Thirty-five human influenza A(H5N1) cases were reported in Cambodia during 2013–2014 after emergence of a clade 1.1.2 reassortant virus. We tested 881 villagers and found 2 cases of pauci- or asymptomatic infection. Seroprevalence after emergence of the reassortant strain (0.2%) was lower than the aggregate seroprevalence of 1.3% reported in earlier studies. Twenty-one cases, including 19 (90.5%) deaths, were reported during 2005–2012. In January 2013, researchers at the Institut Pasteur du Cambodge (IPC) in Phnom Penh, Cambodia, identified a new influenza A(H5N1) clade 1.1.2 reassortant virus in humans exposed to poultry  clade 1.1.2 reassortant virus occurred in a village in Kratie Province ; The cases were in 2 separate households. At the same time, a third confirmed case was identified in a village in Kompong Cham Province . The 3 patients with confirmed H5N1 virus infection were young children who had close contact with sick or dying poultry. Cases of H5N1 virus infection had also been reported in village 2 in April 2007 and December 2009.>2 weeks later to test for a possible increase in H5N1 virus antibody levels. We obtained National Ethics Committee for Human Research approval for all serostudies conducted as part of pandemic risk assessments.We selected these 2 villages to conduct seroprevalence studies within a month of case occurrence to determine point-seroprevalence in the general population . Epidemi>80 and a microneutralization titer >40) were considered H5N1 virus positive  for the second sample were also considered H5N1 positive (We used hemagglutination inhibition (HI) and microneutralization assays as described previously  to double-enter questionnaires. We excluded index cases from the analysis to assess risk of infection due to co-exposure or secondary transmission and calculated prevalence by using Poisson confidence intervals. We used the Fisher exact test to compare our data to those from community seroprevalence studies conducted before emergence of this H5N1 reassortant virus.Paired samples were obtained from 238 (34.2%) of the 695 children and adults in village 1 and from 643 (69.8%) of 921 persons in village 2 . All perSeroprevalence studies to document the incidence and clinical spectrum of H5N1 virus infections help evaluate the effects of emergence of a reassortant virus on virus transmission in affected communities . We therStudies we conducted prior to the emergence of the reassortant strain, including 2 unpublished serosurveys, tested 2,713 persons, of whom 35 (1.3%) were positive for H5N1 antibodies . Our sur,,6). Despite the 2013–2014 surge in cases, there is no evidence of increased H5N1 virus transmission to humans or altered clinical and epidemiologic characteristics associated with the emergence of this reassortant virus. A review of literature showed that with the exception of 3 studies in China (Even if the 2 healthy but untested contacts in village 1 had been found positive, the seroprevalence of H5N1 virus in humans would still be significantly lower following the emergence of the clade 1.1.2 reassortant virus when compared with historical IPC data (Routine biocontrol measures in Cambodia are poor, and poultry surveillance is lacking. Farmers either delay reporting and detection, or sell sick poultry, due in part, to lack of compensation from the government. This enables poultry outbreaks to spread. These factors could explain why prevalence in populations around confirmed cases was 10-fold higher in Cambodia than elsewhere until 2012. The drop in the number of positive cases in the 2014 community serosurveys raises the possibility that villagers’ knowledge of avian influenza and prevention may have improved after 2012, probably as a result of Cambodian authorities’ commitment to and international funding for community education campaigns. Recent studies, however, have shown a >2.5-fold increase in the circulation of avian H5N1 virus in live bird markets in Cambodia during 2013 compared with 2011 (,Our study has limitations. Although there were 9 human cases of infection with H5N1 virus reported in 2014, all followed by direct-contact tracing, only 2 spatiotemporal seroprevalence studies were conducted around H5N1 clusters and foci. There was also some minor loss to follow-up. Transmission risk estimates in villages with clusters might be influenced by the role of host genetics in susceptibility to infection (In conclusion, our findings provide no evidence for increased human-to-human H5N1 virus transmission in Cambodia after emergence of the clade 1.1.2 reassortant virus. Direct-contact tracing is essential, but regular community-wide seroprevalence studies may not be required. However, each new epidemic phase or emergence of a new reassortant virus must be carefully investigated to address specific public health and research questions that can be answered only in an outbreak setting.


Sciurus vulgaris) were found with moist, crusty skin lesions. Histology, electron microscopy, and cell culture isolation revealed an orthopoxvirus-like infection. Subsequent PCR and genome analysis identified a new poxvirus (Berlin squirrelpox virus) that could not be assigned to any known poxvirus genera.Near Berlin, Germany, several juvenile red squirrels ( Sciurus vulgaris) is the only species of tree squirrels endemic throughout most of Europe. Although they are usually abundant, red squirrels are endangered or extinct in some regions in Great Britain and Ireland that are co-inhabited by invasive eastern gray squirrels (Sciurus carolinensis), which were introduced from North America in the late 19th century. One major threat is the transmission of squirrelpox virus (SQPV) from the gray squirrel reservoir host to red squirrels, which succumb to lethal infections (Tamiasciurus hudsonicus) from Canada  and PCR. Three animals that died under care were submitted for necropsy. We obtained samples of all organs for histological and PCR examination. We used 1 sample of a skin lesion for virus propagation in cell culture.EM-negative staining of skin lesions from all animals led to the discovery of brick-shaped poxvirus particles with irregular threadlike surface fibers and an average size of 294 nm × 221 nm . PatholoTo confirm the morphologic diagnosis, we extracted DNA from skin lesions and performed various PCRs. An orthopoxvirus (OPV)–specific PCR showed negative results (Chordopoxvirinae), with BerSQPV forming a unique branch (data not shown). In addition, any partial sequences of SQPV available in GenBank were aligned to BerSQPV, showing a maximal sequence identity of 64.3% to gene E9L (GenBank accession no. AY340976.1), further emphasizing the uniqueness of this newly identified virus.We compared characteristics of BerSQPV to related viruses and found that the EM structure shows features typical for OPV but the genome size of ≈143 kb is more consistent with PPV or SQPV from the United Kingdom  demonstrWe designed a BerSQPV-specific quantitative PCR based on the genome sequence as a tool for future investigations . Sensitivity was <10 genome equivalents per reaction amplifying plasmid dilution rows. We first validated the specificity in silico during the design process, revealing the highest identity of 88% to cowpox virus Kostroma (GenBank accession no. KY369926.1), with mismatches in crucial positions in the primer and probe binding sites. Squirrel poxvirus strain Red squirrel UK (GenBank accession no. NC_022563.1) showed only 84% identity, with additional mismatches in amplification-relevant positions. Practical PCR testing using DNA from cowpox, monkeypox, ectromelia, parapox-ORF, myxoma, avipox, and molluscipox viruses showed no cross-reactivity. The new specific quantitative PCR was subsequently applied to DNA from skin lesions archived from 1 squirrel found dead in 2014 in the Berlin area, 2 live squirrels from 2015, and 5 live squirrels from 2016, as well as various organs from 3 affected squirrels necropsied in 2015 . Organ tWe describe a new poxvirus, BerSQPV, isolated from red squirrels in Berlin, Germany, that causes pathological changes consistent with other epidermal poxvirus infections. Genome analysis revealed a unique sequence within the poxvirus family, as BerSQPV is not clustering to other poxvirus genera, including UK SQPV from red squirrels from Great Britain. In contrast to UK SQPV, which resembles PPV ultrastructurally (BerSQPV is suspected to have been circulating for several years among Eurasian red squirrels in the greater Berlin area. Although diseased animals in care were handled in close contact, caretakers have remained asymptomatic, suggesting a negligible risk for human infection. Further detailed characterization of the isolated virus is ongoing.


The evaluation and interpretation of forensic DNA mixture evidence faces greater interpretational challenges due to increasingly complex mixture evidence. Such challenges include: casework involving low quantity or degraded evidence leading to allele and locus dropout; allele sharing of contributors leading to allele stacking; and differentiation of PCR stutter artifacts from true alleles. There is variation in statistical approaches used to evaluate the strength of the evidence when inclusion of a specific known individual(s) is determined, and the approaches used must be supportable. There are concerns that methods utilized for interpretation of complex forensic DNA mixtures may not be implemented properly in some casework. Similar questions are being raised in a number of U.S. jurisdictions, leading to some confusion about mixture interpretation for current and previous casework.Key elements necessary for the interpretation and statistical evaluation of forensic DNA mixtures are described. Given the most common method for statistical evaluation of DNA mixtures in many parts of the world, including the USA, is the Combined Probability of Inclusion/Exclusion (CPI/CPE). Exposition and elucidation of this method and a protocol for use is the focus of this article. Formulae and other supporting materials are provided.Guidance and details of a DNA mixture interpretation protocol is provided for application of the CPI/CPE method in the analysis of more complex forensic DNA mixtures. This description, in turn, should help reduce the variability of interpretation with application of this methodology and thereby improve the quality of DNA mixture interpretation throughout the forensic community.The online version of this article (doi:10.1186/s12863-016-0429-7) contains supplementary material, which is available to authorized users. A DNA mixture refers to a biological sample that originated from two or more donors and is determined after a DNA profile is generated. Mixture evidence has always been a part of casework; however there are indications that the fraction of samples presenting as a mixture may have increased, presumably due to changes in methodology, sampling strategies, types of cases . A retrospective study over a 4 year period in Spain  found, iIn current forensic investigations DNA mixtures occur commonly . MoreoveAt this time, the most commonly used method for forensic evaluation of DNA evidence is the assessment of short tandem repeat (STR) polymorphisms present at multiple distinct genetic loci –6. The aThe combined probability of inclusion (CPI) , 11, 12 Prior to comparison with known profiles, peak heights are used to determine whether contributors  can be distinguished. When a known individual’s DNA can reasonably be expected to be present, the known contribution can be “subtracted” . When a The advantages of the CPI approach are thought to be its simplicity and the fact that the number of contributors need not be assumed in the calculation. However, even with simplicity, recently, in the U.S., interpretation protocols used for DNA mixtures using the CPI method have been criticized when applied to forensic mixtures for which it is not suited, highlighting issues of effective communication and technology transfer to the end users of the forensic science community . One shoIf the DNA crime stain profile is low level, then possibility of allele drop-out should be considered. If allele drop out is a reasonable explanation for the observed DNA results, then the CPI statistic cannot be used at those loci in which the phenomenon may have occurred. The formulation of the CPI statistic requires that the two alleles at each locus of the donor being considered must be above the analytical threshold. Hence, if a profile, or a component of it, is low level, additional considerations are needed to ensure that allele drop-out has not occurred at this locus.While interpretation of a mixture prior to a statistical calculation requires the direct use of peak heights, the assumed number of contributors, the genotype of known contributors or the genotype of persons of interest (POIs), the CPI calculation, in a strict sense, does not require such consideration , 15, 16.The authors recommend moves in favour of using the likelihood ratio (LR) approaches and laboratories have been embracing LR application –19. Use If a lab chooses not to convert to using LRs, or if it does intend to convert but is using CPI in the interim, it remains necessary to ensure that when the CPI is used it is applied correctly.Herein a more explicit description of a DNA mixture protocol is offered with recommendations for applying the CPI. While the approach described herein overall is not a completely new approach to the use of the CPI, it has become essential to formalize the protocol so that proper statistical analyses can be performed when needed in courtroom proceedings. This protocol is provided as one that should be used for applying the CPI when needed.Calculation of the CPI involves a statistical model that returns an estimate of the sum of the frequencies of all possible genotype combinations included in the observed DNA mixture. While the computation of the statistical estimate, itself, does not require assumptions about the number of contributors, an assumption of the number of contributors is necessary to help inform decisions about whether allele drop-out is likely at particular loci in the evidentiary sample. For example, if only four allelic peaks appear at a locus in a profile assumed to be from two donors, then it is reasonable to assume that allele drop-out has not occurred at that locus.That there is no published unifying protocol for use of the CPI for evaluation of forensic DNA mixtures has led to some confusion among forensic practitioners on its proper use. Accordingly a detailed protocol is provided herein to guide the community to reduce variation in interpretation and to promote a more defensible application of the CPI. Three publications describe the use of the CPI , 20, 21.Given emerging criticism of methods used in forensic DNA mixture analysis, interpretation and statistical evaluation - particularly in the U.S. - it is timely to revisit and reinforce the foundational principles of interpretation of mixtures and subsequent computation as it relates to the CPI (or CPE). The authors recognize and advocate the community as a whole move towards the use of probabilistic genotyping methods , 22, 23 It is not possible to prescribe rules for every conceivable situation; therefore, it is essential that application of the CPI be performed by well-trained professionals using their judgement and knowledge under the spirit of the guidelines provided herein, their professional education, and relevant experience. Lastly, the protocol described herein is a guideline and does not preclude alternate acceptable methods to interpret DNA mixture evidence as long as the rules applied are always held subservient to the foundational principles involved in proper mixture interpretation.Interpretation of a DNA mixture should not be done by simply counting observed alleles. Efforts to deconvolve a mixture into single contributors are advocated where possible , 24–26. One caution is that single source statistics at some loci and CPI statistics at other loci should never be combined into one statistical calculation . Either The procedure for DNA mixture interpretation using the CPI approach assumes that a laboratory has an established valid analytical (or detection) threshold (AT), stochastic threshold (ST), stutter filter values (SF), and minimum peak height ratio(s). As PCR is “semi-quantitative” STR allelic peak heights are approximately proportional to the amount of DNA from each donor , 24. OneDNA mixtures involve two or more donors. It is incumbent upon the DNA analyst to carefully assess and state the assumed number of contributors to a profile, even when using the CPI. The SWGDAM STR Interpretation Guidelines  3.4.1. sEach donor may contribute 0, 1, or 2 alleles at each genetic marker (locus) tested . Any of the observed peaks  may overlap with a peak(s) from the same or another donor of the mixture. When allele or artefact sharing occurs there is an additive effect of the two or more peaks, termed “allele stacking” or “allele masking”. As the number of potential contributors increases, so does the uncertainty in accurately determining the true number of contributors . For exaThese guidelines do not describe in detail how to determine the number of contributors, as a minimum requirement, the number of alleles at each locus and their peak heights should be considered when assigning the number of contributors. Because of the quantity and quality of the DNA being analysed, some loci may meet the determined number of contributors and some may not. For those loci that do not fit the best estimate of the number of contributors, there should be evidence of low signal and/or degradation, which would render the specific locus (or loci) inconclusive for the CPI calculation. Testing additional STR loci may reduce the uncertainty in estimating the potential number of contributors . In addiStutter, the inherent by-product of slippage during amplification of STRs, adds complexity to mixture interpretation. Typically, interpretation of whether a peak is solely stutter or stutter along with an allele from another contributor arises when a minor or trace contributor peak(s) is observed at a locus (or other loci) that is similar in height relative to the stutter of the major contributor alleles at the locus. These peaks and their heights are used to help determine whether to qualify or disqualify the locus for use in the CPI calculation.Sampling of template from the extract for the aliquot used for the PCR ,The greater stuttering and lower amplification efficiency of larger alleles (or template accessibility during PCR), andQuality of the template DNA.Random variation in peak heights is an inherent property of current DNA typing methodologies. These random variations of peak heights within an individual STR profile or between replicate samples are known as stochastic variation. As the quantity and quality of the input DNA decreases stochastic effects can increase. These effects manifest as variation in peak height between the two peaks at the same locus in a heterozygote or the variation of allele peak heights from the same donor at different loci across the degradation slope line. Such allele peak height variation arises from several factors:It is likely that most of the variation in allele peak heights results from the sampling of template , 35 and The ST is the peak height value(s) above which it is reasonable to assume that allele drop-out of a sister allele of a heterozygote has not occurred at a locus , 41. TheApplication of a ST is straightforward for single-source DNA profiles. If a single allele is observed and its peak height is below the ST it is considered possible that a sister allele at that same locus may have dropped out. In contrast to single source samples, in DNA mixtures any given allele peak may actually represent a composite of allele peaks (and depending on position can include stutter peaks). Because of the potential of allele sharing among different contributors to a DNA mixture and the accompanying additive effects in peak heights, a peak height above the ST does not necessarily assure one that a sister allele has not dropped out at that locus. Analysis of the full profile is required to assist in the determination of potential allele drop-out.Laboratories typically apply a ST for interpretation using a peak height threshold determined based on validation experiments. If the same ST peak height is used across all loci in an entire DNA profile, for many cases involving low level or degraded samples, the loci at the low molecular weight end of the profile  can exceed the ST whereas at the higher molecular weight end  they may straddle or fall below this threshold.STR allelic peak heights are approximately proportional to the effective  amount of DNA from the donor , 24. TypEach STR allelic peak may be associated with one backward stutter peak and occasionally a lower signal forward stutter peak , 41–44. Consider a case where it is ambiguous whether a peak is stutter or an allele. In such an instance a contributor with an allele in this ambiguous position would not be excluded. The appropriate inclusion statistic for this locus then includes the allele probabilities for the ambiguous peak positions in the summation for the CPI calculation . SubtracApply a stutter filter as normal and remove any artefacts such as pull-up and spikes.If a single source profile may be deduced from the mixture, then do so. This single-source profile may be a deduced major or minor contributor or a deduced foreign contributor by subtracting an assumed known contributor’s alleles.Approaches for calculating single-source statistical estimates of a profile probability can be found in the National Research Council Report . The ranIf no single-source profile could be deduced or there is some interest in interpreting irresolvable components of the mixture, the CPI approach can be invoked.The generalized approach is described as follows:To formalize the interpretation the overriding principle (P) for use of loci in CPI calculations is:1: Any locus that has a reasonable probability of allele drop-out should be disqualified from use in calculation of the CPI statistic.P1. Failing to consider the potential of allele drop- out when there are no detectable peaks between the AT and the ST has allowed the often misguided concept to develop that if all observed peaks are above the ST, then the locus unequivocally can be used.All guidelines that follow are subservient to PWe cannot prescribe what is a “reasonable probability” as the probability relies on the validation performed by the laboratory and on what ST value has been applied (could be overly conservative). However, if a numerical estimation is sought then one could consider allele drop-out no higher than 0.01 being a reasonable value for addressing uncertainty.1. The exception to P1 (termed modified or restricted CPI) is an interpretation that can apply to a portion of a profile as opposed to the entire profile. This scenario sometimes occurs where the mixture profile is comprised of multiple major contributors and minor (or trace) contributors where the majors can be resolved readily from the lesser contributing alleles  [With one exception the approach to DNA mixture interpretation should never trump Pter, R4) , 24, 30.A locus is included for use in a CPI calculation if allele drop-out is considered to be highly unlikely. Only qualified loci are used in the calculation of the CPI statistic Figs.  and 2.Fi1.Guidance (G) for R1.1: Any locus with an allelic peak height below the ST and above the AT is disqualified for a CPI calculation.G3, it may be possible to re-qualify locus D3S1358).For example, as shown in Fig. A locus disqualified for a CPI statistic may still be suitable for an RMP calculation.1.2: Any locus with an observable peak(s) residing below the AT that is likely to be a true allele(s) is disqualified. A peak below the AT may be deemed to be an allele if there is evidence of low level peaks at other loci, the peak(s) is distinct from the local noise, is not in the “N + 4”  or “N-4”  or “N-8”  stutter position and has Gaussian morphology. While peaks below the AT are not used for comparison purposes, they might be informative to support the possibility of allele drop-out at the locus (or loci) being evaluated, particularly when there are peaks below the ST (and above the AT) at smaller amplicon loci.G1.3: Evaluation of potential allele drop-out is not constrained to observable peaks at a specific single locus. Instead, a global profile evaluation is required. Any locus that has no allelic peaks below the ST and above the AT but may have an unseen allele(s)  is disqualified.G1.3: If there are minor peaks below or close to the ST or below the AT at other loci, these peaks may be indicators of the potential of allele drop-out. These indicator peaks at other loci should be taken into consideration for potential allele drop-out in the specific locus being evaluated.Implementation of G2: Stutter. Additive effects for alleles overlapping with stutter products must be considered in assessing the potential for allele drop-out at a locus and indistinguishable stutter/allele peaks may need to be included in CPI calculations.R2.1 Check if a peak in a stutter position is considered to have an allele contribution.R2.1.1 To determine whether there is an allele contributing to a peak in the stutter position subtract the stutter threshold or stutter filter value (SF) for the locus from the peak height value for the peak in the stutter position (SPH). The remaining value is the minimum allele contribution (MAC).GSPH – SF = MACIf MAC > ST, then the locus can be used for use in the CPI calculation.If MAC ≤ ST, then the locus is disqualified for use in the CPI calculation.The SF value may not represent the true stutter contribution, as this value often is calculated as the mean stutter + 3SDs. There is a reasonable expectation that the true stutter contribution can be less than the SF value. However, since there is no way to determine the precise stutter contribution, using the maximum value of stutter is advocated.2.1.2 The locus may be re-qualified (see exception rule R3 below) even when the MAC ≤ ST, if there is evidence of no allele drop-out at the locus. Evidence of no allele drop-out could come from a deconvolution where all minor or trace alleles have been observed or inferred based on subtraction of an assumed known contributor’s alleles. Determining the number of minor contributors  can be challenging with complex DNA mixtures. A peak in the stutter position that does not exceed the SF may still have been comprised of both stutter and an allele from another contributor. This peak(s) should be considered potentially allelic based on the data in the profile  should be included in the summation for the CPI calculation.RIt is possible to reinstate  some loci for use in the CPI calculation. This qualification can occur for alleles observed at a locus, dependent on the assumption of the number of contributors to that mixture even where the peak height of an allele(s) falls below the ST (and above the AT). As stated above, while the number of contributors is not taken into account when calculating the CPI, it is imperative that the number of contributors be assumed to determine the potential of allele drop-out. For example, consider a two-person mixture with one major and one minor contributor Fig. , and theThis approach can be extended to three-person mixtures if interpretation of the overall profile indicates that allele drop-out has not occurred under an assumed number of contributors.3: If a mixture interpretation suggests no drop-out, then the locus can be used in the CPI calculation.G3.1: If all possible alleles are observed , then the locus can be used in the CPI calculation.G4.1, Fig. If a set of peaks representing more than one donor is distinct from one or more minor or trace peaks then the CPI approach may be applied to the “major cluster” see G4., Fig. 1.4.1: To qualify a locus for use with a major cluster, first there must be a clear visual distinction between a set of large peaks and a set of trace peaks. The principle is that all major peaks must be identifiable and for these major peaks allele drop-out must be deemed unlikely.G4.1.1 Any allele peak assigned to the major cluster must be sufficiently high that it could not have a partner allele in the minor set, andG4.1.2 Allele peaks assigned to the major cluster must be sufficiently high that allele drop-out is unlikely even when consideration is given that the peak might be a composite of major and minor.GThere are two aspects to this principle;4.2: This assessment requires some level of deconvolution and is more straightforward if there are only two major profiles and one trace contributor. Consider a locus with four large peaks and two small ones , comparison of a DNA mixture profile with that of a known suspect/victim or other known POIs, when possible, should not be carried out until the mixture evidence has been fully evaluated as described above. Comparison of the evidence and known profiles for inclusion/exclusion purposes is independent of the CPI statistical calculation. Regardless, All the alleles of the POIs should have a corresponding allelic or potentially allelic peak in the qualifying loci. If the evidence supports an exclusion, the calculation of the CPI is unnecessary for that comparison. If there is a failure to exclude based on genotype possibilities derived from peak heights at qualified or disqualified loci, then a computation is provided. Computation of the CPI does not require examination of the STR profile (genotypes) of the known individuals . At the point of computation of the CPI, the DNA mixture profile is composed of qualified and disqualified loci.G5.2 There can be only one value for the CPI/CPE computed for each DNA mixture profile. The interpretation of potential allele drop-out should be made prior, when possible, to evaluating known reference samples. Adjustments to fit the interpretation to reject or reinstate a locus based on additional information from a person of interest profile  are inappropriate [Gropriate –50.5.3 One exception to using data from a reference profile is where an assumption of one of the contributors is known, such as from an “intimate” sample. The assumption of the individual(s) being a known contributor(s) must be documented. In situations where a contributor(s) is assumed, then subtraction of the alleles of the known contributor(s) is justified .G5.4 Use of the 2p rule for the CPI is not valid.GThe 2p statistic was designed for single-source samples where one allele was present at a locus and there was strong support for allele drop-out.Once the mixture has been evaluated, both the qualified and disqualified loci should be inspected for potential exclusionary evidence. For the qualified loci exclusionary evidence may be based on the absence of alleles or the absence of deconvolved genotypes in the mixture compared with those of the known reference profile. If the deconvolved genotypes of the mixture are different from the genotype of the known comparison profiles, then an exclusion interpretation is supported. If the locus (or loci) was deemed disqualified for the CPI calculation, allele drop-out should be considered when including or excluding a potential donor.6. For the qualified loci exclusionary evidence exists when the POI has any alleles not present in the crime stain profile.RConsider the D3S1358 locus shown in Fig. 7. For loci that can be deconvolved exculpatory evidence exists when the POI has a genotype not amongst the set of supported genotypes.RConsider again the D3S1358 locus Fig. . At this8. For disqualified loci exculpatory evidence can occur but relies on the profile, allowing for missing data, to determine if the POI is unlikely to be a donor.R8. The POI is unlikely to be a donor if the allele(s) consistent with the POI and the total number of observed alleles at a given locus invalidate or do not support the assumed number of contributors to the DNA mixture. The inclusion of the POI would cause a mismatch with the assumed number of contributors .In Texas, the Forensic Science Commission has been working with laboratories to assess the DNA mixture protocols and review the statistical analyses in selected cases using the CPI/CPE. For laboratories or jurisdictions that modify their DNA mixture interpretation protocols, either in light of this document or for other reasons, there may be reason to review a sample of selected pending or previously reported DNA mixture casework. Forensic laboratories can work closely with all stakeholders in their respective jurisdictions to address these issues in a collaborative and constructive manner.


To differentiate exposure to the newly introduced chikungunya virus from exposure to endemic dengue virus and other pathogens in Haiti.Plasmodium falciparum antigens. Most (217) of the blood samples investigated were collected longitudinally, from each of 61 children, between 2011 and 2014 but another 127 were collected from a cross-sectional sample of children in 2014.We used a multiplex bead assay to detect immunoglobulin G (IgG) responses to a recombinant chikungunya virus antigen, two dengue virus-like particles and three recombinant Of the samples from the longitudinal cohort, none of the 153 collected between 2011 and 2013 but 78.7% (48/61) of those collected in 2014 were positive for IgG responses to the chikungunya virus antigen. In the cross-sectional sample, such responses were detected in 96 (75.6%) of the children and occurred at similar prevalence across all age groups. In the same sample, responses to malarial antigen were only detected in eight children (6.3%) but the prevalence of IgG responses to dengue virus antigens was 60.6% (77/127) overall and increased steadily with age. Spatial analysis indicated that the prevalence of IgG responses to the chikungunya virus and one of the dengue virus-like particles decreased as the sampling site moved away from the city of Léogâne and towards the ocean.Serological evidence indicates that there had been a rapid and intense dissemination of chikungunya virus in Haiti. The multiplex bead assay appears to be an appropriate serological platform to monitor the seroprevalence of multiple pathogens simultaneously. Alphavirus in the family Togaviridae, can cause conjunctivitis, debilitating polyarthralgia, diarrhoea, headache, fatigue, fever, myalgia, nausea, rashes and vomiting.Flavivirus in the family Flaviviridae, is also often associated with severe joint pain and can lead to dengue fever or to the potentially deadly severe dengue. Aedes aegypti and Ae. albopictus are the main vectors of both chikungunya and dengue virus.The symptoms of human infections with dengue virus are often so similar to those of infection with chikungunya virus that clinical differentiation of the two types of infection is difficult. Infection with chikungunya virus, which belongs to the genus ––Although the disease we now call chikungunya appears to have been initially described in the 1820s, almost simultaneously in East Africa – the area now known as the United Republic of Tanzania – and India, chikungunya virus was not isolated until 1952.Ae. aegypti and Ae. albopictus occur in Haiti, chikungunya virus was expected to arrive in the country and to be disseminated rapidly.,,Since both ,–Plasmodium falciparum in Haitian children. Our post-hoc testing of blood samples was primarily done to generate epidemiological data about the introduction of chikungunya virus in Haiti and was not aimed at diagnosis or case management.Because the symptoms and epidemiology of chikungunya and dengue fever are similar and occur against a backdrop of other infectious diseases, our objective was to identify and assess immunoglobulin G (IgG) responses to these closely related pathogens. A search, on 6 November 2015, for both “multiplex” and “chikungunya” in the titles and abstracts of the published articles listed by PubMed resulted in a list of 21 articles. Of these articles, nine described laboratory techniques based on the reverse-transcription polymerase chain reaction, two used assays based on the same reaction to identify chikungunya virus in the fieldThe study protocol was reviewed and approved by the Ethics Committee of the Hôpital Sainte Croix, Léogâne, Haiti, and the Institutional Review Boards of both the CDC, Atlanta, United States of America, and the University of Notre Dame, Notre Dame, USA.We collected blood spot samples from a longitudinal cohort of 61 children – all residents in the small coastal town of Ca Ira – at three or all four of four time points: December 2011, February 2013, December 2013 and August 2014. All the blood spot samples collected before 2014 had been collected, from children who were aged 2–10 years in December 2011, for filariasis surveillance. In August 2014, specifically for our investigation of the bead assay, we collected blood from all 61 previously sampled children and from another 127 children from Ca Ira, then aged 2–10 years, who had not been sampled before. We considered the 127 children to represent a cross-sectional sample.Prior to the collection of each blood spot sample, a local community health worker obtained the informed consent from a parent of the sampled child. To prepare each sample, 10 µl whole blood was collected from a finger-prick and transferred to one unused extension of a piece of filter paper with six circular extensions . After collection, the blood spots were allowed to dry and then stored at −20 °C until tested.,P. falciparum.–P. falciparum antigens represented a 42-kDa fragment from clone FVO, a 42-kDa fragment from clone 3D7 and a 19-kDa fragment from clone 3D7  that was linked with glutathione-S-transferase.–We used one recombinant chikungunya virus antigen – that is, mutant A226V envelope 1 antigen  – two dengue virus antigens – propagated using a eukaryotic plasmid vector that expressed the premembrane/membrane and envelope proteins that self-assemble into two different non-infectious virus-like particles known as DENV-2 and DENV-3S-transferase, we coupled 12.5 million activated beads, in 50 mmol/L 2-(N-morpholino) ethanesulfonic acid buffer with 0.85% (w/v) NaCl, at pH 5.0 – with 28 µg of the 19-kDa fragment, from clone 3D7, linked to glutathione-S-transferase, 15 µg of the 42-kDa fragment from clone 3D7, 15 µg of the 42-kDa fragment from clone FVO, and 12 µg of glutathione-S-transferase. Coupling efficiency was evaluated using monoclonal antibodies and reference sera that were known to be highly reactive to the antigens.,1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide  was used to convert the carboxyl groups on carboxylated polystyrene beads  to esters. The esters on the so-called activated beads could then react with amine groups on the antigens to form covalent amide bonds between the beads and the antigens. We coupled 12.5 million activated beads – in phosphate-buffered saline at pH 7.2 – with 30 µg of each dengue virus-like particle and 7.5 µg of the chikungunya virus antigen. For the malaria antigens and glutathione-Escherichia coli – that might have reacted with E. coli proteins inadvertently coupled to the beads – each elution was diluted 1:4 with the same buffer containing sufficient crude E. coli extract to give a final concentration of 3.0 µg extract per mL.E. coli extract.Each dried blood spot was eluted overnight, at 6 °C, in 500 µL of a buffer consisting of phosphate-buffered saline, at pH 7.2, containing 0.5% (w/v) bovine serum albumin, 0.5% (w/v) polyvinyl alcohol, 0.8% (w/v) polyvinylpyrrolidone, 0.5% (w/v) casein, 0.3% (w/v) Tween 20 and 0.02% (w/v) sodium azide. To adsorb any antibodies to E. coli-treated samples were stored overnight at 6 °C and then clarified by centrifugation at 20 238 g for 10 minutes. All incubation steps were conducted at room temperature – i.e. 20–22 °C – in 96-well filter-bottom plates . For each assay, a 50-µl clarified sample was added to a single well containing 1500 antigen-coupled beads from each coupled-bead classification and incubated, with gentle shaking, for 1.5 hours. The assay was then run as previously described.The For setting the threshold for assay positivity for the chikungunya virus antigen, we used assay results for the blood spots collected, before 2014, from the longitudinal cohort of Haitian children. For all the other thresholds, we used the bead assay to test 86 sera from adults who claimed to have never left the United States . Any serP. falciparum if it was found positive for at least two of the three P. falciparum antigens we investigated. A test blood spot was considered positive for an IgG response to dengue virus if it was found positive for one or both of the virus-like particles we investigated.A test blood spot was considered positive for an IgG response to We used both the bead assay and an enzyme-linked immunosorbent assay (ELISA) to test 78 sera – i.e. 26 sera collected from Haitian adults in 1996, when chikungunya was known to be present in Cambodia but not in Haiti,S-transferase. For this analysis, we used a 400-m zone of indifference to reflect the mean range of an adult female Ae. aegypti.Gi statisticThe positions of the 143 households that participated in the blood spot collection in August 2014 were mapped using a global positional system and ArcGIS software . The same software was then used to perform a so-called hot-spot analysis of the corresponding bead assay results for three of the test antigens: chikungunya virus, the DENV-2 virus-like-particle and the 19-kDa malarial antigen linked to glutathione-P-value of less than 0.05 was considered indicative of statistical significance. Inter-plate consistency was evaluated as the percentage coefficient of variation for the diluted positive controls.We used Spearman’s correlation to investigate the levels of association between the IgG responses for the test antigens. A ,,The positivity thresholds are summarized in In the capture ELISA, using chikungunya virus from a mouse, only 50 of the 78 tested sera gave interpretable results.P. falciparum never exceeded 12% throughout the study. No exposure to any of the three pathogens investigated was evident in 4.9% (3/61) of the longitudinal cohort.Within the longitudinal cohort , no evidThe temporal trends in the background-adjusted fluorescence intensities are summarized in The assay results for one child who was sampled at each of the four time points are summarized in P. falciparum, respectively, and 17 (13.4%) showed no evidence of exposure to any of these pathogens.Of the 127 children who each provided a single blood spot in 2014, 96 (75.6%), 77 (60.6%) and eight (6.3%) were found seropositive for chikungunya virus, dengue virus and In the assays based on dengue virus antigens, median intensities  and prevr2 < 0.08; P > 0.12) or P. falciparum  or between dengue virus and P. falciparum .The Spearman’s correlations indicated no significant cross-reactivity among our three study pathogens – i.e. between chikungunya virus and either dengue virus  and between the two dengue antigens .In contrast, in terms of positivity in the bead assays, a strong correlation existed among the three malaria antigens (http://www.who.int/bulletin/volumes/94/11/16-173252). In general, the detected IgG responses to the chikungunya virus antigen and DENV-2 virus-like particle appeared to get weaker the further north-west the sampling site – i.e. the further away from the city of Léogâne and the closer to the ocean. Conversely, the corresponding responses to the 19-kDa malarial antigen linked to glutathione-S-transferase exhibited no discernible spatial pattern – although they were, in general, relatively weak.Clusters identified in the spatial analysis of the study area, which lies immediately outside urban Léogâne, are shown in The results from the bead assay indicate that infection with chikungunya virus only became common in Haiti in 2014 and that there was rapid transmission of the virus after its introduction into an immunologically naive Haitian population. By the first week of 2015, there were 64 695 suspected cases of chikungunya virus infection in Haiti and – since there were over 500 000 cases reported, at the same time, in the neighbouring Dominican Republic – even this high number is likely to have been an underestimate because of underreporting.Ae. aegypti and Ae. albopictus for habitats with relatively lower salinity and/or wind activity.Our bead assay was sensitive enough to show both an initial immune response and an anamnestic response to the DENV-2 and DENV-3 virus-like-particles and it appeared to be unaffected by cross-reactivity among the three pathogens we investigated. It also indicated an increasing gradient, in the level of IgG responses to chikungunya virus and dengue virus antigens, running from an urban area towards the beach. One reason for this gradient may be the preference of Our results relating to dengue virus were consistent with those of previous studies on dengue in Haiti. Prior to 1969, dengue virus had not been reported in Haiti. In a study conducted in Port-au-Prince between 1969 and 1971, however, antibody responses to this pathogen were detected in 43%, 60% and 76% of the subjects aged 1–5, 6–10 and 41–50 years, respectively.Our results relating to chikungunya virus, although based on a relatively small sample, are consistent with those of an unpublished but much larger study conducted in early 2015. The subjects of this larger study, which was focused on malaria, were checked for IgG responses to the chikungunya virus we used in the bead assay. The seroprevalence of such responses was found to be 82% in urban areas throughout Haiti – similar to the 79% found in our urban study site – and only 45% in rural areas . In Haiti, rural areas are generally at higher altitudes than urban areas and the cool temperatures at high altitudes tend to limit vector densities.–,Multiplex bead assays have been found to be at least as sensitive as ELISA


Hepeviridae includes enterically transmitted small non-enveloped positive-sense RNA viruses. It includes the genera Piscihepevirus, whose members infect fish, and Orthohepevirus, whose members infect mammals and birds. Members of the genus Orthohepevirus include hepatitis E virus, which is responsible for self-limiting acute hepatitis in humans and several mammalian species; the infection may become chronic in immunocompromised individuals. Extrahepatic manifestations of Guillain–Barré syndrome, neuralgic amyotrophy, glomerulonephritis and pancreatitis have been described in humans. Avian hepatitis E virus causes hepatitis–splenomegaly syndrome in chickens. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Hepeviridae, which is available at www.ictv.global/report/hepeviridae.The family  T=3 capsids contain flat dimers, with less curvature than those of T=1 virus-like particles [The virions of human hepatitis E virus are icosahedral, non-enveloped, spherical particles with a diameter of approximately 27–34 nm , Fig. 1.7G-capped and the 3′-end is polyadenylated , rats, ferrets, shrews, bandicoots, mink (Orthohepevirus C members), bats (Orthohepevirus D members), and birds (Orthohepevirus B members) [members) . Unclassmembers) .Piscihepevirus. This genus includes a single species whose typical isolate, cutthroat trout virus, infects trout, although its pathogenicity and full host range are unknown [ unknown .www.ictv.global/report/hepeviridae.Full ICTV Online (10th) Report:


Since 2007, Zika virus has spread through the Pacific Islands and the Americas. Beginning in 2016, women in Brownsville, Texas, USA, were identified as possibly being exposed to Zika virus during pregnancy. We identified 18 pregnant women during 2016–2017 who had supportive serologic or molecular test results indicating Zika virus or flavivirus infection. Two infants were evaluated for congenital Zika syndrome after identification of prenatal microcephaly. Despite standard of care testing of mothers and neonates, comparative results were unreliable for mothers and infants, which highlights the need for clinical and epidemiologic evidence for an accurate diagnosis. A high index of suspicion for congenital Zika syndrome for at-risk populations is useful because of current limitations of testing. Aedes aegypti and Ae. albopictus mosquitoes, vectors that also transmit other arboviruses, such as dengue virus and chikungunya virus. Zika virus was discovered in the Zika Forest of Uganda in 1947 in rhesus and macaque monkey populations (http://www.who.int/emergencies/zika-virus/timeline/en/). Until 2007, only 14 cases of human infection were reported in Asia and Africa  who spent the first 4 months of her pregnancy in Matamoros, Mexico. She received prenatal Zika testing while residing there, and results were negative. She moved to Brownsville, where she received prenatal care at 28 weeks’ gestation. She was screened for Zika virus by serum IgM testing; results were negative. She was referred for maternal fetal medicine at 36 weeks’ gestation because of suspected microcephaly. The fetus was found to have microcephaly: head circumference (HC) 251 mm, which was 5 SD below the mean value. The mother denied having any symptoms of Zika virus infection . At 37 weeks’ gestation, transvaginal fetal neuroimaging was performed; results showed calcifications in the cortical white matter–gray matter junction, but no calcifications were observed in the thalami , panel CAn elective primary cesarean delivery was performed at 39 weeks’ gestation. APGAR scores for the baby were 9 at 1 min and 9 at 5 min. At initial examination, the neonate had a vesicular generalized rash, overriding sutures, and microcephaly. The initial HC of the infant was 29 cm, which was 2.63 SD below the mean value for term male newborns. Birthweight was 2.62 kg (4.76 percentile), and birth length was 45 cm (3.2 percentile). On further examination, mild craniofacial disproportion with narrow and laterally depressed frontal bone and mild retrognathia was seen. No limb contractures were observed , panel AToxoplasma spp., and parvovirus.The newborn was transferred to the neonatal intensive care unit (NICU) at Driscoll Children’s Hospital  on day 1 of life. Because of a generalized vesicular rash, concern for herpes simplex virus infection prompted treatment with acyclovir for the first 2 days of life. The rash was diagnosed as neonatal pustular melanosis; it faded by day 1 of life and disappeared by day 2 of life  testing because of new information concerning development of seizures in 30%–50% of infants with congenital Zika syndrome; the EEG result was unremarkable  who lived in Brownsville. She reported weekly travel to Matamoros, Mexico, during the early stages of her pregnancy. She denied any viral symptoms of rash, fever, malaise, arthralgia, or conjunctivitis. She was screened by her obstetrician for Zika virus at 23 weeks’ gestation by a PCR for serum; results were positive. Results were negative for a Zika virus PCR for urine and serum Zika virus IgM. At 28 weeks’ gestation, fetal ultrasonography was performed for growth and anatomy evaluation. The fetus had microcephaly and was referred for maternal fetal medicine evaluation. The HC of the fetus was 203 mm at 29 weeks’ gestation, which was 4−5 SD below the mean value. Coarse calcifications were observed in the basal ganglia and thalami by transabdominal and transvaginal fetal neuroimaging  might have shown a false-negative result, or the infection might have occurred later. However, even without definitive evidence of maternal Zika virus infection at the time of delivery, the neonate showed a positive result for Zika IgM in serum, and a subsequent placental test showed a positive result, which confirmed maternal infection.Maternal diagnosis for case-patient 2 was confirmed with positive PCR results for serum at 23 weeks’ gestation. However, despite this newborn displaying more severe features of congenital Zika syndrome postnatally , results of serum testing for Zika virus infection were negative.In conclusion, results for these 2 case-patients indicate the complexity and challenges of screening and diagnostic testing for congenital Zika syndrome and illustrate the need for clinical findings and epidemiologic history. We advise a high index of suspicion for congenital Zika syndrome for at-risk populations on the basis of current limitations of testing.


During 2015–16 Brazil experienced the largest epidemic of Zika virus ever reported. This arthropod-borne virus (arbovirus) has been linked to Guillain-Barré syndrome (GBS) in adults but other neurological associations are uncertain. Chikungunya virus has caused outbreaks in Brazil since 2014 but associated neurological disease has rarely been reported here. We investigated adults with acute neurological disorders for Zika, chikungunya and dengue, another arbovirus circulating in Brazil.st November 2015 and 1st June 2016. Cerebrospinal fluid (CSF), serum, and urine were tested for evidence of Zika, chikungunya, and dengue viruses.We studied adults who had developed a new neurological condition following suspected Zika virus infection between 1Of 35 patients studied, 22 had evidence of recent arboviral infection. Twelve had positive PCR or IgM for Zika, five of whom also had evidence for chikungunya, three for dengue, and one for all three viruses. Five of them presented with GBS; seven had presentations other than GBS, including meningoencephalitis, myelitis, radiculitis or combinations of these syndromes. Additionally, ten patients positive for chikungunya virus, two of whom also had evidence for dengue virus, presented with a similar range of neurological conditions.Zika virus is associated with a wide range of neurological manifestations, including central nervous system disease. Chikungunya virus appears to have an equally important association with neurological disease in Brazil, and many patients had dual infection. To understand fully the burden of Zika we must look beyond GBS, and also investigate for other co-circulating arboviruses, particularly chikungunya. During 2015–16, Brazil experienced the largest outbreak of the mosquito-borne Zika virus ever reported and saw a subsequent increase in cases of Guillain-Barré syndrome (GBS), a disorder of the peripheral nervous system  that can result in paralysis and sometimes death. In this assessment of adults presenting with suspected Zika virus-associated neurological disease in Rio de Janeiro, Brazil, we add to the growing body of evidence linking Zika to a wider range of neurological disease, including disease of the central nervous system . We also show that many patients initially thought to have neurological disease associated with Zika virus were in fact infected with chikungunya virus, another arthropod-borne virus (arbovirus) that is also associated with a wide range of neurological disease. Importantly, many patients had evidence of infection with more than one virus. We discuss the challenges in diagnosing the infections and how different body fluid samples can be used to help facilitate this. Our results suggest that clinicians and public health officials must look beyond GBS if we are to understand fully the disease burden of Zika virus. In addition, we highlight the need to investigate patients with acute neurological syndromes for other co-circulating arboviruses, particularly chikungunya. Flavivirus, family Flaviviridae) that causes a fever-arthralgia-rash syndrome and is transmitted principally by Aedes mosquitoes. An apparent association between Zika virus and an increase in severe congenital disease and other neurological disorders, particularly Guillain-Barré syndrome (GBS),[Zika virus is an arthropod-borne virus (arbovirus) first isolated in Uganda in 1947, which spread to cause large outbreaks in Micronesia in 2007, French Polynesia in 2014 and Latin America from 2015.[me (GBS),–6 promptA carefully conducted case-control study of the French Polynesian outbreak showed an association between Zika virus infection and GBS, althoughAedes mosquitoes and leads to a fever-arthralgia-rash syndrome; it also occasionally presents with neurological disease, including GBS, encephalitis and myelitis,[Alphavirus, family Togaviridae) there is no serological cross reactivity with the flaviviruses, making diagnosis more straightforward. Although chikungunya virus co-circulates in many Zika-affected areas, including Colombia and Brazil, and can cause neurological disease, its role has not been assessed.Chikungunya is another arbovirus also identified in Africa in the 1950s that has spread to cause epidemics in the tropics in recent years. It was fmyelitis, althoughmyelitis, Because To assess the spectrum of neurological disease associated with Zika virus, we studied adults in Rio de Janeiro with acute neurological syndromes following suspected Zika virus infection. Given their similarities and co-circulation, we also investigated for chikungunya and dengue viruses.We studied patients who had developed a new neurological condition associated with suspected Zika virus infection, whose samples had been submitted to the Flavivirus Reference Laboratory of the Instituto Oswaldo Cruz (Fiocruz), Rio de Janeiro.The study protocol was approved by the Comitê de Ética em Pesquisa do Instituto Nacional de Infectologia Evandro Chagas (reference 59254116.0.1001.5262). Patient-identifying data was anonymised.st November 2015 to 1st June 2016, who had presented with an acute neurological condition associated with a suspected Zika virus infection, as identified by fever, arthralgia or rash illness in the preceding three months. In this evolving epidemic situation we used three approaches to identify patients: using the laboratory database, we retrospectively identified 29 patients who had had either cerebrospinal fluid (CSF) regardless of indication, or serum and/or urine in the context of neurological disease, sent to the laboratory for Zika virus diagnostics; additionally, clinicians from our study hospitals identified six further patients, whose CSF sample was not on the database and whose serum and/or urine request forms did not have an indication  in Rio de Janeiro from 1dication . PatientClinical information was obtained from case notes and discussion with the patients’ clinicians and documented on standardised case report forms by a member of the study team. The information obtained included demographics, past medical history, admission history, examination, investigations, diagnosis, and management. Investigations included brain and spine imaging for patients with suspected central nervous system infection, and nerve conduction studies with or without electromyography for those with peripheral disease. Nerve conduction study results were reviewed by an independent expert neurophysiologist to ensure consistency. The Brighton criteria were used to indicate the level of certainty for diagnosing GBS and similar criteria were applied for radiculitis, encephalitis, myelitis, and meningitis (appendix). We determined whether patients had peripheral nervous system disease , CNS disease  or both.CSF, serum, and urine samples were tested for evidence of Zika, chikungunya, and dengue virus infection at the Fiocruz Flavivirus Laboratory. We considered detection of viral RNA and/or IgM-specific antibody in the CSF as evidence of recent CNS infection as previously; IgM antiAn expanded protocol based on the interim recommendations from the WHO for laboratory testing for Zika virus was followed: RNA was The median time from illness onset to the development of neurological symptoms was compared for those with CNS and peripheral nervous system disease, and for those with or without CNS Zika virus infection, using the Wilcoxon-Mann-Whitney U-test.We identified 35 patients with new neurological disease associated with a suspected Zika virus infection. Evidence for recent arbovirus infection was found in 22 (63%) of them. Twelve (34%) had evidence of Zika virus infection. Three (9%) with evidence of Zika virus infection alone presented with GBS (two patients) or encephalitis (one). Nine (26%) with evidence of Zika also had evidence for another arbovirus; five with chikungunya, three with dengue, one with both chikungunya and dengue. Three of them presented with GBS, including one with the facial diplegia and paraesthesia variant, and six had CNS infection, predominantly encephalitis and/or myelitis. Ten patients (29%) negative for Zika had evidence for another arboviral infection; eight with chikungunya and two with chikungunya and dengue. Two of these presented with GBS and eight with CNS disease.The remaining 13 patients (37%), with no evidence of a recent Zika, chikungunya, or dengue virus infection, presented with GBS (six patients) or CNS disease (seven). Their diagnoses are given in the appendix, but they are not considered further here.All 22 patients with evidence of recent arbovirus infection were Brazilian nationals with no recent travel history, from 18 different districts of Rio de Janeiro. The median (range) age was 51·5 (17–84) years. The male:female ratio was 1:1. Their individual clinical features are summarised in An 80-year-old Brazilian female with a past medical history of hypertension, obesity and osteoarthritis presented with a one-day history of headache, confusion, and symmetrical weakness in all four limbs. Five days earlier, she had fever, rash, arthralgia and general malaise. There was no preceding history of respiratory or gastrointestinal infection, or recent vaccination, and she did not have any bowel or bladder symptoms.On examination, she had a diffuse, centrifugal, erythematous rash, with mild joint swelling of the knees. She was mildly confused with a Glasgow Coma Scale of 14. She had a flaccid tetraparesis, worse proximally  than distally . Her reflexes were present but depressed throughout. As far as assessment would allow, light touch, vibration, temperature and proprioception appeared normal. There was no sensory level apparent, or cranial nerve involvement.9/L and raised creatinine of 160 µmol/L. An MRI scan of her brain and spine showed signal abnormality involving the anterior medulla, anterior cervical and thoracic cord, temporal lobes, amygdala and a small area adjacent to temporal horn of her left lateral ventricle, with pachymeningeal enhancement , rash (68%), malaise (55%) and arthralgia (50%). Two patients had diarrhoea in the month preceding their neurological illness ; no patiThe seven patients with GBS all had a preceding febrile and/or rash syndrome, which was a median (range) 12 (5–41) days before the neurological presentation; there was no statistically significant difference in prodrome length between those with and without CNS Zika infection. The presentations for six patients were similar—typically paraesthesia (five patients) with a rapidly ascending symmetrical flaccid paralysis, involving all four limbs in five patients, or the legs only in one (patient 5). Another (patient 4) presented with a GBS variant, with bilateral lower motor neuron facial nerve palsies and paraesthesia in all four limbs.Fifteen patients presented with CNS disease, including encephalitis and/or myelitis, with or without involvement of the meninges or peripheral nerves. All had a febrile or rash syndrome, many with arthralgia and malaise. The median (range) time delay between this systemic illness and neurological disease was 4 (0–27) days; for these patients with CNS disease, there was no statistically significant difference in prodrome length between those with and without CNS Zika infection.The eight patients with encephalitis (with or without other CNS disease) had a median (range) Glasgow Coma Scale score of 13·5 (3–15); six were confused, and two had seizures. One patient had a supranuclear gaze palsy; two had facial weakness and four had difficulties with speech or swallowing. The ten patients with myelitis  comprised five with paraparesis , four with quadraparesis  and one with a triparesis. Seven of these patients had a sensory level, six had urinary retention and three had urinary incontinence. Three patients with flaccid paresis had signs and symptoms compatible with transverse myelitis, namely a sensory level and urinary retention. However, one patient with encephalomyelitis with flaccid areflexic quadraparesis (patient 9) had extensive imaging changes in the anterior of the cord consistent with poliomyelitis-like anterior horn cell damage.Neurophysiological studies (see below) confirmed the involvement of lower motor neurons for four patients with myelitis: two of those with flaccid paraparesis, one with flaccid quadraparesis, and one with paraparesis and normal tone. The clinical characteristics of the patients with central, peripheral, and mixed nervous system disease are compared in Zika virus RNA was detected in two CSF, one serum and six urine samples (from eight patients); Zika IgM was detected in 10 CSF and two serum samples (10 patients). Chikungunya virus RNA was detected in 11 CSF, six serum, and three urine samples (14 patients); chikungunya IgM was detected in six CSF and seven serum samples (11 patients). Dengue virus RNA was not found; dengue IgM was found in two CSF and six serum samples (six patients).As expected, many patients had serum IgG against dengue virus, consistent with prior exposure (appendix). Of the 13 patients with no evidence of recent arbovirus infection, serum Zika virus IgG was detected in four. Anti-GM1, GD1a, GD1b and GQ1b antibodies were found in the serum of patients with both peripheral and central nervous system disease .Ten out of the 22 patients had a CSF pleocytosis  and 15 had a CSF protein above 0·45 g/L. Four patients had a mild thrombocytopenia and seven had a peripheral leucocytosis, but none had leucopenia. Imaging studies are detailed in Eight  of the 22 patients required admission to an intensive care unit; six (27%) needed intubation . Ten patients were treated with intravenous immunoglobulin, two with corticosteroids, and seven with both; four with encephalitis received aciclovir as presumptive treatment for herpes simplex virus encephalitis. Four patients developed hospital acquired infections, including ventilator-associated pneumonia and sacral osteomyelitis secondary to immobility. Fourteen  patients improved; one with evidence of Zika +/- dengue infection of the CNS deteriorated rapidly and died.With 84 countries or territories now affected by Zika virus, increasiFour patients with evidence of Zika virus infection also had CNS infection with chikungunya virus. Ten further patients negative for Zika tested positive for chikungunya. In South America, reports of neurological disease associated with chikungunya virus are scarce, which may reflect a lack of awareness among clinicians about the potential to affect the nervous system, or the relatively recent arrival of the virus. Interestingly, in one patient (patient 17) virus was detected 30 days after the onset of neurological disease, suggesting a persistent infection or a late coincidental infection. In our study of patients from Rio de Janeiro, chikungunya virus was as important a cause of neurological disease as Zika virus. Its importance in other settings where Zika virus is assumed to be the cause of febrile illness, with or without neurological disease, needs to be assessed urgently.We are only now beginning to understand the full spectrum of neurological syndromes associated with both Zika and chikungunya infections. For example, patient 19 showed clinical signs of myeloradiculitis and had neurophysiological evidence of AIDP, which is consistent with simultaneous diagnoses of both myelopathy and a form of GBS. Another patient 15) had an unusual combination of encephalitis, cerebral microangiopathy demonstrated on MRI and inflammatory myopathy based on neurophysiological studies. We saw evidence of extensive intramedullary myelitis in some patients, and anterior myelitis in another Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


To analyse the proportions of protein identity between Zika virus and dengue, Japanese encephalitis, yellow fever, West Nile and chikungunya viruses as well as polymorphism between different Zika virus strains.k-mers across the proteome, with k ranging from 6 to 100. We assessed accessibility of protein fragments by calculating the solvent accessible surface area for the envelope and nonstructural-1 (NS1) proteins. We used published protein sequences for the Zika virus and obtained protein sequences for the other viruses from the National Center for Biotechnology Information (NCBI) protein database or the NCBI virus variation resource. We used BLASTP to find regions of identity between viruses. We quantified the identity between the Zika virus and each of the other viruses, as well as within-Zika virus polymorphism for all amino acid k-mers) of protein fragments on the list. In total, we identified 294 Zika virus protein fragments with both low proportion of identity with other viruses and low levels of polymorphisms among Zika virus strains. The list includes protein fragments from all Zika virus proteins, except NS3. NS4A has the highest number (190 We provide a candidate list of protein fragments that could be used when developing a sensitive and specific serological test to detect previous Zika virus infections. Target product profiles for better diagnostic tests for Zika virus infection,Monitoring the geographic and the demographic distribution of people infected with Zika virus is important for informing decision-makers and researchers during the ongoing epidemic. Health officials also need further knowledge about the associations between Zika virus infection and its sequelae, such as microcephaly and Guillain–Barré syndrome. However, the absence of a sensitive and specific serological test for detecting prior Zika virus infection impedes research. According to the World Health Organization’s Flaviviridae, while chikungunya virus belongs to the Togaviridae family. Although they belong to different virus families, Zika and chikungunya viruses share some similarities in envelope protein folding and membrane fusion mechanisms.Dengue and Zika viruses belong to the virus family ,–,Active Zika virus infections can be detected by nucleic acid-based diagnostic tools.We hypothesize that immunogenic protein regions with sequence dissimilarity may exist across arthropod-borne viruses (arboviruses) and that antibodies targeting these regions may be less likely to be cross-reactive. Identifying such regions could aid the development of specific microarray-based serological tests, such as a peptide microarray, to detect Zika virus and/or other related viruses. A peptide microarray is a high-throughput method for detecting interactions between peptides and antibodies and is composed of multiple spots of peptides on a solid surface.We used publicly available proteomic sequencing data . For theE)-value threshold of 10, that is the expected number of hits of the observed similarity, by chance, is fewer than 10. The results are robust and we obtained the same results when E-value thresholds were 5 or 50. When comparing the chikungunya and the Zika viruses, we used an E-value threshold of 1000, because chikungunya does not belong to the Flaviviridae family and we could not identify any regions of similarity when using an E-value threshold of 10. For all protein fragments across the proteome, we calculated the proportion of shared amino acids between virus species and polymorphism among different Zika virus strains. We analysed protein fragments of different lengths, so called k-mers (where k is the amino acid length of the protein fragment), with k equal to 6 or ranging from 10 to 100. We used a sliding window approach, where we moved the window one amino acid at a time along the proteome to include every possible k-mer. To be conservative, we identified protein fragment identity between species by the maximum identity among all the pairs of strains for each window considered. For analysing the identity with dengue virus, we used the highest identity between the Zika virus and all four serotypes of the dengue virus for each window considered. To assess if protein identity between the Zika virus and each of the dengue serotype was significantly associated with polymorphism within each dengue virus serotype, we calculated P-values by using Pearson's correlation test.We used BLASTPTo identify polymorphisms within viruses, we used both the average pairwise difference and the proportion of polymorphic sites. Average pairwise difference is calculated by averaging the proportions of differences in peptide sequences from all pairs of the virus strains. We chose to plot the proportion of polymorphic sites in the figures because it is less sensitive to population structure and/or sampling bias.k-mers with low proportion of identity between the Zika virus and other arboviruses as well as low polymorphism between different strains of Zika virus as lead candidate protein fragments. The rationale for this approach was that fragments with low between-species identity and low within-species polymorphism are most likely to have both the required specificity and sensitivity for such tests. We chose k-mers in the bottom quintile of values of identity and polymorphism for each k-mer length.To identify potential protein fragments that could be used for diagnostic tests, we selected 2 as the upper limit for buried residues, as this value corresponds to half the surface area of a single water molecule. The regions at the C-terminal end of the dengue virus envelope protein interact with the viral lipid membraneC-terminus in Zika virus envelope protein is also buried. For the lead candidate list, we excluded the k-mers without any continuous exposed peptides longer than five amino acids in the two proteins, because exposed peptides are more likely to be antigenic. The threshold of five amino acids was chosen because 99.7% of experimentally determined antigenic B-cell epitopes for flaviviruses found in Virus Pathogen Database and Analysis Resource database are longer than five amino acids.http://www.viprbrc.org/.Insights into protein structures are critical for assessing the possible antigenicity of peptides, because buried peptides are less likely to be antigenic.k-mers are available from the corresponding author. As an example, On average, Zika virus shares 55.6% amino acid sequence identity with dengue virus, 46.0% with yellow fever virus, 56.1% with Japanese encephalitis virus, 57.0% with West Nile virus and 1.3% with chikungunya virus. The identity between Zika virus and other viruses and Zika virus polymorphism for all http://www.who.int/bulletin/volumes/95/7/16-182105). The list excluded 10.9% (36/330) of k-mers containing previously identified B-cell epitopes for other flaviviruses than Zika, because they are likely to be cross-reactive. Protein fragments from all Zika virus proteins, except NS3, are present in the list. NS4A has the highest number (190 k-mers) of candidate protein fragments . In contrast, one region (amino acid positions 430–500 in the proteome) in the envelope protein shows both low identity between Zika virus and other flaviviruses and low polymorphism within Zika virus  and the P-values < 0.01 for all dengue serotypes; Protein identity between dengue and Zika viruses is negatively associated with polymorphism within the dengue virus proteins , suggesting that NS1 is not the top candidate protein for low cross-reactivity. Recently, Euroimmun AG  developed a Zika virus ELISA for immunoglobulins (Ig)M and IgG, based on the NS1 protein. Preliminary results show that the test is Zika virus specific.To further dissect the molecular mechanism leading to the Zika virus sequelae not seen with other flaviviruses, the protein fragments presented in the candidate list may be useful. The low polymorphisms in NS2A and NS2B proteins might be good candidates to start investigating the possible molecular link between Zika virus and microcephaly and Guillain–Barré syndrome.Peptide-sequence identity is unlikely to fully predict cross-reactivity due to other factors, such as glycosylation. Nonetheless, this analysis based on publicly available sequences provides a step towards the development of a serological test that can distinguish previous Zika virus and co-circulating arbovirus infections.


Zika virus is currently causing extensive outbreaks in a number of countries in South and Central America and the Caribbean and has been associated with foetal abnormalities. We report an outbreak of Zika virus infection in a Chinese tour-group returning from a nine day holiday in Fiji and Samoa. The index case was a 38-year old male who developed symptoms while travelling back from Fiji to Hong Kong on the 14th February, 2016. A field investigation was initiated to define the epidemiological, clinical and virological characteristics of Zika virus infection in this tour group and revealed two further symptomatic infections and one asymptomatic infection among the 33 travellers; an overall infection attack rate of 12% in these travellers. Active surveillance led to detection of Zika virus RNA in the serum of one case four days prior to onset of symptoms and detection of Zika virus in saliva from one asymptomatic infection. Flaviviridae and the genus Flavivirus1Aedes mosquitoes, such as Aedes aegypti, Aedes albopictus, Aedes africanus, and Aedes luteocephalus4568911Zika virus, related to dengue, yellow fever, Japanese encephalitis, and West Nile viruses, is an arbovirus belonging to the family th 2016, and a total of 9 cases were reported from February 1st to 29th, 2016In mainland China, the first imported Zika case was confirmed on February 9th and similar samples for the other 32 participants were collected on February 17th. Except the serum sample of a three year old boy which was not available, 32 serum, 33 saliva and 33 urine samples were collected from the travel group. Besides the index patient, samples from three people in the group were positive for Zika virus. Serum, saliva, and urine were collected daily when any sample for that person was found positive for Zika virus. Furthermore, saliva and urine were collected from the other participants on February 19th, February 22nd, and February 25th.The travel group comprised 33 persons, 17 male and 16 female. The median age of participants was 37 years . Serum, saliva, and urine of the index patient were collected on February 16th, 2016. They reached Fiji at 7:10 am, February 5th and went sightseeing until February 7th. They reported that they mainly swam and rowed a boat. Although they did not have a history of mosquito bites in Fiji, they wore short-sleeved shirts and short pants and the mosquito density was very high.A total of four Zika virus infections including three patients and one asymptomatic infection were identified. The index case was a 38-year old male. He and the other 32 persons (his family members and friends) travelled to the republic of Fiji (Fiji) by air on February 4th. During the period in Samoa, the index case swam, rowed a boat, hunted in the jungle, attended a torch party  and visited tree houses. The index case reported a history of mosquito bites during the latter three activities. The group returned to Yiwu, China by way of Fiji, Hong Kong, Shenzhen and Guangzhou from February 14th to 15th. On February 14th, the index case developed fever, chills and diarrhea when he was in the airplane from Fiji to Hong Kong and his body temperature was 38.5 °C at 16:48 on February 14th. He had rashes behind his ears on the evening of February 15th and the rashes expanded to his body . The rashes began to subside from February 18th and disappeared on February 20th. His body temperature was 37.3 °C at 16:00 on February 16th and returned to normal from February 17th. Saliva and urine tested positive for Zika virus by Zhejiang Provincial Center for Disease Control and Prevention (ZJCDC) on the evening of February 16th. Then samples were transported to the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC) on February 17th and were confirmed to be positive for Zika virus on February 18th. The index case was hospitalized from February 16th to February 24th  at 18:50, February 7ary 24th . No Zikaary 20th .th and rashes appeared on his back (February 21st) and face (February 22nd). The rashes began to subside on February 23rd and disappeared on February 25th. Zika virus RNA was found in his serum (from February 17th), saliva (from February 19th) and urine (from February 22nd).Case 2, case 3 and the asymptomatic infection were family members. Case 2 was the son, case 3 was the father and the asymptomatic infection was the mother. Case 2 was an 8-year old boy, case 3 was a 36-year old male, and the asymptomatic infection was a 34-year old female. These three cases were in the same travel group . Notablyst. However, Zika virus RNA was found in his serum sample collected on February 17th. Serum, saliva and urine of case 3 which were collected on February 19th were all positive for Zika virus. Serum and saliva samples collected on February 20th were also positive , rashes and conjunctivitis on February 21positive . There wth to February 18th. Saliva collected on February 19th was positive for Zika virus  in GenBank. According to published papers1516th (KU955589), May 18th (KX253996), June 13th (KU866423), 2016, respectively. Phylogenetic analysis showed that complete sequences of Zika virus in our study belong to the same clade (We submitted sequences of the index patient (KU820899) and case 2 KX117076) to GenBank on March 7076 to Geme clade . They weSince early 2015, a widespread outbreak of Zika fever, caused by the Zika virus, is ongoing, primarily in the Americas. The outbreak began in April 2015 in Brazil, and has spread to other countries in South America, Central America, Mexico, and the Caribbean. Travel-related imported infections have also been increasingly reported in many countries. In China, the first Zika case from Samoa and Fiji was identified by the General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (AQSIQ)Aedes mosquitoes, vectors for Zika virus, are found across many provinces of China1819Aedes mosquitoes have occurred frequently in China in recent years2122Due to the fact that most Zika virus infections are asymptomatic and symptoms of Zika fever are non-specific, it is impossible to identify all imported Zika virus infections in China. Till now, Zika virus is reported to be pandemic in only some areas including South America, Central America, and the Caribbean. Moreover, the amount of travel between China and Zika virus endemic areas has increased after Spring Festival. It is inevitable that more and more Zika virus infections will be imported to China and most of them can’t be identified. Furthermore, st, but his serum had been positive for Zika virus since February 17th. One study reported that 42 (3%) of 1505 blood donors were found positive for ZIKAV by PCR although they were asymptomatic at the time of blood donation in French PolynesiaCase 3 developed symptoms of fever on February 21An asymptomatic infection was also identified in our study. Although it is possible that this is a false positive or a result of cross contamination as only the patient’s saliva sample was positive for Zika virus, we can’t rule out that Zika virus could have been detected in serum or urine if these samples had been collected daily. Consequently, we suggest that persons who come from Zika virus endemic areas should reduce their sexual activities and outdoor activities during mosquito active period for 12 days even if they don’t develop symptoms of Zika fever.In our study, Zika virus RNA was found in serum, and also in saliva and urine. Similar to dengue virus and West Nile virus252728293031Consistent with other studies, symptoms of Zika fever were milder than dengue fever and chikungunya feverThere are several limitations of our study. First, serum was not collected daily from the other persons in the travel group. Second, semen of all adult males in the group were not available. Third, detection methods of Zika virus IgM and IgG had not been established in our laboratory. As a result, we don’t know whether other participants have been infected with Zika virus.Aedes mosquito active seasons triggered by imported cases. Our study also indicates cases during the incubation period and asymptomatic infections might be infectious. They might play an important role in the transmission of Zika virus even when they have no symptoms. These results indicate that more research should be done to explore the role of Zika cases during the incubation period and asymptomatic infections in the spread and transmission of Zika virus.In summary, we identified the first cluster of Zika virus infections in China among a group who travelled to Fiji and Samoa. It is likely that the outbreak of Zika virus infection in China might occur in In accordance with the document entitled “The diagnosis and treatment programs of Zika virus disease” issued by the National Health and Family Planning Commission of the People’s Republic of Chinath, 2016, Guangdong Provincial Center for Disease Control and Prevention (CDC) informed ZJCDC that they found a suspected Zika case who had entered China through a port in Guangdong Province and then went to Yiwu City, Zhejiang Province, China. Epidemiologists from ZJCDC and Yiwu Municipal CDC (YWCDC) conducted field investigation of the index case and all persons in the travel group. A standardized questionnaire was used to collect demographic information such as age, gender, occupation, residential address and exposure history. All individuals in the travel group were placed under daily active surveillance for fever, rash, arthralgia or conjunctivitis for 12 days following their departure from Samoa. Individuals were taken to hospital as soon as possible when they developed symptoms such as fever, rash, arthralgia or conjunctivitis.On February 16The research protocol was approved by the ethics committee of Zhejiang Provincial Center for Diseases Control and Prevention. All participants provided written informed consent. For children, parents or guardians of eligible children were informed and asked to provide written informed consent on behalf of their children. The aims of our study were explained to all of them, and their written consent was obtained before inclusion onto this study.th, 2016. The viral RNA of each sample was extracted using the QIAamp viral RNA minikit according to the manufacturer’s instructions . Then the viral RNA was screened with a rRT-PCR assay targeting the Zika virus gene with the primers ZIKVF 9121–9141 (5′-CCT TGG ATT CTT GAA CGA GGA-3′) and ZIKVR 9312–9290 (5′-AGA GCT TCA TTC TCC AGA TCA A-3′). The temperatures and cycling times are as follows: 50 °C 10 minutes, 1 cycle; 95 °C 30 minutes 1 cycle; 95 °C 15 seconds and 55 °C 45 seconds, 40 cycles. All experiments were performed in accordance with relevant guidelines and regulations. The assay was provided by the National Institute for Viral Disease Control and Prevention, China CDC and the institute approved the experiments.Samples including serum, saliva and urine of the index case and two other cases were collected daily once the case was hospitalized. In order to identify whether individuals who had the same exposure history were infected with Zika virus, samples including serum, saliva and urine were also collected once from participants on February 17st, 2016. The Maximum likelihood (ML) phylogenetic trees model was used to generate the phylogenetic trees of complete sequences of Zika virus. We used the General Time Reversible Nucleotide substitution model and Gamma distributed with Invariant site (G + I) rates among sites with a bootstrapping resampling process (500 replications) implemented in MEGA version 7.Viral RNA was extracted from the samples of Zika patients using the QIAamp viral RNA minikit . Samples were sequenced based on the Ion torrent PGM platform. Sequencing data were analyzed with Geneious v. 8.1.7. Complete genomes for the index patient and case 2 were submitted to GenBank . We searched all available Zika full genomes from Asia and the South Pacific as of October 1How to cite this article: Sun, J. et al. A cluster of Zika virus infection in a Chinese tour group returning from Fiji and Samoa. Sci. Rep.7, 43527; doi: 10.1038/srep43527 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Identifying Sexual Transmission of Zika Virus Infection,” should have read “In late January, CDC, in collaboration with Texas health officials, worked to confirm sexual contact as the source of Zika virus infection in a Dallas man whose partner had traveled to Venezuela (


Many proteins and signaling pathways participate in anti-viral host responses. Long non-coding RNAs (lncRNAs), a subset of non-coding RNAs greater than 200 nucleotides in length, have been recently described as critical regulators in viral infections. Accumulating research indicates that lncRNAs are important in the development and progression of infectious diseases. LncRNAs are not only involved in anti-viral responses, but in many different virus-host interactions, some of which may be beneficial to the virus. Here we review the current knowledge regarding host and viral lncRNAs and their roles in viral infections. In addition, the potential of using lncRNAs as diagnostic biomarkers is discussed. Compared with miRNA, much less is known about the function of lncRNAs.Fewer than 2% of genes are transcribed into mRNAs. A large number of non-coding RNAs (ncRNAs) also play important cellular functions. Based on their length, ncRNAs can be broadly classified as either short ncRNAs (<200 nucleotides) or long ncRNAs . Short ncRNAs can be further classified as small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). MiRNAs are the best characterized ncRNAs and are well known to induce mRNA degradation or inhibit mRNA translation LncRNAs are the transcribed and spliced products of RNA polymerase II or III transcription, are 5′capped, and may contain a polyadenylated tail at the 3′end. The expression of lncRNAs is much lower in comparison to mRNAs and lncRNAs are expressed in cell-, tissue-, and developmental stage-specific manners  and inhibiting normal miRNA targeting activities on mRNA Figure . CompetiRecently, lncRNAs have been shown to exert both positive and negative effects on innate immunity and virus replication , differential expression of approximately 500 annotated lncRNAs and 1,000 non-annotated genomic regions after SARS coronavirus infection were identified in mice  and with RNA viruses (SeV and MDRV)  infection. In contrast, overexpression of lncRNA#32 significantly inhibited EMCV replication. LncRNA#32 interacts with hnRNPU and ATF2 to regulate ISG expression , was identified in Tmevp3 locus on mouse chromosome10 through gene mapping, which is next to the IFN-γ coding gene Ifng  was initially identified as an inhibitor of transcription factor NFAT  is encoded by Kaposi sarcoma-associated herpes virus (KSHV) and was first identified as a novel abundant 1.2-kb RNA that is transcribed by RNA Polymerase II , mitochondrial and cellular single-stranded binding proteins (SSBPs) and interferon regulatory factor 4 (IRF4), interact with PAN  is 300–500 nt in length and is derived from the 3′ UTR of the RNA genome of flaviviruses, a large group of single-stranded, positive-sense RNA viruses including several human pathogenic viruses, such as yellow fever virus, JEV, dengue viruses, and West Nile virus  tail was recently discovered. This lncRNA guides a chromatin-remodeling complex consisting of proteins such as DNMT3a, EZH2, and HDAC-1 to the viral promoter driving transcriptional regulation , herpes virus saimiri U-rich RNAs (HSURs) and virus-associated RNA I and II (VA I and II) encoded by adenovirus are sometimes also referred to as viral lncRNAs.via interaction with cellular proteins  and EBER2 (172 nt) in latently EBV-infected cells are produced by EBV Iwakiri, . EBER arvia the miRNA pathway after viral infection.Herpesvirus saimiri, which causes aggressive T-cell leukemia and lymphoma, encodes 7 HSURs (Herpesvirus saimiri (HVS) U-rich RNAs)  are produced from RNA polymerase III (pol III) (Mathews and Shenk, In addition to the cellular lncRNAs and virus encoded lncRNAs, HBx-LINE1 was identified as a chimeric lncRNA, which is produced by viral integration into the host genome leading to activation of a LINE-1 sequence such that a chimeric lncRNA is produced (Lau et al., Although, thousands of lncRNAs are expressed after viral infection, the specific lncRNA with experimentally verified functions is limited, thus the roles and functions of lncRNAs in viral infection require further investigation. A deeper understanding of how the lncRNA transcriptome is altered in the infected cell and how these alterations affect the interaction between the host and virus should also be explored. Such studies may help in the identification novel cellular pathways involved in the antivirus response.In addition to the role of lncRNA associated with the antivirus response, lncRNAs may be both unique diagnostic biomarkers as well as novel targets against which new therapeutics can be developed. Virus-related lncRNAs secreted into the serum may serve as prognostic markers. For example, two serum lncRNAs, uc001ncr and AX800134, have potential as novel potential biomarkers to diagnose HBV-positive HCC, especially in the early stage of disease (Wang et al., WL wrote this manuscript and CD designed this project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.