Mixed Genotype Infections Research Articles

Correlation between Hepatitis B Virus Genotypes and Clinical Outcomes

Hepatitis B virus (HBV) has been classified into 10 genotypes (A-J) according to genome sequence divergence. HBV genotypes have a distinct geographical distribution. As chronic HBV infection is endemic in the Asian region, genotypes B and C prevail there, and genotypes A and D are mainly found in the western world and Europe. Genotypes A, B, C, and D have been studied most extensively. In Europe and Asia, most patients with genotypes A and B have acute hepatitis B; however, some mutants may tend to cause fulminant hepatitis B. Many studies have indicated that the severity and outcomes of chronic hepatitis B infections are more serious in patients with genotypes C and D than in those with genotypes A and B. Cirrhosis and hepatocellular carcinoma (HCC) are more frequently diagnosed in carriers of genotypes C and D than in those of genotypes A and B. Accumulating evidence indicated that higher plasma HBV DNA levels, infection with HBV genotype C, as well as mutations at 1653T, 1753V, and A1762T/G1764A are independently associated with the risk of HCC in Asian men. However, the therapeutic responses differ with regard to the different HBV genotypes. For example, the response to interferon-α treatment in patients with genotypes A and B was better than that in patients with genotypes C, D, and mixed genotypes. Some studies have shown seroconversion after treatment, i.e., genotypes A and C may switch to genotypes D and B, respectively. Some reports indicated a correlation between the emergence of the hepatitis B e antigen-negative variant in patients with genotypes C and D and worsening of liver injury without sustained response. In order to provide better treatment options for these poorly responding patients, further studies, e.g, novel immunomodulatory therapies, are required. Many studies have shown that HBV genotypes have remarkable clinical and epidemical differences; however, HBV sub-genotypes, mixed genotype infections, and the effect of different genotypes on the treatment of HBV infections require further studies.

Mixed Genotype Infections with Hepatitis C Virus, Pakistan

To the Editor: The prevalence of hepatitis C virus (HCV) infection is high (8% of the population) in Pakistan (1). HCV is an RNA virus that has a high mutation rate. This high rate results in extensive genetic heterogeneity, and HCV isolates are found as either quasispecies or genotypes (2). Humans can be co-infected with >1 genotype (mixed genotype infection) of this virus (3). The rate of HCV mixed genotype infections is extremely variable for different regions and for the same group of patients tested by using different assays (4). Thus, it is difficult to determine the prevalence of mixed genotype infections by currently available assays, including direct DNA sequencing, because they are designed to identify only the HCV genotype dominant in that particular population. Consequently, genotypes present at lower frequencies could be missed or mistyped (5). To determine the prevalence of HCV mixed genotype infections, we retrospectively analyzed genotyping data for paired serum samples from 22,125 HCV-infected patients during the past 11 years (March 2000–May 2010) for all regions in Pakistan by using molecular-based genotype-specific methods (6,7). A total of 12,036 (54.4%) were male patients and 10,089 (45.6%) were female patients. The sensitivity and reliability of the assay we used has been assessed and found to be superior to restriction fragment length polymorphism analysis and serotyping methods for detection of mixed genotypes in a viral population. Our method can detect a small amount (8.3%) of HCV RNA in a mixed genotype population (7). Restriction fragment polymorphism analysis can detect 2 genotypes only if 1 of them represents >41.6% of the genotypes in a mixed genotype population. Of 22,125 HCV RNA-positive serum samples, type-specific PCR bands were observed in 18,181 (82.2%) samples and 3,944 (17.8%) were not typeable. A total of 1,007 (5.5%) patients had HCV mixed genotype infections. The distribution of mixed genotype infections in 1,007 patients is shown in Figure A1. Infection with mixed genotype 3a + 3b was most prevalent (43.79%). Age distribution of patients with mixed genotype infections is shown in the Table. Approximately 33% of patients with mixed genotype infections were 31–40 years of age and 22.5% were 41–50 years of age. Table Distribution of mixed genotype infections with hepatitis C virus in 1,007 patients, by age, Pakistan, March 2000–May 2010 Patterns of HCV mixed genotype infections in Pakistan are similar to those reported from India and Iran (8). However, the prevalence of HCV mixed genotype infections was lower (2%) (8) for Iran than for Pakistan. This lower rate may have been caused by use of a genotyping kit that can detect only genotypes 1a, 1b, 2, and 3a. Thus, mixed infections with other genotypes would not have been detected. A recent study in Brazil reported that mixed genotype infections were detected in 3.9% of intravenous drug users and 7.1% of former injecting drug users (9). These rates were similar to those in our study. In contrast, data from Sweden and Russia showed no mixed genotype infections in serum samples of chronically infected intravenous drug users, hemodialysis patients, and patients with hemophilia (10). Women (288/7,390, 3.89%) in Pakistan had significantly fewer HCV mixed genotype infections than men (719/10,791, 6.66%) (p<0.01). This finding might be the result of women having fewer risk factors for contracting mixed genotype infections. Possible risk factors for infection with mixed genotype infections analyzed were blood transfusions and use of blood products (51.3%); multiple use of needles or syringes (18.4%); sharing razors during shaving or circumcision, piercing instruments, nail clippers, and toothbrushes (13.7%); and major or minor dental surgery (9.5%). Mode of transmission was not clear for 7.1% of the patients. In conclusion, the prevalence of HCV mixed genotype infections in Pakistan is higher than previously reported and higher among men (p<0.01). Comprehensive and detailed investigations are warranted to evaluate the clinical role of chronic HCV mixed genotype infections, provide essential information that can be used to determine type and duration of therapy needed, and predict disease outcome.

One Is Enough: In Vivo Effective Population Size Is Dose-Dependent for a Plant RNA Virus

Effective population size (Ne) determines the strength of genetic drift and the frequency of co-infection by multiple genotypes, making it a key factor in viral evolution. Experimental estimates of Ne for different plant viruses have, however, rendered diverging results. The independent action hypothesis (IAH) states that each virion has a probability of infection, and that virions act independent of one another during the infection process. A corollary of IAH is that Ne must be dose dependent. A test of IAH for a plant virus has not been reported yet. Here we perform a test of an IAH infection model using a plant RNA virus, Tobacco etch virus (TEV) variants carrying GFP or mCherry fluorescent markers, in Nicotiana tabacum and Capsicum annuum plants. The number of primary infection foci increased linearly with dose, and was similar to a Poisson distribution. At high doses, primary infection foci containing both genotypes were found at a low frequency (<2%). The probability that a genotype that infected the inoculated leaf would systemically infect that plant was near 1, although in a few rare cases genotypes could be trapped in the inoculated leaf by being physically surrounded by the other genotype. The frequency of mixed-genotype infection could be predicted from the mean number of primary infection foci using the independent-action model. Independent action appears to hold for TEV, and Ne is therefore dose-dependent for this plant RNA virus. The mean number of virions causing systemic infection can be very small, and approaches 1 at low doses. Dose-dependency in TEV suggests that comparison of Ne estimates for different viruses are not very meaningful unless dose effects are taken into consideration.

Heterogeneous Host Susceptibility Enhances Prevalence of Mixed-Genotype Micro-Parasite Infections

Dose response in micro-parasite infections is usually shallower than predicted by the independent action model, which assumes that each infectious unit has a probability of infection that is independent of the presence of other infectious units. Moreover, the prevalence of mixed-genotype infections was greater than predicted by this model. No probabilistic infection model has been proposed to account for the higher prevalence of mixed-genotype infections. We use model selection within a set of four alternative models to explain high prevalence of mixed-genotype infections in combination with a shallow dose response. These models contrast dependent versus independent action of micro-parasite infectious units, and homogeneous versus heterogeneous host susceptibility. We specifically consider a situation in which genome differences between genotypes are minimal, and highly unlikely to result in genotype-genotype interactions. Data on dose response and mixed-genotype infection prevalence were collected by challenging fifth instar Spodoptera exigua larvae with two genotypes of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), differing only in a 100 bp PCR marker sequence. We show that an independent action model that includes heterogeneity in host susceptibility can explain both the shallow dose response and the high prevalence of mixed-genotype infections. Theoretical results indicate that variation in host susceptibility is inextricably linked to increased prevalence of mixed-genotype infections. We have shown, to our knowledge for the first time, how heterogeneity in host susceptibility affects mixed-genotype infection prevalence. No evidence was found that virions operate dependently. While it has been recognized that heterogeneity in host susceptibility must be included in models of micro-parasite transmission and epidemiology to account for dose response, here we show that heterogeneity in susceptibility is also a fundamental principle explaining patterns of pathogen genetic diversity among hosts in a population. This principle has potentially wide implications for the monitoring, modeling and management of infectious diseases.

Alternative splicing of the Anopheles gambiae Dscam gene in diverse Plasmodium falciparum infections

BackgroundIn insects, including Anopheles mosquitoes, Dscam (Down syndrome cell adhesion molecule) appears to be involved in phagocytosis of pathogens, and shows pathogen-specific splice-form expression between divergent pathogen (or parasite) types (e.g. between bacteria and Plasmodium or between Plasmodium berghei and Plasmodium falciparum). Here, data are presented from the first study of Dscam expression in response to genetic diversity within a parasite species.MethodsIn independent field and laboratory studies, a measure of Dscam splice-form diversity was compared between mosquitoes fed on blood that was free of P. falciparum to mosquitoes exposed to either single or mixed genotype infections of P. falciparum.ResultsSignificant increases in Anopheles gambiae Dscam (AgDscam) receptor diversity were observed in parasite-exposed mosquitoes, but only weak evidence that AgDscam diversity rises further upon exposure to mixed genotype parasite infections was found. Finally, a cluster of AgDscam exon 4 variants that become especially common during Plasmodium invasion was identified.ConclusionsWhile the data clearly indicate that AgDscam diversity increases with P. falciparum exposure, they do not suggest that AgDscam diversity rises further in response to increased parasite diversity.

Synchronous attack is advantageous: mixed genotype infections lead to higher infection success in trematode parasites

Co-infecting parasite genotypes typically compete for host resources limiting their fitness. The intensity of such competition depends on whether parasites are reproducing in a host, or using it primarily as a transmission vehicle while not multiplying in host tissues (referred to as 'competition hypothesis'). Alternatively, simultaneous attack and co-infection by several parasite genotypes might facilitate parasite infection because such a diverse attack could present an additional challenge to host immune defence (referred to as 'facilitation hypothesis'). We tested the competition hypothesis by comparing the production of transmission stages (cercariae) from snails infected with one or two genotypes of the trematode Diplostomum pseudospathaceum. We found that cercarial production did not differ between the two groups of snails, suggesting lower per genotype production in double infections, and competition for host resources. Second, we tested the facilitation hypothesis by comparing parasite infection success on fishes (proportion of parasites establishing in the host) using cercariae originating from single-infected snails, double-infected snails and artificial mixtures of the single genotypes. In both cases, we found higher infection success when fishes were challenged with two parasite genotypes instead of one, supporting the facilitation hypothesis. Our results suggest that constraints defining the success of multiple genotype infections in parasites with multiple host life cycles include both between-genotype resource competition in the host and performance of host immune defences against a diverse parasite challenge.

Evaluation of three different hepatitis C virus typing methods for detection of mixed‐genotype infections

To evaluate the clinical applicability of an eligible assay for the true prevalence of hepatitis C virus (HCV) mixed-genotype infections. A newly developed HCV genotyping method targeting all six major HCV genotypes and 12 subtypes, restriction fragment length polymorphism (RFLP) and a serotyping assay were utilized for the detection of HCV mixed-genotype infections using known HCV genotypes and unknown samples. In a defined mix of HCV genotypes, a genotype present at levels as low as 8.3% was detected by our newly developed assay, showing a threefold increase in sensitivity over that of direct deoxyribonucleic (DNA) sequencing. A comparative study of the accuracy among the three genotyping methods was carried out on samples obtained from 50 thalassemic patients who received multiple blood transfusions. The results showed that viruses in approximately 42% of the samples from this group were determined to be infected with mixed genotypes by our newly developed method. A serotyping assay and RFLP analysis, performed with poor results, could identify only 18% and 10% of mixed-genotype infections, respectively. The newly developed assay may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections due to its higher level of sensitivity.

Relative clonal proportions over time in mixed-genotype infections of the lizard malaria parasite Plasmodium mexicanum

Vertebrate hosts of malaria parasites ( Plasmodium) often harbour two or more genetically distinct clones of a single species, and interaction among these co-existing clones can play an important role in Plasmodium biology. However, how relative clonal proportions vary over time in a host is still poorly known. Experimental mixed-clone infections of the lizard malaria parasite, Plasmodium mexicanum, were followed in its natural host, the western fence lizard using microsatellite markers to determine the relative proportions of two to five co-existing clones over time (2–3 months). Results for two markers, and two PCR primer pairs for one of those, matched very closely, supporting the efficacy of the method. Of the 54 infections, 67% displayed stable relative clonal proportions, with the others showing a shift in proportions, usually with one clone outpacing the others. Infections with rapidly increasing or slowly increasing parasitemia were stable, showing that all clones within these infections reproduced at the same rapid or slow rate. Replicate infections containing the same clones did not always reveal the same growth rate, final parasitemia or dominant clone; thus there was no clone effect for these life history measures. The rate of increase in parasitemia was not associated with stable versus unstable relative proportions, but infections with four to five clones were more likely to be unstable than those with two to three clones. This rare look into events in genetically complex Plasmodium infections suggests that parasite clones may be interacting in complex and unexpected ways.

HCMV gB genotype and its association with cytokine levels in hematopoietic stem cell transplantation

Glycoprotein B (gB) has been implicated in determining the pathogenicity and clinical outcomes of human cytomegalovirus (HCMV) disease. The purpose of this study was to assess the prevalence of gB genotypes in allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the relationship between it and cytokine levels in saliva and blood samples. The impact of these parameters on patients' survival was also investigated. Samples were obtained from 63 patients receiving an allo-HSCT. HCMV gB genotyping was carried out by multiplex nested PCR. The cytokine levels were assessed using ELISA assay. A single or mixed genotype infection was detected in the saliva and blood of 36/63 and 52/63 subjects, respectively. Patients with gB2 in their saliva showed lower IL-10 levels in comparison with patients without gB2. Reduced blood levels of IFN-γ and IL-1β were also found in recipients with the HCMV gB4 genotype compared with patients without it. Decreased IL-1β and increased IL-10 blood levels were associated with lower survival. However, HCMV gB genotypes have no impact on patient outcome. Decreased IL-1β and increased IL-10 levels in the blood are associated with lower survival. HCMV genotypes are associated with different cytokine levels in saliva and blood.

Human Cytomegalovirus: an Enormous Variety of Strains and Their Possible Clinical Significance in the Human Host

Human cytomegalovirus (HCMV) does not exist as one defined virus genotype, but as a variety of different strains. Several studies have investigated the significance of specific viral genotypes for the clinical course of HCMV infection. Upon reinfection, patients may acquire additional HCMV strains, and infections with a mixture of HCMV strains appear to be quite common. The analysis of such mixed infections has become increasingly important, not only for investigating the clinical implications of mixed-genotype infections, but also for understanding the pathogenesis of subsequent reinfections with HCMV strains, and this is also of importance for HCMV vaccine development. This article summarizes the clinical implications of infection with individual HCMV genotypes and focuses on infection with mixed populations of HCMV strains.

Hepatitis C virus genotypes circulating in district Swat of Khyber Pakhtoonkhaw, Pakistan.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis worldwide and its subtypes/genotypes are clinically important for clinical management and vaccine development. The present study describes frequency distribution of different HCV genotypes and their treatment status in HCV RNA positive patients from district Swat. A total of 185 HCV infected sera were analyzed by molecular genotyping assay. The most prevalent genotype was 3a (34.1%), followed by 2a (8.1%), 3b (7%) and 1a (5.4%). The samples found untypable by the present method of genotypes was 37.8% while, patients with mixed genotype infections were 7.6%. More than 80% of untypable cases were from those HCV patients who had received interferon plus ribavirin standard therapy in the past and either were non-responders and were relapsed thereafter or were under treatment. In conclusion, genotype 3a is the most prevalent HCV genotype in the region. A high prevalence rate of untypable genotypes is present in treated patients that need further investigation for the successful genotyping by developing new assays or using viral sequencing method.

Overestimation of Incidence of Hepatitis B Virus Mixed-Genotype Infections by Use of the New Line Probe INNO-LiPA Genotyping Assay

The new version of the INNO-LiPA HBV genotyping assay (Innogenetics) developed to identify all hepatitis B virus (HBV) genotypes, A to H, has been evaluated in comparison with sequencing of PCR-amplified HBV DNA from 200 samples before or after cloning. The genotyping data obtained with INNO-LiPA were in agreement with those from direct sequencing in the 179 samples characterized by the two methods. INNO-LiPA revealed 28 mixed infections. However, sequencing after molecular cloning confirmed only 15 of them and did not identify any that were of genotype H (n = 9). Our study demonstrates that INNO-LiPA overestimates mixed infections as a result of erroneous genotype H detection.

Mixed-genotype white spot syndrome virus infections of shrimp are inversely correlated with disease outbreaks in ponds

Outbreaks of white spot syndrome virus (WSSV) in shrimp culture and the relationship between the virus and virulence are not well understood. Here, we provide evidence showing that WSSV mixed-genotype infections correlate with lower outbreak incidence and that disease outbreaks correlate with single-genotype infections. We tested 573 shrimp samples from 81 shrimp ponds in the Mekong delta with outbreak or non-outbreak status. The variable number tandem repeat (VNTR) loci of WSSV were used as molecular markers for the characterization of single- and mixed-genotype infections. The overall prevalence of mixed-genotype WSSV infections was 25.7 %. Non-outbreak ponds had a significantly higher frequency of mixed-genotype infections than outbreak ponds for all VNTR loci, both at the individual shrimp as well as at the pond level. The genetic composition of WSSV populations appears to correlate with the health status of shrimp culture in ponds. The causal relationship between genotypic diversity and disease outbreaks can now be experimentally approached.

Strain filtering and transmission of a mixed infection in a social insect

Mixed-genotype infections have attracted considerable attention as drivers of pathogen evolution. However, experimental approaches often overlook essential features of natural host-parasite interactions, such as host heterogeneity, or the effects of between-host selection during transmission. Here, following inoculation of a mixed infection, we analyse the success of different strains of a trypanosome parasite throughout the colony cycle of its bumblebee host. We find that most colonies efficiently filter the circulating infection before it reaches the new queens, the only offspring that carry infections to the next season. A few colonies with a poor filtering ability thus contributed disproportionately to the parasite population in the next season. High strain diversity but not high infection intensity within colony was associated with an increased probability of transmission of the infection to new queens. Interestingly, the representation of the different strains changed dramatically over time, so that long-term parasite success could not be predicted from short-term observations. These findings highlight the shaping of within-colony parasite diversity through filtering as a crucial determinant of year-to-year pathogen transmission and emphasize the importance of host ecology and heterogeneity for disease dynamics.

Pattern and molecular epidemiology of Hepatitis B virus genotypes circulating in Pakistan

The continuously mutating nature of Hepatitis B virus (HBV) is responsible for the emergence of varying genotypes in different regions of the world affecting the disease outcome. The objective of the current study was to find out the pattern of HBV genotypes circulating in Pakistan. HBV genotypes were determined in HBV chronic patients of different age and gender from all the four different geographical regions (provinces) of Pakistan for a period of 2 years (2007–2009). Out of the total 3137 consecutive patients, 300 (175; 58.3% males and 125; 41.7% females) were randomly selected for HBV genotype A through H determination using molecular genotyping methods. Total 269 (89.6%) isolates were successfully genotyped where as 31 (10.3%) samples failed to generate a type-specific PCR band and were found untypable. Out of the successfully genotyped samples, 43 (14.3%) were with type A, 54 (18%) were with type B, 83 (27.6%) were with type C, 39 (13%) were with type D, 2 (0.6%) were with type E, 4 (1.3%) were with genotype F and total 44 (14.6%) were with mixed HBV infections. Of the mixed genotype infection cases, 16 were with genotypes A/D, 9 were B/C, six were A/D/F, five were with genotypes A/F, two were with A/B/D and B/E and one each for A/C as well as A/E genotypes. Four common genotypes of HBV found worldwide (A, B, C & D) were isolated from Pakistan along with uncommon genotypes E and F for the first time in Pakistan. Overall Genotype C is the most prevalent genotype. Genotypes B and C are predominant in Punjab & Balochistan and Khyber Pakhtoonkhwa, respectively whereas genotype A in Sindh.

INNO-LiPA HBV genotyping is highly consistent with direct sequencing and sensitive in detecting B/C mixed genotype infection in Chinese chronic hepatitis B patients and asymptomatic HBV carriers

Background Different HBV genotypes have effect on the progression and outcome of chronic hepatitis B (CHB) and on the response to antiviral therapy. Accurate and sensitive genotyping methods are needed in clinical practice. Methods In this multicenter retrospective study, 504 HBV DNA positive serum or plasma samples of CHB patients or asymptomatic carriers were genotyped by INNO-LiPA HBV genotyping and direct sequencing. A part of the consistent and discrepant results were confirmed by clonal sequencing. Results Except two samples with indeterminate results by INNO-LiPA, 463 had the same genotype results by INNO-LiPA and direct sequencing with a concordance of 92.23% (463/502). However 24 had completely inconsistent results by the two methods, seven of which were confirmed by clonal sequencing as the same genotype as by direct sequencing. Fifteen samples were determined as B/C mixed genotypes by INNO-LiPA but singe B or C genotype by direct sequencing, five of which were confirmed by clonal sequencing as B/C mixed infection. Further studies showed INNO-LiPA could detect 6% of genotype B and genotype C among mixed genotype samples at HBV DNA ≥3×10 3 IU/ml. Conclusions INNO-LiPA is accurate and sensitive in detecting B, C, D and B/C mixed genotype infection in Chinese CHB patients and asymptomatic carriers.

Relative Clonal Density of Malaria Parasites in Mixed-Genotype Infections: Validation of a Technique Using Microsatellite Markers for Plasmodium falciparum and P. mexicanum

Quantifying the relative proportion of coexisting genotypes (clones) of a malaria parasite within its vertebrate host's blood would provide insights into critical features of the biology of the parasite, including competition among clones, gametocyte sex ratio, and virulence. However, no technique has been available to extract such data for natural parasite-host systems when the number of clones cycling in the overall parasite population is likely to be large. Recent studies find that data from genetic analyzer instruments for microsatellite markers allow measuring clonal proportions. We conducted a validation study for Plasmodium mexicanum and Plasmodium falciparum by mixing DNA from single-clone infections to simulate mixed infections of each species with known proportions of clones. Results for any mixture of DNA gave highly reproducible results. The relationship between known and measured relative proportions of clones was linear, with high regression r² values. Known and measured clone proportions for simulated infections followed over time (mixtures) were compared with 3 methods: using uncorrected data, with uncorrected data and confidence intervals constructed from observed experimental error, and using a baseline mixture of equal proportions to calibrate all other results. All 3 methods demonstrated value in studies of mixed-genotype infections sampled a single time or followed over time. Thus, the method should open new windows into the biology of malaria parasites.

Comparative evaluation of INNO-LiPA HBV assay, direct DNA sequencing and subtractive PCR-RFLP for genotyping of clinical HBV isolates

Genotypes (A to H) of hepatitis B virus (HBV) influence liver disease progression and response to antiviral therapy in HBV-infected patients. Several methods have been developed for rapid genotyping of HBV strains. However, some of these methods may not be suitable for developing countries. The performance of INNO-LiPA HBV Genotyping assay (LiPA), direct DNA sequencing and subtractive PCR-RFLP of genotype-specific HBV genome regions were evaluated for accurately determining the HBV genotypes by analyzing sera (n = 80) samples from chronic HBV patients. Both, LiPA and DNA sequencing identified 63, 4 and 13 HBV strains as belonging to genotype D, genotype A and mixed genotype A and D, respectively. On the contrary, the PCR-RFLP-based method correctly identified all 4 genotype A but only 56 of 63 genotype D strains. Seven genotype D strains yielded indeterminate results. DNA sequence comparisons showed that a single nucleotide change in the target region generated an additional restriction site for Nla IV that compromised the accuracy of this method. Furthermore, all the mixed genotype A and D strains were identified only as genotype A strains. The data show that the PCR-RFLP-based method incorrectly identified some genotype D strains and failed to identify mixed genotype infections while LiPA and DNA sequencing yielded accurate results.

Taqman Real-Time PCR Detects Avipoxvirus DNA in Blood of Hawaìi `Amakihi (Hemignathus virens)

Background Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawaìi, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds.Methodology/Principal FindingsHere, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawaìi `amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawaìi but were observed in two individuals in this study.Conclusions/SignificanceWe anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.

Deep Sequencing Reveals Highly Complex Dynamics of Human Cytomegalovirus Genotypes in Transplant Patients over Time

In lung transplant patients undergoing immunosuppression, more than one human cytomegalovirus (HCMV) genotype may emerge during follow-up, and this could be critical for the outcome of HCMV infection. Up to now, many cases of infection with multiple HCMV genotypes were probably overlooked due to the limitations of the current genotyping approaches. We have now analyzed mixed-genotype infections in 17 clinical samples from 9 lung transplant patients using the highly sensitive ultradeep-pyrosequencing (UDPS) technology. UDPS genotyping was performed at three variable HCMV genes, coding for glycoprotein N (gN), glycoprotein O (gO), and UL139. Simultaneous analysis of a mean of 10,430 sequence reads per amplicon allowed the relative amounts of distinct genotypes in the samples to be determined down to 0.1% to 1% abundance. Complex mixtures of up to six different HCMV genotypes per sample were observed. In all samples, no more than two major genotypes accounted for at least 88% of the HCMV DNA load, and these were often accompanied by up to four low-abundance genotypes at frequencies of 0.1% to 8.6%. No evidence for the emergence of new genotypes or sequence changes over time was observed. However, analysis of different samples withdrawn from the same patients at different time points revealed that the relative levels of replication of the individual HCMV genotypes changed within a mixed-genotype population upon reemergence of the virus. Our data show for the first time that, similar to what has been hypothesized for the murine model, HCMV reactivation in humans seems to occur stochastically.