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We report the rare finding of BCG reactivation in a child with confirmed measles infection. Although BCG reactivation is known to be an extremely important and highly specific clinical manifestation of KD, this case suggests that BCG reactivation may also occur in other childhood diseases or infections. A thorough search for an infective aetiological agent may be indicated in children presenting with this clinical finding.
Human exposure may occur in many ways – preparing and consuming animal products, washing with, and drinking well water contaminated with animal fecal coliform, animal bites/scratches, and working in occupations involving regular contact with animals, manure, soil, and/or by-products (e.g., farmers, slaughtering plant workers). Even living down-wind of a farm field fertilized with animal manure poses a potential risk. A list of major sources and exposure routes of animal-to-people transmission of viruses and bacteria is shown in Figure 2. Factors influencing the probability of disease transmission involve the proximity and temporal contact with the infectious organism, length of time that the infectious agent is present, virulence of the agent, incubation period, stability of the agent under varying environmental conditions, population density of carrier animals, husbandry practices, and control of wild rodents and insects (31). The type and maintenance of animal housing also may affect the extent to which individuals working in or around such facilities are exposure to zoonotic viruses and bacteria. Often, animal containment structures (e.g., hen houses, pig pens, cattle barns, and horse stables) may be inadequately ventilated and/or have poor waste removal systems, increasing the exposure of animals and their caretakers to dust, fecal matter, and microbes (32).
Animal bacteria also have been implicated in cancer. The occurrence of gliomas in the brain of fowl have been noted in several reports (17–19) and these tumors have been described as having the pathognemonic encephalitic features of a pleomorphic parasite infection (e.g., hypertrophy and hyperplasia of blood-vessels; perivascular infiltration by lymphocytes, plasma cells, and monocytes; and the presence of A-D bodies) (20). Chickens spontaneously and experimentally infected with toxoplasma have been observed to develop glioma-like tumors (21, 22). A study of 16 human brain tumors observed bodies indistinguishable from the C and D phases of the fowl parasite (23). Epizootic outbreaks of toxoplasmosis have been reported in various avian species and mammals (22, 24, 25). Furthermore, toxoplasma antibodies have been isolated in the blood of exposed sheep farmers, flock animals, herder dogs, mice, and rats (26). Potential cellular mechanisms by which animal viruses and bacteria lead to tumorgenesis are shown in Figure 1.
A novel paramyxovirus, feline morbillivirus (FmoPV), has recently been detected in domestic cats [1–8]. FmoPV is genetically most closely related to viruses such as canine distemper virus (CDV), measles virus (MV), rinderpest virus (RPV), peste-des-petits-ruminants virus (PPRV), phocine distemper virus (PDV) and cetacean morbillivirus (CMV), belonging to the genus morbillivirus in the family Paramyxoviridae [1–5]. FmoPV showed genetic diversity among isolates [3–5], and a natural recombination in the envelope protein region between viruses in different clades was also found. In Germany, three groups of feline paramyxoviruses (FPaV) have been detected, and these were associated with feline chronic kidney diseases (CKD) including lower urinary tract diseases (LUTD). Phylogenetically, the first group of these viruses belongs to the same cluster of FmoPV with 99 % homology, whereas the second group represents a new cluster between FmoPV and other morbilliviruses. The third group represents a group that is distinct from FmoPV and other morbilliviruses. A seroepidemiological survey of CDV infection in Asian countries showed that domestic cats were susceptible to CDV infection, but CDV was not virulent in domestic cats. At the moment, it is not yet confirmed that FmoPV is classified in the genus morbillivirus or in a novel genus separate from the genus morbillivirus.
Kidney failure is one of the most important and common diseases in domestic cats. It can be divided into acute kidney disease (AKD) and chronic kidney disease (CKD), or inherent kidney disease and acquired kidney disease [10–13]. AKD, which could be caused by toxins, trauma, infection, shock, blockage of the blood flow and heart failure, is reversible and can affect cats of all ages. CKD affects domestic cats, especially middle-aged or older cats, and its prevalence increases according to age, affecting up to half of cats older than 15 years. CKD could result from infection, blockages, dental disease, high blood pressure and cancer. In particular, idiopathic CKD such as pyelonephritis, glomerulonephritis and chronic tubulointerstitial nephritis (TIN) due to unknown causes has been reported extensively [10, 11, 15–17]. It is suspected that FmoPV is one of the causative agents of CKD [1, 5], such as chronic TIN. Therefore, it is important to clarify the characteristics or the pathogenicity of FmoPV and the pathogenesis in domestic cats as the natural host. In this respect, large-scale epidemiological investigation is considered to be indispensable.
In this study, epidemiological and pathological studies were performed to demonstrate the seroprevalence of FmoPV and the relationship between FmoPV and CKD in Japan. These studies revealed that the infection rate of FmoPV was considerable and FmoPV might be related to urinary tract diseases.
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
, 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.
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.
In the present study, 22 out of 100 cats in Japan were shown to be FmoPV RNA positive in urine and/or kidney. Phylogenetic analysis of the FmoPV detected in Japan showed FmoPV was clustered in three groups. However, a phylogenetic relationship with geographic distribution of FmoPV in Japan and Hong Kong was not observed. Further, a significant relationship between FmoPV cluster and renal disease in the cat was not observed (data not shown). It is, however, necessary to compare the FmoPV sequences in cats from other regions in future to confirm this hypothesis. Recently, several partial sequences of FmoPV detected in cats in Germany were reported, however, we could not compare our data with them because the region of the sequences were not overlapped.
RNAs and proteins of FmoPV were detected in cat urine, sera and/or renal tissues. Anti-FmoPV Abs were also demonstrated in cat sera. FmoPV-infected cats in the present study were divided into three groups: RNA+/Ab+, RNA+/Ab- and RNA-/Ab+. Cats in the RNA+/Ab- group were considered to be in an acute phase of FmoPV infection. Cats in the RNA+/Ab + group were considered to be in either a subacute or a chronic phase of the infection. Since it is not confirmed that the nested R-PCR used in the present study detected all the FmoPV strains because of the genetic diversity of the viruses, thus it is possible that there are some false negative results in the RT-PCR. Since humoral immune response in addition to cellular immune response to the virus are thought to eliminate the virus from animals, cats in the RNA-/Ab + group were considered to be in a convalescent phase. Consequently, it appears that FmoPV could establish as an acute, subacute or chronic infection, and could be eliminated. Additionally, there were cases in which RNA of FmoPV was negative in urine but positive in renal tissues. Since approximately half of the infected cats (14 out of the 29 infected cats) were positive for viral RNA and Ab, it seems likely that cats are easily chronically or persistently infected with the virus. However, further long-term follow-up studies in FmoPV-infected cats are necessary to confirm this hypothesis.
Cat samples were collected randomly from healthy cats and sick or wounded cats, including stray cats in Tokyo. No relationships among the clinical data, except age and sex, with the infection by FmoPV could be found. The infection rate of FmoPV was significantly high in unneutered male cats (P < 0.005) in this study (Fig. 6). These tendencies could be due to differences of behavior patterns, such as more aggressive or active behavior in unneutered male cats than neutered male cats and female cats, and such behavior might result in an increase in opportunity of infection with FmoPV from other infected cats. The transmission of FmoPV between cats could occur in veterinary hospitals, boarding kennels, breeding facilities, outdoors or at home. Given the high positive rate, it seemed that FmoPV might be maintained between their transmissible cats for a long time.
In a pathological examination, 25 out of 29 infected cats had renal lesions, from mild mononuclear cell infiltration to chronic tubulointerstitial nephritis. Half of them were mediate to severe cases. As a result of IHC, 19 out of 29 cats showed FmoPV-N protein in their kidney tissues. Four of the 19 cats showed severe lesions with FmoPV-N protein in the inflammatory lesions. The FmoPV-N protein-positive cells were identified to be renal tubular epithelial cells in the renal cortex, medulla and pelvis. FmoPV-N protein-positive mononuclear cells could not be found in this study, different from a report in Hong Kong. These cases had RNA of FmoPV both in urine and renal tissues and relatively high IgG titer of 1: 640 to 20,480, except one case, representing the chronic phase. However, the IHC-positive sites were focally located, not distributed widely in the lesions.
In the other 15 cases, normal renal tubular cells or transitional cells of the renal medulla or pelvis were IHC-positive. No statistically significant correlations were found between the grades of lesions and the infection of FmoPV. However, a statistically significant relationship with the presence of inflammatory lesions and the infection of FmoPV was found, regardless of TIN. Consequently, no meaningful direct relationship between infection by FmoPV and chronic TIN was confirmed in this study. It might be plausible that FmoPV antigens in the lesions were already eliminated by host immune responses in the case of severe chronic TIN. A similar phenomenon was reported in other virus infectious diseases such as hemorrhagic fever with renal syndrome (HFRS) caused by hantavirus and severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) [19, 20]. In these cases, uncontrolled responses in cytokines and chemokines are considered to be responsible for the pathogenesis. In this regard, it would be necessary to analyze innate immune responses in cats with FmoPV.
It is well known that many viruses such as feline immunodeficiency virus, feline foamy virus, feline infectious peritonitis virus and feline leukemia virus can affect the kidneys of domestic cats, resulting in severe CKD including glomerulosclerosis, TIN, amyloidosis and pyrogranulomatous nephritis [21–27]. FmoPV is a recently identified virus, but it could affect the kidneys in infected cats. Thus, the existence or pathogenicity of FmoPV could have been underestimated.
In addition, a correlation between lower urinary tract disease (LUTD) and infection of FmoPV has been reported recently. LUTD including urethral obstruction (UO) and interstitial cystitic (IC) is also an important and common disease in domestic cats [10, 11, 15–17, 28–30]. Feline calicivirus, feline herpesvirus type II and feline foamy virus have been detected in some cats with LUTD [21, 28–32]. In this study, lower urinary tract tissues or related clinical symptoms were not examined. However, it could not be excluded that FmoPV may also be related to LUTD, for two reasons: 1) only four cases had FmoPV antigens in inflammatory lesions of the renal cortex, even focal, 2) normal tubular cells of the renal medulla and transitional cells of the renal pelvis in proximity to the lower urinary tract were FmoPV antigen-positive.
Genetic diversity of FmoPV has been described extensively [3–5]. In Hong Kong, FmoPV seemed to be related to chronic TIN. In Germany, it was detected in a LUTD case. In addition, they detected FmoPV in a LUTD case showing 86 ~ 99 % homology compared to reported FmoPV. At the moment, we could not detect FmoPV belonging to genetically diverged group found in Germany among cats in Japan. We can not rule out the possibility that our RT-PCR and IHC could not detected these FmoPV RNA and N protein and their antibodies could not be detected using N proteins of FmoPV belonging to cluster C. However, it is unlikely since N proteins of paramyxoviruses in the same species are highly conserved and cross-reactive.
In our study, no clear relationship between FmoPV and chronic TIN or LUTD could be clarified although many types of FmoPV-infected cases are described.
It was suggested that FmoPV infection has a significant relationship with inflammatory reactions in the feline kidney and may be associated with urinary tract diseases. This was the first epidemiological report including seroprevalence and pathological examination of FmoPV among domestic cats in Japan. Considering the high infection rate and the genetic diversity of FmoPV, it seemed likely that FmoPV has evolved in Japan over a long period. Further retrospective and larger scale studies, including pathological studies, in other organs of cats are needed to elucidate the pathogenicity of FmoPV in cats.
All of the owners of the CDV PCR-positive dogs were instructed by the first author (BW) to quarantine the dogs until they tested CDV PCR-negative. The owners of the CDV-positive dogs in multidog households (Dogs 3, 6, 7, 8 and 9) were instructed to separate the infected dog from the other dogs in the household. However, several dogs (Dogs 6, 7, 8 and 9) had already had contact with adult dogs within the household at the time when the CDV diagnosis was made. All of the contact dogs had been vaccinated against CDV, although several dogs had only received the initial vaccination series as puppies and had received no booster vaccinations (data not shown). Two dogs that were in close contact with Dogs 7 and 9 were tested for the CDV infection with PCR one month and two months after the initial CDV diagnosis in Dogs 7 and 9. One contact dog exhibited a single, very weak CDV-positive result in the conjunctival swab in the first sampling but was negative in all of the swabs collected one month later (data not shown). The other contact dog tested PCR-negative in all of the collected samples (data not shown). In all of the other multidog households, no samples were collected for CDV PCR, but no clinical signs of the disease were noted in the 6-month follow-up period.
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.
HeV and NiV are currently classified as BSL-4 agents and consequently human efficacy studies for testing potential therapeutic products are not easily achievable. In 2002, the U.S. Food and Drug Administration (FDA) implemented the Animal Efficacy Rule for the development of therapeutic products under these circumstances. Specifically, FDA can rely on evidence derived from animal studies in the evaluation of product effectiveness when particular criteria are met, such as a well-understood mechanism for both the pathogenicity of the agent and the underlying mode of action of the product. Importantly, the therapeutic effect must also be demonstrated in more than one animal species. Once these criteria have been met, human clinical trials could commence, most likely in populations at high risk of natural infection by HeV and NiV.
A strong epidemiological association existed in Malaysia between human NiV infection and close direct contact with pigs, especially sick and dying animals. No direct association with flying foxes was made. An epidemiological link has been noted between HeV infection in people and horses dying from HeV disease. Again, neither HeV disease nor seroconversion has ever been identified in wildlife carers who came into close and regular contact with sick and injured bats. While both horses and pigs have been experimentally infected with HeV and NiV, respectively [42, 44], neither represents a practical option for multiple experimental efficacy studies. Large animals such as horses are difficult to manage in sufficient numbers under BSL-4 conditions and in pigs NiV infection is mostly inconsequential from a clinical perspective. For the purposes of this review we will focus on the smaller animal model systems that have been explored (Table 1). There was no serological evidence of NiV infection in rodents in Malaysia [9, 45]. However, attempts were made to infect mice experimentally. NiV and HeV do not cause disease in mice after subcutaneous administration (5,000 TCID50 HeV) (Crameri, G and Eaton, B.T., unpublished observations) or with either an intranasal (6x105 pfu NiV) or intraperitoneal (107 pfu NiV) challenge of NiV, although the HeV is lethal if administered intracranially. Rabbits are not susceptible to HeV associated disease when challenged subcutaneously (5,000 TCID50 HeV). Guinea pigs were experimentally infected with HeV soon after its discovery (5,000-50,000 TCID50 HeV) and developed generalized vasculitis affecting lung, kidney, spleen, lymph nodes, gastrointestinal tract, and skeletal and intercostal muscles [36, 38]. However, in spite of vascular involvement of the lung, there was little or no pulmonary edema. NiV infection in the guinea pig (50,000 TCID50 NiV) clinically manifested as ruffled fur and abnormal (less fearful) behavior with gross pathology limited to edema of the mesentery, broad ligament, and retroperitoneal tissues. Significant microscopic pathology included vasculitis with fibrinoid necrosis and endothelial syncytial cell formation in the myocardium, kidney, lymph node, spleen, myometrium, retroperitoneal tissues, and submucosal vessels of the bladder. Oophoritis and the presence of hemorrhagic corpora lutea were also noted together with endometrial degeneration and necrosis accompanied by multinucleated cells.
A golden hamster animal model for acute NiV virus infection has been established where, importantly, encephalitis and neuron infection were demonstrated similar to those seen in NiV-infected humans. The LD50 values for hamsters inoculated by intraperitoneal and intranasal routes were 270 pfu and 47,000 pfu, respectively. Notably, the brain was the most severely affected organ in terms of vascular and parenchymal lesions. Neurons in the vicinity of vascultis showed numerous cytoplasmic eosinophilic inclusion bodies and both viral antigen and RNA were found extensively throughout neurons. Ultrastructural analysis revealed cytoplasmic inclusions composed of defined herringbone nucleocapsids typical of paramyxoviruses. However, although HeV and NiV clearly cause systemic disease in humans, NiV was not detected in serum samples from infected hamsters. Moreover, the pathology seen in the lungs and kidneys of NiV-infected hamsters differed from that seen in HeV-infected horses and NiV-infected humans. HeV infection of hamsters has not been reported.
Experimental HeV infection of cats has been performed and findings from those studies were similar to those of horses and humans, namely generalized vascular disease with the most severe effects seen in the lung [36, 44]. NiV infection in cats was comparable to that observed with HeV except that with NiV there was also extensive inflammation of the upper and lower respiratory tract epithelium, associated with the presence of viral antigen, similar to the severe respiratory disease observed in humans in the recent NiV outbreaks in Bangladesh. Cats succumb to infection 6 to 10 days following parenteral inoculation of as low as 500 TCID50 NiV, or oronasal administration of 50,000, TCID50 of low passage, plaque purified HeV [41, 42, 46] or NiV (Bossart, K., Bingham, J. and Middleton, D., unpublished data). Gross pathology common to both HeV and NiV infection of cats consisted of hydrothorax, dense purple-red consolidation in the lung with fluid accumulation and froth in the bronchi (reviewed in. Histologically cats infected with NiV develop a necrotizing alveolitis, with necrotic foci developing within a range of other organs, particularly kidney, spleen, lymph nodes, bladder, ovaries, adrenal and meninges. Studies on cats that were euthanized early in the course of the infection (1 day after the onset of fever, as determined by radiotelemetry measurement of body temperature) indicated that alveolar infection by NiV preceded vascular infection suggesting an increased tropism for alveolar epithelium as compared to vascular tissue (Bingham, J., unpublished observation). Thus evidence to date indicates that the cat represents an animal model in which henipavirus induced pathology closely resembles the lethal respiratory disease caused by HeV and NiV in humans.
Experimental NiV infection in Pteropid bats has been attempted. All bats that were challenged with 50,000 TCID50 NiV remained clinically well throughout the study period and no febrile responses were recorded following NiV inoculation via a parenteral route. Challenged bats developed a sub-clinical infection characterized by episodic viral shedding in urine, limited presence of virus within selected viscera and seroconversion. No gross abnormalities were identified on post-mortem examination of animals at various times post exposure; all bat tissues were negative upon immunohistochemical labeling for NiV antigen.
Recently, we have been evaluating the potential of the ferret as an improved animal model for NiV infection. Ferrets have emerged as important animal models for several major respiratory diseases including highly pathogenic avian influenza, severe acute respiratory syndrome (SARS), and also morbilliviruses, the closest relatives to HeV and NiV. Significant similarity exists between ferret and human lung physiology and morphology and ferrets have been used previously for toxicology and biological safety assessment studies. We have confirmed that ferrets are susceptible to NiV infection with disease developing 6 to 10 days following oronasal administration of 500 to 50,000 TCID50 (Bossart, K., Bingham, J. and Middleton, D., unpublished data). A lower dose of 50 TCID50 failed to produce infection as defined by fever, illness, detection of virus, viral antigen or viral genome, histopathological lesions or seroconversion. Infection first manifested as fever and this was followed by inappetance and severe depression. A mild increase in respiratory rate in some animals was attributable to fever with one ferret showing tremors and hindlimb weakness. Gross pathology included subcutaneous edema of the head, hemorrhagic lymphadenopathy of submandibular, retropharyngeal and sometimes visceral lymph nodes, numerous diffuse pin-point hemorrhagic nodules scattered throughout the pulmonary parenchyma and petechial hemorrhages of the renal cortex. Histopathological lesions included focal necrotizing alveolitis, vasculitis, degeneration of glomerular tufts, and focal necrosis in a range of other tissues, including lymph nodes, spleen, adrenal cortex, bladder and ovary. Lesions in each case were associated with significant quantities of viral antigen, as determined by immunohistochemical staining (Fig. 1). Syncytial cells were also frequently present in lesions. NiV genome was detected in blood, brain, liver, testes, adrenal, kidney, lung, lymph node and spleen. These preliminary results are encouraging and indicate that the ferret may be another suitable model for human infection.
The golden hamster, cat and ferret are practical laboratory animal models that can be used to evaluate aspects of disease caused by either HeV or NiV, and each is more amenable to therapeutic efficacy testing than large domestic animals particularly for these BSL-4 pathogens. In hamsters NiV-associated encephalitis was unique among the small animal models; however, it is unclear if this was due to the length of clinical course, where animals were kept until death occurred naturally. In all other studies, most animals were euthanized before advanced disease onset (Table 1). In summary, exploration and validation of such animal models is critical for the future exploration of therapeutic intervention strategies as is the development of non-human primate models for both HeV and NiV infection which has yet to be attempted in earnest.
Hendra virus (HeV) and Nipah virus (NiV) are closely related highly pathogenic paramyxoviruses that have emerged independently in the past 15 years and continue to emerge in new locations. Flying foxes in the genus Pteropus are considered to be the natural reservoir for both viruses as demonstrated by seroconversion and isolation of HeV- and NiV-like viruses from bat tissues and secretions [1, 2]. Additionally, extensive serological surveys have not demonstrated the presence of HeV- or NiV-specific antibodies in other species. Indeed the flying fox geographic range encompasses all locations where HeV and NiV have been found. Paramyxoviruses are large, enveloped, negative-sense ssRNA viruses, and include such well-known members as measles virus (MeV), simian virus 5 (SV5), and respiratory syncytial virus (RSV). It is a diverse virus family, with various members causing common upper and lower respiratory tract infections to less common manifestations of neurological disease. In contrast, NiV and HeV are distinguished from all other paramyxoviruses most notably by their broad species tropism and high case fatality, and they have been classified into the new Henipavirus genus within the family Paramyxoviridae. HeV has appeared sporadically in Australia since 1994 where infection has been transmitted from horses to humans (reviewed in). Presumably horses become infected through spillover events from flying foxes, although no virus has been isolated from flying foxes during outbreaks. In horses, the disease presented as a severe respiratory infection, while one of the two reported human mortalities had severe respiratory disease, and the other succumbed to encephalitis 13 months following the presumed time of exposure. Recent outbreaks where horse fatalities were documented include 1999, 2004 and 2006 and although no human mortalities have occurred, one mildly ill, seroconverting, human case has been reported. NiV first appeared in peninsular Malaysia and Singapore in 1998-9 and the majority of infections occurred in pigs with subsequent transmission to humans (reviewed in [9, 10]). In pigs, infection was largely subclinical; however, where clinical disease was observed, it manifested as respiratory and encephalitic disease with low fatality ratios for respiratory cases. In contrast, humans developed severe febrile encephalitis with high case fatality and up to 25% of NiV cases also exhibited respiratory signs including non-productive cough. Interestingly, both relapsing and late-onset encephalitis syndromes with significant fatality (∼18%) have been recognized following either acute NiV encephalitic episodes or non-encephalitic/ asymptomatic infection. NiV has re-emerged numerous times since 1998: twice in 2001 in Bangladesh and West Bengal India, again in 2003 in Bangladesh, three times in Bangladesh in 2004 and 2005, and most recently in 2007 in Nadia, India. Significant observations in all of the Bangladesh and Indian NiV outbreaks have included a higher incidence of acute respiratory distress syndrome in conjunction with encephalitis, epidemiological findings consistent with person-to-person transmission, and higher case fatality ratios (∼75%). Furthermore, no intermediate or amplification host has been identified and direct transmission of NiV from the reservoir host to humans has been suggested. In West Bengal in 2001 there was no concurrent illness in animals and in Bangladesh in 2004 the common epidemiological link among cases was drinking fresh date palm sap [12, 17]. In general, it is believed that date palm sap is regularly contaminated by flying foxes and their excretions.
NiV and HeV are classified as zoonotic biosafety level 4 (BSL-4) viruses and infectious virus can only be studied at a handful of laboratories worldwide. Both viruses have also been included among the various pathogenic agents of biodefense concern and each are classified as priority pathogens in category C by the Centers for Disease Control and Prevention (CDC) and the National Institute of Allergy and Infectious Diseases (NIAID). The category C agents include high emerging pathogens with the potential for causing morbidity and mortality with major economic and health impacts. Henipaviruses in particular, could be engineered for mass dissemination because of their availability from natural sources and their relative ease of propagation and dissemination. Currently there are no specific antiviral therapies or vaccines available for treating or preventing NiV or HeV infection resulting from a natural outbreak, laboratory accident or deliberate misuse.
Few studies have documented the negative results from PCR testing of European bats for other human-pathogenic viruses. For instance following generic PCR screening for flavi-, hanta- and influenza-A viruses in 210 European bats in 2011, testing of another 1369 Central European bats for influenza-A viruses and testing 42 European bats for hepadnaviruses in 2013 did not lead to the detection of any viral nucleic acids. PCR screening of 468 European bats for orthopoxviruses has not revealed any known or novel virus sequences.
Marine mammals are susceptible to strandings, which is defined by the Marine Mammal Protection Act as a marine mammal that is dead or alive on the shore or beach. Infectious disease is highly associated with marine mammal stranding events. For instance, in Massachusetts, a survey of 405 stranded pinnipeds and cetaceans concluded that diseases were linked to the largest proportion (37%) of animal deaths. Although there are efforts to examine the roots of some of these stranding events, many go undetermined. More thorough examination of the infectious base of marine mammals mortalities should be conducted, since 44% of stranded marine mammals die from unknown causes.
Stranded animals can supply an ideal source of information for the identification of emerging infectious diseases in marine mammal populations. For example, investigations of stranded harbor seals Phoca vitulina in 1998 and 2002 concluded that morbillivirus caused the death of 23,000 and 30,000 harbor seals, respectively. Unfortunately, harbor seals have not been the only marine mammals affected by this virus; strandings of pinnipeds and cetaceans has led to the discovery of four new morbillivirus types (PDV, CMV, CDV, and MSMV). The importance of these discoveries is evident in the number of morbillivirus cases that are now easily diagnosed, and that better treatments to prevent outbreaks are currently underway. Yet, although morbillivirus infections can now be readily identified, marine mammal stranding events still remain poorly characterized in terms of their etiology.
Alexander von Humboldt discovered the latitudinal gradient in species diversity as early as 1799: The richness of species is subject to a global diversity gradient, abating from the species-rich tropics toward the higher latitudes. Bats influence this gradient significantly. More than 1100 bat species have been described worldwide. Although they are abundant worldwide except for the polar regions, a steep diversity gradient is present from the tropics towards the poles. Are fewer viruses prevalent in European bats because of the lower abundance of species in the more temperate Europe? And is the zoonotic risk posed by bats decreased accordingly?
Only few studies on the biogeography of microorganisms are available. These studies indicate that the latitudinal diversity gradient has either no or a top-down effect on microbial diversity. Two studies hypothesized that the local diversity and dispersal of viruses is very high, though overall, the viral diversity is limited on the global scale. Therefore, no assumptions can be made regarding the viral diversity in species abundant in temperate climates. As the total number of abundant species might not be essential, the change in biodiversity might play a role.
The effect of decline in biodiversity on the emergence of diseases is subject of numerous publications. Basically, there are arguments in favor of two controversial theories; reduced biodiversity could either increase (dilution effect) or decrease the risk of disease transmission. For almost half of the zoonotic diseases that have newly emerged by spillover since 1940, a preceding change in land-use, agriculture, and wildlife hunting was reported. All of the above-mentioned effects contribute to changes in biodiversity and increased contact situations between human and animal hosts, also in Europe. Once spillover in novel hosts has occurred, a high density of the novel host population eventually facilitates the establishment in the novel niche. Thus, human overpopulation and a decreased biodiversity might be mutual factors promoting the establishment of emerging infectious diseases.
In conclusion, the Baas Becking hypothesis from 1932 might still be appropriate: “Everything is everywhere, but the environment selects”.
To the best of our knowledge, this is the first case report to describe BCG reactivation in a child with confirmed measles infection. A search of the PubMed/MEDLINE database using the key terms “Bacillus Calmette-Guérin”, “Bacille Calmette-Guérin”, “BCG” and “measles” revealed no reports of similar cases in the published literature. Although the detection of BCG inoculation site erythema and induration did prompt a thorough investigation for KD in our patient, diagnostic criteria for the disease was not met. All clinical and laboratory findings were however consistent with the diagnosis of measles, and a complete and rapid recovery ensued with supportive therapy.
The diagnosis of measles in the patient was confirmed by both measles virus isolation from the throat swab and also detection of measles-specific antibodies (IgM) in the serum. Having been exposed to an unvaccinated relative who had laboratory-confirmed measles, the patient, a 7-month old infant, then developed the classical manifestations and progressed through the typical natural history of measles infection, without developing any other complications. Measles infection is caused by measles virus, a Morbillivirus in the family paramyxoviridae. The disease remains an important childhood affliction, causing 89 780 deaths globally in 2016, despite the availability of an effective vaccine. In Malaysia, measles vaccination is given to infants aged 9-months. With a vaccination coverage of 94% in 2016, the incidence of measles in Malaysia was reported to be 5.0 per 100,000 population while the mortality rate from measles was 0.02 per 100,000 population. Although the diagnosis is usually made based on the characteristic clinical manifestations, confirmation of measles infection may be obtained by detection of measles specific IgM antibodies in the serum, and also through measles virus identification from throat-swab and urine specimens.
Other than fever, the rash and the BCG inoculation site erythema and induration, no other features of KD were present in the patient. The clinical symptoms and signs resolved without KD specific therapy, with no coronary artery complications detected on follow up. KD is an acute vasculitic syndrome of unknown etiology, occurring mainly in children. As there are no confirmatory tests, the diagnosis of KD is made based on the presence of fever lasting for 5 days or more, and 4 of 5 principal clinical criteria that include conjunctivitis without exudates, cervical lymphadenopathy, polymorphous rash, changes in the lips or oral mucosa, and changes of the extremities. A major challenge in the diagnosis and management of KD, however, is the recognition that some infants present with only 2–3 clinical features, and therefore do not fulfill the diagnostic criteria. Laboratory investigations may be of some use in this situation of incomplete KD. BCG reactivation has repeatedly been described as an important sign in incomplete KD, although it is not included in any of the diagnostic algorithms [14, 15]. For example, in South Korea, 85% of children aged < 1 year who were diagnosed with incomplete KD had erythema, induration or crust formation at the BCG inoculation site. The unexpected finding of BCG reactivation in our patient correctly raised the concern of a possible diagnosis of incomplete KD. Although echocardiography was not performed in the initial phase, no additional supporting clinical or laboratory criteria, other than mild anemia, were found. Reassuringly, the rapid resolution of symptoms precluded any further concerns with regard to the need for KD therapy in the patient.
Apart from those to confirm the measles infection, no laboratory investigations were undertaken to determine if additional viral pathogens were present in the patient and contributed to the development of the BCG reactivation. Human herpes virus 6 infection has been reported in an infant with a viral exanthem presenting with BCG reactivation. In addition, various respiratory viruses including enterovirus, adenovirus, human rhinovirus and coronavirus have been isolated significantly more frequently in children with KD than in controls, although no direct association with BCG reactivation have been documented. As viral co-infections have been identified in children with measles, a more extensive virology work-up may have been warranted in our patient when the unusual finding of BCG reactivation was discovered.
The mechanism leading to the BCG reactivation in our patient with measles is not known. One possible mechanism is the reactivation and multiplication of live but dormant Mycobacterium bovis BCG bacilli that may have been present at the inoculation site, facilitated by immune suppression induced by measles infection. In one previous case report, a severely malnourished 2.5-year old boy developed ulceration of a healed BCG scar and possible disseminated BCG infection 2 weeks after measles infection. Acid-fast bacilli was detected from a swab of the BCG ulcer. This mechanism is, however, unlikely in our patient in view of the rapid onset and resolution of the BCG site erythema/induration with the appearance and resolution of the measles rash. Immune suppression induced by measles would normally be expected to persist for a longer duration. In addition, the lack of malnutrition in the patient, the otherwise uncomplicated measles course, and the absence of secondary infections normally seen in severe immunosuppressed states further renders this mechanism unlikely. Another possible mechanism for the BCG reactivation, which appears more probable, is an immune-mediated reaction. Cross-reactivity between specific epitopes of mycobacterial and human heat shock proteins (HSP), in particular mycobacterial HSP 65 and human homologue HSP 63, have been postulated as the cause of BCG reactivation in children with Kawasaki disease [22, 23]. HSP are ubiquitous molecules present in all organisms, including humans, and mediate important functions in protein folding, assembly and transport essential for cell survival. Synthesis of HSP are increased during conditions of cellular stress, including infection, ischaemia and other physical stresses. In humans, increased production and serum concentrations of HSP, especially HSP 70, have been reported during viral infections, including measles [25, 26]. As homology between HSP 70 and mycobacterial antigens have been demonstrated, a similar cross-reactivity as thought to occur in children with Kawasaki disease could possibly also explain the BCG reactivation in our patient. These mechanisms are especially intriguing as measles infections typically cause suppression of delayed type hypersensitivity and anergy to tuberculin.
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
, 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.
The use of high throughput sequencing can identify and yield new insights into the virome and microbiome of wildlife. This technique does not require prior information about the disease agents and is therefore a promising approach for pathogen identification and surveillance in stranded marine mammals. In this study, we use deep sequencing of cDNA to examine the role of possible pathogenic viruses and bacteria in a stranding event of several harbor seals.
Previous metagenomic studies of marine mammals have focused on the viral and microbial community in the gut, skin, and respiratory tissue [21–23]. As some of the worst marine mammal epidemics have been due to neurotropic diseases (morbillivirus), here, for the first time, we looked at the viral and microbial community present in the brain tissue of harbor seals to identify possible neurotropic pathogens. For this study we sampled seven harbor seals that stranded along California, USA, in the spring of 2009. These animals had abnormalities in the brain that may have been caused by an unknown virus, or an abiotic source. As a comparative group, seven other harbor seals with known causes of death were sampled. We targeted both DNA/RNA viruses to identify the possible viral pathogens in this stranding event. Additionally, we looked at microbial RNA to identify opportunistic or secondary bacterial infections in these animals.
∘Several types: A, B, C, D, E.∘Clinical findings: abdominal pain, nausea, anorexia, fevers progressing to jaundice, transaminitis.•Hepatitis A (Picornaviridae, +RNA virus):
∘Acute only. Not chronic.∘Spreads rapidly in emergency conditions.∘Can last 1 year in a minority of cases.∘Low fatality rate.∘Incubation: 30 days average∘Transmission: Fecal-oral route.∘Prevention: sanitation, hygiene, water supply, vaccine if in epidemic area, immunoglobulin in special cases.∘Management: supportive.•Hepatitis B (Hepadnavirus, dsDNA virus);
∘Acute infection, but can progress to chronic. Can result in hepatocellular carcinoma, cirrhosis.∘Diagnosis: analysis of antigens and antibodies to different components of the virus in the serum.∘2 billion persons infected globally by WHO estimates.∘Incubation: 2-3 months.∘Transmission: bodily fluid exposure, and even up to 7 days outside of human reservoirs on objects.∘Prevention: vaccine, blood bank control.∘Treatment: supportive. In some cases, although high cost, anti-viral medications can be given.•Hepatitis C (Hepacivirus, of the Flaviviridae, enveloped RNA virus):
∘75% become chronic infections. 20% of these will develop cirrhosis over 20 years. Some will develop hepatocellular carcinoma.∘Diagnosis: analysis of antigens and antibodies to HCV.∘150 million people are chronically infected with HCV according to WHO.∘Incubation: 6-9 weeks average.∘Transmission: parenteral (at-risk populations: drug abusers sharing needles, those receiving blood products frequently, hemodialysis patients)∘Prevention: no vaccine. Blood bank control. Needle exchange.∘Management: supportive. Anti-viral medications can clear the infection in some cases.•Hepatitis D (delta antigen of Hepatitis B)
∘Requires co-infection with Hepatitis B.∘More severe form of Hepatitis B.•Hepatitis E (Hepeviridae, single-stranded RNA virus)
∘Similar to Hepatitis A, but shorter course.∘High mortality in pregnant women.∘Co-infection indicates higher infectivity of Hepatitis B in adults.
Paramyxoviridae is a large and diverse family whose members have been isolated from many species of avian, terrestrial, and aquatic animal species around the world. Paramyxoviruses are pleomorphic, enveloped, cytoplasmic viruses that have a non-segmented, negative-sense RNA genome. The family is divided into two subfamilies, Paramyxovirinae and Pneumovirinae, based on their structure, genome organization, and sequence relatedness. The subfamily Paramyxovirinae contains five genera: Respirovirus, Rubulavirus, Morbillivirus, Henipavirus, and Avulavirus, while the subfamily Pneumovirinae contains two genera, Pneumovirus and Metapneumovirus. All paramyxoviruses that have been isolated to date from avian species can be segregated into two genera based on the taxonomic criteria mentioned above: genus Avulavirus, whose members are called the avian paramyxoviruses (APMV), and genus Metapneumovirus, whose members are called avian metapneumoviruses. The APMV of genus Avulavirus are separated into nine serotypes (APMV-1 through -9) based on Hemagglutination Inhibition (HI) and Neuraminidase Inhibition (NI) assays. Various strains of APMV-1, which is also called Newcastle disease virus (NDV), have been analyzed in detail by biochemical analysis, genome sequencing, and pathogenesis studies, and important molecular determinants of virulence have been identified. As a first step in characterizing the other APMV serotypes, complete genome sequences of one or more representative strains of APMV serotypes 2 to 9 were recently determined, expanding our knowledge about these viruses.
APMV-1 comprises all strains of NDV and is the best characterized serotype because of the severity of disease caused by virulent NDV strains in chickens. NDV strains vary greatly in their pathogenicity to chickens and are grouped into three pathotypes: highly virulent (velogenic) strains, which cause severe respiratory and neurological disease in chickens; moderately virulent (mesogenic) strains, which cause mild disease; and non-pathogenic (lentogenic) strains, which cause inapparent infections. In contrast, very little is known about the comparative disease potential of APMV-2 to APMV-9 in domestic and wild birds. APMV-2 strains have been isolated from chickens, turkeys and wild birds across the globe. APMV-2 infections in turkeys have been found to cause mild respiratory disease, decreases in egg production, and infertility. APMV-3 strains have been isolated from wild and domestic birds. APMV-3 infections have been associated with encephalitis and high mortality in caged birds. APMV-4 strains have been isolated from chickens, ducks and geese. Experimental infection of chickens with APMV-4 resulted in mild interstitial pneumonia and catarrhal tracheitis. APMV-5 strains have only been isolated from budgerigars (Melopsittacus undulatus) and cause depression, dyspnoea, diarrhea, torticollis, and acute fatal enteritis in immature budgerigars, leading to very high mortality. APMV-6 was first isolated from a domestic duck and was found to cause mild respiratory disease and drop in egg production in turkeys, but was avirulent in chickens. APMV-7 was first isolated from a hunter-killed dove and has also been isolated from a natural outbreak of respiratory disease in turkeys. APMV-7 infection in turkeys caused respiratory disease, mild multifocal nodular lymphocytic airsacculitis, and decreased egg production. APMV-8 was isolated from a goose and a feral pintail duck. APMV-9 strains have been isolated from ducks around the world. APMV types -2, -3, and -7 have been associated with mild respiratory disease and egg production problems in domestic chickens. There are no reports of isolation of APMV-5, -8 and -9 from poultry. But recent serosurveillance of commercial poultry farms in USA indicated the possible prevalence of all APMV serotypes excluding APMV-5 in chickens.
APMV-1 (NDV) is known to replicate in non-avian species including humans, although its only natural hosts are birds. APMV-1 infections in non-avian species are usually asymptomatic or mild. Clinical signs in human infections commonly involve conjunctivitis, which usually is transient and self-limiting. Presently, APMV-1 is being evaluated as a vaccine vector against human pathogens. When administered to the respiratory tract of non-human primates, NDV is highly restricted in replication, but foreign antigens expressed by recombinant NDV vectors are moderately to highly immunogenic. One of the major advantages of this approach is that most humans do not have pre-existing immunity to APMV-1. Pre-existing immunity is a potential drawback to using vectors derived from common human pathogens, and also can be a concern for any vector if two or more doses are necessary to elicit protective immunity. Therefore, we are investigating APMV types 2 to 9, which are antigenically distinct from APMV-1, as alternative human vaccine vectors. Also, some of these additional APMV types likely will have differences in replication, attenuation, and immunogenicity compared to APMV-1 that may be advantageous. However, the replication and pathogenicity of APMV-2 to -9 in non-avian species has not been studied. As a first step, we have evaluated the replication and pathogenicity of APMV-2 to -9 in hamsters. In this study, groups of hamsters were infected with a prototype strain of each APMV serotype by the intranasal route and monitored for virus replication, clinical symptoms, histopathology, and seroconversion. Our results showed that each of the APMV serotypes replicated in hamsters without causing adverse clinical signs of illness, although histopathologic evidence of disease was observed in some cases, and also induced high neutralizing antibody titers.
The Paramyxoviridae family within the order of Mononegavirales includes a large number of human and animal viruses that are responsible for a wide spectrum of diseases. Measles virus (MV) is one of the most infectious human viruses known, and has been targeted by the World Health Organization for eradication through the use of vaccines. The paramyxovirus family includes several other viruses with high prevalence and public health impact in humans, like respiratory syncytial virus (RSV), human metapneumovirus (HMPV), mumps virus (MuV), and the parainfluenza viruses (PIV). In addition, newly emerging members of the Paramyxoviridae family – hendra and nipah virus – have caused fatal infections in humans upon zoonoses from animal reservoirs,,. In animals, Newcastle disease virus (NDV) is and Rinderpest virus (RPV) was among the viruses with the most devastating impact on animal husbandry. Members of the Paramyxoviridae family switch hosts at a higher rate than most other virus families and infect a wide range of host species, including humans, non-human primates, horses, dogs, sheep, pigs, cats, mice, rats, dolphins, porpoises, fish, seals, whales, birds, bats, and cattle. Thus, the impact of paramyxoviruses to general human and animal welfare is immense.
The Paramyxoviridae family consists of two subfamilies, the Paramyxovirinae and the Pneumovirinae. The subfamily Paramyxovirinae includes five genera: Rubulavirus, Avulavirus, Respirovirus, Henipavirus and Morbillivirus. The subfamily Pneumovirinae includes two genera: Pneumovirus and Metapneumovirus
. Classification of the Paramyxoviridae family is based on differences in the organization of the virus genome, the sequence relationship of the encoded proteins, the biological activity of the proteins, and morphological characteristics,. Virions from this family are enveloped, pleomorphic, and have a single-stranded, non-segmented, negative-sense RNA genome. Complete genomic RNA sequences for known members of the family range from 13–19 kilobases in length. The RNA consists of six to ten tandemly linked genes, of which three form the minimal polymerase complex; nucleoprotein (N or NP), phosphoprotein (P) and large polymerase protein (L). Paramyxoviruses further uniformly encode the matrix (M) and fusion (F) proteins, and – depending on virus genus – encode additional surface glycoproteins such as the attachment protein (G), hemagglutinin or hemagglutinin-neuraminidase (H, HN), short-hydrophic protein (SH) and regulatory proteins such as non-structural proteins 1 and 2 (NS1, NS2), matrix protein 2 (M2.1, M2.2), and C and V proteins,.
Routine diagnosis of paramyxovirus infections in humans and animals is generally performed by virus isolation in cell culture, molecular diagnostic tests such as reverse transcriptase polymerase chain reaction (RT-PCR) assays, and serological tests. Such tests are generally designed to be highly sensitive and specific for particular paramyxovirus species. However, to detect zoonotic, unknown, and newly emerging pathogens within the Paramyxoviridae family, these tests may be less suitable. Development of virus family-wide PCR assays has greatly facilitated the detection of previously unknown and emerging viruses. Examples of such PCR assays are available for the flaviviruses, coronaviruses, and adenoviruses. For the Paramyxoviridae, Tong et al. described semi-nested or nested PCR assays to detect members of the Paramyxovirinae or Pneumovirinae subfamily or groups of genera within the Paramyxovirinae subfamily. Although these tests are valuable for specific purposes, nesting of PCR assays and requirement for multiple primer-sets are sub-optimal for high-throughput diagnostic approaches, due to the higher risk of cross-contamination, higher cost, and being more laborious.” Here, a PCR assay is described that detects all genera of the Paramyxoviridae with a single set of primers without the requirement of nesting. This assay was shown to detect all known viruses within the Paramyxoviridae family tested. As the assay is implemented in a high-throughput format of fragment analysis, the test will be useful for the rapid identification of zoonotic and newly emerging paramyxoviruses.
Hantaviruses are negative-sense RNA viruses transmitted to humans from small animal hosts. Different viral species are associated with one of two disease syndromes: hemorrhagic fever with renal syndrome (HFRS), or hantavirus pulmonary syndrome (HPS). Hantaan virus (HTNV), primarily found in Asia, is among the most prevalent HFRS-causing hantaviruses with a case fatality rate of between 1–15%. Puumala virus (PUUV) causes most HFRS cases in Europe, though its case fatality rate is lower at <1% [3, 4]. There are currently no FDA licensed vaccines or therapeutics for either HFRS or HPS.
The Syrian hamster (Mesocricetus auratus) is the typical animal used to model hantavirus infection and disease. Andes virus (ANDV), an HPS-causing hantavirus, causes lethal disease in immunocompetent hamsters, while numerous other HPS-causing hantaviruses including Sin Nombre Virus (SNV) and Choclo virus cause lethal disease in hamsters immunosuppressed with dexamethasone and cyclophosphamide [7, 8]. In contrast to HPS-causing hantaviruses, exposure of hamsters to HFRS-causing hantaviruses such as HTNV, PUUV, Dobrava (DOBV) and Seoul (SEOV) leads to asymptomatic infection, despite viral dissemination, even when immunosuppressed (Hooper Lab, unpublished data) [8–11]. In these studies hamsters were exposed to high doses of HTNV and PUUV, far exceeding the infectious dose 99% (ID99) for the virus. Development and characterization of a uniformly infective, low-dose challenge model, enhances the hamster model’s usefulness in vaccine and therapeutic testing. In this report we present a low-dose hamster infection model for both HTNV and PUUV infected animals.
Ferrets (Mustela putorius furo) have become a popular animal model for a number of respiratory pathogens including influenza, coronavirus, Nipah virus, and morbillivirus, due to the similarity in lung physiology to humans. In addition, they have recently been described as a disease model of two hemorrhagic fever viruses, Bundibugyo virus and Ebola virus [16, 17], supporting viral replication without prior adaptation. Most hantavirus-related human disease occurs by aerosolized transmission of the virus from the excreta or secreta of infected rodents [18, 19], a model of viral infection for which the ferret is well suited. In this study we demonstrate that ferrets are capable of being infected by high titers of HTNV and PUUV, though aside from gradual weight loss infected animals exhibit no clinical symptoms or impaired renal function.
It has been established that infection of rhesus macaques (Macaca mulatta) with HFRS-causing hantaviruses (DOBV, SEOV, HTNV, and PUUV) leads to asymptomatic infection and seroconversion, while infection of cynomolgus macaques (Macaca fascicularis) with PUUV leads to a mild disease characterized by lethargy, mild proteinuria and hematuria, and kidney pathology, similar to mild HFRS in humans. However, the macaques’ large size and cost limits their usefulness in therapeutic studies, especially when test article availability is limited, as is often the case in passive transfer studies. The common marmoset (Callithrix jacchus) is becoming more popular for infectious disease studies. Its genetic similarity to humans, cost, relative safety, and small size make it an attractive alternative to traditional non-human primate species. Marmosets have been used as a disease model for other viral agents including Dengue virus, Hepatitis C virus, influenza virus, Lassa fever virus, orthopox viruses [26–28], Rift Valley Fever virus, Eastern Equine Encephalitis virus, and filoviruses. In this study we demonstrate that exposure of marmosets to HTNV leads to asymptomatic infection characterized by high levels of neutralizing antibodies. This is the first report of hantavirus infection in marmosets.
Medical countermeasures are products including biologics (e.g., vaccines and antibodies) and small molecule drugs that can be used to prevent or combat infectious disease outbreaks. This study presents three animal models of HTNV infection, and two models of PUUV infection that can be used to evaluate the efficacy of medical countermeasure that are intended to prevent or mitigate infection (e.g., vaccines) by these viruses through induction of sterile immunity.
All twelve dogs that were tested for CPV at the initial presentation were PCR-negative (Table 5). Vector-borne infections were detected in 4 dogs (31 %, Table 5): infection with Babesia spp. was detected in Dogs 3 and 8; infection with L. infantum was diagnosed in Dog 4 and infection with Dirofilaria immitis was found in Dog 13. Dog 13, which tested positive in D. immitis antigen and Knott tests, had received a certificate from a laboratory in Budapest, Hungary, that stated a negative result in the Knott test in August 2013. None of the rescue dogs tested positive for Ehrlichia canis (Table 5).
Feline leukemia virus, a retrovirus of domestic cats, displays a prevalence of 1–8% among feral cats worldwide. Transmission is usually by direct contact, and outcome after exposure depends on several host and viral factors. In approximately one third of exposed cats, viremia is persistent and eventually results in clinical syndromes including some combination of immunosuppression, anemia and/or neoplasia. Mortality among persistently infected domestic cats is high as 83% die within 3.5 years.
Like other Type C retroviruses, FeLV induces immune suppression making the cats susceptible to opportunistic infections and cancers. There are four naturally occurring exogenous FeLV strains FeLV-A, -B, -C, and -T, that are distinguished genetically by sequence differences in the env gene and by receptor interactions required for cell entry. FeLV-A is the predominant subgroup circulating in feral cats and is often only weakly pathogenic. The endogenous feline leukemia provirus sequences are transmitted vertically though the germ line as integrated provirus nested on several cat chromosomes. Among infected cats the pathogenic subgroups, FeLV-B, -C, and -T, are generated de novo by mutation or recombination in the env region between exogenous subgroup A virus and endogenous proviral sequences.
FeLV infection among non-domestic cats of the Felidae family is rare. Most reported infections involved captive animals that acquired FeLV by physical contact with FeLV-infected domestic cats, and in nearly all cases that were followed, the virus was cleared by the infected individuals. Therefore, it was postulated that FeLV pathogenicity did not occur in exotic felids, simply because there were no endogenous FeLV present in species outside the domestic cat lineage. The outcome with a Florida panther FeLV outbreak in 2001–2006 was unexpected and served to change this hypothesis.
The Florida panther (Puma concolor coryi) is an endangered subspecies whose range was contiguous with other puma populations. By the late 20th century, however, depredation, exploitation, human population growth and habitat destruction had reduced the population to an isolated relict population of fewer than 30 individuals. In 1995, a Florida panther restoration management action relocated eight Texas cougars (Puma concolor stanleyii) to the Florida habitat in a hopeful rescue of the threatened subspecies. The population rebounded to over 100 individuals, doubling panther numbers, density, survival parameters and fitness.
Florida panthers have undergone continued surveillance from 1978 -2001 and routinely tested for several pathogens, including FeLV. However, for the first time in early 2001, 23 panthers were discovered to carry antibodies for FeLV by ELISA that was confirmed by Western Blot. Clinical symptoms including lymphadenopathy, anemia, septicemia and weight loss rapidly appeared. Five panthers shown to carry FeLV antigens in their sera subsequently died of diseases compatible with FeLV etiology.
The rapid appearance and spread of FeLV in this Florida panther population was unprecedented among large cats and caused concern in the Felidae conservation community. FeLV was not thought to cause serious disease in species other than in cats closely related to domestic cats (F. catus, F. sylvestris, F. margarita, F. nigripes and F. bieti) because only these Felis species carry endogenous FeLV sequences in their genome, a prerequisite for in situ development of recombinant and virulent FeLV strains.
To explore the origins and the unusual virulence of the emerging FeLV strain in pumas, Brown et al. obtained infectious FeLV gene sequence (LTR and env genes; 2851 bp) from several FeLV-infected Florida panthers. Alignment and phylogenetic analysis of panther FeLV gene sequences and those from known domestic cat FeLV strains revealed three important aspects: (1) The panther FeLV was clearly aligned with FeLV domestic cat type FeLV-A, the strain that is largely avirulent until after recombination with endogenous sequences; (2) There was no evidence of endogenous FeLV sequences within in the panther FeLV; and (3) The panther FeLV was closely aligned with a highly virulent FeLV from domestic cats, FeLV-945. Although FeLV-9545 is considered an FeLV-A strain, it has a distinctive envelop and LTR sequence that are different from other FeLV-A strains. FeLV-945 is unusual is that its severe pathogenicity in domestic cats does not involve recombination with the endogenous FeLV sequences. A vaccination campaign was initiated in 2006 and 52 Florida panthers were captured and vaccinated with no major FeLV incidence reported to date.
An interesting corollary to the Florida panther FeLV outbreak is that FIVPco is endemic in this population. Two distinctive strains were present in 2001, one from the original authentic Florida panther and a second accidentally introduced in 1995 from FIVPco infected Texas cougars. FIV incidence in the population was low (~15% in 1999–2000;). By contrast, 13 of 17 panthers tested during 2004–2005 in the FeLV-endemic region (76%; Figure 1) were FIV positive. This apparent elevation in FIV incidence among FeLV afflicted panthers raises the possibility of a role for FIV-mediated immune depletion in FeLV pathogenesis. In domestic cats, FIV and FeLV co-infections have resulted in conflicting interpretations.
The conclusion here is that domestic cat strains of viruses can cross species barriers with potentially devastating consequences to fragile wild populations of large felids. In this case, the requirement for endogenous FeLV recombination was abrogated and perhaps the resultant virulence was accelerated by FIV immune suppression in Florida panthers. As in lions, FIV depletes puma CD4-T lymphocytes, so the possibility of FIV accessory role is feasible. Unfortunately, a similar outbreak has recently occurred in wild populations of Iberian lynx, confirming that FeLV is capable of causing disease in non-domestic felids, contrary to conventional wisdom.
Feline Immunodeficiency Virus (FIV) was first discovered 25 years ago as a cat lentivirus with structural, genomic, and pathogenic parallels to HIV. Infected domestic cats develop symptoms of immune depletion including a precipitous drop in CD4 bearing T-lymphocytes, neutropenia, lymphadenopathy and susceptibility to normally harmless bacteria, fungal lesions, wasting, and rare cancers. FIV is endemic in feral cat populations and has diverged into several phylogenetic clade types across the world.
FIV has infected many of the 37 described species of the Felidae family (Table 2). It is speculated that most cat species (Table 2) acquired the virus within the last 10–20,000 years, but patterns of evolution within both virus and host genomes, suggest FIV may have existed far longer in some species such as lion. Phylogenetic analysis of individual FIV-isolates in a dozen or more species of felids demonstrates reciprocal monophyly of FIV among various species (that is, every lion strain has as its closest relative another lion isolate rather than FIV from a different cat species) (Figure 2). These phylogenetic results supported the notion that although FIV occasionally can move from species to species, these events are exceedingly rare, leading to a monophyletic expansion of viral genome sequence diversity within every species, so that most cat species carry their own distinct version of FIV.
Originally, the absence of clear clinical pathology among FIV infected felids in zoological collections, and field observations of seemingly healthy (or asymptomatic) FIV in natural populations of felids fostered the view that FIV is pathogenic in domestic cats but not in other free ranging species of Felidae. However, that conclusion now seems premature and over-simplified. For example, Roelke et al.. mounted a detailed physical examination and associated clinical measures among 64 free ranging lions in Botswana and Tanzania between 1999 and 2006. They examined a suite of biochemical, clinical, and pathogenic manifestations of immune suppression and disease analogous to pathogenesis observed in FIV infected domestic cats, in HIV-infected AIDS patients and in simian immunodeficiency virus (SIV)-infected macaques (see citations).
Multiple indications and sequelae of AIDS defining conditions were manifest amongst FIV infected lions compared to FIV negative lions; the statistical associations are summarized in Table 3. First, a marked depletion of CD4 bearing T lymphocytes was apparent in FIV infected lions, a prelude to immune collapse in well defined AIDS. In addition there were multiple elevations in opportunistic infections (papilloma, gingivitis, dehydration during wet conditions, anemia, hyperalbuminemia, weight loss in the face of abundant prey, abnormal red cell parameters, depressed serum albumin, liver pathogenesis, and elevated gamma globulin). Further, spleen and lymph node biopsies from nine free ranging lions revealed evidence of lymphoid depletion, the hallmark of AIDS disease in human, cats and macaques. These findings strongly suggest FIV is contributing to the loss of immune competence in these lions. A similar pathogenic study of wild SIV-infected chimpanzees also revealed definitive evidence of pathology in that species after a decade of pronouncing chimps as resistant to SIV.
As most people infected with HIV do not actually die of HIV infection per se, rather from subsequent opportunistic infections (e.g., pneumocystis, CMV, Kaposi’s sarcoma, candidiasis and other infections) it seemed fair to ask whether FIV in large cats might contribute to secondary infection pathogenesis. An opportunity to inspect this occurred during the mid-1990s in Tanzania when an outbreak of canine distemper virus (CDV; a morbillivirus) eliminated ~1000 lions from the large Serengeti populations in a 10 month interval. Because FIV prevalence in East African and Botswana lions approaches 100% in adults, the potential influence of FIV on CDV pathology was to us an interesting question.
Lions harbor six genetically distinct strains, or subtypes, of lion FIV (FIVPle) resolved by phylogenetic analyses (Figure 2). These strains have distinct phylogeographic distributions, suggesting prolonged host association, perhaps predating the Late-Pleistocene expansions of lions roughly 325,000 years ago. Two lion FIVPle strains, FIVPle E and FIVPle A, circulate in Botswana; while three very divergent strains FIVPle A, B, and C occur in the Serengeti. Perhaps consequent of the highly social nature of lions, FIVPle infected lion populations have high prevalence of seropositive individuals, approaching 100% in adult animals (Figure 3a).
Troyer et al. recently examined the association of FIV strains with relative survival (from death) in the Serengeti lions during the CDV outbreak. A rather striking difference was seen in that FIVPle B infected lions were twice as likely to survive CDV compared to lions infected with alternative strains FIVPle A and FIVPle C (Figure 3b). The apparent FIVPle B associated protective influence was evident whether individuals were infected with a single strain or with multiple strains (Figure 3b). These observations would suggest that infection with FIVPle A or C might have increased the risk of mortality upon secondary CDV infection. This inference that certain FIVPle strains predispose carriers to CDV pathogenesis has some parallels with FIV strain-specific pathogenicity in domestic cats. Further, the higher CDV mortality among of FIVPle A and C carrying individuals actually altered FIV strain incidence causing a rise in FIVPle B and a drop in FIVPle C during the course of the CDV outbreak (Figure 3c).
The statistical rigor associated with these conclusions is rather weak since the number of lions was limited (total = 119 lions) and should be interpreted cautiously. Nonetheless, the striking influence of FIV on lion immune function (Table 3), clinical disposition, and a potential ancillary role in CDV mortality (Figure 3b,c) affirms that FIV is likely pathogenic in lions. However, the degree to which viral pathogenicity is influenced by host genomics underlying the immune response, the role of secondary infections, stochastic events due to ecological and environmental factors, has yet to be described. Nonetheless, FIV is a potentially harmful agent in free ranging lions, as for housecats, and deserves further scrutiny in the other free ranging species afflicted with FIV.
The emergence of novel infectious diseases continues to represent a threat to global public health. Emerging pathogens have been defined as those newly recognised infections of humans following zoonotic transmission or those increasing in incidence and/or geographic range. High-profile examples of emerging pathogens include the discovery of the novel Middle East respiratory syndrome (MERS) coronavirus from cases of respiratory illness in 2012 and the expansion of the range of Zika virus across the South Pacific and the Americas. The emergence of previously unseen viruses means that the set of known human viruses continually increases by around two species per year. Initial comparative studies identified trends among emerging human pathogens, e.g., increased risk of emergence for pathogens with broad host ranges and RNA viruses [6–9]. However, more recent comparative analyses have focused on risk factors for specific pathogen traits such as transmissibility [10–12]. Here, we focus on understanding the ecological determinants of pathogen virulence, using all currently recognised human RNA viruses as a study system.
Emerging RNA viruses vary widely in their virulence, with some never having been associated with human disease at all. For example, Zaire ebolavirus causes severe haemorrhagic fever with outbreaks, including the 2014 West African outbreak, showing case fatality ratios (CFRs) of approximately 60% or more. In contrast, human infections with Reston ebolavirus have never exhibited any evidence of disease symptoms. Applying the comparative approach to understand the ecology of virulence could offer valuable synergy with studies of emergence towards prioritisation and preparedness in the detection of potential new human viruses.
Few comparative analyses have addressed the risk factors driving human pathogen virulence to date (but see [17–19]), and none have investigated virulence across the entire breadth of currently recognised human RNA viruses. Of relevance here is an ongoing, largely theoretical debate about the possibility of an evolutionary tradeoff between virulence and transmissibility, which has proven challenging to empirically characterise [20–22]. We also note that in the absence of coevolution, a zoonotic virus may demonstrate ‘coincidental’, nonadapted virulence. We therefore compared viruses with different levels of transmissibility in human populations. Transmission route is another potential predictor of virulence; higher mortality rates have been observed in earlier comparative analyses for vector-borne pathogens and pathogens with greater environmental persistence. We therefore hypothesised vector-borne transmission or routes with environmental components (e.g., faecal–oral or food-borne transmission) would be associated with higher virulence than direct, contact-based transmission.
Several studies have suggested a link between host range breadth and virulence, in which higher virulence has been predicted for pathogens with a narrower, specialist host range. Virulence (or host exploitation) has also been predicted to vary with host relatedness through phylogenetic distance or in phylogenetic clustering. We therefore hypothesised that a narrow host range, and specifically, infection of nonhuman primate hosts, may also predict virulence. Finally, we hypothesised that a broader tissue tropism could predict higher virulence. This idea is largely unexplored, although experimental studies have demonstrated a broader tissue tropism for more virulent strains of Newcastle disease virus.
We aimed to determine patterns of virulence across the breadth of all known human RNA viruses. We then aimed to use predictive machine learning models to ask whether ecological traits of viruses can act as predictive risk factors for virulence in humans. Specifically, we examined hypotheses that viruses would be more highly virulent if they lacked transmissibility within humans, had vector-borne or faecal–oral transmission routes, had a narrow host range or infected nonhuman primates, or had greater breadth of tissue tropisms.