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Deep Learning Technology: Sebastian Arnold, Betty van Aken, Paul Grundmann, Felix A. Gers and Alexander Löser. Learning Contextualized Document Representations for Healthcare Answer Retrieval. The Web Conference 2020 (WWW'20)
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Disease incidence for any given island was compared to the average incidence for the whole country, using the z-score test for two population proportions available at https://www.socscistatistics.com/tests/ztest/default2.aspx. P values < 0.05 were considered statistically significant.
Prior to testing samples, a small study was performed to determine if alternative samples types such as mouth swabs or urine samples from patients suspected of DENV infection could be used as a less invasive alternative sample type. Four matched urine, plasma and mouth swab samples were taken and these extracted using standard procedures as described. Although DENV RNA could be detected in all three sample types, a significant Ct lag was observed for urine and mouth swab compared to plasma (S1 Fig). The data indicated there was approximately 100,000 fold more virus in serum/plasma samples compared to other matrix, further demonstrating that serum/plasma are the preferred test sample types. These data also agree with a previous study using viral RNA and NS1 antigen assays.
A total of 187 samples were tested (Table 5) during the 2016–2017 Vanuatu dengue-2 outbreak. The original testing algorithm consisted of screening each sample for pan-flavivirus, pan-alphavirus and pan-dengue only. During the first day of testing, pan-flavivirus/pan-dengue positive samples were subsequently typed with dengue typing primers that showed the serotype responsible for the current outbreak was DENV-2. In order to provide a more rapid diagnostic, DENV-2 primers were included at the request of the hospital in the initial screening assay to negate the need to perform subsequent reflex testing on the samples but still having the ability to detect new viruses if they emerged.
No molecular tests were available at Port Vila Central hospital for the detection of dengue virus. Thus samples previously tested for NS1 antigen/IgG and IgM were obtained from patients in the months December 2016 to February 2017. From March 2017 patients attending the hospital with suspected dengue infection were screened with the pan-flavivirus/pan-alphavirus/pan-dengue/dengue-2 assays. Fig 5 shows the positivity by week from December 2016 to March 24th 2017. Molecular testing was carried out between the 12th to the 24th of March.
Fig 6 shows the number of dengue infections by age and the age of the general population. As can be seen from the figure the number of dengue positive samples mirrors the age of the population.
Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been one of the most important and widely used techniques for the identification and characterization of new viruses. Two TEM techniques are usually used for this purpose: negative staining on an electron microscopic grid coated with a support film and (ultra) thin section TEM of infected cells, fixed, pelleted, dehydrated, and embedded in epoxy plastic. Negative staining can be conducted on highly concentrated suspensions of purified virus or cell culture supernatants. For some viruses, TEM can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or concentrated stool material (rotaviruses and noroviruses). For successful detection of viruses in ultrathin sections of infected cells, at least 70% of cells must be infected, and so either high multiplicity of infection (MOI) or rapid virus multiplication is required.
Viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location or, for some viruses, from the ultrastructural cytopathology and specific structures forming in the host cell during virus replication. Usually, ultrastructural characteristics are sufficient for the identification of a virus at the level of a family. In certain cases, confirmation can be obtained by immuno-EM performed either on virus suspension before negative staining or on ultrathin sections. This requires virus-specific primary antibodies, which might be not available in the case of a novel virus. For on-section immuno-EM, OsO4 post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic (usually LR White). The ultrastructure of most common viruses is well documented in good atlases and book chapters and many classical publications of the 1960s, 1970s, and 1980s. Several excellent reviews were recently published on the use of TEM in the detection and identification of viruses.
The diagnostic sensitivity and specificity of the CHIKV RT-RPA assay was assessed with plasma samples from 58 suspect CF cases and compared to two CHIKV specific real-time RT-PCR tests during a field trial in Bangkok, Thailand. Both CHIKV real-time RT-PCR detected 36 out of 58 sample (62%) positive. The Ct values obtained by real-time RT-PCR for positive samples ranged from 20.19 to 36.02 (1.6x104 -1x108 GC/rxn). In comparison to real-time RT-PCR, RT-RPA correctly identified all 36 positive samples and did not detect the 22 negative samples with 100% sensitivity and specificity (PPV: 1, NPV: 1, Fig 4). Additionally, we tested 20 sera from acute CHIK patients from France. All three methods efficiently detected 20 out of 20 CHIKV positive samples.
The sensitivity of the RT-RPA assay was assessed by testing 18 CHIKV strains representing all three genotype (Table 1), the specificity was tested with eleven different alphaviruses, ten flaviviruses and one phlebovirus (Table 2). The CHIKV RT-RPA assay utilizing RF+RR3 efficiently detected all 18 CHIKV strains and the O'nyong'nyong virus (ONNV), while RT-RPA assay using RF2+RR2 amplified only the CHIKV strains. There was no cross reactivity found to other viruses of the cross reactivity panel (Table 2 and S2 Fig).
Using specifically designed RT-PCR primers, we detected viral RNA in 223 of the 285 acute serum samples tested (Table 5). The specificity of the RT-PCR was confirmed by sequencing selected PCR products. None of the 80 sera from patients with respiratory diseases or the 50 sera from healthy subjects was positive using the novel virus-specific RT-PCR.
The concept of viruses developed from the observations of Ivanovsky and Beijerinck of “filterable agents”, with the discovery of the causative agent of tobacco mosaic in the 1890s. Yellow fever virus and dengue virus were the first two arboviruses to be isolated early in the 20th century. The pioneering work of Alexis Carrel on the development of many cell and tissue culture methods in the 1910s at the Rockefeller Institute, and later refinements by Maitland, Eagle, and Enders, led to the widespread use of various culture systems as indispensable tools for virus studies. Although these tools have since been used extensively for the in vitro characterization of viruses, they were inadequate for the identification and classification of a flood of novel viruses collected through the YFV surveillance program supported by the Rockefeller Foundation. Jordi Casals, among others, led the use of the complement fixation (CF) test in order to study viruses affecting the central nervous system. The CF test exploits the unique affinity of complement for antigen–antibody complexes. The original assay was developed in the 1920s for the serologic study of YFV, was improved in the 1930s, and its sensitivity and specificity improved again in the 1950s. Using CF tests, Casals and his colleagues were able to classify viruses into antigenic groups. However, the inherent complexity and labour consuming aspects of the assay (titrations of antigen, complement, and hemolysin for optimal outcomes), technical demands (accurate interpretation of outcomes) and the development of alternative assays (see below), have restricted its applicability in laboratories worldwide.
Hirst observed in 1941 that chicken erythrocytes agglutinated in the presence of the influenza A virus and that virus-specific antibodies inhibited agglutination, forming the foundation for the hemagglutination inhibition (HI) test. A decade later and on Theiler’s suggestion, Casals showed that many arboviruses also agglutinated erythrocytes, establishing the HI test as a diagnostic tool for arbovirus infection and identification. The gold standard for arbovirus identification, the plaque reduction neutralization test (PRNT), has its origins in the observations of Stokes and colleagues, dating back in the 1920s when monkeys could be protected against YFV by the inoculation of convalescent sera from patients who had recovered from the disease. By the early 1930s, Max Theiler had adapted the assay for use in mice, in which mixed serum and virus was inoculated intracerebrally. The cell culture adaptation of the test was first demonstrated by Itoh and Melnick in 1957 for studying the seroconversion of Chimpanzees infected with echoviruses, and a year later by Henderson and Taylor to detect antibodies to the eastern equine encephalitis virus. Versions of this assay are now widely used, including the microPRNT, the virus reduction neutralization test (VRNT), the focus reduction neutralization test (FRNT), the rapid fluorescent inhibition test (RFFIT), the flow-cytometry neutralization, the colorimetric micro-neutralization assay (CmNt), and the reporter virus particle-based neutralization assays.
Historically, these methods (virus isolation, HI, CF, and neutralization tests) served as the basis of arbovirus diagnosis for many years, augmenting electron microscopy (see section below), which allowed the visualization of viruses in infected tissues and cell cultures. However, an inherent limitation of the serology-based assays has been their inability to determine whether antibodies in the examined serum were the result of a recent or past infection. This conundrum was solved by determining whether antibodies were IgM (recent infection) or IgG (past infection), using the enzyme-linked immunosorbent assay (ELISA). The introduction of ELISA revolutionized the field by also offering increased specificity and sensitivity for the accurate detection of many viruses.
Overall, although identification of pathologic agents through serologic assays is quite straightforward, there are instances where accurate identification may not be possible due to cross-reactivity. For example, cross-reactivity among flaviviruses poses a challenge in their identification, even when the “gold standard” of PRNT for arbovirus detection is applied, especially in hyper-endemic settings of flavivirus circulation. Several diagnostic labs faced this challenge during the recent emergence and explosive spread of the Zika virus in the Americas. A similar challenge is also common for the serologic diagnosis of bunyavirus infections, which is attributed to their ability to reassort. In this scenario, a novel bunyavirus may be misidentified as a known pathogen due to the presence of the M segment (contributed by the known pathogen), which encodes the immune-reactive envelope proteins (reviewed in).
IgG antibodies to the novel bunyavirus were detected in 80 of 285 acute-phase serum samples from patients with FTLS (Table 5). Of 95 patients from whom paired acute- and convalescent-phase sera were available, 52 had seroconversions and 21 had greater than 4-fold increases in antibody titer to the virus. Six had less than a 4-fold increase in antibody titer to the virus, but all paired sera tested positive. Sixteen patients tested negative to the virus, suggesting that some non-FTLS patients with similar symptoms were included in this study, a situation that is not surprising given that FTLS is a newly emerging disease. The acute-phase sera of four patients from whom the virus was isolated tested negative for IgG antibody to the virus. All convalescent sera obtained 2 months later from the same four patients contained IgG antibody to the virus. None of the 130 sera from patients with respiratory diseases or healthy subjects had detectable antibody.
Total RNA was extracted from both plasma specimens and spent media, and tested by real-time RT-PCR (rtRT-PCR) following published protocols for CHIKV vRNA for confirmation of previous tests performed in Haiti. RNA extracted from plasma specimens were then screened for DENV serotypes 1–4 and ZIKV vRNAS by rtRT-PCR. Cycle threshold (Ct)-values under 38 were considered positive. Viral genomic RNA that was extracted from CHIKV, DENV1-4, and ZIKV strains that were obtained from the Biodefense and Emerging Infections Research Resource Repository (BEI Resources, Manassas, VA) were used as positive control materials for rtRT-PCR. Cell cultures were also tested for the presence of DENV and ZIKV vRNAs by rtRT-PCR, even if the corresponding plasma specimen tested negative. Additionally, spent media from cultures displaying non-CHIKV CPE, that were DENV and ZIKV negative by rtRT-PCR, were screened with a duplex RT-PCR for the vRNAs of other alphaviruses (Venezuelan equine encephalitis -, Eastern equine encephalitis -, Western equine encephalitis -, Aura—and Mayaro viruses) and flaviviruses (Yellow fever -, Saint Louis encephalitis -, Bussaquara -, Ilheus -, and Rocio viruses). The DENV-1 strain from BEI and the 2015 MAYV sample from our laboratory were used as the flavivirus and alphavirus positive controls, respectively, in the duplex RT-PCR protocol.
Other candidate dengue vaccines have been developed in USA by the Johns Hopkins University and National Institute of Allergy and Infectious Diseases (NIAID) and have reached advanced clinical trials. Four live-attenuated DENV/delta-30 were generated each containing 30 nucleotides deletion of the 3’-untranslated region of genomic RNA (delta-30). These vaccines efficiently impaired viral growth in human liver carcinoma cells. To improve the attenuation of DENV-2/delta-30 and DENV-3/delta-30, chimeric DENV were developed by substitution of the prM-E gene region of DENV-4/delta-30 virus with the prM-E genes of DENV-2 and DENV-3 [72, 77]. The results from phase I clinical trial showed that all four live-attenuated DENV/delta-30 are safe and immunogenic with minor side effects such as faint rash and transient leucopenia only after higher dose [78, 79].
Sera were tested for hantavirus IgG and IgM antibodies using the recomLine hantavirus assay (Mikrogen, Munich, Germany), coated with specific hantavirus nucleocapsid antigens of Puumala virus, Dobrava-Belgrade virus, Hantaan virus and Seoul virus. Patients were included in the study if both, hantavirus IgG and IgM were positive. Positive results of IgG and negative results of IgM for hantavirus may also indicate an infection with mild clinical symptoms. However, for these patients with positive IgG and negative IgM for hantavirus, reference samples were available indicating no acute but past hantavirus infection. Consequently, these patients had been excluded from the study. PCR for the detection of hantavirus RNA was performed on the L-segment. For phylogenetic analysis positive samples were subsequently sequenced in the S-segment. Characteristics of the sequence analysis are given in the legend to the figure.
Whole genome sequence data from 10 of the CHIKV samples (7 from children with co-infections, 3 from selected randomly mono-infections) were obtained by Sanger sequencing and a primer-walking approach, as previously described. Similarly, we designed sequencing primers for MAYV and ZIKV that also amplify approximately 800bp overlapping segments, and used a primer walking method for whole genome sequencing of those viruses. For the sequencing of DENV, primers described by Christenbury et al were utilized. Amplification of each segment was performed using an Accuscript high-fidelity first-strand cDNA synthesis kit (Agilent Technologies, Santa Clara, CA) followed by PCR with Phusion polymerase (New England Biolabs, Ipswich, MA). The 5’ and 3’ ends of the viral genomes were obtained using RNA-ligase mediated (RLM) systems for 5’ and 3’ Rapid Amplification of cDNA Ends (RACE) per the manufacturer’s protocols (Life Technologies, Carlsbad, CA). Amplicons were purified, sequenced bi-directionally, then the sequences assembled with the use of Sequencher DNA sequence analysis software v2.1 (Gene Codes, Ann Arbor, MI), and subsequently analyzed in comparison to DENV, MAYV, and ZIKV sequences available in GenBank for nucleotide sequence comparisons. The vRNA sequences we obtained differed from those of the corresponding viruses in our repository, confirming the newly analyzed sequences did not arise from laboratory contamination.
Till-date, there is no effective, commercially available, therapy/vaccine for dengue virus. Numerous groups have already made intensive efforts and made good progress to develop a safe, affordable and effective vaccine against all serotypes for global public health [63–69]. Vaccines which are being developed use various approaches such as live attenuated viruses, inactivated viruses, subunit vaccines, DNA vaccines, and chimeric viruses using yellow fever vaccine and attenuated dengue viruses as backbones (Table 2).
In this retrospective study, we analyzed clinical and laboratory data of 77 hospitalised patients diagnosed with acute hantavirus infection at the University Hospital Heidelberg. Glomerular filtration rate (GFR) provides the best index of overall kidney function and creatinine concentration is the most widely used parameter for estimation of GFR. Based on GFR, we divided patients into two groups: patients with mild disease (GFR ≥ 30 ml/min) and patients with severe disease (GFR < 30 ml/min). The first blood sample of each patient was obtained on admission to the hospital, on average 7 days after onset of symptoms. An additional blood sample was obtained one week after admission. All samples were analysed for cytokine and lymphocyte, monocyte/macrophage activating and inflammatory marker expression including transforming growth factor (TGF)-β1, −β2, −β3, sCD30 and neopterin levels. The mean age of patients was 40.8 years, 52 were male (68%), five (6%) were under the age of 18 years.
FCV, FHV-1, or both were detected in 55 from the 302 cats examined in this survey (Tables 1 and 2). FCV alone was isolated in 52.7% (29/55) of the cats that tested positively, FHV-1 alone in 38.2% (21/55) and double infection was detected in 9.1% (5/55) (Table 2). Virus isolation was confirmed in all cases by PCR and RT-PCR for FHV-1 and FCV, respectively.
The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses and animal dietary viruses can also be found in human stool, as well as bacteriophages and viruses infecting protozoa. Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine, and rhinoviruses, bocaviruses and coronaviruses originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus (HIV)-1 can also be detected in fecal samples. Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells.
Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen. His postulates can be summarized in three points: (1) The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; (2) the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and (3), after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that “the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered”. For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured, and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described, reviewed in the literature. Although attempts have been made to adjust the Koch’s postulates specifically for viruses and the current methodologies deployed, fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature.
Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture. Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) which uses ~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates. However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified.
The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs (RT-PCR), in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease?
In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies.
The feline kidney cell line CRFK (Crandell-Rees feline kidney) was used for virus isolation and amplification. Cells were routinely maintained in Eagle's minimal essential medium (MEM) containing penicillin (1.6 mg/L), streptomycin (0.4 mg/L), amphotericin B (2.0 mg/L), and 10% fetal calf serum.
Swabs were kept in microtubes with MEM medium (0.5 ml) and stored at - 70°C until use in experiments. The swabs were briefly agitated in a vortex and the content was then transferred to microcentrifuge tubes and centrifuged at 10.000 x g for 5 min. The supernatants (0.15 ml) were inoculated onto CRFK cell monolayers grown in 24-well plates and were submitted to three passages of five days each while the cells were monitored for cytophatic effect (CPE). Cultures exhibiting CPE were investigated for the presence of feline calicivirus (FCV), feline herpesvirus type 1 (FHV-1), or both viruses using a polymerase chain reaction (PCR) assay. Three blind passages were performed for cultures not exhibiting CPE, and the cultures were considered negative for virus isolation.
To date, four MPXV small animal models have been used for the testing of antiviral drugs Cidofovir, CMX001 and ST246 (tecovirimat). Herein we will summarize those studies, efficacy data, and discuss the advantages, and limitations, of the animal models used.
Sbrana et al. utilized ground squirrels to test the efficacy of ST-246 against a MPXV challenge. The authors used 100 pfu of MPX-ZAI-1970 (200 × LD50) via a subcutaneous route of inoculation. Squirrels (8–9 per group) were divided into five treatment groups; drug was given either at 0 hours of infection, 24 hours, 48 hours, 72 hours or 96 hours p.i. 100 mg/kg of drug was given once a day for 14 days. Two animals in each group were sacrificed at day 7 to measure objective morbidity; the remainder of the animals were used to calculate survival rates. Animals in the placebo group, that were not given ST-246, showed signs of illness beginning on day 4 and all died between days 6–9. Signs of disease included lethargy, anorexia, nosebleeds, and terminal respiratory distress. At day 7, a sampling of placebo-treated animals exhibited significant leukocytosis, transaminitis, and coagulopathy; almost 105 pfu/mL of infectious monkeypox was found in blood; at this time, between 107 and 108 pfu /mL of infectious MPXV was observed in 10% organ homogenates of liver, spleen and lung. Animals treated on days 0, 24, 48 or 72 hours, before symptomatic disease onset, all survived infection and showed no signs of disease. At day 7, in a sampling of animals treated at hour 0, 24, 48 or 72 p.i., no virus was found in the liver, spleen, lung, or blood; although some abnormal values were apparently recorded, no clear trends in leukocytosis, transaminitis or coagulopathy were noted with delay in treatment onset. In animals initiating treatment at 96 hours p.i., concurrent with symptomatic disease onset, 67% of animals survived infection. 2/4 survivors showed signs of disease. In those animals that succumbed to infection, ST-246 prolonged the time to death; the mean time to death was day 7 for animals receiving placebo and day 13 for those receiving ST-246 in the 96 hour p.i. treatment group. The sampling of animals at day 7, initiating ST-246 at 96 hour p.i., demonstrated lower levels of viremia (∼3 log decrease) and ∼5 logs less virus in liver, spleen and lungs than that seen on the placebo treated animals at day 7. Although some evidence of transaminitis was present, leukocytosis and coagulopathy were not observed in this treatment group. Pathologic examination of tissues in general showed greater tissue necrosis in animals treated at later times p.i. This study was able to demonstrate a survival benefit in animals treated prior to, or at the onset of disease symptoms, in a disease model that has a time course attenuated with respect to what is seen in human disease.
Schultz et al. infected African dormice with a lethal challenge of Congo Basin clade virus MPXV-ZAI-79 via an IN route of infection to evaluate the efficacy of Cidofovir as post exposure prophylaxis. Four hours post intranasal infection with 75, 4 × 103, or 5 × 103 pfu of MPXV, animals were intraperitoneally administered 100 mg/kg cidofovir (the calculated LD50 for the dormouse MPXV model was 12 pfu). Aggregate data from all challenges showed animals treated with cidofovir had a mortality rate of 19% (7/36), whereas vehicle treated animals all (41/41) succumbed to disease. Treatment initiation at later times p.i. was not evaluated; effects on viral load or histopathologic changes were not reported.
As inbred mice have historically shown little disease symptomatology or pathogenesis post monkeypox infection, Stabenow et al. utilized a laboratory mouse strain lacking STAT1 (C57BL/6stat-/-), which has been found to be sensitive to a range of viruses including SARS, murine norovirus 1, respiratory viruses, dengue virus and MPXV [19,22–25]. These animals are deficient in their ability to transcribe many of the Type I and Type II receptor interferon response genes. The authors used the Congo Basin clade virus MPX-ZAI-79, evaluated disease and the protective efficacy of CMX001 and ST246. In untreated mice, 0% mortality was observed with 4.7 pfu challenge, 90% mortality with 470 pfu of virus and 100% mortality with 4,700 pfu. Over 25% total body weight loss, and mortality was observed on or prior to day 10 p.i. in untreated animals. Animals in the treatment studies were subsequently challenged with 5,000 pfu via an IN infection. Animals were then treated with 10 mg/kg of CMX001 by gastric gavage on the day of challenge followed by every other day with 2.5 mg/kg until day 14 p.i. All C57BL/6 stat-/- mice that were treated with drug survived infection, demonstrated <10% body weight loss between days 10 and 20, and developed a serologic response to monkeypox. Similarly, mice treated daily, starting at the day of virus challenge, with 100mg/kg of ST246 for 10 days also survived infection and manifest <10% body weight loss between days 10 and 20. In this system, antiviral treated animals rechallenged with monkeypox at day 38 post initial infection (at least 10 days post reinitiation of steady weight gain), manifest 20% mortality. The model—again one with a short disease course—is useful for demonstrating immediate post exposure efficacy of antiviral treatment in the absence of a functioning interferon response system. Additionally, in this animal model system, perhaps due to the immune defect, a monkeypox protective immune response was not elicited in all animals receiving antiviral treatment. This observation merits further observation in other animal model systems.
Smith et al. tested the efficacy of ST246 in a prairie dog MPXV model. MPXV challenged prairie dogs have previously been shown to have an asymptomatic period followed by symptoms of disease including lethargy, nasal discharge, inappetence, weight loss and systemic lesion development most commonly between days 9–12. In the current study, animals were inoculated via an IN challenge with the Congo Basin clade virus ROC-2003-358. This is a different strain of MPXV than that used in the previous described studies, but is also a strain belonging to the Congo Basin clade. The challenge dose was 3.8 × 105, equal to 65 × LD50 for the prairie dog model. Animals were divided into three treatment groups; prophylactic (day 0), post exposure (day 3) and therapeutic (varying day based on rash onset), and a control vehicle treated group. ST246 was formulated at 30 mg/mL and administered daily, by oral gavage, for 14 days. Animals initiating treatment at day 0 or 3 were protected from death and apparent signs of illness. Animals treated at rash onset had symptoms similar to the placebo control group; however symptoms were less severe in the treated animals. Although all animals treated at rash onset survived infection, animals lost 10–24% of body weight and did develop generalized rash (however, lesions resolved more quickly when compared to untreated prairie dogs in previous studies. Although asymptomatic, viable virus was shed sporadically from animals in the prophylaxis and post exposure groups (from two oropharyngeal samples in the day 0 prophylaxis group, and five samples from the day 3 post exposure group). More, sustained virus was detected in the oropharyngeal samplings of the animals in the therapeutic treatment group, but levels were less than the virus levels in the untreated group. 1/4 sham-treated animals survived infection. Signs of disease and viral titers were all increased in this group of animals compared to the animals treated with ST-246. This is the first small animal study where a treatment and survival benefit has been demonstrated when animals are treated at later stages of illness. Initiation of treatment at rash onset is similar to expectations of a human treatment regimen. The observation of virus shedding after treatment cessation in the prophylactically or post exposure treated animals merits further study to assess whether this reflects viral resistance or a blunted and delayed immune recognition and ultimate clearance of virus.
The number of MERS-CoV-infected individuals is low, the availability of clinical samples is limited, and no autopsies have been reported. It is therefore crucial to begin development of an animal model for MERS, but thus far, experimental infection has been reported only in rhesus macaques (18). MERS-CoV-infected macaques develop a nonfatal mild pneumonia. The absence of severe respiratory disease and kidney disease in these nonhuman primates makes it imperative that additional animal models be developed. Thus far, there have been no reports of successful infection of mice or ferrets, but infection of these animals may be initiated or enhanced if the receptor for the virus, human DPP4, is expressed in lieu of the mouse protein. Notably, similar efforts to introduce the human receptor for SARS-CoV resulted in a transgenic mouse that developed an overwhelming neuronal infection (19, 20). These mice were useful for studies of vaccines and antiviral therapies but not for studies of pathogenesis. As mice engineered to express human DPP4 are developed, it will be important to minimize the likelihood of brain infection by careful attention to tissue-specific expression.
Animal models permit an advance beyond what can be gleaned from tissue culture evaluation of an antiviral effect. The evaluation of an antiviral, in the context of a host with a functioning immune system, enables better understanding of therapeutics’ potential efficacy. The evaluation of an antiviral in the context of an impaired immune system enables better understanding of therapeutic use in a particular immunosuppressed population. Pathogen host range, especially if not a simple issue of receptor utilization, can confound the ability to interpret, and extrapolate to the human, some of the nuances of the host pathogen interaction and prediction of potential human therapeutic benefit. Of the small animal models used to evaluate antiviral efficacy, all have used stringent virus challenges (all greater than 10 × LD50) and shown survival benefit. Routes of infection have used methods that attempt to simulate potential human routes of infection and resultant human illness courses. Given the uncertainties of what a human infectious or lethal monkeypox dose is, it is difficult to extrapolate the potential “best fit” of any of these models for human disease. The clinical time course of disease in the prairie dog model, however, has a temporal relationship that is close to what has been described with human systemic orthopoxvirus (variola or monkeypox) disease. However, a limitation of the prairie dog and some of the other described animal systems, with the exception of the mouse model, is a paucity of immune reagents. There are a handful of antiviral compounds which show promise in these small animal models using monkeypox virus as the challenge. Additional studies evaluating treatment benefit when used in later stages of disease, their effect on elicitation of a protective immune response, evaluation of antiviral resistance, and their effect on viral shedding will improve our understanding of how they may be used in treatment of human disease, or in response to epidemic disease.
At this point, while the number of infected individuals is low, it is important to determine the extent and source of the infection. This information not only will provide a framework for establishing the relative importance of the disease and the potential for epidemic spread, but it may also facilitate direct interventions to eliminate reservoirs of infection. These analyses will require well-validated diagnostic reagents and access to human and animal blood samples in Al-Ahsa governorate in Saudi Arabia and elsewhere in the Middle East.
A second major goal will be to better understand the unique features of MERS. Does the high mortality rate reflect infection primarily of patients with substantial comorbidities? Does lack of recognition by host innate immune sensors result in high levels of virus in the lung and a dysregulated immune response, as occurred in patients with SARS? Does MERS-CoV inhibit interferon induction by novel mechanisms not utilized by other coronaviruses? Do the accessory proteins have novel mechanisms of action?
Third, development of a useful animal model that reproduces some of the features of the human infection will be critical for testing antiviral therapies and vaccines. A fourth goal will be to develop MERS-CoV-specific drugs and vaccines. The public health community learned a great deal from the SARS epidemic about approaches to treatment and prevention that might be effective against MERS-CoV.
Arguably, the most important outcome will be the development of drugs or vaccines that target a broad array of coronaviruses and not just a single virus. Events over the past 10 years show that coronaviruses are not only widespread in nature but also able to cross species to infect new hosts and can cause severe disease in humans. Having tools in hand before such events occur is critical for public health.
Flavivirus serology has been historically challenging due to the cross-reactivity of viral epitopes to circulating antibodies. Therefore, the results of serologic surveillance studies must be interpreted cautiously. Further, multiple methods exist for antibody detection (e.g., HI, PRNT, ELISA), and the biological significance of neutralizing vs. non-neutralizing antibodies must be taken into account.
In 2010, the serum of 140 Mexican bats from three species (Glossophaga soricina, Artibeus jamaicensis, and Artibeus literatus) was assayed by PRNT using WNV, SLEV, and DENV 1–4, and 26 were positive for flavivirus-specific antibodies (19%). None of the titers exceeded 80, and all samples were also negative when tested for flavivirus nucleic acid by RT-PCR. In a 2015 serosurvey, eight bats (2.6%) displayed non-specific hemagglutination-inhibition (HI) results indicating cross-reactivity or antibodies against an undetermined flavivirus. Kading and colleagues performed a serosurveillance study in Ugandan bats and identified 13.6% (85/626) had non-specific flavivirus antibodies by plaque reduction neutralization assay (Chaerephon pumilus, Hipposideros ruber, Mops condylurus, Nycteris macrotus, Eidolon helvum, Epomophorus minor, and Rousettus aegyptiacus). Still, results generally supported the widespread exposure of bats in Uganda to flaviviruses.
In 2018, Sotomayor-Bonilla and colleagues reported that liver and spleen samples from 12 Mexican bat species tested negative using pan-flavivirus NS5 primers. A recent study in Brazil suggested a lack of arboviral circulation in bat populations, as 103 individuals from 9 species were tested for molecular and serologic evidence of alphavirus and flavivirus infection and all were negative. Results of experimental infection of Egyptian rousette bats with WNV and of Angolan free-tailed bats (Mops condylurus) with Ntaya virus resulted in very low levels of viremia, while infection of African straw-colored fruit bats with Ntaya virus resulted in neither pathology nor detectable viremia.
The MERS-CoV infection is considered to be a new respiratory disease with a dire global concern. MERS-CoV infections are caused by a newly emerging coronavirus (CoV), belonging to the designated lineage C of Betacoronavirus of the RNA family Coronaviridae. With respect to viral origin and transmission, bats are thought to be the reservoir host of Betacoronaviruses, and the African Neoromicia bats in particular are the natural reservoir of MERS-CoV.
Since its emergence in 2012 in Saudi Arabia, when an elderly patient (60 years old) with respiratory illness died after admission to a hospital in Jeddah, the disease was subsequently reported to have been transmitted to several countries worldwide, and has affected more than 1000 patients with over 35% fatality.
Moreover, a 60-year-old Saudi man was admitted to a private hospital in Jeddah, Saudi Arabia in June 2012 with a history of fever, severe acute respiratory syndrome with cough, expectoration, and shortness of breath. He did not smoke; and for the disease, which was suggested to be due to an animal transmission of coronaviruses, he was treated with oseltamivir, levofloxacin, and piperacillin-tazobactam. On day 11, he died. After this, a 61-year-old Saudi male with hypertension and diabetes with no history of smoking, reported for surgery. At the time of admission, he was asymptomatic. He was initially screened using nasopharyngeal swab, endotracheal aspirate, and serum sample for MERS-CoV per protocol with the MERS RRT-PCR assay. The results confirmed MERS-CoV infection. He died three days after admission. It was discovered that the patient owned a dromedary camel barn in Saudi Arabia, and had a history of close contact with camels, as well as a habit of raw milk consumption of an unknown duration.
Two studies have suggested a relationship between the infection and contact with dromedary camels. In addition to this, serological diagnostic methods have been used to confirm MERS-CoV infections in dromedary camels for at least 2–3 decades and has thus confirmed camels as an intermediate host for this virus. Thus, in 2012, a novel coronavirus (MERS-CoV) was isolated from two fatal human cases in Saudi Arabia and Qatar; and since then, more than 1400 clinical cases of MERS-CoV have been identified, and the great majority of the cases were from Saudi Arabia. This previous report author raised a thoughtful comment related to the emerging viral diseases “Why We Need to Worry about Bats, Camels, and Airplanes”. Moreover, another study suggested that MERS-CoV infection is usually transmitted from human’s direct contact with dromedary camels, especially when people drink the milk or use camel’s urine for medicinal purposes. More recently, a metagenomics sequencing analysis of nasopharyngeal swab samples from 108 MERS-CoV-positive live dromedary camels marketed in Abu Dhabi, United Arab Emirates, showed at least two recently identified camel coronaviruses, which were detected in 92.6% of the camels in that study. However, limited human-to-human infections have been reported.
The prevalence of MERS-CoV infections worldwide still remains unclear. In addition to this, the WHO reported about 1797 cases of these infections since June 2012, with about 687 deaths in 27 different countries, worldwide. Recently, a study was conducted from June 2012 to July 2016, during which samples were collected from MERS-CoV infected individuals, from the National Guard Hospital in Riyadh (the Saudi Arabian capital city), the MOH in Saudi Arabia, and other Gulf Corporation Council countries, to determine the prevalence of MERS-CoV. The epidemiologic data that were collected, showed that the highest number of cases (about 1441 of 1797 patients) were reported from Saudi Arabia (~93%). Among the 1441 MERS-CoV cases from Saudi Arabia, Riyadh was the worst-hit area with 756 infected cases (52.4%), followed by the western region of Makkah where 298 cases (20.6%) were reported.
Furthermore, this study also showed that the incidence of MERS-CoV infections was highest among elderly people aged ≥60 years; with speculation that there might be certain conditions or factors involved. It is considered that MERS-CoV infection might have a peculiar gender predisposition. Recent data examined the mortality in patients with MERS-CoV and the gender relationships, looking at the survival of cases among females and males. It was suggested that males have a higher risk of death; however, this was contradicted by the findings from two other studies which suggested that males have a low risk of death; while another survey which examined the influence of gender on 3-day and 30-day survival, found a low risk of death especially in the older age group. On the other hand, Badawi et al., suggested that MERS-CoV infections could be mild and may only result in death among patients suffering from any kind of immune system disorder and/or any chronic disease.
More recently, data regarding the mortality in patients with MERS-CoV have been published. According to Saudi Arabia’s MOH daily statements, dated from February 26 through March 3, laboratory-confirmed new cases of MERS-CoV and 2 deaths occurred. Recently, on February 26, patients infected while hospitalized at Riyadh included two men (23 and 59 years old) in stable condition, who were not healthcare workers. According to a February 27 update, a new case involved a 71-year-old man from the city of Buraydah who later died. Meanwhile, on March 1, another MERS-CoV infection in a Riyadh hospital patient, a 64-year-old man who was listed in critical condition and who likewise had contact with camels, as the other two patients, was reported. Thus, the MOH stated that the spillover from camels is thought to be the main source of MERS-CoV in Saudi Arabia, since all these patients were exposed to the animals before reporting ill.
Furthermore, an 83-year-old patient from Riyadh, and other two patients who had camel contacts from Hail city in the north central part of Saudi Arabia were listed in critical condition. The illness in these patients was reported on March 1. According to a March 3 statement, another patient, a 74-year-old man from Najran located in southern Saudi Arabia, was reported. The man was listed in a stable condition. Of these new cases, only one death, involving the 83-year-old man from Riyadh, according to the March 3 MOH statement, was reported. Still, much work is needed to detect the MERS-CoV infection risk in Saudi Arabia, because data showed increasing number of cases exist among the eight countries including Saudi Arabia. Thus, the emergence of MERS-CoV in the region and its continuing transmission from 2012–2017, currently poses one of the biggest threats to global health security. Most cases (over 85%) reported to date have been from countries in the region (e.g., Egypt) notably from Saudi Arabia, with 1527 cases including 624 deaths.
Western equine encephalitis (WEE) is an uncommon viral illness of horses and human. WEE virus (WEEV) is an Alphavirus of the family Togaviridae which is maintained between birds and mosquitoes, occasionally causing disease in humans and equids [135, 136]. This is an arbovirus transmitted by mosquitoes of the genera Culex and Culiseta. It is a recombinant between Sindbis and Eastern equine encephalitis like viruses. It has also been reported to cause disease in poultry, game birds and ratites. WEEV is normally maintained between Culex tarsalis mosquitoes and birds. WEE has several subtypes consisting Sindbis, Aura, Ft. Morgan and Y 62–33. WEEV previously isolated in the south and eastern USA has been shown to belong to the HJ virus serogroup.
Horses and humans are often referred to as “dead-end” hosts as the virus does not build to high enough levels in blood to infect other mosquitoes. Most people infected with WEE virus will have either no symptoms or a very mild illness. A small percentage of people, especially infants and elderly people to a lesser extent, may develop encephalitis. Approximately 5-15% of these encephalitis cases are fatal, and about 50% of surviving infants will have permanent brain damage.
Geographically, WEEV exists throughout uine deaths were estimated in central America and northern portions of South America, Mexico and Canada. In the US, WEEV exist in the western two third of the country. Outbreaks of the disease have been recorded since 1847. In 1930 about 6000 horses and mules were infected leading to about 50% mortalities in California. The largest epidemic was recorded in 1937 and 1938 in USA and Canada. In 1938 outbreak an estimated 264000 equids were infected with a morbidity of 21.4%. In the USA, WEE is seen primarily in provinces west of the Mississippi River. During 1941, there was an outbreak of WEE in several states of US and Canada causing 300,000 cases of encephalitis in mules and horses and 3336 cases in humans. The 1970s saw 209 human cases; 87 were reported during the 1980s, only 4 cases during the 1990s, and no cases have been reported in the USA or Canada since 1998. The last documented human case in North America occurred in 1994, and the virus has not been detected in mosquito pools since 2008. In human, WEEV infections tend to be asymptomatic or cause mild disease after a short incubation period of 2–7 days with nonspecific symptoms, e.g., sudden onset of fever, headache, nausea, vomiting, anorexia and malaise. In some cases, additional symptoms of altered mental status, weakness and signs of meningeal irritation may be observed. In a minority of infected individuals, encephalitis or encephalomyelitis occurs and may lead to neck stiffness, confusion, tonic-clonic seizures, somnolence, coma and death. WEEV is considered as agent that the US researched as potential biological weapons before the nation suspended its biological weapons program.
In horses, infections with WEEV begin with fever, inappetence and lethargy, progressing to various degrees of excitability and then drowsiness, ultimately leading to paresis, seizures and coma in 5-10 day course of the disease. The WEEV mortality rate in horses is higher than humans. Mortality of horses showing clinical signs of WEE is 20–50%. These symptomatic horses either progress to recumbency or die from WEE infections.
There is no treatment for WEE other than supportive care. Formalin-inactivated whole viral vaccines for EEE, WEE, and VEE are commercially available in mono-, bi-, or trivalent form. Previously non vaccinated adult horses require booster. For adult horses in temperate climates, an annual vaccine within 4 wk of the start of the arbovirus season is recommended. However, for horses that travel between areas affected by the virus, 2 or even 3 times vaccination in a year is recommended. Mares should be vaccinated 3–4 wk before foaling to induce colostral antibody.
In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster. For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found. In addition an antibody response was observed against some but not all novel astrovirus types. Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis, suggesting that astrovirus infection may not be limited to the gastrointestinal tract.