Inhibition of Hazara nairovirus replication by small interfering RNAs. Part 3
HAZV titration
Monolayers of Vero E6 cultured in 12-well microplates were infected with serial 10-fold dilutions of supernatant from infected cells. After 1 hr incubation at 37°C, 3.2% carboxymethylcellulose (CMC) sodium salt (VWR International Ltd, Poole, England) were added into each well. CMC overlay was removed five days post-infection and cells were fixed with 4% formaldehyde for 20 min at room temperature (RT) and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich, St Quentin-Fallavier, France) for 5 min. Viral foci were detected by probing with mouse anti-HAZV hyperimmune ascitic fluid (1:2000) for 1 hr at 37°C, followed by horseradish peroxydase-conjugated goat anti-mouse IgG (1:2000, Interchim, Montluçon, France) at 37°C for 1 hr. The cell monolayer was then incubated with 0.7 mg/ml of 3,3′-diaminobenzidine (DAB) solution (Sigma-Aldrich, St Quentin-Fallavier, France) diluted in PBS 1X for 10 min at RT. Once clusters of infected cells were visible (dark stain), the reaction was stopped by removing the DAB solution followed by water washing. The foci were counted manually under the light microscope.
Design and synthesis of siRNAs
The sequences of HAZV L, M and S genomic segments [GenBank:DQ076419.1, DQ813514.1 and M86624.1, respectively] were used to design the siRNAs. Duplexes of 21-nucleotide siRNAs with short 3′ overhangs were synthesized by Qiagen (Courtaboeuf, France). For each viral mRNAs, four lyophilized siRNAs were produced (Table 1) and their sequence was subjected to a BLAST search against GenBank to minimize off-target effects. In the present study, a non targeting siRNA (siNT) showing no complementarities neither with HAZV mRNAs nor with any human, mouse or rat mRNAs was used as a negative control. The TOX siRNA (siTOX) (Dharmacon RNAi technologies, Lafayette, USA) was used to determine transfection efficacy (see below). Before use, all siRNAs were reconstituted in rehydration buffer to obtain 20 μM solutions according to the company’s instructions.
Twenty four hours before transfection, A549 cells were seeded in 24-well microplates at a density of 8 × 104 cells/well to achieve 60% confluent cell monolayers the day after. Various siRNAs concentrations (ranging from 0.01 to 100 nM) were complexed with the Lipofectamine 2000 transfection reagent (Invitrogen, Cergy Pontoise, France) in Opti-MEM I medium (Gibco, Invitrogen Corporation, Paisley, United Kingdom). The final volume of Lipofectamine 2000 was 1.5 μl/well. The transfection mixture was incubated for 20 min at RT to allow the formation of siRNA/transfection reagent complexes and 100 μl of the solution were added in each well. One day post-transfection, cells were gently washed twice with F12K medium and infected with HAZV at a MOI of 0.1. The inoculum was incubated for 1 hr. Cells were then cultivated in F12K medium supplemented with 0.4% FCS for 48 hrs. Infected cells supernatants were tittered as described above. The EC50 was calculated as the mean of two independent experiments using the GraphPadPrism version 4.00 software (GraphPad Software, San Diego California, USA) for non linear regression.
For the post infection treatment studies, 60% confluent A549 cell monolayers were infected with HAZV for 1 hr at a MOI of 0.01. At 1 hr, 8 hrs or 24 hrs post-infection, 100 μl of the transfection mixture (containing 100 nM of siRNA) were added. One day after transfection, cells were washed, and grown in F12K medium with 0.4% FCS for 48 hrs. The supernatant from infected cells were then harvested and tittered.
For each experiment, transfection efficiency was monitored by transfecting A549 cells with 100 nM of siTOX under the same experimental conditions as described above. Cells successfully transfected with siTOX undergo apoptosis and cell death within 24-48 hrs. After 3 days of incubation, siTOX-transfected cells were trypsinized and manually counted using a hematocytometer (Trypan blue exclusion assay). Transfection efficiency was calculated as the ratio between the number of viable siTOX-transfected cells versus non-transfected cells. In our experiments, transfection efficiency was routinely above 90%.
Detection of HAZV nucleoprotein by Western blot
A549 cells, seeded in 6-well microplates at a density of 3.2 × 105 cells/well, were transfected with siRNA and infected with HAZV as described above. Protein extraction was performed 48 hrs post-infection as follow: confluent cells were washed twice in fresh phosphate-buffered saline 1X (PBS 1X) and lysed in buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 0.6% NP40, 0.5 mM EDTA and protease inhibitors cocktail (Complete EDTA-free, Roche Diagnostics GmbH, Mannheim, Germany) for 10 min on ice. After removal of cellular debris by centrifugation at 12,000 × g for 10 min, 20 μl of protein extracts were boiled for 5 min in Laemmli buffer and separated on a 10% SDS-PAGE. Proteins were then electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Marne-la-Coquette, France). The PVDF membrane was saturated with 5% dry milk in PBS 1X containing 0.1% Tween-20 and incubated overnight at 4°C with mouse anti-HAZV hyperimmune ascitic fluid (1:250) or with mouse anti-GAPDH monoclonal antibody (1:1000, Ambion, Austin, TX, USA). Horseradish peroxydase-labeled goat anti-mouse IgG (1:10000, Interchim, Montluçon, France) was used as secondary antibody followed by chemoluminescent (ECL) revelation (Amersham GE Healthcare, Orsay, France).
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ELISA-based assay for interferon-β detection
A549 cells were cultured in 24-well microplates at a density of 8 × 104 cells/well and transfected as previously described with either 100 nM (i.e. 1.48 μg/ml) of siNT, siS1 and siS2 or 0.25 μg/ml of poly(I:C) dsRNA (Sigma-Aldrich, St Quentin-Fallavier, France). Cell culture supernatants were harvested 24 hrs post-transfection to detect human beta interferon (IFN-β). The cytokine measurement was performed using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (PBL Biomedical Laboratories Tebu-Bio, Le Perray-en-Yvelines, France), according to the manufacturer’s instructions.
Antiviral assays with ribavirin and combination with siS1 or siS2
Confluent monolayers of A549 cells in 24-well plates were infected with HAZV at a MOI of 0.1. Cells were then cultivated with F12K medium supplemented with 0.4% FCS or treated with 0.4% FCS F12K medium containing serial dilutions of ribavirin (1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) (Sigma-Aldrich, St Louis, Missouri, USA). Forty eight hours post-infection, the supernatant of each well was collected and virus titer was performed. The EC50 value for ribavirin was determined as the mean of two independent experiments.
The combination assay required 60% confluent monolayers of A549 cells in 24-well microplates transfected with siS1, siS2 or siNT at a concentration of 1 nM and 10 nM. One day post-transfection, cells were washed twice, infected with HAZV at a MOI of 0.1. One hour after infection, the inoculum was removed and transfected cells were cultured for 48 hrs in 0.4% FCS F12K medium containing 0, 25 or 50 μM of ribavirin. The cell supernatants were then tittered.
Statistical analysis
In this study, we compared the antiviral effect of selected siRNAs to the negative control (siNT) to detect significant variations using the Student’s t-test (P ≤ 0.05 was regarded as significant difference between the two groups of transfected/infected cells).
Inhibition of Hazara nairovirus replication by small interfering RNAs
Background
The genus Nairovirus in the family Bunyaviridae contains 34 tick-borne viruses classified into seven serogroups. Hazara virus (HAZV) belongs to the Crimean-Congo hemorrhagic fever (CCHF) serogroup that also includes CCHF virus (CCHFV) a major pathogen for humans. HAZV is an interesting model to study CCHFV due to a close serological and phylogenetical relationship and a classification which allows handling in a BSL2 laboratory. Nairoviruses are characterized by a tripartite negative-sense single stranded RNA genome (named L, M and S segments) that encode the RNA polymerase, the Gn-Gc glycoproteins and the nucleoprotein (NP), respectively. Currently, there are neither vaccines nor effective therapies for the treatment of any bunyavirus infection in humans. In this study we report, for the first time, the use of RNA interference (RNAi) as an approach to inhibit nairovirus replication.
Results
Chemically synthesized siRNAs were designed to target the mRNA produced by the three genomic segments. We first demonstrated that the siRNAs targeting the NP mRNA displayed a stronger antiviral effect than those complementary to the L and M transcripts in A549 cells. We further characterized the two most efficient siRNAs showing, that the induced inhibition is specific and associated with a decrease in NP synthesis during HAZV infection. Furthermore, both siRNAs depicted an antiviral activity when used before and after HAZV infection. We next showed that HAZV was sensitive to ribavirin which is also known to inhibit CCHFV. Finally, we demonstrated the additive or synergistic antiviral effect of siRNAs used in combination with ribavirin.
Conclusions
Our study highlights the interest of using RNAi (alone or in combination with ribavirin) to treat nairovirus infection. This approach has to be considered for the development of future antiviral compounds targeting CCHFV, the most pathogenic nairovirus.
Background
Hazara virus (HAZV) is a member of the genus Nairovirus of the family Bunyaviridae which also includes Orthobunyavirus, Hantavirus, Phlebovirus and Tospovirus. Nairovirus comprises 34 tick-borne viruses classified into seven serogroups. The main representative serogroups are the Nairobi sheep disease group containing Nairobi sheep disease virus (NSDV) and Dugbe virus and the Crimean-Congo hemorrhagic fever (CCHF) group including HAZV and Crimean-Congo hemorrhagic fever virus (CCHFV). While NSDV induces acute hemorrhagic gastroenteritis in sheep and goats, CCHFV is responsible of severe hemorrhagic fever in humans associated with elevated levels of mortality (up to 50%). Due to its high pathogenicity for humans and because of the lack of therapeutics, CCHFV must be handled in BSL4 (biosafety level 4) laboratory. Widely distributed throughout Eastern Europe, Asia and Africa, CCHFV represents a major public health problem and is now considered as an emerging disease. HAZV was isolated for the first time in 1954 from Ixodes ticks collected in Pakistan. Although its natural host is not known, antibodies against HAZV were detected in rodent sera. While non pathogenic for humans, it is lethal in new-born mice and elicits cross-protection against CCHFV challenge in adult mice. HAZV represents an alternative model to study CCHFV due to its close serological and phylogenetical relationship. Furthermore, it can be handled in BSL2 laboratories.
Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus. Discussion
Discussion
Infectious bronchitis (IB) is one of the most common and difficult-control poultry diseases in China, caused persistent but infrequent outbreaks in commercial chicken farms. Commercial vaccines based on H120, H52, 28/86, Ma5, W93 and M41 strains, have been widely used to control the disease. Natural outbreaks of IBV often are the result of infections with strains that differ serologically from the vaccine strains. Come to the rapid and complicated evolutionary of IBV, it is imperative to learn profoundly the circulating IBVs, facilitate selecting the candidate vaccine strain against the infections.
In this study, 80 IBV strains were isolated from the vaccinated chicken flocks, with a wide age range of IB outbreak. The chickens infected before the age of 5 days which might be caused by the vertical transmission of IBVs or the maternal antibody could not provide pertinent protection against the prevalent strains. Furthermore, there was accumulating evidence indicated that the nephropathogenic IBVs have become prevalent in China in last several years. Through clinical records and the virus recovery trials, 70 identified isolates mainly caused typical swollen kidney, different from the respiratory type strains isolated in earlier years, including the major vaccine strains. These findings indicated that all 80 isolated IBV strains from China during 2008-2009 were evolutionarily distant from the vaccine strains used for current, resulting in vaccination failure cases.
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The S1 protein determined the serotypic evolution, the phenotype change and the genetic diversity of IBVs. In the present study, nucleotide and derived amino acid sequences of S1 protein genes of the 80 field strains were aligned and compared to the representative strains, to determine the relationship of circulating field isolates, vaccine strains and previously described variant strains. Newly isolated strains shared between 75.4% to 100% nucleotide sequence similarity with each other, higher similarity than the vaccine strains and other representative IBVs. Although the IBVs all over the world shared some common antigenic types, virus strains within a geographic region were unique and distinct, even in different provinces of China. The variants were mostly located in the first 300 amino acids in the N-terminal of the S1 protein of IBV, even though the mutants consisted of insertions, deletions and point mutations were complicated and detailedly different, the hypervariable regions in S1 protein in this study were similar to previous studies.
The phylogenetic analysis showed that there were five subgroups of IBVs co-circulating in China, and multiple strains might cause the constant IB outbreaks. The newly isolated strains were mostly derived from A2, 4/91 and HN08. Only CK/CH/Chongqing/0908 belonged to the branch of Gray. The phylogenetic distributions were closely relative to geographical factors. Most of the recently isolated IBVs in this study formed the distinct cluster related to the A2 type. However, the routine vaccine strains mainly belong to M41-type branch. A2 strain is closely related to 4/91 serotype, spreading over Europe since its first isolation in UK in 1991. In this study, 61.3% (49/80) field isolates belonged to the A2-type branch, which included 85.7% (42/49) nephropathogenic field isolates of this study. The QXIBV, first isolated in China and reported associated predominantly with various forms of renal pathology in China, was also representative A2-type strain. The analysis results were according to the prevalence of nephropathogenicity IB. To date, the QX-like IBV strains have been widely isolated in many European countries, and become a dominant genotype. Through IB surveys, the European QX-like IBV strains have been reported that caused 86% respiratory signs, 22% litter or enteric problems, only 2% had swollen kidneys. Absorbingly, the QX-like IBV strains have undergone divergent evolution paths, brought out different variants in Europe and China. Similarly, seven exceptional strains located in the A2-type branch caused evident respiratory problems, including three isolates from Zhejiang province (QZ1, QZ2 and QZ3) and three isolates from Guangdong province (XD2, XD3 and LZ2), and GL from Guangxi province. The results of our study indicated the strain grouping, such as phenotype and genotype, were not only depended on the geographical factors. The evolutionary pace and the epidemiology characteristics of the IBV were complicated.
Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus. Mutation analysis
Mutation analysis
S1 genes of the newly strains contain mutations, insertions and deletions, resulting in different lengths of nucleotides. S1 genes of these strains were generated and confirmed from three time sequencing results, contained 1641, 1647, 1650, 1653, 1656, 1659 and 1662 nucleotides, amino acids sequences ranging from 547 (LC strain) to 554 (LC strain). The length differences indicated amino acid insertions and deletions exist among the different strains.
Most variations in the deduced amino acid sequences of Chinese IBVs were observed among residues 63-69, 211-212 and 354-358 (numbering was with reference to S1 sequence of the Mass41 strain).
The precursor protein of S glycoprotein is cleaved into amino-terminal S1 and S2 protein by the protease during viral maturation [9]. In this study, the most common cleavage recognition sites of S1 gene were RRF(S/L) RR (49/80) or HRRRR (28/80) in the China field strains. The exceptional ones included CQ8 (RRTGR), HY52 (RRSKR), and HY2 (RRSKR). The cleavage sites of these two strains containing amino acids K, T, and G, were novel motifs compared to the reference strains, and quite different with the other isolates of the cleavage site.
Phylogenetic analysis of the isolated strains
A phylogenetic tree was constructed from the nucleotides sequences of the S1 glycoprotein genes. The 80 isolates IBV strains were clustered into five distinct genetic groups or genotypes which were considerably heterogeneous, including A2-type (49 newly isolated strains), 4/91-type (9 newly isolated strains), HN08-type (20 newly isolated strains), Gray-type and M41-type. The newly isolated strains mainly belonged to A2-type, 4/91-type and HN08-type branch. The phylogenetic relationship of strains at different times and geographical regions displayed complexity and diversity.
Strains isolated from Hubei, Zhejiang, Jiangsu, Guangdong, Guangxi and Fujian province mainly belonged to the A2 branch, also including other seven published IBV strains from China (QXIBV, CK/CH/LJL/07II, CK/CH/LJS/07IV, CK/CH/LSD/08-12, IBVSX4, LZ05 and LZ07). The isolated strains of Hainan province and a few isolated strains from Guangdong and Fujian province belonged to the HN08 branch, included PSH050513 and CK/CH/LCQ/08II. Group Gray-type was correlative with the American strain (Gray), included other two classical American strains (ARK99 and Holte), one Japanese strain (JP9758), and the exceptional field strain (CQ08). Most of the current vaccine strains (H120, H52, Ma5, M41, W93, 4/91 and 28/86) were belonged to the M41 branch, which including one field strains (NJ). However, the current pandemic strains were mostly 4/91-type, A2-type (QXIBV-type) and HN08-type, indicating that the field IBVs co-circulating in chicken flocks in China were evolutionarily distant from the known vaccine strains.
Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus. Part 2
It was documented that nephropathogenic type IB has become more and more prevalent in China. The unprecedented economic losses caused by the nephropathogenic IB suggested that selecting the appropriate vaccine strain against the IB outbreaks is of great importance. However, the integrated natures of novel circulating IBV strains in mainland China were not well-learned.
The previous study by other researchers has been revealed that the variation in S1 sequences was closely confirmed relative to the emergence of novel strains, and S1 gene sequence was a good predictor of challenge of immunity in chickens. This study was conducted to identify the IBV strains that have escaped immune defenses conferred by vaccination in China. The genetic characterization of recent IBV field isolates in China was performed by sequencing the whole S1 genes, sequence alignment and phylogenetic analysis compared with other reference strains.
Results
Eighty IBV strains isolated during 2008-2009 in China
From unhealthy birds suspected of IBV infection in the vaccinated chicken flocks from Guangdong, Guangxi, Fujian, Hainan, Jiangsu, Zhejiang, Chongqing, Hubei, Sichuan and Jiangxi province of China, 80 filed IBV strains were isolated during 2008-2009. The isolation rates in the two years were season-dependent to some extent, 30 strains were isolated in October, while only seven strains were isolated in summer (from June to August). The ages of flocks at the time of the outbreak varied between 4 and 69 days. Most of the strains were isolated from the chickens between 10 to 30 days of age.
After three passage propagation, IBVs of all isolates induced peripheric lesions and growth retardation of embryo at 72 h post-inoculation. Since the fourth day post-inoculation, most of the chicks were listless and huddled together, showed ruffled feathers. The results of virus recovery in chicks indicated 87.5% (70/80) isolates caused serious kidney lesions, which were presented with swollen specked kidney and distended ureters filled with uric acid were nephropathogenic type, and the other ten isolates in the study caused respiratory system signs, which were consistent with the clinical record of each strain.
Homologies among S1 nucleotide and deduced amino acid sequences
The obtained strains were characterized phylogenetically by nucleotide sequence analysis of the hyper-variable S1 gene of IBV. The nucleotide and amino acid sequence similarities between the eighty IB strains were ranging from 75.4% (strain CQ8 and HY) to 100% (strain PT1 and PT3) and 73.9% to 100%, respectively. Compared to the 28 reference strains published in the GenBank, the identity of the nucleotide and amino acid sequence among the 108 isolates (including the 80 isolates in this study plus the 28 reference strains) were 75.1 to 99.8% and 73.1 to 99.8%, respectively, indicating low homology and high variation among the isolated and reference strains.
Infection of human monocyte-derived dendritic cells. Part 2
The hemorrhagic viruses, including the members of the Bunyaviridae as well as dengue viruses, target endothelial cells and immune cells, mainly monocyte-derived cells such as the professional antigen-presenting cells, Dendritic cells (DCs). DCs activation triggers their maturation and trans-endothelial migration occurring during wound healing or inflammation. These processes require extracellular matrix remodeling and involve changes in endothelial permeability regulated by the production of matrix metalloproteases (gMMPs) or vascular endothelial growth factor (VEGF). However, in excess, these soluble factors can have deleterious effects on endothelial cell integrity. Data from different reports show that endothelial cells infected by dengue virus trigger secretion of soluble factors such as VEGF and the decrease of VEGF-R2 receptor. We have recently reported in vitro and in vivo showing that soluble factors secreted from DV-infected DCs enhance endothelial permeability and down-regulate expression of endothelial junction proteins, Pecam-1 and VE-cadherin in a gMMP-9-dependent manner. More recently, complementary and convergent studies, to our own previous data on dengue, have reported that Hantavirus-infected endothelial cells enhances the permeability via the reduction of VE-cadherin expression due to its dissociation with VEGF-receptor2 (VEGF-R2) which, in turn, become associated with VEGF. An accurate understanding of Hantavirus pathogenesis is pivotal to design de novo therapeutic or vaccine approaches that are still lacking against this hemorrhagic viral infection. In this study, we show that ANDV-infected DC are quickly activated and rapidly progress to an intermediate maturation and pro-inflammatory state that contributes to the increase of soluble factors in their supernatant able to trigger the enhancement of endothelial permeability.
Methods
Virus and cells
The primary isolate, ANDV strain CHI-7913 was propagated in the epithelial Vero-E6 cell line (ATCC CRL 1586). Titrated supernatants of these cells were used to infect, at a MOI of 1 for 2 h, human iDCs derived from peripheral blood monocytes (PBMC), as previously described. In these experiments, UV (λ: 250 nm; 15 min)-irradiated ANDV was used as the negative control. Four days post-DC infection, ANDV N-protein was detected by indirect immunofluorescence (IFA) using a well characterized anti-ANDV N monoclonal antibody (MAb). Total RNA was extracted using the High Pure viral nucleic acid kit (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacture’s protocol and 1 μl of total RNA was amplified in a one step RT-PCR (SuperScript III One-Step RT-PCR with Platinum Taq, Invitrogen) using primers that recognize the nucleocapsid coding region (forward primer: 5′ ACA CGA ACA ACA GCT CGT GAC ‘3 and reverse primer: 5′ AGG CTC AAG CCC TGT TGG ATC ‘3). To assess the viral infectivity, from ANDV-positive DCs, their supernatants were used to infect Vero-E6 cells.
Infection of human monocyte-derived dendritic cells
Background
Andes virus (ANDV), a rodent-borne Hantavirus, is the major etiological agent of Hantavirus cardiopulmonary syndrome (HCPS) in South America, which is mainly characterized by a vascular leakage with high rate of fatal outcomes for infected patients. Currently, neither specific therapy nor vaccines are available against this pathogen. ANDV infects both dendritic and epithelial cells, but in despite that the severity of the disease directly correlates with the viral RNA load, considerable evidence suggests that immune mechanisms rather than direct viral cytopathology are responsible for plasma leakage in HCPS. Here, we assessed the possible effect of soluble factors, induced in viral-activated DCs, on endothelial permeability. Activated immune cells, including DC, secrete gelatinolytic matrix metalloproteases (gMMP-2 and -9) that modulate the vascular permeability for their trafficking.
Methods
A clinical ANDES isolate was used to infect DC derived from primary PBMC. Maturation and pro-inflammatory phenotypes of ANDES-infected DC were assessed by studying the expression of receptors, cytokines and active gMMP-9, as well as some of their functional status. The ANDES-infected DC supernatants were assessed for their capacity to enhance a monolayer endothelial permeability using primary human vascular endothelial cells (HUVEC).
Results
Here, we show that in vitro primary DCs infected by a clinical isolate of ANDV shed virus RNA and proteins, suggesting a competent viral replication in these cells. Moreover, this infection induces an enhanced expression of soluble pro-inflammatory factors, including TNF-α and the active gMMP-9, as well as a decreased expression of anti-inflammatory cytokines, such as IL-10 and TGF-β. These viral activated cells are less sensitive to apoptosis. Moreover, supernatants from ANDV-infected DCs were able to indirectly enhance the permeability of a monolayer of primary HUVEC.
Conclusions
Primary human DCs, that are primarily targeted by hantaviruses can productively be infected by ANDV and subsequently induce direct effects favoring a proinflammatory phenotype of infected DCs. Finally, based on our observations, we hypothesize that soluble factors secreted in ANDV-infected DC supernatants, importantly contribute to the endothelial permeability enhancement that characterize the HCPS.
Background
Hantaviruses are rodent-born enveloped RNA-viruses belonging to Bunyaviridae family. Two major severe pathologies associated to Hantaviruses have been reported: hemorrhagic fever with renal syndrome (HFRS) in the Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. HCPS is more frequently associated (40%) to fatal outcomes than HFRS (<1%). Andes Hantavirus (ANDV) is the major etiological agent of the HCPS in South America, syndrome characterized by the presence of high amounts of pulmonary fluids leading to an edema evolving to a cardiogenic shock that synergistically acts with hypovolemia due to capillary leakage resulting in an abrupt cardiopulmonary collapse. Although disease severity directly correlates with the viral RNA load [3], considerable evidence exists suggesting that immune mechanisms rather than direct viral cytopathology are indeed responsible for the massive vascular dysfunction and plasma leakage of HFRS and HCPS.
Ultra-violet radiation is responsible for the differences in global epidemiology of chickenpox. Part 3
In Australia widespread preventative measures are taken limit exposure to UVR in schools by having large, shaded playground areas.
In urban Brazil, man-made biomass burning and in rural areas, the forest canopy and high humidity act together to reduce UVR.
In the Congo, the first ever demonstration of transmission of temperate virus, occurred in only one family all living in the same house, the implication being that temperate virus is rapidly inactivated by UVR after leaving the confines of the family home.
Finally, the detection of temperate virus genotypes from cases of chickenpox in Mexico City may be explained because it is one of the most heavily polluted cities in the world which reduces UVR, allowing temperate genotypes to survive.
Proving the hypothesis
The hypothesis is biologically plausible because UVR is virucidal against many viruses, yet the effect of UVR on survival of VZV in vitro has never been tested. However, the effect of UVR on virus transmission in vivo was demonstrated over 60 years ago when artificial UVR was used successfully to reduce virus transmission in US schools to limit spread of chickenpox. Epidemiological evidence to support the hypothesis could be provided by correlating the transmission of different virus genotypes with ambient UV radiation. Genotyping VZV in cases of chickenpox could determine if there are seasonal differences in genotype transmission in temperate areas. The hypothesis would predict that tropical virus genotypes should predominate during summer in temperate countries since they would have the selective advantage of increased resistance to UVR.
If different genotypes of VZV possess different tolerances to UVR this could be demonstrated in vitro by exposing virus to UVR and quantifying the surviving virus by either plaque forming units or quantitative mRNA RT-PCR. Finally, it may also be possible to make hybrid viruses by exchanging those regions of the VZV genome which are significantly different between genotypes and determine for the first time the molecular markers that underlie transmission or reactivation of VZV.
Implications of the hypothesis
The principal difficulty with the hypothesis is explaining how an ancestral tropical virus genotype, inherently more resistant to UVR, migrated with man out of Africa 200,000 years ago only to lose the selective advantage of resistance to UVR, form a temperate virus genotype lineage and as result become less transmissible. The solution to this paradox could be that loss of the selective advantage of resistance to UVR and reduced transmissibility was offset by an increased propensity to reactivate as zoster. This could indicate that the areas of the VZV genome which confer resistance to UVR are the same as are involved in latency and reactivation.
I suspect this to be the case because as the transmission environment is so harsh in the tropics, random mutation and natural selection should have brought about a tropical virus genotype which reactivates much more frequently to counter-act the lower transmissibility of chickenpox. The fact that the data on zoster epidemiology from tropical countries (in the pre-AIDS era) are virtually absent suggests that the tropical genotype reactivates only in severely immune suppressed individuals. Potentially it may have implications for VZV vaccine since if it was made from a tropical genotype which reactivated much less frequently, it might be possible, in years to come, to significantly reduce the disease burden from zoster.
Ultra-violet radiation is responsible for the differences in global epidemiology of chickenpox. Part 2
Background
Chickenpox epidemiology is unique among human herpes viruses. In the tropics primary infection is often delayed into later childhood whereas in temperate zones most infection occurs before leaving school. Indeed, in some tropical countries 30-50% of adults are susceptible, compared with only 5-10% from temperate areas.
Conventionally, transmission has been considered to occur by shedding of virus from the upper respiratory tract 1-2 days before the rash. The papers which claim to show such virus transmission however, also conclude that the titres of virus in vesicular fluid are considerably greater than those present in the pharynx and that vesicular virus makes the greatest contribution to spread. Indeed, the few papers cited as providing epidemiological evidence for airborne spread are either mis-quoted, based on case reports or do not reflect the normal transmission environment. In this regard chickenpox appears similar to smallpox, which also had a distinct winter-spring seasonal peak in incidence and was spread partly by the vesicular eruption.
Why such a common, global infection should be less common in children from the tropics when infections are generally more common remains unknown. Although previously suggested factors such as heat, humidity, viral interference, population density or infection with cross-protecting viruses, have been suggested as possible causes of the epidemiological differences, a unified, coherent explanation has eluded discovery. The climatic factor which I propose to show is responsible for the geographical differences in transmission is ultra-violet radiation (UVR). Furthermore, as varicella-zoster virus (VZV) exists only in man, I propose that UVR has been involved in the co-evolution of virus as man migrated out of Africa. The evolution of varying degrees of resistance to UVR among the different genotypes may also have implications for virus reactivation as zoster.
Chickenpox is seasonal in temperate zones, with the highest incidence seen in winter and spring. One explanation for this seasonality could be the significantly higher levels in ultra-violet radiation (UVR) of approximately 10-25-fold seen in summer in temperate zones, which could inactivate virus either in vesicular lesions or after their rupture. Chickenpox is not seasonal in the same way in the tropics possibly because UVR differs only by a factor of two during the year. The tropics however, do experience peaks in chickenpox incidence when the climate is hot, dry and sunny with a rapid decline to very low levels during the rainy season. This appears difficult to reconcile with UVR inactivating virus until the effects of atmospheric pollution on ambient UVR are considered. For example, the Indo-Asian haze, a continent-wide increase in air pollution during the dry season from December to April, has been shown to reduce significantly the level of ambient UVR. As the Monsoon arrives, atmospheric particles and pollutants are washed out, increasing the UVR which inactivates virus more effectively. This correlates very well with the observed chickenpox incidence in Sri Lanka and south India. Furthermore, outbreaks of varicella have been terminated in certain African countries by the arrival of the rainy season. Increased atmospheric pollution might partly explain, in association with locally increased population density, why chickenpox is commoner in urban environments compared with rural communities in adjacent geographic areas.
Further support for the hypothesis derives from sequence analysis which has classified VZV into distinct genotypes. In the largest published study, 348 genotypes of VZV were given geographic locations based on where the virus was originally detected. In the temperate zones which were studied (N America, Argentina, Europe, S Africa, N China, N Asia) a total of 35/259 (13.5%) genotypes were tropical. In contrast, of the 89 isolates from tropical countries/regions (India, Nepal, Bangladesh, Chad, DRC, Southern China, Western Australia, Brazil, Cote D’ Ivoire, Ethiopia, Thailand, Vietnam, Zimbabwe), only 5 (5.6%) were temperate. This difference was statistically significant by Chi-square testing (p < 0.0001). Nevertheless, temperate virus genotypes, which should be more sensitive to UVR than tropical strains, and so would be out-competed in terms of transmission, have been detected in tropical areas, namely Australia, Brazil, Congo, and Mexico City. However, survival of temperate genotypes in these regions is still consistent with the hypothesis when it is considered how reducing ambient UVR allows temperate genotypes to transmit.