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The past decade and a half has been punctuated by multiple global infectious threats (Table 1). Epidemics of new influenza variants, novel coronaviruses and enteroviruses, new strains of Ebola virus, and the emergence of Zika virus and Chikungunya in regions of the world previously unaffected has created significant concerns in healthcare about minimizing the time from identification to disease control. Globalization of tourism and business have further complicated disease epidemiology that may have once been more localized but now poses greater potential for international spread.
The approach to emerging infectious disease (EID) mitigation differs based on the respective pathogen. For example, the recent H3N2 and H7N9 outbreaks were associated with porcine and avian exposure as a risk for infection. Additionally, introduction of a pathogen into new regions may alter the epidemiology of disease. Although Ebola virus outbreaks occurred sporadically since 1976, its appearance in the major population centers of West Africa resulted in a significant amplification of transmission not seen with the prior, geographically limited outbreaks. The Middle East Respiratory syndrome coronavirus (MERS-CoV) represented a new viral entity, related to other minimally pathogenic coronaviruses but causing a highly lethal syndrome. And whereas, Zika virus had been recognized in African and East Asia for almost 6 decades, its emergence into the Western hemisphere and the recognition of heretofore unrecognized complications including congenital microcephaly and Guillain Barre Syndrome (GBS).
Vaccines are considered as a critical component of disease prevention for EIDs, especially since in some cases treatment options are limited or non-existent, or rapid clinical deterioration may limit the effectiveness of therapeutics. However, for EID vaccine development the desire for rapid deployment of vaccines for newly emergent diseases is tempered by the realities of the life-cycle for drug development.
In this paper, we review the epidemiology and clinical presentation of MERS-CoV and Zika virus with regard to vaccine development. In particular, the challenges in clinical trial design of efficacy studies are considered and discussed - in particular for diseases that may be limited in scope and/or for which the epidemiology is changing in real-time.
The Zika virus, discovered in Uganda in 1947, was shown to be endemic through Sub-Saharan Africa and tropical areas of Southeastern Asia in studies through the second half of the 20th century. Isolated outbreaks occurred in Yap Island in 2007 and on French Polynesia in 2014. Starting in mid-2015, Zika virus infection achieved epidemic status, spreading rapidly through South America, Central America, and the Caribbean Islands. It was soon recognized that Zika virus infection occurring during pregnancy caused microcephaly and other congenital disorders in the developing fetus, the latter being the primary reason for the World Health Organization (WHO) labelling Zika as an international threat in early 2016. Beginning in late 2015, numerous academic labs and pharmaceutical companies initiated work to develop a vaccine against Zika, however, by the time the first vaccines entered clinical trials, the Zika epidemic had started to wane creating significant challenges to vaccine assessment that has engendered discussion of other regulatory pathways to licensure.
In this paper, we provide a brief update of current progress in Zika virus vaccine development and explore the challenges to vaccine assessment and eventual licensure.
Vaccinia virus (VV) is a member of the Poxviridae, which constitute a large family of enveloped DNA viruses and replicate entirely in the cytoplasm of the infected cells with a linear double-stranded DNA genome of 130–300 kilo base pairs. Poxviruses have a broad range of eukaryotic hosts including mammals, birds, reptiles and insects and can grow in many cell lines in vitro. Some poxviruses are causative agents of human diseases. Variola virus caused a deadly human disease smallpox until its global eradication in 1977, in which VV was used as a vaccine. Other poxviruses causing human diseases are molluscum contagiosum virus and the zoonotic monkeypox virus. Notably, variola and monkeypox viruses are transmitted to humans by respiratory route, whereas molluscum contagiosum virus is mainly transmitted through the skin. Variola and monkeypox viruses cause systemic infections with high levels of lethality, but the details of their pathogenesis are not well-understood.
Intranasal inoculation of different VV strains in mice shows different levels of virulence and only neurovirulent strains cause lethality. Western Reserve (WR) strain was generated by intracerebral mouse passages, and an intranasal inoculation results in an acute infection of the lung followed by dissemination of the virus to various organs. Intranasal infection with a low dose of WR strain induces an inflammatory infiltrate in the lung, and the virus was cleared 10 to 15 days after infection; however, infection with a high dose of WR strain caused lethality, which has been used as a challenge model to study the effect of antiviral drugs, immune IgG, soluble viral proteins and other vaccine strains. In one report intranasal infection with the WR strain caused pneumonia showing severe alveolar edema and acute necrotizing bronchiolitis and peribronchiolitis as well as neutrophilic infiltrates in the interstitium of the lung. The mechanisms of lethality in mice infected with the lethal dose of WR strain are, however, not well-understood.
In this study, we focused on the differences in virus replication and host immune responses between lethal and non-lethal respiratory infections with VV. We used two VV strains; neurotropic virulent WR strain and the less virulent Wyeth strain. Although BALB/c mice are frequently used for intranasal challenge of vaccinia virus, we used the C57BL6/J strain of mouse in these experiments for two reasons. One is that most knockout mice lacking genes involved in immune responses have been made with C57BL6/J genetic background. The other is that we and one other group have characterized cellular immune responses, especially CD8+ T cell responses, to vaccinia virus in C57BL6/J mice, when this study was planned. Infection of C57BL/6J mouse with a high dose (106 p.f.u. (plaque-forming units)) of the WR strain was lethal, whereas a high dose (106 p.f.u.) of Wyeth strain and a lower dose (104 p.f.u.) of WR strain were not lethal. The WR strain replicated and produced higher titers of virus in the lung and the brain compared to the Wyeth strain. There was, however, no difference between the virus titers in brains of mice infected with the high or low dose of WR strain. Lethal infection with WR strain resulted in fewer lymphocytes and an altered phenotype of T cells in the lung compared to non-lethal infection and uninfected controls, and induced severe thymus atrophy with a marked reduction of CD4 and CD8 double positive (DP) T cells.
Influenza caused by highly-pathogenic avian H5N1 virus has been one of the most important zoonotic viral infections of humans during the last decade [1–3], with human fatality rates more than 50 percent in some areas of 15 affected countries, where outbreaks continue. Influenza viruses belong to the family Orthomyxoviridae, of which the H5N1 type has a broad range of hosts. Although H5N1 naturally infects poultry and wild birds, transmission occurs to mammalian species, including humans [6–9]. H5N1-infected humans often develop severe clinical respiratory and gastrointestinal symptoms and signs; in some cases, symptoms of the central nervous system (CNS) ensue [10–13]. Infection of the CNS by H5N1 may be severe, causing encephalopathy and other serious neurological complications and sequelae [14–16]. Recent reports found that mice experimentally infected with H5N1 virus developed encephalitic lesions during the acute phase, which was associated with a degree of neuronal degeneration and necrosis. Interestingly, CNS inflammation has not generally been observed in the chronic phase of infection, despite the detection of virus in such lesions. Furthermore, Jang et al. (2009) reported protein aggregation mediated by the influenza virus within degenerated and necrotic neurons. These findings suggest the hypothesis that pathogenic influenza virus might induce a “hit and run mechanism”, in which virus infection triggers a cytokine storm resulting in acute CNS inflammation, with consequent chronic Parkinson-like disease, encephalitis lethargica, or other neurodegenerative diseases. Nevertheless, the exact pathologic mechanism(s) of H5N1-induced encephalopathy remains unclear.
Neurogenesis in the adult CNS is regulated by the neural stem/progenitor cells residing in the subventricular zone (SVZ) of the lateral ventricles, and subgranular zone (SGZ) of the hippocampus. This neurogenesis was shown to be de-regulated by infection with neurovirulent viruses causing Borna disease and Varicella-zoster. Thus, it would be essential to determine whether infection by H5N1 virus occurs in neural progenitor cells, and if so, whether such infection plays a role in the pathogenesis of H5N1-induced encephalopathy. In order to shed light on these questions relevant to the pathogeneses of influenza virus-induced neuropathology, we infected human neural progenitor cells (hNPCs) with H5N1 avian influenza virus and examined the resulting virus-cell interactions, the induction of cellular mediators, and the phenotypic characterizations of hNPCs following H5N1 virus infection in vitro.
Arboviruses are arthropod-borne viruses that exhibit worldwide distribution and are a constant threat, not only for the public health but also for wildlife, domestic animals, and even plants. The rise in global travel and trade as well as the changes in the global climate conditions are facilitating the expansion of the vector transmitters, including mosquitoes, ticks, sandflies, and midges among other arthropods, from endemic to new areas, augmenting the number of outbreaks around the world at an unprecedented rate. Arboviruses need multiple hosts to complete their cycle (i.e., host and vector), making it possible to impact disease by targeting either the arthropod vector and/or the pathogen. For some of these pathogens, efficient antivirals or vaccines are not available, in some cases due to the genetic variability of these viruses. Moreover, there are a limited availability of animal models to study infections, and some of them display a poor immunogenicity and some others viral infections cause neglected diseases that have not been deeply studied. Transmission between the vector and the host occurs when the vector feeds on the blood of the host by biting. However, the vector does not act as a simple vehicle that passively transfer viruses from one individual to another. Instead, arthropod-derived factors found in their saliva have an important role in infection and disease, modulating (positively and negatively) replication and dissemination within the host. In addition, the inflammatory response that the host mounts against these vector molecules can enhance the severity of arbovirus infection.
To study disease pathogenesis and to develop efficient and safe therapies to prevent (vaccines) or treat (antivirals) viral infections, the use of an appropriate animal model is a critical concern. The use of mice as small animal models to study immunity, pathogenesis, as well as to test candidate vaccines and antivirals against a largely variety of viral diseases is widely spread. They are cost effective, being affordable for most of research laboratories. They reproduce quickly, are easy to handle, do not require specialized facilities to house, and multiple inbred strains of genetically identical mice are available. In many cases such as Crimean Congo Hemorrhagic Fever (CCHFV), Bluetongue (BTV), Middle East respiratory syndrome (MERS), or Ebola (EBoV) viruses, the pathogenesis of disease in humans is also partially mimicked. Furthermore, optimal reagents have been developed for in vivo and in vitro studies in mice, a fact which allows the study of other animal viruses apart from those which are human specific. Also, it is possible to manipulate the mouse genome and generate transgenic, knock-out, knock-in, humanized, and conditionally mutant strains to interrogate protein function in physiological and pathological signs.
Immunocompetent wild-type mice are susceptible to infections with a number of viral pathogens such as influenza virus; severe acute respiratory syndrome coronavirus (SARS-CoV); and Rift Valley fever virus (RVFV). Unfortunately, immunocompetent mice are not susceptible to many other viruses with outbreak potential, and thus alternative strategies are needed.
Hantaviruses are negative-sense RNA viruses transmitted to humans from small animal hosts. Different viral species are associated with one of two disease syndromes: hemorrhagic fever with renal syndrome (HFRS), or hantavirus pulmonary syndrome (HPS). Hantaan virus (HTNV), primarily found in Asia, is among the most prevalent HFRS-causing hantaviruses with a case fatality rate of between 1–15%. Puumala virus (PUUV) causes most HFRS cases in Europe, though its case fatality rate is lower at <1% [3, 4]. There are currently no FDA licensed vaccines or therapeutics for either HFRS or HPS.
The Syrian hamster (Mesocricetus auratus) is the typical animal used to model hantavirus infection and disease. Andes virus (ANDV), an HPS-causing hantavirus, causes lethal disease in immunocompetent hamsters, while numerous other HPS-causing hantaviruses including Sin Nombre Virus (SNV) and Choclo virus cause lethal disease in hamsters immunosuppressed with dexamethasone and cyclophosphamide [7, 8]. In contrast to HPS-causing hantaviruses, exposure of hamsters to HFRS-causing hantaviruses such as HTNV, PUUV, Dobrava (DOBV) and Seoul (SEOV) leads to asymptomatic infection, despite viral dissemination, even when immunosuppressed (Hooper Lab, unpublished data) [8–11]. In these studies hamsters were exposed to high doses of HTNV and PUUV, far exceeding the infectious dose 99% (ID99) for the virus. Development and characterization of a uniformly infective, low-dose challenge model, enhances the hamster model’s usefulness in vaccine and therapeutic testing. In this report we present a low-dose hamster infection model for both HTNV and PUUV infected animals.
Ferrets (Mustela putorius furo) have become a popular animal model for a number of respiratory pathogens including influenza, coronavirus, Nipah virus, and morbillivirus, due to the similarity in lung physiology to humans. In addition, they have recently been described as a disease model of two hemorrhagic fever viruses, Bundibugyo virus and Ebola virus [16, 17], supporting viral replication without prior adaptation. Most hantavirus-related human disease occurs by aerosolized transmission of the virus from the excreta or secreta of infected rodents [18, 19], a model of viral infection for which the ferret is well suited. In this study we demonstrate that ferrets are capable of being infected by high titers of HTNV and PUUV, though aside from gradual weight loss infected animals exhibit no clinical symptoms or impaired renal function.
It has been established that infection of rhesus macaques (Macaca mulatta) with HFRS-causing hantaviruses (DOBV, SEOV, HTNV, and PUUV) leads to asymptomatic infection and seroconversion, while infection of cynomolgus macaques (Macaca fascicularis) with PUUV leads to a mild disease characterized by lethargy, mild proteinuria and hematuria, and kidney pathology, similar to mild HFRS in humans. However, the macaques’ large size and cost limits their usefulness in therapeutic studies, especially when test article availability is limited, as is often the case in passive transfer studies. The common marmoset (Callithrix jacchus) is becoming more popular for infectious disease studies. Its genetic similarity to humans, cost, relative safety, and small size make it an attractive alternative to traditional non-human primate species. Marmosets have been used as a disease model for other viral agents including Dengue virus, Hepatitis C virus, influenza virus, Lassa fever virus, orthopox viruses [26–28], Rift Valley Fever virus, Eastern Equine Encephalitis virus, and filoviruses. In this study we demonstrate that exposure of marmosets to HTNV leads to asymptomatic infection characterized by high levels of neutralizing antibodies. This is the first report of hantavirus infection in marmosets.
Medical countermeasures are products including biologics (e.g., vaccines and antibodies) and small molecule drugs that can be used to prevent or combat infectious disease outbreaks. This study presents three animal models of HTNV infection, and two models of PUUV infection that can be used to evaluate the efficacy of medical countermeasure that are intended to prevent or mitigate infection (e.g., vaccines) by these viruses through induction of sterile immunity.
Our experiments were designed to compare pathogenic processes that occur during H1N1 pdm and H5N1 HPAI viral infection in mice. Naïve BALB/c mice were infected with either H1N1 pdm virus or H5N1 HPAI virus at the same infectious dose (1 × 104 PFU in a volume of 50 μL per mouse) (Fig. 1). The H5N1-infected mice exhibited marked decreases in body weight through day 9 (Fig. 1a), and all of these animals died or were humanely euthanized since symptoms reached a humane endpoint (see Methods) by 10 days post-infection (dpi) (Fig. 1b). In contrast, mice infected with H1N1 pdm virus exhibited moderate decreases in body weight before recovering without mortality (Fig. 1a,b). Pathological analyses demonstrated that the mice infected with H5N1 HPAI virus developed severe pneumonia with diffuse alveolar damage at 7 dpi and that the symptoms were markedly aggravated at 9 dpi (Fig. 1c). In contrast, the mice infected with H1N1 pdm virus showed only partial inflammation (Fig. 1c). The day-9 histopathological scores in the mice infected with H5N1 HPAI virus were significantly higher than those in the mice infected with H1N1 pdm virus (5.2 ± 0.8 vs. 2.2 ± 0.7 (mean ± SD), respectively; Fig. 1d).
To investigate differences in immune responses against H5N1 HPAI virus and H1N1 pdm virus infection in mice, we measured half-maximal titer of neutralizing antibody (NT50) and titer of hemagglutination inhibition (HI) against each virus. In mice infected with H1N1 pdm virus, the NT50 titer increased markedly from 5 dpi, reaching a mean value of 3487 ± 1250 at 9 dpi (Fig. 1e). The HI titer achieved a mean value of 207.5 ± 1.7 at 9 dpi (Fig. 1f). In contrast, the NT50 and HI titers in all H5N1 HPAI-infected mice (with the exception of one H5N1-infected mouse) remained below the limits of detection (4 in NT50 titer and 10 in HI titer; Fig. 1e and f) during the experimental period.
The ferret, Mustela putorius furo, is a superlative animal model for respiratory infections and the influenza ferret infectome has recently been published212223242526272829. Ferrets show respiratory illness similar to humans and clinical features of disease are easily observed where fevers can persist days following infection of viruses such as 2009 H1N1pdm influenza212330. As well as fever, nasal discharge and sneezing can also be observed in animals infected with influenza viruses21. Although the genetic evolution of H1N1 viruses between 1918 and 2008 has been studied9, the clinical features of these viruses have not been previously investigated and compared including the most recent 2009 H1N1 pandemic virus. Here we used ferrets to experimentally monitor and compare the clinical immune responses during infection with human historical H1N1 influenza strains.
We infected ferrets with six influenza A/H1N1 strains; A/AA/Marton/43 (Marton/43), A/FortMonmouth/1/1947 (FM/47), A/USSR/90/1977 (USSR/77), A/Taiwan/1/1986 (Taiwan/86), A/NewCaledonia/20/1999 (NCal/99), and A/NewYork/18/2009 (NY/09). These influenza A viruses were chosen due to their emergence and influence in H1N1 genetic history (Fig. 1, strains used in this study are marked with an asterisks) as covered in the introduction. Following infection, ferrets were monitored for body temperature, weight, inactivity level, sneezing and nasal discharge from each group were observed daily until Day 14 post-infection (pI). Infection by all strains produced an increase in temperature; the normal range for ferret temperature is indicated by the shaded area of each graph23 (Supplementary Fig. S1a). The pandemic H1N1 strain NY/09 induced the greatest fever on Day 2 to a temperature of 104% (of baseline). NCal/99 and Marton/43 infection also caused a high temperature of 103% from baseline, which peaked on Day 1 and Day 2 pI, respectively (Table 1). USSR/77 and Taiwan/86 had moderate fevers and FM/47 had the smallest increase in temperature reaching only 101% above baseline (Table 1).
Analysis of weight loss showed that animals infected with of all viruses except pandemic NY/09 were able to recover to original weight or greater following infection (Supplementary Fig. S1b and Table 1). NY/09 infected ferrets had the most significant weight loss compared to normal weight fluctuations (shaded area)23 which peaked at 91% of baseline weight on Day 6 and Day 7. USSR/77 and NCal/99 reached less than 95% and 95% of baseline weight, respectively, on Day 2 pI (Table 1). Infection with Taiwan/86 produced the smallest amount of weight loss and animals infected with FM/47 did not lose any weight at all (Table 1).
Secondary clinical signs were also measured and analysed for all infections, including nasal discharge, sneezing, and inactivity level (Supplementary Table S1). USSR/77 infected ferrets had the highest amount of nasal discharge and NY/09 ferrets had the greatest amount of sneezing and lethargy. Taken together, analysis of the complete clinical signs for each H1N1 strain infection suggested a unique clinical picture for each strain: NY/09 infection had the most severe with significant increase in temperature and an unrecovered weight loss compared to mildest strain, FM/47, which did not produce any weight loss and only a slight increase in temperature.
Adult ferrets (weight, 500–1500 g) were housed at bioCSL under a Support Services Agreement with the Victorian Infectious Diseases Reference Laboratory. Ferrets were seronegative (hemagglutination inhibition [HI] titer, <10) to currently circulating influenza virus strains before use. Experiments were conducted with approval from the CSL Limited/Pfizer Animal Ethics Committee, in accordance with the National Health and Medical Research Council, Australia, code of practice for the care and use of animals for scientific purposes.
Since LACV replicated to high titers in the nasal turbinates, we sought to determine if intranasal inoculation of mice with LACV could lead to infection. Three-week-old Swiss Webster weanling mice (n = 6/dose) were inoculated intranasally (IN) (10 μl volume) or intraperitoneally (IP) (100 μl volume) with serial dilutions of LACV/human/1960, and the LD50 and 50% infectious dose (ID50) were determined. Clinical disease served as a surrogate for lethality and mice were promptly euthanized prior to succumbing to LACV disease. In both groups, clinical disease was first noted on day 6 (Figure 3). Twenty days post-inoculation, the LD50 was determined. All surviving mice were tested for the development of a neutralizing antibody response. To determine the ID50 titer, mice were considered infected if they either developed clinical disease or a serum neutralizing antibody titer. The LD50 was similar in both the IN and IP groups (2.4 and 2.3 log10 PFU, respectively) with the LD50 following IP injection in agreement with previous experiments. The ID50 titers (1.5 and 1.6 log10 PFU for the IN and IP routes, respectively) were slightly lower than the LD50 titers, indicating LACV can cause a subclincal infection in weanling mice, but only at low doses.
La Crosse virus (LACV), family Bunyaviridae, is a mosquito-borne pathogen endemic in the United States. The LACV genome consists of three single-stranded, negative-sense RNA genome segments designated small (S), medium (M), and large (L). The S segment encodes two proteins in overlapping reading frames: the nucleoprotein (N) and a non-structural protein (NSS) which suppresses type 1 interferon (IFN) in the mammal host. The M segment encodes a single polyprotein (M polyprotein) that is post-translationally processed into two glycoproteins (GN and GC), and a non-structural protein (NSM). GN and GC are the major proteins to which neutralizing antibodies are directed. The L segment encodes a single open reading frame for the RNA-dependent RNA polymerase (L).
The virus is transmitted by hardwood forest dwelling mosquitoes, Aedes triseriatus, which breed in tree holes and outdoor containers. Ae. triseriatus feed on Eastern chipmunks (Tamias striatus grinseus) and Eastern gray squirrels (Sciurus carolinensis) which serve as amplifying hosts for LACV. Interestingly, the virus can be maintained in the mosquito population in the absence of vertebrate hosts by transovarial (vertical) transmission, thus allowing the virus to over-winter in mosquito eggs. More recently, LACV has been isolated from naturally infected, non-native Aedes albopictus mosquito. The infection of Ae. albopictus may represent a shift in virus ecology and increases the possibility for generation of new reassortants.
LACV was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl from Minnesota who suffered meningoencephalitis and later died in La Crosse, Wisconsin. In humans, the majority of infections are mild and attributed to the "flu" or "summer cold" with an estimated 300,000 infections annually, of which there are only 70–130 serious cases reported. Isolation of virus is rare and has been made from post-mortem brain tissue collected in 1960, 1978, and 1993. Two isolates from non-fatal LACV cases were collected in 1995, one from a brain biopsy of a child and one from cerebrospinal fluid.
Histopathologic changes in two human cases, one from 1960 and one from 1978, were characteristic of viral encephalitis. Inflammatory lesions consisted of infiltration of mononuclear leukocytes either diffusely or as microglial nodules. The largest inflammatory foci were observed in the cerebral cortex, including the frontal, parietal, and temporal lobes, and foci could also be found in the basal ganglion and pons. In these two cases, there was a lack of inflammatory lesions in the posterior occipital cortex, cerebral white matter, cerebellum, medulla, and spinal cord. Brain biopsy from a non-fatal LACV infection contained areas of perivascular mononuclear cuffing and focal aggregates of mononuclear and microglia cells. RT-PCR analysis of neural tissues from the 1978 case could only detect viral RNA in the cerebral cortex and not in the medulla, cerebellum, spinal cord, basal ganglion, or temporal lobe.
In children and adults, severe LACV encephalitis clinically mimics herpes simplex virus encephalitis with fever, focal signs, and possible progression to coma. Confirmatory diagnosis has been made by RT-PCR of cerebrospinal fluid to exclude herpes simplex virus and by fluorescent staining for LACV antigen in brain biopsy sections. Children who recover from severe La Crosse encephalitis may have significantly lower IQ scores than expected and a high prevalence (60% of those tested) of attention-deficit-hyperactivity disorder. Seizure disorders are also common in survivors. Projected lifelong economic costs associated with neurologic sequelae range from $48,775 – 3,090,398 per case. Currently, a vaccine or specific antiviral treatment is not available, but could serve to reduce the clinical and economic impact of this common infection.
Although evidence of LACV infection has been reported for several species, only limited research has been done to understand LACV pathogenesis in its natural host or experimental rodents. LACV administered subcutaneously to suckling mice first replicates in muscle, and viremia develops with virus invading the brain across vascular endothelial cells. Virus replication in muscle was confirmed by immunohistochemical (IHC) staining, and the predominant cell type infected in the CNS is the neuron. The virulence of LACV for mice decreases with increasing age, similar to humans in which it causes CNS disease predominantly in pediatric populations. As an initial step in the establishment of animal models useful for vaccine development for humans, we sought to better characterize the tissue tropism of LACV in mice by identifying the tissues that support LACV replication after peripheral inoculation. We have previously described Swiss Webster mice as suitable for characterization of LACV infection at birth and at 3-weeks of age. Here we inoculated 3-week old Swiss Webster mice with either 1 or 100 LD50 of virus intraperitoneally. Twenty tissues were individually collected for six consecutive days and processed for virus titration, immunohistochemical staining, and histopathology studies.
Since experimental infection of non-human primates with LACV has not been reported, we also sought to determine if rhesus monkeys were susceptible to LACV infection. Rhesus monkeys were chosen since they are susceptible to a variety of neurotropic arboviruses, including flaviviruses. In this study, rhesus monkeys were infected intramuscularly or subcutaneously with a mosquito or human isolate of LACV. These two isolates were used since preliminary genomic sequence analysis indicate that there are host specific differences between LACV isolated from humans and mosquitos. LACV was found to be highly infectious for rhesus monkeys, but infection did not result in viremia, disease, or significant changes in blood chemistries or cell counts. However, high titers of neutralizing antibodies developed in all monkeys indicating that rhesus monkeys, although not optimal, will be useful for studying the infectivity and immunogenicity of LACV vaccine candidates.
Here we briefly summarize the data collection method and data of Liu et al.. Investigations were conducted on all influenza like illness (ILI) cases identified during the outbreak. Epidemiological, clinical and contact tracing data were collected by interviewing patients and retrieving medical records. Viruses were identified by reverse-transcription polymerase chain reaction assays followed by sequence analysis. The heamagglutination inhibition tests were used to detect antibodies to both viruses. The outbreak is reported to have taken place within three buildings (two dormitories and one college clinic). No other cases at the college were reported. Buildings 1 and 2 (with a total membership of 235 and 191 persons, respectively) are next to each other and there is restricted access between the two and to the wider community. Forty five ILI cases were reported from 31 August to 10 September and forty (N = 40) had laboratory-confirmed influenza A infection. Three different types of infection were reported: 22 infected with pandemic A/H1N1 virus, 12 infected with seasonal A/H3N2 virus and six co-infected with both influenza A viruses. In their sequences no substantial differences were observed between patients with mixed and single infections in either pandemic A/H1N1 or seasonal A/H3N2 virus. The clinical features were similar for patients with different infections and the six co-infected patients showed no more severe symptoms than the singly infected patients. Contacts between infected people are shown in Fig. 2 of Liu et al. but this only extends to the contact network within one dormitory. The ‘index’ case with pandemic influenza A/H1N1 infection was a college student whose symptoms first occurred on 31 August, 2 days after his returning to college. Except for the index case, all patients with A/H1N1 infection had not left the campus during the previous week. In contrast, the source of seasonal A/H3N2 virus infection cannot be determined exactly although available data indicate that A/H3N2 virus might have been prevailing in the college when the pandemic H1N1 virus was introduced. Before the isolation of cases and the initiation of prophylaxis among the campus population (5 September 2009), several patients visited the college clinic and the mixing between students of different dormitories was not frequent in comparison to the mixing between students within each dormitory.
Adult female C57BL6/J mice were inoculated with various doses of the WR and Wyeth strains intranasally. Weight change and survival of infected mice were recorded daily. Inoculation with higher doses (106 p.f.u. and 105 p.f.u.) of the WR strain induced rapid and severe weight loss, which became obvious at 3 days post-infection (Fig. 1A), and most of mice died at 7–10 days post-infection (with 106 p.f.u. all mice died by 8 days post-infection) (Fig. 1B). Lower doses (104 p.f.u. and 103 p.f.u.) of the WR strain caused mild weight loss, and all mice survived. These mice recovered their weight after 6–8 days post-infection (Fig. 1A). The 50% lethal dose (LD50) of WR strain was calculated as 4.2 × 104 p.f.u., which is similar to the LD50 reported for BALB/c mouse. Wyeth strain did not kill mice (Fig. 1B) or cause weight loss (Fig. 1A) even when 106 p.f.u. of the virus was inoculated.
Viral encephalomyelitis is an important cause of morbidity and mortality worldwide, and many encephalitic viruses are emerging and re-emerging due to changes in virulence, spread to new geographic regions, and adaptation to new hosts and vectors. The term encephalomyelitis refers to inflammation in the brain and spinal cord that results from the immune response to virus infection. In humans, the viruses most commonly identified as causes of viral encephalomyelitis are herpesviruses and RNA viruses in the enterovirus (e.g., polio, enterovirus 71), rhabdovirus (e.g., rabies), alphavirus (e.g., eastern equine, Venezuelan equine, and western equine encephalitis), flavivirus (e.g., West Nile, Japanese encephalitis, Murray Valley, and tick-borne encephalitis), and bunyavirus (e.g., La Crosse) families. Other virus families with members that can cause acute encephalitis are the paramyxoviruses (e.g., Nipah, Hendra) and arenaviruses (e.g., lymphocytic choriomeningitis, Junin). However, this is certainly not a complete list, because for most cases of human viral encephalitis the etiologic agent is not identified, even when heroic attempts are made.
The primary target cells for most encephalitic viruses are neurons, although a few viruses attack cerebrovascular endothelial cells to cause ischemia and stroke or glial cells to cause demyelination, encephalopathy, or dementia–. Widespread infection of neurons may occur or viruses may display preferences for particular types of neurons in specific locations in the central nervous system (CNS). For instance, herpes simplex virus (HSV) type 1 often infects neurons in the hippocampus to cause behavioral changes, while poliovirus preferentially infects motor neurons in the brainstem and spinal cord to cause paralysis and Japanese encephalitis virus infects basal ganglia neurons to cause symptoms similar to those of Parkinson’s disease.
Because infections with encephalitic viruses are initiated outside the CNS (e.g., with an insect bite, skin, respiratory, or gastrointestinal infection), innate and adaptive immune responses are usually mounted rapidly enough to prevent virus entry into the CNS. Therefore, most viruses that can cause encephalitis more often cause asymptomatic infection or a febrile illness without neurologic disease, and encephalomyelitis is an uncommon complication of infection.
A/Tasmania/2004/2009 (A[H1N1]pdm09), A/Perth/16/2009 A(H3N2) and B/Brisbane/1/2007 (B/Florida/4/2006-like; B/Yamagata lineage) viruses were passaged in the allantoic cavity of embryonated hen eggs and stored at −80°C. Infectious virus was measured by 50% tissue culture infectious doses (TCID50) assays, using hemagglutination as the read-out.
In 2012, cases of a progressive pulmonary infection related to individuals who reside in or traveled to the Arabian Peninsula were determined as caused by a novel Group C, β-coronavirus MERS-CoV.1,2 In contrast to the majority of human pathogenic coronaviruses that cause self-limited upper-respiratory illness, the mortality rate of early MERS-CoV cases was approximately 60%,3 and has remained greater than 35% -approximating that seen during the West African Ebola virus outbreak. In contrast, the mortality rate during Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) epidemic was 10%.
Following an incubation period of about 1 week, MERS-CoV causes a rapidly progressive lower respiratory infection with a prodromal illness characterized by fever, cough, and mild shortness of breath. Clinical deterioration is typical leading to the need for intensive care and ventilator support within days of presentation to hospital.4,5 Complications of MERS-CoV include renal failure and cardiac arrhythmias.
The MERS-CoV epidemic has been punctuated by large healthcare associated6-9 and dialysis unit10 outbreaks. Person-to-person spread between family members, while documented,11 represents a small minority of transmission events. Contact with camels is considered a significant risk for infection,12 and while direct evidence of camel-to-human transmission has been reported13 others have questioned the certainty of direct transmission suggested by this report.14 For most cases, sources of infection are unknown.15
Humans have served as the vector for global spread of MERS. Cases across Europe, North America, and Asia have emanated from travel to Saudi Arabia, Qatar, Oman, the UAE, and Kuwait.3,16,17 Secondary infections were infrequently reported in early travel-associated cases.5 However, the global epidemic potential for MERS-CoV was exemplified by the fact that a businessman returning from the Middle East to Seoul Korea served as the index case for 185 subsequent cases of MERS-CoV with a 20% mortality rate despite early diagnosis and intensive supportive care. The latter outbreak was in large part due to a breakdown in basic infection control.9,18 Further spread beyond the Arabian peninsula appears to have been avoided through active screening and quarantine of returning travelers.
Human influenza virus infection replicates primarily in the respiratory epithelium. Other cell types, including many immune cells, can be infected by the virus and will initiate viral protein production. However, viral replication efficiency varies among cell types, and, in humans, the respiratory epithelium is the only site where the hemagglutinin (HA) molecule is effectively cleaved, generating infectious virus particles. Virus transmission occurs when a susceptible individual comes into contact with aerosols or respiratory fomites from an infected individual.
The ferret has traditionally been used as a model of influenza transmission as most human influenza viruses do not need any adaptation to infect and transmit among ferrets. Studies in ferrets have identified the soft palate as a major source of influenza viruses that are transmitted between individuals. Notably, the soft palate is enriched in α2,6-linked sialic acids, which are preferred by the hemagglutinin proteins currently found in circulating human influenza viruses. This enrichment also occurs in the soft palate of humans.
The primary mechanism of influenza pathophysiology is a result of lung inflammation and compromise caused by direct viral infection of the respiratory epithelium, combined with the effects of lung inflammation caused by immune responses recruited to handle the spreading virus (Table 1). This inflammation can spread systemically and manifest as a multiorgan failure, but these consequences are generally downstream of lung compromise and severe respiratory distress. Some associations have also been observed between influenza virus infection and cardiac sequelae, including increased risk of myocardial disease in the weeks following influenza virus infection. The mechanisms of this, beyond a general inflammatory profile, are still unresolved [5, 6].
Data that were used in the analysis of this study were extracted from a previous study and hence did not require Human Resource Ethics committee approval.
Next, all of the above findings were considered in conjunction with the literature data on the transmission ecology collated for each of the 36 viruses in Figure S1a. Pieced together on this basis was an outer- to inner-body line-up of viruses by organ system or combination of organ systems, guided by the one-to-four virus infiltration score, the corresponding virus organ system tropism, the matching virus transmission modes, length of the infection and shedding periods, infection severity level, and virus environmental survival rate, see Figure 3 and, also, Figure S1d.
It is estimated by the World Health Organization (WHO) that approximately 36.9 (34.3~41.9) million persons worldwide are infected with human immunodeficiency virus (HIV), which is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS), including 1.7 million in the United States. Influenza A virus can cause acute respiratory infection in humans and animals throughout the world, and has continued to be a significant public health threat, leading to substantial global morbidity and mortality and an average of approximately 23,600 deaths annually in the United States alone.
The impact of influenza virus on HIV infection has not been well investigated. Little is known about influenza virus infection in HIV-positive individual. HIV infection has been shown to be related to worse prognosis of influenza. HIV-infected patients in Canada who also had pandemic 2009 influenza A (H1N1) virus (pH1N1) infection had more severe illness than those who did not have the co-infection, and fatality was higher than for patients who were not co-infected in California (USA). Recent reports indicate that adults with AIDS experience substantially elevated influenza-associated mortality.
Influenza virus infection has been associated with viremia in human and animal models. Viral RNA has been detected in blood in severe human pH1N1 infection. Encapsidated pH1N1 RNA is stable in blood derived matrices and influenza viruses can be transmitted by blood transfusion in ferrets. Normally, influenza A virus infection is confined to the airways where the virus replicates in respiratory epithelial cells. Cumulated reports indicate that influenza viruses can infect and replicate in blood cells, such as dendritic cells, primary monocytes/macrophages, and T cells.
Infection with HIV-1 can result in apoptotic cell death through activation of both death receptor-mediated and Bax/mitochondrial-mediated apoptotic pathways, which cause a progressive depletion of a select group of immune cells namely the CD4+ T helper cells leading to immunodeficiency. While HIV directly and selectively infects CD4+ T cells, the low levels of infected cells in patients is discordant with the rate of CD4+ T cell decline and argues against the role of direct infection in CD4 loss. A viral protein, neuraminidase (NA), derived from the human influenza virus was reported to enhance the level of HIV-1-mediated syncytium formation and HIV-1 replication. However, it is not known whether influenza A virus in blood affects HIV-1 replication, or reactivates HIV-1 replication in HIV-1-infected cells. Here, we showed that pandemic influenza A (H1N1) virus infection increased apoptotic cell death and HIV-1 replication in HIV-1 infected Jurkat cells.
Zika virus infection presents with a symptom complex consisting of a diffuse maculo-papular rash, fever, asthenia, myalgias, arthralgias, headache, and retroorbital pain. The frequency and degree of symptomatology has, however, varied between studies. A retrospective study immediately following the outbreak on Yap Island found that only 19% of survey participants reported symptoms consistent with Zika virus infection, a figure that has been cited to suggest that Zika virus infection is asymptomatic in as many as 80% of individuals. However, of 557 individuals who provided blood samples, representing 16% of households, 38% were symptomatic. A second retrospective study of the larger outbreak in French Polynesia, similarly appeared to have a low rate of symptomatology based on a sample of blood donors. However, a subsequent seroprevalence study found that 43% of those with evidence of prior infection had symptoms consistent with Zika virus infection. A more recent meta-analysis of 23 studies noted that between 0 and 83% of cases were reported as asymptomatic, although as the authors note, assessment of the prevalence of symptoms was not the goal of many studies.
In adults, the most common reported complication of Zika virus infection is a Guillain–Barré-like illness that had a prevalence of 0.24 cases per 1000 cases of infection in the French Polynesian epidemic. Of interest, a recent meta-analysis has questioned this causal association. Other less common reported neurologic complications in adults include meningoencephalitis and an ADEM illness.
In contrast, infection during pregnancy has been associated with fetal microcephaly and a number of other congenital illnesses including visual deficits, hearing disorders, neural calcifications, learning disabilities, and arthrogryposis that may affect as many as 30% children born to mothers infected during pregnancy. Additionally, Zika can directly invade the placenta and has been associated with prolonged maternal viremia.
Zika virus can also be transmitted through sexual contact, first reported for a researcher returning from Senegal in 2008. Numerous subsequent case reports were published among travelers as part of the epidemic affecting the Americas. The frequency of sexual transmission in endemic regions is unknown and could not be differentiated from mosquito-borne transmission. Zika virus carriage in the male urogenital tract is common through 90 days post infection and may persist in some to 9 months.
In mice, Zika virus infection causes testicular atrophy and significantly decreases spermatic function and fertility rates. In one study of a Zika virus DNA vaccine, the adverse effects in the male reproductive system were prevented by vaccination. The question of whether Zika virus can adversely affect human reproductive potential and, if so, whether such effects would be age-related is unknown.
Females may also excrete Zika virus RNA for extended times. A prospective study of five women showed that Zika RNA was detected in vaginal fluid for as long as 6 months and a month or more in three of the five. Murine studies showed that Zika virus caused infection of the ovaries of non-immunosuppressed C57Bl/6 mice and induced a T-cell inflammatory reaction, but without affecting reproductive potential. In contrast to the above human study, female macaques rapidly cleared virus from the genital tract.
Thus, while Zika virus infection is mildly symptomatic and self-limited for the vast majority of individuals, with infrequent neurologic complications in adults, vaccination of the general population may not be warranted. However, vaccination of females at or entering reproductive age and their male partners is prudent. As discussed previously, vaccination during pregnancy is non-ideal due to the time to generate protective immunity and unknown vaccine safety. The one unknown aspect is whether vaccination of males of any age may be beneficial to protect against testicular complications. Calls for generalized vaccination programs have, however, been put on hold due to the decreasing incidence of Zika infection rates, as discussed in the next section.
For the epithelial, outer-body viruses it turned out that the length of the infection and shedding periods, as well as the virus environmental survival rate generally increased from respiratory tract to alimentary tract to skin. The respiratory viruses transmitted on the basis of aerosols, direct contact or fomites. Alimentary tract viruses were found to transmit on the basis of a fecal-oral cycle, through direct contact, contamination of feed and water, or involving fomites, persons and vehicles. Viruses infecting both respiratory and alimentary tract featured a mix of these transmission modes. Mostly, these viruses caused rather severe infections. Among the skin viruses, the more infiltrative viruses affecting all layers of the skin caused slowly healing lesions. The transmission of these deep-rooted skin viruses was found to rely on abrasion or biting flies rather than on direct touch or on indirect contact, more typical for superficial skin lesions. Some of the epithelial viruses are shed in feces over a prolonged time period, also in the absence of clinical signs, and these infections were considered to feature a systemic component. Next, the epithelial herpesviruses establishing latently in peripheral nerves and ganglia were found to cause a recurrence or persistence of the mucosal and/or skin infection, including of the distal urogenital tract and external genitalia.
The influenza pandemic from 1918 to 1919 was the most devastating infectious disease pandemic ever documented in such a short period of time, killing nearly 50 million people worldwide. Unlike the epidemiological profiles of most influenza infections, young adults aged 18–35 yrs old had the highest mortality rate, so much so that the average life expectancy during those years was lowered by 10 years. In 1918, severe destruction of lung tissue observed by pathologists at autopsy was unlike that typically seen in cases of pneumonia and histopathological analysis of lung tissue showed severe tissue consolidation with unique destruction of the lung architecture,. Human infections with highly pathogenic avian influenza (HPAI) strains of subtype H5N1 since the first outbreak in 1997 have also been particularly severe for children and young adults–. Assessing pulmonary infiltrates in response to influenza H5N1 virus infection has been difficult due to the lack of autopsy material. The basis for the high morbidity and mortality associated with the 1918 virus and recent H5N1 viruses remains inconclusive based on viral genetic analysis alone and accounts of patient lung pathology provide only qualitative information about the host factors contributing to disease,,. Great concern about a pandemic caused by a novel avian H5 subtype virus warrants comparative studies to better understand the cellular pathology caused by a pandemic virus and potentially pandemic viruses. Identification and quantification of the inflammatory cell types associated with highly pathogenic respiratory infections represent prospective targets for modulation of host innate immune responses.
Recent studies using animal models to investigate the mechanism(s) of severe influenza virulence have implicated the innate immune system in complicating lung tissue recovery–. Mouse models of highly pathogenic (HP) H5N1– and 1918, influenza virus infection confirm histological observations of severe lung pathology in human patients, however, the types of immune cells present during the peak of lung pathology have not been fully elucidated. Excessive immune cell infiltration during an acute lung injury may impair tissue restoration directly by interfering with gas exchange, or indirectly through the release of soluble immune mediators. In the present study, we determined key immune cellular components in the murine lung following infection with matched H5N1 and H1N1 virus pairs that represent high and low virulence infections of each influenza subtype as previously determined in the mouse model,. The two H5N1 viruses used in this study (A/Thailand/16/2004 and A/Thailand/SP/83/2004) were isolated in 2004 from fatal human cases in Thailand but have a differential pathogenic outcome in mice, specifically a low and high mouse lethal does 50 (LD50 = 1.7 and 5.6 log10 PFU respectively). For relevant comparison, we also used a contemporary (non lethal) seasonal H1N1 human isolate from 1991 (A/TX/36/91) and the reconstructed 1918 pandemic virus. A detailed flow cytometry evaluation of lung cells demonstrated that macrophages and neutrophils are the prominent cell types associated with and potentially mediating the severe lung pathology following infection with the highly virulent H5N1 and 1918 viruses. Moreover, inoculation of macrophages and dendritic cells with the HP viruses in vitro or ex vivo reveals that some innate immune cells can themselves serve as targets of viral infection.
Vaccinia virus (VV), a member of the Poxviridae family, is an enveloped, DNA virus with a genome of 192 kb encoding about 200 proteins. Various cell lines can be infected by VV, including HeLa, CV-1, mouse L, and chicken CEF cells. VV causes major changes in host cell machinery shortly after infection, and cytopathic effects (CPE) are observed several hours after infection with VV. VV infection modulates host cell gene expression: several previous studies have shown that mRNA synthesis in the host cells was inhibited immediately after VV infection. Microarray analysis showed that around 90% of the host genes were down-regulated after VV infection, including genes involved in DNA replication, transcription, translation, apoptosis, and the proteasome-ubiquitin degradation pathway. Only a smaller fraction of host genes were up-regulated after VV infection, including WASP protein, and genes implicated in immune responses.
Several viral factors of VV utilize ATP and several steps in viral multiplication of VV require ATP. ATP is also required for DNA packaging and capsid maturation of herpes simplex virus, for capsid assembly and release of type D retrovirus, for capsid assembly of human immunodeficiency virus, and for budding of influenza virus. Therefore, it was expected that viral factors would modulate cellular energetics to benefit the virus, though this area is understudied.
In this study, the possible up-regulation of host cell genes after VV infection was analyzed by differential display-reverse transcriptase-polymerase chain reaction (ddRT-PCR), a simple technique with high sensitivity and specificity . Two mitochondrial genes involved in the electron transport chain (ND4 and COII) to generate ATP were found to be up-regulated after VV infection using this assay.
Given its continuous exposure to the outside environment, the respiratory mucosa is highly susceptible to viral infection. The human respiratory tract can be infected with a variety of pulmonary viruses, including respiratory syncytial virus (RSV), influenza virus, human metapneumovirus (HMPV), rhinovirus (RV), coronavirus (CoV), and parainfluenza virus (PIV) (1). The severity of disease associated with respiratory viral infection varies widely depending on the virus strain as well as the age and immune status of the infected individual. Symptoms can range from mild sinusitis or cold-like symptoms to more severe symptoms including bronchitis, pneumonia, and even death. RSV is the leading cause of severe lower respiratory tract infection in children under 5 years of age (2). RSV is commonly associated with severe lower respiratory tract symptoms including bronchiolitis, pneumonia, and bronchitis and is a significant cause of hospitalization and mortality in children, the elderly, and immunocompromised individuals (2–6). Similarly, PIV commonly infects children and is a major cause of croup, pneumonia, and bronchiolitis (7, 8). Seasonal influenza infections, most often of the influenza A virus (IAV) subtype, are responsible for 3–5 million cases of severe infection annually (9). Seasonal IAV infections also result in approximately 290,000–650,000 deaths per year, most commonly in either young children or elderly populations (9–11). However, infection with emerging pandemic IAV strains, such as the 2009 H1N1 pandemic strain, primarily induces severe disease and mortality in otherwise healthy adults younger than 65 years of age (12). In contrast, respiratory infection with HMPV, RV, and CoV are most commonly associated with symptoms of the common cold (13–15). Two notable exceptions are severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome (MERS) CoV, which cause acute respiratory distress and mortality in infected individuals (16–18).
Despite their profound impact on human health, most common respiratory viruses lack an approved vaccine. The strategy employed most often in vaccine development is the induction of robust neutralizing antibody responses. However, the hallmark of many respiratory viral infections, including RSV, HMPV, and RV, is the ability for reinfections to occur frequently throughout life (19–21). This suggests that the antibody response to these respiratory viruses may wane over time. Indeed, despite a correlation between pre-existing nasal IgA and protection from reinfection, the development of long-lasting RSV- and RV-specific mucosal IgA responses was poor in infected adults (22, 23). Although IAV-specific neutralizing antibodies are elicited efficiently through either infection or vaccination, IAV vaccine formulations must be redeveloped annually to account for the rapid mutations of HA and NA genes in seasonal strains (24). Therefore, vaccinations that solely promote the induction of neutralizing antibodies may not be optimal in providing protection against many respiratory virus infections. The induction of cellular immune responses has thus far received little attention in respiratory virus vaccine development. CD8 T cells play a critical role in mediating viral clearance following many respiratory virus infections including RSV, IAV, and HMPV (25–27). In addition, recent murine studies utilizing CD8 T cell epitope-specific immunization strategies observed significantly reduced lung viral titers following IAV, RSV, or SARS challenges (28–30). Therefore, the induction of virus-specific CD8 T cell responses has the potential to improve upon the efficacy of current vaccination strategies. Here, we review the current literature on CD8 T cell responses following respiratory virus infections and discuss how this knowledge may best be utilized in the development of future vaccines.