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In comparison to non-viral infected individuals, viral-infected SARI ones had significantly predominant signs and symptoms at presentation. Particularly, they had significant viral prodromal symptoms, as well as tachypnea, wheezes, and convulsions (p=0.000 each). Among individual viral pathogens, SARI patients with influenza had more significant tachypnea (p= 0.038), wheezes (p=0.000), and abnormal breath sounds (p= 0.023), than those with non-influenza viral infections. Patients whose specimens were collected within 5 days of the onset of symptoms were more likely to have a viral pathogen detected than those whose specimens were collected later (73% versus 36%, p = 0.047).
Fifty-three percent of patients had at least one underlying medical condition. These comorbidities included chronic respiratory disorders (asthma, COPD, bronchiectasis, and immotile cilia syndrome), cardiac disorders (heart failure congenital heart diseases, and cardiomyopathy), neuromuscular disorders (epilepsy, cerebral palsy, and myopathies), hematological disorders (thalassemia), endocrine disorders (diabetes mellitus, hypothyroidism, and morbid obesity), renal disorders (end-stage renal disease), and liver disorders (liver cirrhosis and hepatic failure).
Patients with comorbidities (n = 570, 53%) were significantly older compared to those with no comorbidities (median age: 54 versus 3, p <0.001). Additionally, they were significantly more likely to be symptomatic.
In terms of comorbidities, patients with and without viral detection differed significantly in the frequencies of chronic respiratory (p=0.002), endocrine (p=0.000), hepatic (p=0.002), and neuromuscular disorders (p=0.001). Among individual viral pathogens, SARI patients with para-influenza virus had significant endocrine (p= 0.004), and neuromuscular disorders (p=0.012), than those with non-para-influenza viral infections.
For influenza vaccination history; 832/1,075 (77.4%) cases did not receive the vaccine within the 12 months prior to hospital admission, while 243/1,075 (22.6%) were reported as unknown for an influenza vaccination status. Table 1 details these results.
Humans and animals have coexisted since the beginning of time, sharing viruses, bacteria, and perhaps the etiology of cancers. Approximately 75% of viruses and 50% of bacteria known to cause disease in humans are zoonotic and can be transmitted between animals and people (1). While evolution has provided adaptive immunity against microbes and cancer, the ability to defend against infection is sometimes absent or compromised. Excluding ionizing radiation, sunlight, and tobacco, infection represents the main known cause of human cancers throughout the world. The list is long including cancers of the anogenital track (HPV), stomach (H. pylori), liver (HBV, HCV, liver flukes), bladder (schistosoma hematobium), prostate (XMRV), and other specific cancers such as adult T-cell leukemia (HTLV-1), Kaposi sarcoma (HHV-8), Merkel Cell Carcinoma (MCPyV), and Burkitt’s lymphoma (EBV) (2, 3). The prevalence and persistence of tumor viruses varies in different parts of the world. Nearly 30% of cancers in developing and tropical countries are attributable to infectious causes compared with 10% in developed countries (4). However, the connection between viruses, bacteria, and cancer, and the role of animals, remains unclear or paradoxical in nature.
An infectious etiology for cancer was first documented in animals during the early part of the nineteenth century with the diagnosis of pulmonary adenocarcinoma in sheep (later attributable to jaagsiekte sheep retrovirus) (5). Animals are the host species for many oncogenes. Among the most studied are rodent (Abl, Int1/Wnt1, Int2, Notch1, Pim1/2, Runx, Tpl2), fowl (Erb-b, Fos, Myc, Src), feline (Myc), and fish (cyc) (6). For example, reticuloendothesliosis virus readily induces cancer in chickens (avian leucosis/sarcoma). The virus has been found in eggs intended for human consumption and vaccines prepared in eggs (7). A wide variety of viruses, mirroring their human analogs, are ubiquitous among animals in nature and their habitat (e.g., fecal coliform contamination) (8–10). Common types include viruses in the polyoma, adeno, retro, and papilloma family.
Animal viruses potentially express oncoproteins in human cells even though stringent replicate restrictions exist in the latter (11). The “hit and run” hypothesis posits that certain viruses interfere with the hosts immune system to cause cancer, yet do not integrate into the victims DNA (leaving no detectable fingerprints) (12). Newborn hamsters infected with polyoma virus have been shown to develop cancers, even though the cells of this species do not support virus replication (13). Similarly, tumors induced in immunocompetent mammals with Rous sarcoma virus do not present neutralizing antibodies (14). In contrast, some animal viruses [e.g., feline leukemia virus (FeLV)] have been observed to replicate in vitro in human cells (15, 16). Sera collected from 69% of 107 persons among 46 households with at least 1 FeLV gs-a positive cat tested positive for antibodies against FeLV (15). Although it is unclear exactly how antibodies directed toward animal viruses could have oncogenic or mitogenic effects on host cells, these findings support the idea that long-lasting “biological memory” of animal virus exposure can exist within the host in the absence of direct effects on host DNA.
The World Health Organization (WHO) estimates that acute respiratory infections (ARI) cause annual deaths approaching 4 million, at a rate of more than 60 deaths/100,000 populations. Viruses are responsible for 30-70 % of ARI where respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), human Bocavirus, human metapneumovirus (hMPV), adenovirus, rhinovirus, enterovirus and Coronaviruses account for the majority of these cases [1, 2]. The 2009 influenza pandemic had highlighted the need for more global data on severe influenza disease, so the WHO recommended conducting surveillance for hospitalized severe acute respiratory infection (SARI), as well as influenza-like illness (ILI) in outpatients [3–6]. SARI surveillances are now conducted in many countries around the world; however, because of limited resources, they are only conducted in limited settings in the Middle East and Egypt [7–9]. Furthermore, the role of individual viral or atypical bacterial infection in causing ARI is not usually documented [10, 11].
In the current study, we analyzed surveillance data from Egyptian patients with SARI, enrolled at Cairo University Hospital (CUH) from 2010 to 2014. We aimed to calculate proportions of positive samples for different viral pathogens, to determine which pathogens were related to severe outcomes, and to address the impact of SARI on the clinical outcomes of enrolled patients, in terms of morbidity and mortality.
Acute respiratory infections (ARIs) are common and contribute significantly to morbidity and mortality. They are the leading causes of outpatient visits and hospitalizations in all age groups, especially for children under 5 years of age.1 Most ARIs in children and outpatients are caused by nine common respiratory viruses, including respiratory syncytial virus (RSV), influenza virus A, influenza virus B, rhinovirus, adenovirus, parainfluenza virus, coronavirus, human metapneumovirus, and boca virus2, 3 Additionally, atypical pathogens, such as Mycoplasma pneumoniae, are also major causes of ARIs in children. The symptoms caused by these pathogens are largely similar, thus definitive diagnosis requires effective laboratory testing. By using multiplex assay targeting these pathogens, early diagnosis can be made in a timely manner. Consequential antimicrobial or antiviral therapy may thus be administrated promptly and appropriately.4 Most importantly, the early diagnosis of influenza viruses, which are contagious, is beneficial for early isolation of patients, thus reducing the spread of influenza viruses.
The routine clinical laboratory testing for respiratory viruses is largely conducted by direct fluorescent‐antibody assays and rapid antigen tests in China. Given the poor sensitivity and complicated manual operation, these methods have been gradually replaced by nucleic acid amplification tests (NAATs), which are more sensitive and more specific. However, majority of the NAAT kits are based on real‐time polymerase chain reaction (PCR), which can only detect one or two pathogens of ARIs within a single tube, thus are not syndromic testing.5 The clinical and economic impacts of syndromic testing for respiratory pathogens have been evaluated in several studies. Overall, the implementation of syndromic testing can decrease the time of diagnosis,4 decreased healthcare resource utilization,6 decrease inpatient length of stay and time in isolation,7 and improve antiviral use for influenza virus‐positive patients.8
SureX 13 Respiratory Pathogen Multiplex Kit (ResP) is a syndromic multiplex molecular test for simultaneous detection of 13 pathogens in a single tube. The aim of this study was to evaluate the application of the ResP for detection of respiratory pathogens in outpatients with flu‐like manifestations.
Acute respiratory viruses cause substantial morbidity and mortality worldwide. Most respiratory viral infections induce self-limiting disease. However, the disease range can vary from common cold, croup, and bronchiolitis to pneumonia, with an array of possible etiological agents, such as parainfluenza, influenza, RSV, adenovirus, rhinovirus, bocavirus, human metapneumovirus and coronavirus. Coronaviruses (CoV) are responsible for a broad spectrum of diseases, including respiratory and enteric illnesses, in humans and animals. Human coronaviruses (HCoV) were identified as the cause of acute respiratory tract disease in the early 1960’s, but their correlation with mild respiratory tract infection outweighed the importance of severe forms of the infection. The emergence of SARS-CoV in humans in 2003 increased scientific interest in CoVs and emphasized the ability of highly pathogenic CoVs, most importantly those of animal origin, to infect humans. Consequently the importance of monitoring circulating coronavirus strains in humans has been reemphasized with the emergence of SARS and Middle East Respiratory Syndrome (MERS) CoV in humans.
The family Coronaviridae was recently subdivided into four genera according to their antigenic and genetic characteristics: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus (http://ictvonline.org/virusTaxonomy.asp?version=2012). Alphacoronavirus (HCoV-229E and HCoV-NL63) and Betacoronavirus (HCoV-OC43, SARS-CoV, HCoV-HKU1 and HCoV-MERS) infect a wide range of mammals [4,7–11], whereas members of the genus Gammacoronavirus and Deltacoronavirus usually infect birds, although a Gammacoronavirus was isolated from a Beluga whale. Feline CoV, an Alphacoronavirus, infects wild and domestic cats causing mild enteritis. However, a lethal systemic disease known as feline infectious peritonitis (FIP) is also associated with FCoV. Feline CoV is closely related to CCoV, TGEV and human coronavirus HCV-229E, especially the Feline aminopeptidase N, which can be used as a functional receptor by these viruses.
The CoVs have a positive-sense, single-stranded RNA genome of 27–32 Kb. Nine to fourteen open reading frames (ORF) have been identified in the CoV genome. ORF1a and ORF1b encode the highly conserved replicase complex. Most RT-PCR assays described in the literature to screen for CoV target the ORF1b region. CoVs show a high frequency of nucleotide mutation and RNA recombination through copy-choice mechanism which, associated with broad receptor and co-receptor usage allow the virus to increase pathogenicity and possibly shift its host range.
Before the SARS-CoV outbreak, only two HCoV respiratory strains, HCoV-229E and HCoV-OC43, had been described. Due to the increased interest highlighted by the SARS outbreak, three new strains were described afterwards; HCoV-NL63, HCoV-HKU1 and HCoV-MERS. This study aimed to investigate the circulation of human respiratory CoV and to determine the genetic variability of HCoV in Arkansas.
Acute respiratory infections (ARIs) are a major public health problem causing approximately 1.9 million child deaths in 2000. About 20% of child mortality (<5 years) is due to pneumonia, bronchitis or bronchiolitis and 90% of them are attributed to pneumonia. Community acquired pneumonia (CAP) is a major cause of morbidity and hospitalization in developed countries and a major cause of mortality among children living in developing countries where socio-economic issues such as malnutrition are aggravating factors. To date, it is difficult to reliably predict the pathogen based on clinical signs and symptoms. Respiratory pathogens etiologies could help to better understand acute febrile illnesses in malaria endemic area and orient adapted therapies. Viruses were considered as causative agents of acute lower respiratory infections (ALRIs) and have been investigated in several studies. Since 2001, several new respiratory viruses have been described such as metapneumovirus (HMPV), human coronavirus (HCoV), NL63 and HKU1 and human bocavirus (HBoV). Various respiratory viruses that caused epidemics and pandemic, such as swine lineage influenza ALRIs A (H1N1) virus infection, in 2009, have heightened the need to develop sensitive and rapid diagnostic test. The development of molecular methods such as multiplex Real-Time PCR (RT-PCR) greatly facilitates the etiological study of respiratory infections but it does not, especially in developing countries, assist the clinician in the care of patient. Madagascar is a country with low HIV prevalence (estimated at 0.2% in 2010) and malaria is endemic with stable transmission during all year on the East Coast. Recent studies on surveillance of fever among child result in malaria over-diagnosis with consequent under diagnosis of other fever-causing disorders such as pneumonia. Little is known about the pathogens responsible for ARIs especially in rural areas. A recent study shows that respiratory viruses play an important role in children under 5 years old consulting in public and private clinics in Antananarivo with Influenza-Like Illnesses (ILIs) symptoms. In Ampasimanjeva, a small village located in a rural area endemic for malaria, a recent study showed that 68% of acute fever illnesses among children are not explained by malaria (Ratsimbasoa, personal communication). The objective of this study is to determine the prevalence and seasonal distribution of a large panel of respiratory pathogens including viruses and atypical bacteria among a well clinically defined cohort of acute febrile children between 2 to 59 months of age presenting clinical ARIs symptoms in Ampasimanjeva.
Feline calicivirus (FCV) is a common viral pathogen in cats worldwide. The prevalence ranges from 10% up to 70% depending on the cat population sampled. Usually, FCV causes painful oral ulcerations, salivation, gingivitis/stomatitis, inappetence, fever and depression with a high morbidity, but low lethality. However, FCV can occur as a very virulent form, causing severe clinical signs, due to systemic infection and inner organ involvement and high mortality rates up to 60% are reported. FCV is a non-enveloped RNA virus with a high tenacity that can remain infectious for approximately one month on dry surfaces at room temperature, and for weeks at colder temperatures. Additionally, the stability of FCV is strain-dependent, and common disinfectants do not inactivate FCV. Viral transmission occurs via direct cat-to-cat contact or indirectly via fomites (e.g., food bowls, contaminated coats). Reports from animal hospitals encountering outbreaks of virulent FCV infections emphasize that indirect transmission is a key factor in viral spread. In human hospitals, environmental testing for common human pathogens is routinely performed to assess the hygienic management. In veterinary facilities or research catteries, nobody has so far investigated environmental contamination with FCV.
It was the aim of this study to investigate the presence and viability of FCV in the environment of a research cat facility following two sequential experimental infections of cats with two different FCV field strains. Oropharyngeal shedding of FCV was tested in parallel. The findings of this study are important to optimize the hygienic management of private practices and veterinary hospitals and to prevent accidental, iatrogenic FCV infections in cats.
A total of 420 oropharyngeal swabs were enrolled from 10 hospitals and 10 CDCs in Guangzhou from 2017 to 2018. Samples were collected from a wide range of ages, with the average age of 27.2 (Table 1). About 55% specimens were from male.
A pathogen‐positive result was determined when the pathogen‐specific fragment(s) was positive, as shown in Figure 1. A negative result was determined when none of the 13 pathogen‐specific fragment was positive, while the controls (huDNA, huRNA, and IC) were positive (Figure 2). In this study, the ResP detected positive results in 141 samples, accounting for 33.6%, while the comparator tests detected positive results in 127 samples, with positive rate 30.2%. Among the detected pathogens, rhinovirus was the most common, followed by adenovirus and influenza virus A pdmH1N1 (2009) (Table 2). Of the 420 specimens, the ResP yielded consistent positive results in 121 specimens (86.5%, 121/141), and consistent negative results in 273 specimens (97.8%, 273/279) comparing with pathogen‐specific PCRs, leading to an overall agreement of 93.8%.
No specimen was detected positive with coronavirus or Chlamydia. In six of the ten detected pathogens, the Cohen's kappa values were over 0.8 with P value <.01 (Table 2). The lowest kappa (0.70) was observed on human metapneumovirus.
This study was approved by the National Ethics Committee of the Malagasy Ministry of Health (CE/MINSAN n° 019). A briefing note explaining the purpose of the project and the informed consent form was given to each of the parents involved in the study who signed the consent forms to provide written informed consent.
All respiratory samples positive for coronavirus by real-time RT-PCR were screened for other respiratory viruses. Among the real-time RT-PCR CoV positive samples, 13% were also positive for Respiratory Syncytial Virus, Parainfluenza 2 and 3, and Rhinovirus (Data not shown). Among the patients harboring respiratory CoVs, detected by qRT-PCR and confirmed by RT-PCR, six (17%) were co-infected with a second respiratory virus.
Influenza viruses pose major threats to public health, as they are responsible for epidemics and pandemics resulting in high morbidity and mortality worldwide. Several pandemics, such as the Spanish flu (1918), Asian flu (1957), and Hong Kong flu (1968), caused millions of deaths in the last century (1). Currently, only two therapies, targeting the viral proteins neuraminidase and M2, are approved to treat influenza. Therefore, novel viral or host targets for antiviral strategies to block viral replication or inhibit cellular proteins necessary for the virus life cycle are urgently needed (2). In this context, host proteases are a group of very promising antiviral targets, because proteolytic cleavage of the precursor hemagglutinin (HA0) into HA1 and HA2 subunits by host proteases is essential for fusion of HA with the endosomal membrane and thus represents an essential step for infectivity of the virus (3, 4). Due to the potential risk of side effects after application of broadband protease inhibitors, the specific inhibition of a single enzyme would convey a huge therapeutic benefit.
The majority of influenza viruses, including low pathogenic avian and human influenza viruses, carry a single arginine (R) residue at the cleavage site. These HAs are cleaved by host trypsin-like proteases (5–8). In vitro studies with cultured human respiratory epithelial cells demonstrated the involvement of several membrane-associated proteases (9). Cell culture studies further identified, among others, the transmembrane serine proteases TMPRSS2, TMPRSS4, and TMPRSS11D as enzymes able to cleave the HAs of influenza virus subtypes H1 and H3 (10–12). We previously showed that deletion of Tmprss2 in knockout mice strongly limits viral spread and lung pathology after H1N1 influenza A virus infection (13). An essential role for TMPRSS2 in cleavage activation and viral spread was also reported for H7N9 influenza A virus (14, 15). We also demonstrated that deletion of Tmprss2 slightly reduced body weight loss and mortality in mice after H3N2 virus infection compared to those for wild-type mice but did not protect mice from lethal infections (13, 15). Therefore, it is likely that in addition to TMPRSS2, other trypsin-like proteases of the respiratory tract are able to cleave the hemagglutinin of H3 influenza viruses.
In this study, we investigated the role of Tmprss4 in the context of influenza A virus replication and pathogenesis in experimentally infected mice. We showed that knockout of Tmprss4 alone did not protect mice from lethal H3N2 influenza A virus infections. In contrast, Tmprss2−/−
Tmprss4−/− double-knockout mice showed massively reduced viral spread and lung pathology and also had reduced body weight loss and mortality.
(Part of this work was performed as Ph.D. thesis work by Nora Kühn at the University of Veterinary Medicine, Hannover, Germany.)
Most of the well-known human viruses persist in the population for a relatively long time, and coevolution of the virus and its human host has resulted in an equilibrium characterized by coexistence, often in the absence of a measurable disease burden.
When pathogens cross a species barrier, however, the infection can be devastating, causing a high disease burden and mortality. In recent years, several outbreaks of infectious diseases in humans linked to such an initial zoonotic transmission (from animal to human host) have highlighted this problem. Factors related to our increasingly globalized society have contributed to the apparently increased transmission of pathogens from animals to humans over the past decades; these include changes in human factors such as increased mobility, demographic changes, and exploitation of the environment (for a review see Osterhaus and Kuiken et al.). Environmental factors also play a direct role, and many examples exist. The recently increased distribution of the arthropod (mosquito) vector Aedes aegypti, for example, has led to massive outbreaks of dengue fever in South America and Southeast Asia. Intense pig farming in areas where frugivorous bats are common is probably the direct cause of the introduction of Nipah virus into pig populations in Malaysia, with subsequent transmission to humans. Bats are an important reservoir for a plethora of zoonotic pathogens: two closely related paramyxoviruses—Hendra virus and Nipah virus—cause persistent infections in frugivorous bats and have spread to horses and pigs, respectively.
The similarity between human and nonhuman primates permits many viruses to cross the species barrier between different primate species. The introduction into humans of HIV-1 and HIV-2 (the lentiviruses that cause AIDS), as well as other primate viruses, such as monkeypox virus and Herpesvirus simiae, provide dramatic examples of this type of transmission. Other viruses, such as influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), may need multiple genetic changes to adapt successfully to humans as a new host species; these changes might include differential receptor usage, enhanced replication, evasion of innate and adaptive host immune defenses, and/or increased efficiency of transmission. Understanding the complex interactions between the invading pathogen on the one hand and the new host on the other as they progress toward a new host–pathogen equilibrium is a major challenge that differs substantially for each successful interspecies transmission and subsequent spread of the virus.
Influenza continues to pose a global health problem, as highlighted by the 2009 swine influenza pandemic and sporadic human infections with avian H5N1 influenza viruses. Antigenic changes in influenza virus, primarily in the surface antigens hemagglutinin (HA) and neuraminidase (NA), are referred to as antigenic shift (subtype changes by reassortment) and antigenic drift (mutation). This variability among influenza viruses hinders vaccination efforts. Currently, annual surveillance is necessary to identify circulating viral strains for use in vaccine production. New vaccines are often required, and take about 6 months to become available. Thus new approaches are needed.
In contrast, so-called “universal” vaccines targeting relatively conserved components of influenza virus can provide protection regardless of strain or subtype of virus, and may provide an alternative to the use of traditional vaccines. This immunity to conserved antigens would not necessarily prevent infection completely, but might decrease severity of disease, speed up viral clearance, and reduce morbidity and mortality during the initial stages of an outbreak until strain-matched vaccine became available. Furthermore, vaccines based on T cell immunity could be used in combination with a seasonal vaccine to improve efficacy, especially in the elderly who are at high risk of severe disease and show reduced responses to current flu vaccines.
Peptide scanning of T cell responses of healthy human individuals has shown that matrix 1 (M1) and nucleoprotein (NP) are among the prominent targets of CD8+ and CD4+ T cell cross-recognition, so they are of interest as vaccine candidates. By sequence homology, NP is >90% conserved among influenza A isolates. Both murine and human cytotoxic T lymphocytes induced by NP of one virus strain have been shown to cross-react with NP from different influenza A strains. The strong immune responses to NP in mice contribute to protection against challenge via CD8+ T cells,, as well as contributions from CD4+ cells, and antibodies–. The influenza A matrix (M) gene encodes two highly conserved proteins: an ion channel protein, M2, and the capsid protein, M1. M1 is not a major protective antigen in the mouse and is not well recognized by mouse T cells, but has long been known to be recognized by human T cells. Thus its potential contribution to vaccine protection may be underestimated by mouse studies.
While epitopes providing targets widely shared among influenza viruses have been identified in multiple viral proteins, not all of them are highly immunogenic when presented by classical vaccines. More potent immunization can be achieved using recombinant vectors to express the influenza antigens and focus immunity on these targets. Recombinant adenovirus vectors are especially effective at eliciting strong T cell responses to transgene products–. Recombinant adenovirus vectors expressing NP or both NP and M2, can protect mice against a range of influenza virus challenges, including highly pathogenic avian H5N1 strains. While potential interference by prior immunity to human adenoviruses has been suggested as a barrier, this issue can be circumvented by use of vectors based on animal adenoviruses–. Chimpanzee adenoviruses have been shown to be useful vaccine vectors in a variety of animal studies–, and the prevalence of neutralizing antibodies against chimpanzee adenoviruses is low in human populations–, but not all of them are equally immunogenic.
In this study, we use a simian adenovirus, PanAd3, isolated from the bonobo Pan paniscus. This novel adenovirus strain was identified in a study of more than 1000 adenoviruses isolated from chimpanzees and bonobos in order to increase the available repertoire of vectors. In the large scale screening experiments, PanAd3 was among the most potently immunogenic in mice and was also among the least frequently recognized by neutralizing antibodies in human sera.
We have generated a replication incompetent PanAd3 vector deleted of E1 and E3 regions and expressing a fusion protein of the NP and M1 antigens of influenza A, chosen as targets of broad and cross-reactive T cell immunity in humans. The PanAd3-based vaccine was tested for induction of antibody and T cell responses in the systemic and mucosal compartments in mice, as well as for protection against lethal influenza virus challenge. We demonstrate that PanAd3 expressing conserved influenza virus antigens provided highly effective protection after a single intranasal administration. Thus it shows considerable promise as a vaccine candidate.
activation site in a cat with feline infectious peritonitis-associated
Feline infectious peritonitis (FIP) is a fatal disease in cats caused by feline coronavirus (FCoV). FCoV is an enveloped, positive-stranded RNA virus belonging to genus Alphacoronavirus, family Coronaviridae, within the order Nidovirales. The genome size of FCoV is approximately 28.9 kb, including a non-structural replicase gene; four structural genes encoding the spike (S), envelope, membrane and nucleocapsid proteins; and five accessory genes 3abc and 7ab.
Feline coronaviruses cause mild to inapparent and transient infections of the gut and are ubiquitous in cat populations worldwide. They exist in two serotypes, I and II. Type I FCoV is predominant in the field, whereas type II virus represents only 2-30% of infection. Accumulating genetic evidence indicates that type II FCoV have arisen by two homologous recombinations between type I FCoV and canine CoV (CCoV). Both serotypes can mutate in the host to acquire macrophage tropism and cause a systemic disease known as feline infectious peritonitis. Due to a lack of virus-shedding in studies of FIP cats, the mutant FIP viruses (FIP causing FCoV, FIPV) are presumably contained only within the diseased tissues and not transmitted by cat-to-cat contact under natural circumstances.
In this paper, we report herein on an epizootic of FIP in a Taiwanese shelter that was caused by a novel type II FCoV. Epidemiological and molecular studies of isolates from various healthy and affected cats in this shelter strongly suggest that this virus was brought in by the introduction of kittens from another shelter with subsequent horizontal spread to co-housed adult cats.
Influenza A virus is an enveloped virus belonging to the Orthomyxoviridae family. It can cause annual epidemics and infrequent pandemics. The Spanish flu pandemic of 1918 as well as the Asian flu of 1957 and the Hongkong flu in 1968 pandemics caused the death of millions of people. In 2009 the pandemic swine origin influenza A H1N1 virus as well as the outbreak of H7N9 in China in 2013 has reminded the world of the threat of pandemic influenza [3–6].
The genome of influenza virus consists of eight segmented negative RNA strands. The envelope bilayer harbors the two spike glycoproteins hemagglutinin (HA) and neuraminidase (NA), and the M2 proton channel. The homotrimeric HA is the most abundant protein on the viral surface. It mediates attachment to the host cell surface via binding to sialic acid (SA) residues of cellular receptors, and upon endocytic virus uptake it triggers fusion of the envelope with the endosomal membrane releasing the viral genome into the cytoplasm. NA cleaves glycosidic bonds with terminal SA facilitating the release of budding virions from the cell.
In diagnostics, antibodies against spike proteins are the preferred tool for identification and serotyping of viruses. Development of therapeutic antibodies against influenza is a challenge, as the high viral mutation rate (antigenic drift) and genetic reassortment of the virus genome (antigenic shift) continuously lead to new strains escaping from neutralization by antibodies [7, 8]. This goes along with adaptation to small molecule inhibitors (e.g. oseltamivir).
Vaccines can only temporarily control the recurring epidemics of influenza, because antigenic changes are typical for HA and NA. 16 avian and 2 bat serotypes of influenza A virus HA (H1—H18) are known, but only three (H1, H2, and H3) have been adapted to humans. Antibodies binding to regions of hemagglutinin conserved among serotypes have been developed which demonstrated broad specificity and neutralization potency [10–15]. However, development, production and quality control of antibodies is expensive and time consuming.
As an alternative, short peptides binding specifically to the spike proteins can be produced in automated high-throughput synthesis at low costs. HA-binding peptides have been recently obtained by phage display, lead structure optimization of natural products and specific toxins, bioinformatics tools and discovery from side effects of known anti-inflammatory peptides [16–23]. Some of them showed antiviral activity [17, 19–23]. A more epitope-oriented accession to binding peptides is the search for paratope-derived peptides from variable regions of specific antibodies. Antibodies against HA have been described, and at least 6 antigenic sites (A-F) on the HA-trimer have been identified, localized either at the receptor binding site, the interface of the three HA-monomers, or at other sites like the stalk [8, 11, 25]. Several structures of HA–antibody complexes have been published deposited in the protein data bank (PDB) [11–14]. Indeed, an antibody was described, whose HA binding is mediated mainly by one CDR, namely HCDR3.
Inspired by this finding, we chose linear peptides corresponding to the CDRs of VH of monoclonal antibody HC19, having the majority of contacts with the HA1 domain of the strain A/Aichi/2/1968 [26, 27]. The antibody and the derived peptides bind to HA at the SA binding site, in particular to the 130-loop and the 190-helix, which belong to the antigenic sites A and B, respectively. This binding site is conserved among several HA serotypes providing a basis for a peptide with broader specificity.
We used complementary experimental and theoretical approaches to select HA binding VH-CDR peptides and to improve their potential to inhibit binding, and finally, infection of cells by influenza A virus. The inhibitory potential of the most efficient CDR-peptide was improved by microarray-based site-directed substitutions of amino acids. We could demonstrate a broader specificity of the selected peptides as they bound to HA of human and avian pathogenic influenza strains.
Zoonotic diseases are those infections that can be transmitted between animals and humans with or without vectors. There are approximately 1500 pathogens, which are known to infect humans and 61% of these cause zoonotic diseases (1). The unique dynamic interaction between the humans, animals, and pathogens, sharing the same environment should be considered within the “One Health” approach, which dates back to ancient times of Hippocrates (2, 3).
Bacterial zoonotic diseases can be transferred from animals to humans in many ways (4): (i) The transfer may occur through animal bites and scratches (5); (ii) zoonotic bacteria originating from food animals can reach people through direct fecal oral route, contaminated animal food products, improper food handling, and inadequate cooking (6–8); (iii) farmers and animal health workers (i.e., veterinarians) are at increased risk of exposure to certain zoonotic pathogens and they may catch zoonotic bacteria; they could also become carriers of the zoonotic bacteria that can be spread to other humans in the community (9); (iv) vectors, frequently arthropods, such as mosquitoes, ticks, fleas, and lice can actively or passively transmit bacterial zoonotic diseases to humans. (10); (v) soil and water recourses, which are contaminated with manure contains a great variety of zoonotic bacteria, creating a great risk for zoonotic bugs and immense pool of resistance genes that are available for transfer of bacteria that cause human diseases (11, 12).
Bacterial zoonotic infections are one of the zoonotic diseases, which can, in particular, re-emerge after they are considered to be eradicated or under control. The development of antimicrobial resistance due to over-/misuse of antibiotics is also a globally increasing public health problem. These diseases have a negative impact on travel, commerce, and economies worldwide. In most industrialized countries, antibiotic resistant zoonotic bacterial diseases are of particular importance for at-risk groups such as young, old, pregnant, and immune-compromised individuals (13).
Almost 100 years ago, prior to application of hygiene rules and discovery of neither vaccines nor antibiotics, some bacterial zoonotic diseases such as bovine tuberculosis, bubonic plague, and glanders caused millions of human deaths. The spread and importance of some bacterial zoonoses are currently globally increasing. That is precisely why most of the developing countries are sparing more resources for a better screening of animal products and bacterial reservoirs or vectors for an optimal preventative public health service (14).
Improvements in surveillance and diagnostics have caused increased recognition of emerging zoonotic diseases. Herein, changes in our lifestyles and closer contacts with animals have escalated or caused the re-emergence of some bacterial infections. Some studies lately have revealed that people have never been exposed to bacterial zoonotic infection risks as high as this before (15). It is probably due to closer contact with adopted small animals, which are accepted and treated as a family member in houses. On the other hand, more intensified animal farms, which have a crucial role in the food supply, are still one of the greatest sources of food-borne bacterial zoonotic pathogens in today’s growing world (4, 8).
People who have closer contact with large numbers of animals such as farmers, abattoir workers, zoo/pet-shop workers, and veterinarians are at a higher risk of contracting a zoonotic disease. Members of the wider community are also at risk from those zoonoses that can be transmitted by family pets.
The immune-suppressed people are especially at high risk for infection with zoonotic bacterial diseases. People can be either temporarily immuno-suppressed owing to pregnancy, infant age, or long-term immuno-suppressed as a result of cancer treatment or organ transplant, diabetes, alcoholism or an infectious disease (i.e., AIDS).
This manuscript reviews the most common bacterial zoonoses and practical control measures against them.
Industrialized livestock operations contain dense populations of animals and humans that are frequently in close contact. As these practices have become more prevalent, the risk of emerging zoonotic diseases has escalated [1–3]. This is evidenced by the 2009 Influenza A (H1N1) pandemic virus which contained genetic components from swine-reservoired viruses, and rapidly spread to at least 214 countries causing at least 151,700 human deaths within a cumulative 12 months of outbreak in each country. Southeast Asia, in particular, is considered a hot-spot for the generation of viruses that have zoonotic potential [5, 6]. This environment calls for increased surveillance for emerging diseases to help ensure that biosecurity measures are in place to protect humans, animals, and the environment. Hence, through this One Health pilot study set in Sarawak, Malaysia, we sought to conduct surveillance for respiratory and diarrheal pathogens that may potentially cross the species barrier between pigs and humans. More information on Sarawak can be found in the supplemental introduction (S1 Text). The primary aim of this study was to determine if viruses could be detected in bioaerosols collected at the animal-interface and to ascertain if these viruses were present in the nasal passages of humans working in these environments. Respiratory and swine pathogens with known or suspected transmission between pigs and humans were selected. More information on selection criteria can be found in the supplemental introduction (S1 Text). While finding molecular evidence of such animal viruses at the human-animal interface or in animal workers’ nasal passageways does not demonstrate active human infection, it does add to our understanding of the possible exposure risk. A secondary aim was to assess the understanding of zoonotic pathogens and their transmission to humans among animal workers, with the overall objective of aiding agriculture industries in controlling these pathogens in their livestock as well as informing cross-species infection prevention mechanisms.
The Orthomyxoviridae family member influenza A virus is the causal agent of acute respiratory tract infections suffered annually by 5–20% of the human population. There is a significant impact on morbidity, concentrated in people younger than 20 years, with economic consequences running into the billions of dollars during large epidemics. In addition, viral infections are associated with development of chronic asthma and disease exacerbation in both children and adults. In particular, acute influenza infection can amplify airway inflammation in asthmatic patients and induce alterations in epithelial and stromal cell physiology contributing to allergen sensitization, exaggerated bronchoconstriction, and remodeling of airway epithelia. Mortality rates associated with seasonal flu are low, but the aging population is at risk for development of severe congestive pneumonia which kills ∼35,000 people each year in the U.S.. Of continual concern is the threat of emergent high virulence strains such as the Spanish flu (H1N1), Asian flu (H2N2) and Hong Kong flu (H3N2) pandemics which claimed millions of lives world-wide.
Current treatments are focused on vaccines and drugs that target viral proteins. However, both of these approaches have limitations as vaccines require yearly development and lag detection of new strains, while viral proteins have a stunning capacity to evolve resistance to targeted agents. The genome of the influenza A virus consists of 8 negative single-strand RNA segments that encode 11 functional peptides necessary for viral replication and virulence. Thus the viral-autonomous repertoire of gene products is extremely limited and influenza A replication is dependent upon hijacking host-cell biological systems to facilitate viral entry, replication, assembly, and budding. The recognition that a suit of human host proteins are required for IVA infection and replication presents additional targeting strategies that may be less prone to deflection by the highly plastic viral genome.
Here we have employed the cytopathic effects of H1N1 infection in bronchial epithelial cells as a mechanism to isolate host genes that represent intervention target opportunities by virtue of their contribution to H1N1 infection and replication, or by virtue of their contribution to viral virulence factor-dependent evasion of innate immune responses. A primary whole-genome arrayed siRNA screen identified gene depletions that either deflected or promoted bronchial epithelial cell death upon exposure to the H1N1 A/WSN/33 influenza virus and were not cytotoxic to mock infected cells. Integration with orthogonal data sets, describing host gene function–, parsed collective ‘targets’ into four functional classes. 1) Targets that, when depleted, enhance bronchial epithelial cell survival upon H1N1 exposure, and are required for viral replication. This class presumably represents host factors that facilitate viral infection and/or are required to support viral replication. 2) Targets that, when depleted, reduce bronchial epithelial cell survival upon H1N1 exposure, and are required for viral replication. This important and initially unanticipated class, likely represents proviral host factors that deflect cell death checkpoint responses that would otherwise engage upon detection of viral infection. 3) Targets that, when depleted, reduce bronchial epithelial cell survival upon H1N1 exposure and enhance viral replication relative to controls. Recently discovered innate immune pathway components, such as IFITM3 that are responsive to H1N1 infection, are members of this class, which presumably represent antiviral restriction factors that normally oppose infection. 4) Targets, that when depleted, enhance bronchial epithelial cell survival upon H1N1 exposure and enhance viral replication as compared to controls. These host factors are likely responsible for influenza virus-mediated cytopathic effects. Chemical inhibition of gene products from two classes, RABGGTASE and CHEK1, indicated these targets might be pharmacologically addressable for H1N1 intervention in an epithelial cell autonomous context.
A number of different poxviruses can infect both people and domestic animals; with cowpox being the best described and most commonly encountered poxvirus infection of cats. Cowpox virus (CPxV) is a member of Orthopoxviridae family and is endemic in Northern Europe and western areas of the Soviet Union.1
The usual route of infection is via skin inoculation from infected rodent bites, typically voles, or rarely via oronasal infection.2 Reflective of this transmission route, skin lesions are commonly found on the head, neck and forelimbs. However, systemic illness such as pyrexia, anorexia, lethargy and/or pneumonia can occur during the viraemic phase and is usually associated with immune dysfunction and death.3,4
It is important that clinicians recognise the signs of potential CPxV infection and perform appropriate diagnostics early. A significant factor contributing to the prognosis of CPxV infection in cats is the speed at which appropriate therapy is instituted, so rapid recognition is crucial. Cytological analysis of the affected organs can be misleading, with the secondarily dysplastic cells having the potential to mimic neoplasma. Instead, electron microscopy, virus isolation and PCR of tissues (including skin scabs, bronchoalveolar or pleural fluid, or pulmonary aspirates) are the easiest ways to confirm the diagnosis.5 Serum assays (including virus neutralisation, haemagglutination inhibition, complement fixation and ELISA) can be utilised to detect a humoral response to orthopoxvirus, although a rising titre is required to support active infection.4
Treatment includes broad-spectrum antibiotics to control secondary bacterial infection and recombinant feline interferon omega (rFeIFN-ω) to modulate the immune response. Antiviral drugs, such as famciclovir, commonly used for the treatment of feline herpesvirus (FeHV-1) disease in cats,6 could also be considered; however, their efficacy against CPxV is unknown.
Our previous case series detailed cats that had pulmonary CPxV-infection, complicated by concomitant infection with FeHV-1, Bordetella bronchiseptica and/or Mycoplasma species.4 The cases described herein have novel findings which have not previously been described in relation to this infection, including central nervous system (CNS) involvement and presentation as a laryngeal mass.
Ageing is often characterised by a progressive decline in immune response, resulting in increased susceptibility to infections and poor response to vaccination. Clinical observations suggest that lower respiratory tract infections may contribute to the pathogenesis of asthma and are important triggers for asthma exacerbations. Asthma in the elderly seems to be a distinct phenotype with higher severity and exacerbation rates, which may be associated with the deterioration of immune function observed in the airways in the aged population.
The airway microbiome might have a profound role on the development, persistence, and clinical course of chronic inflammatory diseases like asthma and chronic obstructive pulmonary disease. The presence of respiratory viruses during childhood has been identified as a significant risk factor for asthma in adolescences and adults, while similar observations have been made for atypical bacteria. Viruses, such as rhinovirus, respiratory syncytial virus, or parainfluenza viruses, are the most common causative factors for asthma exacerbation. In clinically stable periods of asthma, both viruses and bacteria are detected in the airways [9, 10], but the significance of the persistent presence of those pathogens in the airways remains unclear. While the majority of studies indicate that the detection rate of respiratory viruses in patients with asthma and healthy subjects is similar, they have been found to differ with regard to the bacterial composition of the airways.
Information on the respiratory pathogens of the lower airways and their relationship with the nature of asthma in elderly asthmatics is limited, mostly because of the risk and inconvenience associated with direct and invasive methods of airway lumen sampling. Induced sputum (IS) allows for non-invasive assessment of lower airway inflammation and can be employed to detect respiratory pathogens in the lower respiratory tract. This technique was chosen for the present study, to test the hypothesis that elderly patients with asthma display a different profile of respiratory pathogens in the airways as compared to non-elderly asthmatics, and that this profile may be related to local airway and/or systemic inflammation.
This case report describes a young cat with neurologic FIP in which detailed clinical
and molecular characterization of the associated FCoV infection was performed. While
the etiology of FIP remains complex and likely involves multiple mutations in the
viral genome, our results indicate that a specific mutation of the viral spike
protein can be associated with infection of the CNS, which may explain the tropism
to the CNS as opposed to other organ systems.
The pandemic H1N1 outbreak of 2009 and global threat of H5N1 in recent years have been accompanied by a large amount of laboratory-based experimental research activity using purified viruses and infected tissues and animals. The majority of these studies have focused on either natural infections from the community or induced infections in the laboratory, with very few studies considering the infection risk or outcome of laboratory personnel handling the samples. The potential of an accidental laboratory-acquired infection is well-recognized among laboratory staff and researchers. In 1941, Meyer and Eddie published the first report of laboratory infections due to the Gram-negative bacteria Brucella. In 1949, Sulkin and Pike published a report that summarized 222 laboratory-acquired infections due to viruses. Since then, significant efforts have been made by the oversight committees of research institutes and governmental bodies to establish occupational and environmental safety guidelines to protect workers and the local community alike from laboratory-acquired infections; however, these infections have yet to be eradicated and many have been reported over the past eight decades.
The total number and relative frequency of bacterial laboratory-acquired infections has, in fact, declined dramatically over time. In contrast, the relative frequency of viral laboratory-acquired infections has increased by 60%. Research into the underlying factors responsible for these infections have indicated that the main route of infection, for both bacteria and viruses, is through mucous membranes that are contaminated by inhaling pathogens, not dissimilar from the natural route of infection. Traditionally, the risk of laboratory infection has been minimized by simply practicing good laboratory practice (GLP), which is otherwise necessary for reliability of the laboratory work itself. A detailed examination of the publically available information on all reported laboratory-acquired infections, however, indicated that ~80% were not the result of overt "accidents"; it is, thus, likely that inhalation of aerosolized infectious particles that are liberated by normal laboratory techniques account for a large portion of laboratory-acquired infections.
Research into this theory has indicated that influenza virus transmission and infection can be achieved through aerosols. Many of the routine procedures used to process influenza virus for laboratory research, such as centrifugation or mixing, have a high potential of producing aerosols, and the particle load of each has been estimated to be up to 1–5 μm. In addition, it is expected that larger particles will tend to fall out of the air and contaminate surfaces, by which individuals may be infected by contact or may transmit the particles to a secondary aerosol. Fundamentally, aerosols are suspensions in the air of solid or liquid particles small enough that they will remain transmissible and airborne for a prolonged period of time. Particles of 5 μm or less increase the risk of establishing an infection upon airborne transmission, as they are remarkably capable of penetrating the physical cellular barrier of the respiratory tract and traveling all the way to the alveolar region. As with naturally-acquired infections, most individuals are not diagnosed before onset of symptoms, impeding the time to initiation of treatment.
The A type influenza viruses are commonly spread by the airborne route in normal circumstances. Accordingly, more research on the potential and character of aerosol spread of influenza virus has been carried out, and many studies have used experimental animal models of aerosol infection to mimic the natural process. However, less information is available on the features of laboratory-produced aerosolized influenza virus.
The Australian researcher, Adrian Gibbs, suggested that the A/H1N1 flu virus currently circulating around the world was created in a laboratory. Although the World Health Organization (WHO) eventually dismissed this theory, suspicion and panic were aroused in the general public about laboratory safety. There are many situations that may facilitate the spread of a pathogen from the laboratory, ranging from aerosols produced by routine procedures or misuse of laboratory equipment to uncontrollable natural disasters that impact the structural integrity of the laboratory, such as earthquake or fire. In order to regulate the potential of pathogen transmission from the laboratory, we must first gain a detailed understanding of the experimental operations that produce aerosols. To this end, this study was designed to monitor the presence of aerosolized H5N1 virus produced by normal procedures used to process the virus for experimental research and by the most frequently associated “accidents” for each, such as container breakage and accidental subcutaneous injection. This information will help to guide future experimental practice standards to ensure the safety of laboratories and laboratory personnel, thereby increasing community confidence in laboratory bio-safety.
Feline calicivirus (FCV) is a RNA virus that occurs worldwide in domestic cats and exotic felids [1, 2]. FCV infections are commonly associated with oral ulcerations and salivation [3, 4]. Other clinical syndromes that have been attributed to FCV infection include chronic stomatitis [3, 5] and a limping syndrome [6–8]. Some years ago, highly virulent systemic FCV infections associated with fatal disease have been reported in several countries, initially in North America [9, 10] and subsequently in Europe [11–13]. Outbreaks of severe FCV infections associated with edema and skin ulcerations have also been described in cats in Switzerland (Willi et al., submitted for publication).
FCV has also been assigned to the upper respiratory tract disease (URTD) complex (‘cat flu’) [14–17]. In addition to FCV, at least four other pathogens have been shown to be associated with this syndrome, including feline herpesvirus type 1 (FHV-1), Mycoplasma felis, Chlamydophila felis and Bordetella bronchiseptica [14, 16, 18–24]. Cats with URTD are commonly presented to veterinary practitioners and show symptoms such as lethargy, pyrexia, anorexia, sneezing, nasal discharge, ocular discharge, conjunctivitis and keratitis [14, 19].
Conventional, nested and real-time reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays have been developed to amplify FCV-specific RNA from clinical specimens [25–29]. The high genetic variability of FCV can hamper the diagnostic sensitivity of FCV-specific molecular assays. Virus isolation has been advocated as an alternative diagnostic tool, but this method is not routinely available. Virus isolation is less sensitive to genomic variation, but the method might fail due to virus inactivation during transport, thus resulting in reduced diagnostic sensitivity.
FCV vaccines have been shown to be efficacious in protecting cats from the development of severe disease; however, they do not induce sterilizing immunity [30, 31]. Moreover, most commercially available vaccines have been based on only a few FCV strains (i.e. FCV F9 and 255) for many decades. Therefore, the protective potential of FCV vaccines against different circulating FCV isolates has been controversially discussed in recent years [32–35]. Efforts are made for the identification of new vaccine strains with broader cross-reactivity, the development of bi- or polyvalent vaccines and the inclusion of local FCV isolates [35–38]. Along these lines, a vaccine containing two novel FCV strains has become commercially available in Switzerland (FCV G1 and 431) [35, 36].
Despite the introduction of FCV vaccines several decades ago, cats with FCV-related symptoms are still commonly presented in veterinary practices. A presumptive clinical diagnosis relies on the presence of FCV-related symptoms. However, clinical signs induced by FCV and other URTD-associated pathogens can overlap and co-infections are common [4, 14–17]. Molecular assays to detect URTD-associated pathogens in clinical specimens might aid the diagnosis. Because asymptomatic carriers have been reported, results of molecular assays must be interpreted together with clinical presentation.
The aims of this study were to investigate the frequency of FCV in cats suspected FCV infected by veterinary practitioners and in clinically healthy cats in Switzerland, and to address potential risk and protective factors for infection in both groups of cats, such as signalment, housing conditions, vaccination, and co-infection with URTD-associated pathogens. Furthermore, the association between a number of different clinical symptoms and FCV infection was assessed.