV-1. This virus is a member of genus Varicellovirus, which belongs to the Herpesviridae family. Clinical signs infection includes symptoms of inflammatory reactions in respiratory, genital tracts, abortion, and neurological disorders. Betancur et al. found a statistical association between seropositive animals for BHV-1 with respect sex and age in Colombia, while Ochoa et al. reported higher infection in cows older than 5 years of age. BPI-3V is in the genus Respirovirus of the family Paramyxoviridae, which cause serious economic losses in small and large ruminants. Clinical disease is usually mild, with symptoms of fever, nasal discharge, and cough. Betancur et al. reported a statistical association between seroprevalence values for BPI-3V and age groups.

Aguachica, Rio de Oro, and La Gloria municipalities are located in Cesar department, which, in turn, is located in the Northeast of Colombia, and is very important agricultural and fish raising region, being the dual-purpose cattle husbandry one of the most important agricultural components of the regional economy, with a participation of 8% in the cattle national inventory. According to the National Agricultural Institute, the state has a population of 1,305,984 heads of cattle, being 30% located in the three municipalities.

Information about the prevalence of these viral pathogens is available from several countries in which these diseases have been reported. Nevertheless, there is very little epidemiological information on viral pathogens in cattle, mainly in the Northeast region of Colombia. Therefore, the present study was conducted to estimate the seroprevalence of respiratory viral pathogens in dual-purpose cattle and evaluate risk factors in the municipalities of Aguachica, Rio de Oro, and La Gloria in the department of Cesar.

HBoV is a putative member of the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. Before identification of HBoV, parvovirus B19 of the genus Erythrovirus was the only known human pathogen in the family of parvoviruses. Parvovirus B19 is widespread and manifestations of infection vary with the immunologic and hematologic status of the host. In immunocompetent children, parvovirus B19 is the cause for erythema infectiosum. In adults it has been associated with spontaneous abortion, non-immune hydrops fetalis, acute symmetric polyarthropathy, as well as several auto-immune diseases.

Based on its genomic structure and amino acid sequence similarity shared with the namesake members of the genus, bovine parvovirus (BPV) and canine minute virus (MVC), HBoV was classified as a bocavirus and therefore provisionally named human bocavirus.

Other subfamily Parvovirinae members known to infect humans are the apathogenic adeno-associated viruses of the genus Dependovirus and parvovirus 4. Parvovirus 4 has not yet been assigned to a genus, but it was proposed to allocate it to the genus Hokovirus as it shares more similarities to the novel porcine and bovine hokoviruses than with other parvoviruses. Recently a second human bocavirus has been identified, HBoV2, with 75.6 % nucleotide similarity to HBoV. HBoV2 was found in stool samples from Pakistani children as well as in samples from Edinburgh (1 of the 3 positive samples was derived from a patient >65 years old), indicating that it is not restricted to one region or to young children.

Bovine corona virus (BCV) and bovine respiratory syncytial virus (BRSV) are two worldwide distributed viruses. BCV causes diarrhoea in calves, winter dysentery in adults and various degrees of respiratory symptoms. BRSV is regarded as one of the most important causes of respiratory tract disease, especially in young calves. An infection can cause respiratory distress, fever, anorexia and subcutaneous emphysema and can lead to secondary bacterial pneumonia and death. Outbreaks of BCV and BRSV occur mainly in autumn and winter. These infections are common in dairy herds; in a nationwide survey in England and Wales the prevalence of antibodies to these viruses in bulk tank milk (BTM) was 100%. Swedish studies have shown a prevalence of 70-100% for BCV and 41-89% for BRSV, with the higher prevalence in southern parts. In a more recent study in a high animal-density area in south-west Sweden, the prevalence in BTM was 100% for both BCV and BRSV.

Previous studies have shown that BRSV and BCV infections are effectively spread within the herd. It has also been shown that acquired antibodies remain detectable for years, even without reinfection, whereas maternal antibodies are only detectable for a few months. Spot samples from a few young animals can thus be used to reflect recent infections of BRSV and BCV in a herd, whereas bulk tank milk samples mirror the long-term history. Spot sampling has previously been described for bovine virus diarrea virus (BVDV).

Despite the importance of these viruses and the fact that they are widely spread, little is known about transmission routes and management risk factors. Introduction of new animals and indirect spread via people and equipment are believed to be important and airborne transmission has been shown to occur for BRSV, at least under experimental conditions. Studies have been carried out to determine the relationship between herd health, reproduction efficiency and milk production and seropositivity to other viruses, for example bovine viral diarrhoea virus and bovine leukemia virus. Similar studies for BRSV and BCV have, as far as we know, not been conducted and it is therefore difficult to quantify their effect on the farm efficiency and economy. The purpose of this study was to explore if there were any associations between antibody status to BCV and BRSV and disease incidence, reproduction and some herd characteristics in dairy herds. A secondary aim was to investigate if there were any difference in proportion antibody positive herds between two neighbouring areas.

Bovine respiratory syncytial virus (BRSV) is a pneumovirus of the paramyxoviridae family (Valarcher and Taylor 2007). The infection is prevalent worldwide (Elvander 1996, Paton and others 1998, Uttenthal and others 2000, Klem and others 2013), and BRSV is considered one of the major pathogens of the bovine respiratory disease complex (Griffin 1997, Snowder and others 2006, Brodersen 2010). To diagnose the infection, different strategies can be chosen according to the given situation and the purpose. For larger epidemiological studies, antibody analyses are used (Hägglund and others 2006, Beaudeau and others 2010, Ohlson and others 2010, Klem and others 2013). Infected animals seroconvert and IgG can be measured in serum and milk long after the virus is no longer present. Studies on the longevity of antibodies against BRSV are scarce, but Elvander (1996) found that such antibodies could be detected in the serum of adult cattle for at least two years postinfection.

The virus is reported to spread effectively in a herd during outbreaks, resulting in high within-herd prevalence of antibody positive animals (Rossi and Kiesel 1974, Stott and others 1980, Verhoeff and van Nieuwstadt 1984, Bidokhti and others 2009). Antibodies in bulk tank milk (BTM), pooled milk samples from a selected number of cows or serum from several animals at a selected age have been used to classify a herd or given population (Paton and others 1998, Uttenthal and others 2000, Klem and others 2013, Ohlson and others 2013). Since animals are known to be seropositive for a long time, classification of herds based on serology in young animals (Klem and others 2013), or pooled milk samples from primiparous cows (Ohlson and others 2013), will give a more up-to-date picture than BTM testing. Recent studies show that testing of antibodies in BTM is a reliable tool for identification of BRSV-negative herds (Klem and others 2013, Ohlson and others 2013).

For several important infections in dairy cattle, testing of antibodies in BTM is used as an effective and inexpensive method to determine a herd's exposure to infectious agents (Niskanen 1993, Booth and others 2013). Cost-effective methods to classify the infection status of herds are of interest for several reasons. Such knowledge can be used to reduce the risk of virus transmission when animals are traded and as a diagnostic tool in the investigation of herd health problems. It may also be used in large-scale studies, such as screenings for surveillance or control purposes.

Several methods are available for detection of antibodies against BRSV, including virus neutralisation tests and different ELISAs, of which the indirect ELISA is most commonly used. In a neutralisation test, the level of antibodies in a sample is measured quantitatively. The indirect ELISA is not developed as a quantitative assay; its function is primarily to differentiate between negative and positive samples, and to be semiquantitative at antibody levels where seroconversion in individual animals is usually seen. Outside this range, there may be a weaker correlation between the ELISA optical density (OD) values and the actual level of antibodies in the sample.

The aim of the present study was to see how the level of antibodies against BRSV in BTM can be interpreted with respect to the time at which herds are infected with BRSV. Subsidiary aims were to investigate

A total of 1,926 samples of patients with respiratory sym-ptoms were included in the present study. Ninety-three (4.8%) samples were found to be positive for HBoV by PCR and subsequent sequencing. Other respiratory viruses detected during the study period were as follows: influenza A virus (IFA), 56 patients (2.9%); influenza B virus (IFB), 50 patients (2.6%); parainfluenza viruses (PIV), 142 patients (7.4%); respiratory syncytial virus (RSV), 97 patients (5.0%); and adenovirus (ADV), 40 patients (2.1%). HBoV was more prevalent in men (73.1%) than in women (P=0.005).

HBoV was detected in patients ranging from 3 months to 65.6 yr (mean=36.2 months, median=19 months) with a peak prevalence between the ages of 6 and 12 months (8.6%, 22/257). Children aged 5 yr or less constituted 92.5% (86/93) of the HBoV-positive patients (Table 1).

HBoV was detected in the samples obtained throughout the course of the study with the detection rate being the highest in June (16/129, 12.4%), followed by August (9/110, 8.2%) and May (13/162, 8.0%). From April to June 2006, 43% of the HBoV-positive cases were observed (Fig. 1).

All the 93 HBoV-positive cases detected by conventional PCR were also confirmed by real-time PCR performed using the TaqMan probe. The viral load detected in the HBoV-positive samples by using real-time PCR was in the range of 1.3×103-4.6×109 copies/mL (median, 1.82×105 copies/mL). The HBoV-positive cases were categorized in 2 groups: low-viral-load group (viral load ≤1.0×106 copies/mL, N=58) and high-viral-load group (viral load >1.0×106 copies/mL, N=35) (Table 1). The HBoV-positive patients aged less than 3 yr had a significantly higher viral load than that in the patients aged more than 3 yr (P=0.001).

The other respiratory viruses found in 17 (18.3%) of the total HBoV-positive samples were as follows: IFA, 1 sample; IFB, 1 sample; PIV, 10 samples; RSV, 4 samples; and ADV, 1 sample. Most of the cases (88.2%, 15/17) belonged to the low-viral-load group. RSV was isolated from the remaining 2 samples with HBoV copy numbers of 1.09×106 copies/mL and 2.65×108 copies/mL.

Patients positive for HBoV alone had a higher viral load than that in the patients who were positive for both HBoV and another respiratory virus (median 3.78×105 copies/mL vs. 1.94×104 copies/mL, P=0.014). A high-viral-load was almost exclusively seen in the HBoV-positive patients alone (94.3%, 33/35) (Fig. 2).

The high-viral-load group had a significantly higher pulse rate and respiratory rate than the corresponding rates in the low-viral-load group (P=0.007 and P=0.0231, respectively; Table 2). Although the duration of hospital stay was not significantly different between the 2 groups (Table 2), in cases of patients with less than 10 days of hospital stay, the high-viral-load group had a longer hospital stay than the low-viral-load group did (5.2±1.5 days vs. 4.1±1.4 days, P=0.009). Most of the other clinical characteristics had no significant correlation with the viral load of HBoV. The HBoV-positive patients presented with cough (80.6%), sputum (63.4%), fever (62.4%), rhinorrhea (50.5%), crackle (44.1%), wheezing (33.3%), diarrhea (14.0%), and dyspnea (6.5%). No significant difference was observed in the HBoV viral loads in the cases of upper and lower respiratory tract infections (P=0.077). Twenty-one out of 26 patients with underlying conditions had low-viral-loads.

Clinical examination of the HBoV-positive patients showed pneumonia, bronchiolitis, bronchitis, croup, asthma, sinusitis, and pharyngotonsilitis (Fig. 3). HBoV was detected in 8.4% (N=48) and 5.6% (N=45) of the samples obtained from patients with and without pneumonia, respectively (N=572 and 805, respectively; P=0.049). However, no significant difference was observed in the viral load between the patients with and without pneumonia (P>0.05).

Acute respiratory tract infections are major causes of morbidity and mortality. In 2000, lower respiratory tract infections were globally the number one infectious cause of disability adjusted life-years. The commonest respiratory viruses that cause acute upper and lower respiratory tract infections and which are routinely tested for in most virus diagnostic laboratories are: influenza A virus (FLA); influenza B virus (FLB); respiratory syncytial virus (RSV); parainfluenza virus type 1 (PF1); parainfluenza virus type 2 (PF2); parainfluenza virus type 3 (PF3) and adenovirus (ADV). Additionally, human rhinoviruses (HRV) and coronavirus 229E (CoV-229E) are also linked to acute respiratory infection but less commonly included in laboratory reports; human metapneumovirus (hMPV) is not yet part of most United Kingdom virus laboratory test repertoires (personal feed-back from the United Kingdom Clinical Virology Network).

As part of service development it was necessary to provide an alternative to virus culture for testing immunofluorescence negative respiratory specimens. Historically and indeed currently immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Culture is accepted as more sensitive than immunofluorescence but slower and therefore less useful for direct patient management decisions. Using a standard culture technique for the culture of respiratory viruses our median reporting times for culture positive and culture negative specimens were 6 days (based on 407 specimens) and 7 days (based on 2159 specimens) respectively; virus identification by this technique required the use of monoclonal antibody staining of the cell monolayer in addition to observation for viral cytopathic effect. We therefore wished to develop a test capable of reporting on immunofluorescence negative specimens within a 24 hour period.

Increasingly however, the sensitivity of nucleic acid amplification techniques for diagnosis has become recognised. However widespread concerns about contamination issues and perceived cost have slowed their widespread adoption. An added problem for acute respiratory tract infections is the relatively large number of viruses that need to be accounted for, a problem which presents specific technical challenges.

One such challenge is the different optimal annealing temperatures of the primer sets for each prospective virus target. The ABI PRISM 7000 real-time facility from Applied Biosystems addresses this by using bundled software to design primer/probe combinations that use a common amplification protocol. However this approach is compromised by the inability of software to allow for target heterogeneity. In addition it does not allow users to adopt clinically validated primer sets from the literature.

To address these problems we adopted an alternative approach through the development of a generic touchdown amplification protocol. Touchdown protocols involve a pre-designed stepped reduction in the annealing temperature used for primer-to-template binding, which introduces a competitive advantage for specific base-pair priming over non-specific priming. A detailed knowledge of the optimum annealing temperature is therefore not required. The study protocol was empirically constructed and proved robust when applied to a large range of respiratory viral and bacterial targets, without compromising individual test sensitivity. It was designed for use with in-house primer master-mixes that recognise 12 common respiratory viruses.

Before deciding on the layout of the molecular strip, as described in the methods, we undertook a wide range of preliminary validation steps for each primer set. The complexity of the strip makes it impossible to fully evaluate using the classical approach of applying an individual gold standard to each virus type. Classically this approach works well where a single target is under investigation. However although the strip is putatively designed to identify 12 viruses, the actual number of individual types targeted is over one hundred and sixty because of the inclusion of generic primer sets for HRV and ADV respectively. The classical approach is further compounded for viruses (a) that cannot be grown or grown easily; (b) for which commercial IF sera are not available; (c) for which specimen panels are not available. We therefore adopted a phased validation, culminating in the present study. Sensitivity was ascribed by undertaking copy number determination on cloned targets and these ranged form 6 × 103 copies per ml for human rhinovirus type 1b to 4.2 × 103 copies/ml for RSV-A. Specificity was ascribed through reproducibility, i.e. specimens which were repeatedly positive, following our standard clinical reporting algorithm, were regarded as true positives; a similar approach was recently described for hMPV. In addition amplicon sequencing was used as an initial specificity check. The primers sets were tested on clinical respiratory specimens arising from a number of ethically approved studies. These included respiratory specimens from patients: (a) with chronic obstructive pulmonary disease; (b) with acute asthma; (c) on assisted ventilation in intensive care. They were also tested on respiratory specimens collected as part of an influenza spotter program as well as on laboratory specimens of known virus reactivity.

To test the feasibility of its routine use we needed to clinically validate its performance in a routine setting on specimens tested in parallel with our standard immunofluorescence protocol for the diagnosis of acute virus respiratory infections. Although the routine immunofluoresence panel lacked capacity for the detection of rhinoviruses, human metapneumovirus and CoV-229E, these were included on the strip for clinical reasons during the period of the study. These findings and their implications are reported.

Along with equine rhinitis virus (ERV) and foot and mouth disease virus (FMDV), bovine rhinitis A and B viruses (BRAV and BRBV, respectively) are species in the genus Aphthovirus, family Picornaviridae. Two serotypes of BRAV have been identified, BRAV1 and BRAV2, while BRBV consists of a single serotype. The BRAV1 strain SD-1 was isolated in Germany in 1962 from nasal secretions from a calf with rhinitis. Additional BRAV1 strains were subsequently isolated from both healthy and diseased bovines in England, Japan, Italy and the U.S. and shown to cross react in serum neutralization assays [3–6]. The sole BRBV isolate EC-11 was isolated in England in 1964 by Reed from the lung of a specific pathogen free calf with respiratory disease. Likewise, BRAV2 consists of a single specimen, strain H-1, isolated from an outbreak of respiratory disease in cattle in 1984. Despite numerous studies on bovine rhinitis viruses (BRV) in the 1960’s through mid-1980’s, little work has been published on their epidemiology and ecology the past several decades.

Bovine respiratory disease complex (BRDC) is the most economically significant disease of the cattle industry, leading to losses due to mortality, morbidity, treatment costs and feed inefficiency in excess of $750 million dollars per year in the U.S. alone. BRDC has a multifactorial etiology involving a variety of bacteria and viruses in addition to host and environmental factors. Numerous commercial vaccines including both killed and attenuated live bacteria are available. Viruses commonly included in commercial vaccine include bovine viral diarrhea virus (BVDV), bovine herpes virus 1 (BHV1), parainfluenza virus 3 (PI3) and bovine respiratory syncytial virus (BRSV). Despite their widespread use, BRDC incidence has increased over the past 20 years. BRDC pathogenesis often involves a primary viral infection which damages respiratory mucosa and alters host immune responses leading to secondary bacterial pneumonia caused by commensal bacteria already present in the respiratory tract.

Both BRAV and BRBV are established but rarely studied etiologic agents of BRDC. Experimental inoculation of calves with BRAV1 via intranasal (IN) or intratracheal (IT) routes, either singly or in combination, resulted in variable clinical signs of respiratory disease and histologic lesions consistent with pneumonia. BRAV1 was also recovered from nasal swabs of IN inoculated animals and all animals inoculated or exposed by contact seroconverted to BRAV1 by day seven post inoculation. A similar experiment using a different BRAV1 strain (RS 3x) and colostrum deprived calves failed to reproduce clinical disease but was successful in isolating BRAV1 from nasal swabs post inoculation and found histological lesions of focal rhinitis and a neutralizing antibody response in all inoculated calves. BRBV pathogenesis was investigated using intranasal inoculation of gnotobiotic calves. Clinical signs including fever, nasal discharge and increased respiration rate were observed. Foci of epithelial necrosis were observed histologically in the turbinates and trachea and interstitial pneumonia was evident in the lungs. Virus was isolated from multiple tissues and was neutralized by convalescent antiserum. In addition to controlled studies, numerous investigations of acute respiratory disease in cattle resulted in the isolation of bovine rhinitis viruses where paired acute and convalescent sera suggested a causative role for bovine rhinitis virus.

Metagenomic sequencing on nasal swabs obtained from BRDC diagnostic submissions were performed to survey viruses present. Contigs with high identity to BRAV2 and BRBV were identified in one swab. To further our understanding of the epidemiology and ecology of bovine rhinitis viruses in BRDC, a more comprehensive survey was performed.

Acute respiratory infections (ARI) are one of the major causes of morbidity and mortality in young children throughout the world especially in developing countries. Data from WHO estimated the burden of ARI at 94,037,000 disability-adjusted life years (DALYs) and 3.9 million deaths in 2001. Similar report from a meta-analysis study demonstrates that throughout the world 1.9 million (95% CI 1.6-2.2 million) children died from ARI in 2000, 70% of them in Africa and Southeast Asia. A further systematic analysis also estimated 1.575 million (uncertainty range: 1.046 million - 1.874 million) deaths of children worldwide in 2008 as due to ARI.

Majority of acute lower respiratory tract infections(ALRTI) in developed countries have been reported to be often due to viral pathogens of which most common are RSV, PIV, influenza viruses, Adv, human Coronaviruses and Bocaviruses. On the contrary, information on these viruses in developing countries is limited probably due to paucity of modern diagnostic molecular techniques. These infections are therefore treated unsuccessfully with antibiotics based on suspicion of bacterial causes.

Apart from the public health concern of nosocomial infections that are associated with viral respiratory infections, significant costs derived from long duration of hospitalization and several healthcare visits could also aggravate the poor socio-economic status and increased child mortality in developing countries including Ghana.

Lessons from the outbreak of the Severe Acute Respiratory Syndrome (SARS) epidemics which resulted in the death of 776 individuals and the recent emergence of a novel swine flu pandemic emphasize the risk posed by respiratory viral infections in humans.

This study was done to determine the burden of respiratory viruses among children hospitalized at the Komfo Anokye Teaching Hospital for acute lower respiratory illness using the Real Time Polymerase Chain Reaction (RT-PCR).

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.

Bovine coronavirus (BCoV) and bovine respiratory syncytial virus (BRSV) are contagious pathogens detrimentally affecting production and animal welfare in the cattle industry. The viruses are part of the bovine respiratory disease complex and are endemic worldwide. BRSV and BCoV can cause epidemics of respiratory disease and additionally BCoV cause diarrhea in calves and adult cattle (winter dysentery) [1–4]. The traditional way of handling and preventing these diseases is through metaphylactic antibiotic treatment, use of vaccines, or changes in management to improve calf health in herds. The within-herd prevalence and morbidity of BCoV and BRSV infections are high [6, 7] and once the virus enters a herd, circulation is difficult to mitigate. An additional preventive strategy is therefore to reduce inter-herd transmission of virus. Movement of live animals between herds is an important transmission route. If this risk is under control, the next question concerns the contribution of indirect spread of virus between herds. Indirect spread can occur via e.g. personnel travelling between herds, their clothes or equipment.

Important risk factors for indirect spread are the level of virus contamination of relevant surfaces and the infectivity of the viruses. Enveloped respiratory viruses like BCoV and BRSV are generally fragile outside the host. However, as related viruses like human respiratory syncytial virus (HRSV) and human coronavirus 229E remain infective for several hours on contaminated surfaces like countertops and surgical gloves [10, 11], there is a potential for indirect transmission. Epidemiological studies also point out the importance of indirect transmission; Ohlson et al. found that lack of boot provision for visitors was a risk factor for infections with both viruses and Toftaker et al. found that a herd’s BCoV and BRSV antibody status was influenced by the status of its neighboring herds.

Human nasal mucosa might also be a vector for inter-herd virus transmission, as traffic of personnel between herds is common. Carriage of BCoV and BRSV in human nostrils has not been studied. Generally, there are few studies on indirect transmission of these viruses, and no experimental studies have been performed. Molecular methods and virus isolation in cell culture can be used to study the level of virus carriage and infectivity, which are determinants for virus transmission. Combined, these methods provide sensitive quantification of viral genomes and assessment of virus infectivity.

Consequently, the aim of the present study was to investigate whether personnel (nostrils) and fomites carry viral RNA and infective viruses after exposure to BCoV or BRSV infected animals.