Self-compassion and self-coldness and their relationship with psychological distress and subjective well-being among community-based Hazaras in Australia.

Hazaras are a newly emerging community in Australia and limited research has explored their mental health. The first aim of this study was to explore levels of psychological distress and subjective well-being reported by Hazaras in Australia, and whether scores on psychosocial variables (self-compassion, self-coldness, acculturation, resilience, spirituality), psychological distress and domains of subjective well-being differed by sociodemographic groups. The second aim had two parts: (a) to examine bivariate relationships between the psychosocial variables, psychological distress and subjective well-being; and (b) to examine whether the psychosocial predictor variables independently contributed to subjective well-being and psychological distress when controlling for sociodemographic characteristics. Seventy-two Hazaras (58 men and 14 women), with a mean age of 28.82 years (SD = 8.84) and average length of time residing in Australia of 10.17 years (SD = 4.11), completed an online survey. There were sociodemographic differences in relation to key variables of interest; for example, participants who did not have family members in Australia reported lower levels of global life satisfaction. Moderate negative relationships were found between self-compassion and psychological distress and between self-coldness and subjective well-being. Self-coldness, self-compassion, resilience and acculturation contributed uniquely to psychological distress and subjective well-being when controlling for sociodemographic variables. Although migration programmes that provide permanent residency and allow family members to join refugees in Australia are limited, they appear important. Many of the difficulties facing Hazaras are ongoing, external and beyond their control (e.g. visa status); however, there is a possibility that self-compassion can play a role as a protective factor.

Nairovirus polymerase mutations associated with the establishment of persistent infection in human cells.

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne virus of the Orthonairovirus genus, persistently infects tick cells. It has been reported to establish persistent infection in non-human primates, but virological analysis has not yet been performed in human cells. Here, we investigated whether and how nairoviruses persistently infect human cells using Hazara orthonairovirus (HAZV), a surrogate model for CCHFV. We established a human cell line that was persistently infected with HAZV. Surprisingly, virions of persistently infected HAZV (HAZVpi) were not observed in the culture supernatants. There were five mutations (mut1, mut2, mut3, mut4, and mut5) in L protein of HAZVpi. Mutations in L protein of HAZVpi contribute to non-detection of virion in the supernatants. Lmut4 was found to cause low viral growth rate, despite its high polymerase activity. The low growth rate was restored by Lmut2, Lmut3, and Lmut5. The polymerase activity of Lmut1 was extremely low, and recombinant HAZV carrying Lmut1 (rHAZV/Lmut1) was not released into the supernatants. However, genomes of rHAZV/Lmut1 were retained in the infected cells. All mutations (Lmut1-5) found in L protein of HAZVpi were required for experimental reproduction of HAZVpi, and only Lmut1 and Lmut4 were insufficient. We demonstrated that point mutations in viral polymerase contribute to the establishment of persistent HAZV infection. Furthermore, innate immunity was found to be suppressed in HAZVpi-infected cells, which also potentially contributes to viral persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells. IMPORTANCE: We investigated whether and how nairoviruses persistently infect human cells, using Hazara orthonairovirus (HAZV), a surrogate model for Crimean-Congo hemorrhagic fever virus. We established a human cell line that was persistently infected with HAZV. Five mutations were found in L protein of persistently infected HAZV (HAZVpi): mut1, mut2, mut3, mut4, and mut5. Among them, Lmut1 and Lmut4 restricted viral growth by low polymerase activity and low growth rate, respectively, leading to inhibition of viral overgrowth. The restriction of viral growth caused by Lmut1 and Lmut4 was compensated by other mutations, including Lmut2, Lmut3, and Lmut5. Each of the mutations found in L protein of HAZVpi was concluded to cooperatively modulate viral growth, which facilitates the establishment of persistent infection. Suppression of innate immunity also potentially contributes to virus persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells.

Hazara orthonairovirus nucleoprotein facilitates viral cell-to-cell spread by modulating tight junction protein, claudin-1.

Background: Tight junctions act as a barrier that prevents invasion of pathogens through epithelial cells. This study aims to elucidate the correlation between tight junctions and nairoviruses using Hazara orthonairovirus (HAZV) as a surrogate model for Crimean-Congo hemorrhagic fever virus. Methods: mRNA, total protein, and cell surface protein levels of tight junction proteins were examined by quantitative real-time reverse transcription polymerase chain reaction, immunoblot and flow cytometry, respectively. HAZV growth was measured by plaque assay. Immunofluorescence assay was used to examine viral cell-to-cell spread. The interaction between HAZV nucleoprotein and claudin-1 was analyzed by immunoprecipitation. Results: HAZV infection induced mRNA of several tight junction proteins, especially claudin-1. HAZV infection also induced cell surface expression of claudin-1 protein. Claudin-1 overexpression inhibited the growth of HAZV by blocking its cell-to-cell spread. In contrast, HAZV nucleoprotein completely inhibited HAZV-induced cell surface expression of claudin-1, and this inhibition required interaction between HAZV nucleoprotein and claudin-1. Conclusion: HAZV nucleoprotein was shown to bind to claudin-1 to negatively regulate its cell surface expression, and so can promote cell-to-cell spread of HAZV. This is the first presentation of a possible mechanism behind how nairoviruses counteract tight junction barrier function.

The migrant Hazara Shias of Pakistan and their social determinants for PTSD, mental disorders and life satisfaction.

Background: Ensuring safety and wellbeing of all the minority populations of Pakistan is essential for collective national growth. The Pakistani Hazara Shias are a marginalized non-combative migrant population who face targeted violence in Pakistan, and suffer from great challenges which compromise their life satisfaction and mental health. In this study, we aim to identify the determinants of life satisfaction and mental health disorders in Hazara Shias and ascertain which socio-demographic characteristics are associated with post-traumatic stress disorder (PTSD). Methods: We used a cross-sectional quantitative survey, utilizing internationally standardized instruments; with an additional qualitative item. Seven constructs were measured, including household stability; job satisfaction; financial security; community support; life satisfaction; PTSD; and mental health. Factor analysis was performed showing satisfactory Cronbach alpha results. A total of 251 Hazara Shias from Quetta were sampled at community centers through convenience method based on their willingness to participate. Results: Comparison of mean scores shows significantly higher PTSD in women and unemployed participants. Regression results reveal that people who have low community support, especially from national and ethnic community, religious community, and other community groups, had higher risk of mental health disorders. Structural equation modeling identified that four study variables contribute to greater life satisfaction, including: household satisfaction (ß = 0.25, p < 0.001); community satisfaction (ß = 0.26, p < 0.001); financial security (ß = 0.11, p < 0.05); and job satisfaction (ß = 0.13, p < 0.05). Qualitative findings revealed three broad areas which create barriers to life satisfaction, including: fears of assault and discrimination; employment and education problems; and financial and food security issues. Conclusions: The Hazara Shias need immediate assistance from state and society to improve safety, life opportunities, and mental health. Interventions for poverty alleviation, mental health, and fair education and employment opportunities need to be planned in partnership with the primary security issue.

Identification of suitable habitat for Taxus wallichiana and Abies pindrow in moist temperate forest using maxent modelling technique.

Conservation of any species necessitates knowledge of its biology and natural history, as well as prospective locations or newer adaptive landscapes where the species can survive and thrive. This study presents habitat suitability and local conservation status of Taxus wallichiana and Abies pindrow in moist temperate forest of Hazara division, Pakistan. Data was collected through field surveys based on 363 samples from field, topographical and bioclimatic variables. In the present study, we employed the MaxEnt model exclusively for each tree species along with 23 independent or environment variables (19 bioclimatic and 4 topographic). The jackknife test was used to demonstrate the significance of variables with the highest gain, and it was found that overall tree cover, annual temperature range was the factors with the highest gain, while slope was amongst the least important. The MaxEnt model produced high accuracy for each tree species, with receiver operating characteristic (ROC), area under the curve (AUC), training mean testing values for Taxus wallichiana was 0.966 followed by 0.944 for Abies pindrow. Local conservation status of Taxus wallichiana and Abies pindrow was evaluated using IUCN criteria 2001. Taxus wallichiana was declared critically endangered locally as the population size reduced by 87%. In contrast, Abies pindrow was declared as endangered as population size reduced by 69% falling under endangered criteria A of IUCN. The decline in population size of Taxus wallichiana and Abies pindrow species were due to human cause anthropogenic activities such as exploitation and loss of habitat, the extent of occurrence, and slow regeneration of tree species. Results and field-based observation revealed that suitable habitat modeling showed unsuitable (0.0-0.2), less suitable (0.2-0.4), moderately (0.4-0.6), highly (0.6-0.7), and very highly (0.7-1.0) suitable habitat for Taxus wallichiana and Abies pindrow. Results also revealed that both species were distributed irregularly in the moist temperate forest of Hazara division. Habitat suitability of Taxus wallichiana and Abies pindrow can be considered one of most significant points toward conserving these tree species. Habitat loss is a major threat to their occurrence, which should be overcome by ensuring the protection of suitable habitat and conservation approaches. Considering the species ecological and economic value, it is essential to understand how the species distribution may vary as a result of climate change to establish effective conservation policies. This study also includes significant environmental elements that influence species distribution, which could help locate regions where the species could be planted. Forest tree species require effective, scientific, and long-term management and conservation techniques in the study area. Furthermore, the formulation and implementation of protective laws and policies are required to conserve and protect both the conifer species.

Hazara Orthonairovirus Nucleoprotein Antagonizes Type I Interferon Production by Inhibition of RIG-I Ubiquitination.

Viruses have evolved various strategies to evade the host innate immune system. The relationship between nairoviruses and the interferon (IFN) system is poorly understood. We investigated whether and how nairoviruses antagonize host innate immunity using Hazara orthonairovirus (HAZV) as a surrogate model for Crimean-Congo hemorrhagic fever virus. HAZV nucleoprotein (N) was found to interact with the tripartite motif-containing protein 25 (TRIM25). The N-terminal region of N protein and the C-terminal region of TRIM25 are important for their interaction. Overexpression of N protein results in weakened interaction of TRIM25 with retinoic acid-inducible gene I (RIG-I). Furthermore, K63-linked polyubiquitination of RIG-I is inhibited in the presence of N protein. Our data collectively suggest that HAZV N protein interferes with the binding of TRIM25 to RIG-I and subsequent K63-linked polyubiquitination of RIG-I, which leads to inhibition of type I IFN production.

Flood in mountainous communities of Pakistan: how does it shape the livelihood and economic status and government support?

The agriculture sector remains under attack by the consistent flooding events in Pakistan as flooding water is sweeping away crops and livelihood opportunities, thus dragging the already vulnerable communities into poverty. This research has studied the adverse impacts of consistent floods (2010 to 2018) on the rural mountainous agriculture, agrarian occupation, economic system, and rise in poverty in ten districts of Hazara and Malakand divisions, Khyber-Pakhtunkhwa province, Pakistan. In total, 1000 self-administered questionnaires were deployed at the household level through judgment sampling studying the flood-affected households. Primary data for the impacts of floods on the livelihood, economic status, and poverty and correlation among them was estimated through three parallel logistic regression equations based on indexes for three separate models for the study variable to describe the flood-induced vulnerability at HH level. The results of the interrelationship of the selected variables declared Malakand division more prone to loss of livelihood, degradation of low-economic status, and increased in poverty due to flooding than Hazara division. It is concluded that floods have curtailed the agricultural livelihood on damaging crop production that has degraded locals' economic system by reducing household's income and savings, ending up in borrowing of money and leading to debt due to non-repayment. Households' vulnerability has increased due to increased poverty as being more exposed to natural disasters. It calls for government intervention to safeguard poor mountainous communities of Pakistan through financial help, seeds, livestock, and technical help in times of disaster.

Virus-Derived DNA Forms Mediate the Persistent Infection of Tick Cells by Hazara Virus and Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever (CCHF) is a severe disease of humans caused by CCHF virus (CCHFV), a biosafety level (BSL)-4 pathogen. Ticks of the genus Hyalomma are the viral reservoir, and they represent the main vector transmitting the virus to its hosts during blood feeding. We have previously shown that CCHFV can persistently infect Hyalomma-derived tick cell lines. However, the mechanism allowing the establishment of persistent viral infections in ticks is still unknown. Hazara virus (HAZV) can be used as a BSL-2 model virus instead of CCHFV to study virus/vector interactions. To investigate the mechanism behind the establishment of a persistent infection, we developed an in vitro model with Hyalomma-derived tick cell lines and HAZV. As expected, HAZV, like CCHFV, persistently infects tick cells without any sign of cytopathic effect, and the infected cells can be cultured for more than 3 years. Most interestingly, we demonstrated the presence of short viral-derived DNA forms (vDNAs) after HAZV infection. Furthermore, we demonstrated that the antiretroviral drug azidothymine triphosphate could inhibit the production of vDNAs, suggesting that vDNAs are produced by an endogenous retrotranscriptase activity in tick cells. Moreover, we collected evidence that vDNAs are continuously synthesized, thereby downregulating viral replication to promote cell survival. Finally, vDNAs were also detected in CCHFV-infected tick cells. In conclusion, vDNA synthesis might represent a strategy to control the replication of RNA viruses in ticks allowing their persistent infection. IMPORTANCE Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease caused by CCHF virus (CCHFV). Ticks of the genus Hyalomma can be persistently infected with CCHFV representing the viral reservoir, and the main vector for viral transmission. Here we showed that tick cells infected with Hazara virus, a nonpathogenic model virus closely related to CCHFV, contained short viral-derived DNA forms (vDNAs) produced by endogenous retrotranscriptase activity. vDNAs are transitory molecules requiring viral RNA replication for their continuous synthesis. Interestingly, vDNA synthesis seemed to be correlated with downregulation of viral replication and promotion of tick cell viability. We also detected vDNAs in CCHFV-infected tick cells suggesting that they could represent a key element in the cell response to nairovirus infection and might represent a more general mechanism of innate immunity against RNA viral infection.

Cranberry (Vaccinium macrocarpon) Extract Impairs Nairovirus Infection by Inhibiting the Attachment to Target Cells.

Hazara virus (HAZV) belongs to the Nairoviridae family and is included in the same serogroup of the Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is the most widespread tick-borne arbovirus. It is responsible for a serious hemorrhagic disease, for which specific and effective treatment and preventive systems are missing. Bioactive compounds derived from several natural products may provide a natural source of broad-spectrum antiviral agents, characterized by good tolerability and minimal side effects. Previous in vitro studies have shown that a cranberry (Vaccinium macrocarpon Ait.) extract containing a high content of A-type proanthocyanidins (PAC-A) inhibits the replication of herpes simplex and influenza viruses by hampering their attachment to target cells. Given the broad-spectrum antimicrobial activity of polyphenols and the urgency to develop therapies for the treatment of CCHF, we investigated the antiviral activity of cranberry extract against HAZV, a surrogate nairovirus model of CCHFV that can be handled in Level 2 Biosafety Laboratories (BSL-2). The results indicate that the cranberry extract exerts an antiviral activity against HAZV by targeting early stages of the viral replication cycle, including the initial adsorption to target cells. Although the details of the molecular mechanism of action remain to be clarified, the cranberry extract exerts a virucidal effect through a direct interaction with HAZV particles that leads to the subsequent impairment of virus attachment to cell-surface receptors. Finally, the antiviral activity of the cranberry extract was also confirmed for CCHFV. As a whole, the evidence obtained suggests that cranberry extract is a valuable candidate to be considered for the development of therapeutic strategies for CCHFV infections.

The Integration of Human and Veterinary Studies for Better Understanding and Management of Crimean-Congo Haemorrhagic Fever.

Outbreaks that occur as a result of zoonotic spillover from an animal reservoir continue to highlight the importance of studying the disease interface between species. One Health approaches recognise the interdependence of human and animal health and the environmental interplay. Improving the understanding and prevention of zoonotic diseases may be achieved through greater consideration of these relationships, potentially leading to better health outcomes across species. In this review, special emphasis is given on the emerging and outbreak pathogen Crimean-Congo Haemorrhagic Fever virus (CCHFV) that can cause severe disease in humans. We discuss the efforts undertaken to better understand CCHF and the importance of integrating veterinary and human research for this pathogen. Furthermore, we consider the use of closely related nairoviruses to model human disease caused by CCHFV. We discuss intervention approaches with potential application for managing CCHFV spread, and how this concept may benefit both animal and human health.

Forensic features and genetic legacy of the Baloch population of Pakistan and the Hazara population across Durand line revealed by Y-chromosomal STRs.

The Hazara population across Durand line has experienced extensive interaction with Central Asian and East Asian populations. Hazara individuals have typical Mongolian facial appearances and they called themselves descendants of Genghis Khan's army. The people who speak the Balochi language are called Baloch. Previously, a worldwide analysis of Y-chromosomal haplotype diversity for rapidly mutating (RM) Y-STRs and with PowerPlex Y23 System (Promega Corporation Madison, USA) kit was created with collaborative efforts, but Baloch and Hazara population from Pakistan and Hazara population from Afghanistan were missing. In the current study, Yfiler Plus PCR Amplification Kit loci were examined in 260 unrelated Hazara individuals from Afghanistan, 153 Hazara individuals, and 111 Balochi individuals from Baluchistan Pakistan. For the Hazara population from Afghanistan and Pakistan overall, 380 different haplotypes were observed on these 27 Y-STR loci, gene diversities ranged from 0.51288 (DYS389I) to 0.9257 (DYF387S1), and haplotype diversity was 0.9992. For the Baloch population, every individual was unique at 27 Y-STR loci; gene diversity ranged from 0.5718 (DYS460) to 0.9371(DYF387S1). Twelve haplotypes were shared between 178 individuals, while only two haplotypes among these twelve were shared between 87 individuals in Hazara populations. Rst and Fst pairwise genetic distance analyses, multidimensional scaling plot, neighbor-joining tree, linear discriminatory analysis, and median-joining network were performed, which shed light on the history of Hazara and Baloch populations. The results of our study showed that the Yfiler Plus PCR Amplification Kit marker set provided substantially stronger discriminatory power in the Baloch population of Pakistan and the Hazara population across the Durand line.

Sheep and Cattle Are Not Susceptible to Experimental Inoculation with Hazara Orthonairovirus, a Tick-Borne Arbovirus Closely Related to CCHFV.

Hazara orthonairovirus (HAZV) is a tick-borne arbovirus closely related to Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). Whereas CCHFV is a biosafety level (BSL) 4 agent, HAZV is classified as BSL 2, as it is not known to cause any disease in humans. Belonging to the same serogroup as CCHFV, HAZV might act as a model which can provide a better understanding of this important zoonosis. Furthermore, the serological relatedness may cause diagnostic problems if antibodies against HAZV interfere with current CCHFV serological assays. Therefore, sheep and cattle-important natural hosts for CCHFV-were experimentally infected with HAZV to prove their susceptibility and evaluate potential antibody cross-reactivities. According to this study, neither sheep nor cattle are susceptible to experimental HAZV infections. Consequently, the HAZV infection in ruminants is clearly distinct from CCHFV infections. Sera of immunized animals weakly cross-reacted between HAZV and CCHFV in immunofluorescence and immunoblot assays, but not in commercial CCHFV ELISAs commonly used for field studies.

Forensic and phylogenetic characterization of 15 autosomal STRs in Hazara population of Pakistan.

In the current study, 217 unrelated individuals of the Hazara population were genotyped for 15 autosomal short tandem repeats to generate parentage and forensic efficacy parameters. Hazaras belong to the Shi'a sect and are recognized by their Turko-Mogholi features. We found that D2S1338 was the most discriminatory locus with a maximum power of exclusion and high value of polymorphism information content. Whilst the Combined Power of Discrimination (CPD), Combined Matching Probability (CMP) and Combined Power of Exclusion (CPE) were 0.999999999999999, 2.76796338879E-17 and 0.999999040733479 respectively. Furthermore, the pattern of genetic affinity with genetically assumed related populations was demonstrated through Heat Map and Phylogenetic analysis, which revealed a great level of genetic closeness of Hazaras with Mongol population and descendants of Genghis Khan. The resulting data can be used for forensic applications and anthropological studies.

Mutagenic Analysis of Hazara Nairovirus Nontranslated Regions during Single- and Multistep Growth Identifies both Attenuating and Functionally Critical Sequences for Virus Replication.

Hazara nairovirus (HAZV) is a member of the family Nairoviridae in the order Bunyavirales and closely related to Crimean-Congo hemorrhagic fever virus, which is responsible for severe and fatal human disease. The HAZV genome comprises three segments of negative-sense RNA, named S, M, and L, with nontranslated regions (NTRs) flanking a single open reading frame. NTR sequences regulate RNA synthesis and, by analogy with other segmented negative-sense RNA viruses, may direct activities such as virus assembly and innate immune modulation. The terminal-proximal nucleotides of 3' and 5' NTRs exhibit extensive terminal complementarity; the first 11 nucleotides are strictly conserved and form promoter element 1 (PE1), with adjacent segment-specific nucleotides forming PE2. To explore the functionality of NTR nucleotides within the context of the nairovirus multiplication cycle, we designed infectious HAZV mutants bearing successive deletions throughout both S segment NTRs. Fitness of rescued viruses was assessed in single-step and multistep growth, which revealed that the 3' NTR was highly tolerant to change, whereas several deletions of centrally located nucleotides in the 5' NTR led to significantly reduced growth, indicative of functional disruption. Deletions that encroached upon PE1 and PE2 ablated virus growth and identified additional adjacent nucleotides critical for viability. Mutational analysis of PE2 suggest that its signaling ability relies solely on interterminal base pairing and is an independent cis-acting signaling module. This study represents the first mutagenic analysis of nairoviral NTRs in the context of the infectious cycle, and the mechanistic implications of our findings for nairovirus RNA synthesis are discussed.IMPORTANCE Nairoviruses are a group of RNA viruses that include many serious pathogens of humans and animals, including one of the most serious human pathogens in existence, Crimean-Congo hemorrhagic fever virus. The ability of nairoviruses to multiply and cause disease is controlled in major part by nucleotides that flank the 3' and 5' ends of nairoviral genes, called nontranslated regions (NTRs). NTR nucleotides interact with other virus components to perform critical steps of the virus multiplication cycle, such as mRNA transcription and RNA replication, with other roles being likely. To better understand how NTRs work, we performed the first comprehensive investigation of the importance of NTR nucleotides in the context of the entire nairovirus replication cycle. We identified both dispensable and critical NTR nucleotides, as well as highlighting the importance of 3' and 5' NTR interactions in virus growth, thus providing the first functional map of the nairovirus NTRs.

Exploring threatened traditional knowledge; ethnomedicinal studies of rare endemic flora from Lesser Himalayan region of Pakistan

ABSTRACT Himalayas are one among the world biodiversity hotspots harboring many endemic medicinal plants. Despite augmentation in the documentation of ethnopharmacological knowledge of medicinal plant species, information regarding endemic species is still underway. Current paper highlights the traditional medicinal uses of rare endemic and unexplored group of plants having potential for novel chemical constituents with effective pharmacological activities. In total, 142 informants (91 male and 51 female) including seventeen traditional healers were interviewed using semi-structured questionnaire, personal observations and group discussions. Interviews were taken in field or otherwise photographs were shown for identification. Females were interviewed indirectly through male family members. For data analysis, quantitative analytical approach was adopted using ethnopharmacological indices as Relative frequency of citations and Fidelity Level. In total, 38 endemic plant species belonging to nineteen families were utilized by the local inhabitants. Highest number of endemics was belonging to Ranunculaceae (7), followed by Gentianaceae and Rosaceae (4 each) with respect to number of species. Highest number of endemics was used in fever, wound healing, throat infection and tonic (4 species each). Root was the most widely used part (36.17%) in cure of diseases and the leading mode administered was decoction (25.49%). Highest use reports and RFC values were recorded for Pimpinella stewartii (58 citations, 0.41 RFC), Caltha alba var. alba (52 citations, 0.37 RFC). Endemic plant species considerably contribute toward ethnomedicinal knowledge and despite rarity, the communities prefer their utilization. Conservation of endemics is necessary for future availability to the local communities.

Population background exploration and genetic distribution analysis of Pakistan Hazara via 23 autosomal STRs.

Background: Short tandem repeats (STRs) have gained considerable attention in family search (Y-chromosomal STRs), complex paternity identification (X-chromosomal STRs), routine forensic personal identification (autosomal STRs) and population genetics.Aim: To explore the forensic characteristics of 23 autosomal STRs included in the Huaxia Platinum system in the South Pakistan Hazara population and investigate the genetic similarities and differences between Hazara and 54 worldwide reference populations.Subjects and methods: Variation of the 23 autosomal STRs included in the Huaxia Platinum system was first investigated and reported in a sample of 261 Quetta Hazara in Balochistan Province, Southwest Pakistan.Results: The combined power of discrimination is 0.999999999999999999999999999 and combined power of exclusion is 0.99999999989596 in Quetta Hazara. Comprehensive population comparisons between Hazara and another 13 Eurasian populations based on genotype data, as well as between Hazara and 54 worldwide populations based on the allele frequency distribution, were conducted. Multidimensional scaling plots, principal component analysis, and neighbour-joining phylogenetic trees consistently demonstrated that Pakistan Hazara harbours close affinities with neighbouring Turkic-speaking populations. Model-based genetic structure analysis further suggests that Quetta Hazara derives about half its ancestry directly from the East Asians.Conclusion: Twenty-five forensic-related markers included in the Huaxia Platinum system can be used for forensic practice in the Central Asia Hazara population. Quetta Hazara has a close genetic relationship with the Turkic-speaking populations of Uyghur and Kazakh. Further whole-genome sequencing of Hazara needs to be conducted to validate the observed genetic structure and reconstruct the fine-scale population history of Hazara.

A comprehensive exploration of the genetic legacy and forensic features of Afghanistan and Pakistan Mongolian-descent Hazara.

Afghanistan and Pakistan are rich with a complex landscape of culture, linguistics, ethnicity and genetic legacy at the crossroads between Indian-Subcontinent and Central Asia. Hazara people have historically been suggested to be Mongolian decedents but seldom been genetically studied. To dissect the genetic structure and explore the forensic characteristics of Hazara people, we first genotyped 30 Insertion/deletion (Indel) markers in 468 samples from 2 aboriginal Hazara populations from Afghanistan and Pakistan, and 100 East Asian comparative Bouyei samples using the Investigator® DIPplex kit. Subsequently, we carried out a comprehensive population genetic analysis from four different datasets: 8895 30-Indel genotype data from 51 populations, 15,895 30-Indel allele frequency data from 98 populations, 1048 genotypes of 993 STRs and Indels from 53 HGDP populations and 2068 whole-genomes (621,799 single nucleotide polymorphisms) from 165 worldwide Human origin reference populations, to further unravel the genetic complexity between Hazara and worldwide human populations using various statistical analysis. We find that 30 Indels are in accordance with HWE, and informative and polymorphic in both Central Asians Hazara and East Asian Bouyei populations. The forensic combined probability of exclusion is larger than 0.9943 and the cumulative power of discrimination is larger than 0.99999999999936. These forensic parameters show the high level of diversity, which makes the Indel amplification system suitable for forensic routine work and may be used as a supplementary assay for routine forensic investigation. The results from pairwise genetic distances, MDS, PCA, and phylogenetic relationship reconstruction demonstrate that present-day Hazaras are genetically closer to the Turkic-speaking populations (Uyghur, Kazakh, and Kyrgyz) residing in northwest China than with other Central/South Asian populations and Mongolian. Outgroup and admixture f3,f4, f4-ratio, qpWave, and qpAdm results further demonstrate that Hazara shares more alleles with East Asians than with other Central Asians and carries 57.8% Mongolian-related ancestry. Overall, our findings suggest that Hazaras have experienced genetic admixture with the local or neighboring populations and formed the current East-West Eurasian admixed genetic profile after their separation from the Mongolians.

Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage.

The Nairoviridae family of the Bunyavirales order comprises tick-borne, trisegmented, negative-strand RNA viruses, with several members being associated with serious or fatal diseases in humans and animals. A notable member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is the most widely distributed tick-borne pathogen and is associated with devastating human disease, with case fatality rates averaging 30%. Hazara virus (HAZV) is closely related to CCHFV, sharing the same serogroup and many structural, biochemical, and cellular properties. To improve understanding of HAZV and nairovirus multiplication cycles, we developed, for the first time, a rescue system permitting efficient recovery of infectious HAZV from cDNA. This system now allows reverse genetic analysis of nairoviruses without the need for high-level biosafety containment, as is required for CCHFV. We used this system to test the importance of a DQVD caspase cleavage site exposed on the apex of the HAZV nucleocapsid protein arm domain that is cleaved during HAZV infection, for which the equivalent DEVD sequence was recently shown to be important for CCHFV growth in tick but not mammalian cells. Infectious HAZV bearing an uncleavable DQVE sequence was rescued and exhibited growth parameters equivalent to those of wild-type virus in both mammalian and tick cells, showing this site was dispensable for virus multiplication. In contrast, substitution of the DQVD motif with the similarly uncleavable AQVA sequence could not be rescued despite repeated efforts. Together, these results highlight the importance of this caspase cleavage site in the HAZV life cycle but reveal the DQVD sequence performs a critical role aside from caspase cleavage.IMPORTANCE HAZV is classified within the Nairoviridae family with CCHFV, which is one of the most lethal human pathogens in existence, requiring the highest biosafety level (BSL) containment (BSL4). In contrast, HAZV is not associated with human disease and thus can be studied using less-restrictive BSL2 protocols. Here, we report a system that is able to rescue HAZV from cDNAs, thus permitting reverse genetic interrogation of the HAZV replication cycle. We used this system to examine the role of a caspase cleavage site, DQVD, within the HAZV nucleocapsid protein that is also conserved in CCHFV. By engineering mutant viruses, we showed caspase cleavage at this site was not required for productive infection and this sequence performs a critical role in the virus life cycle aside from caspase cleavage. This system will accelerate nairovirus research due to its efficiency and utility under amenable BSL2 protocols.

Hazara nairovirus elicits differential induction of apoptosis and nucleocapsid protein cleavage in mammalian and tick cells.

The Nairoviridae family within the Bunyavirales order comprise tick-borne segmented negative-sense RNA viruses that cause serious disease in a broad range of mammals, yet cause a latent and lifelong infection in tick hosts. An important member of this family is Crimean-Congo haemorrhagic fever virus (CCHFV), which is responsible for serious human disease that results in case fatality rates of up to 30 %, and which exhibits the most geographically broad distribution of any tick-borne virus. Here, we explored differences in the cellular response of both mammalian and tick cells to nairovirus infection using Hazara virus (HAZV), which is a close relative of CCHFV within the CCHFV serogroup. We show that HAZV infection of human-derived SW13 cells led to induction of apoptosis, evidenced by activation of cellular caspases 3, 7 and 9. This was followed by cleavage of the classical apoptosis marker poly ADP-ribose polymerase, as well as cellular genome fragmentation. In addition, we show that the HAZV nucleocapsid (N) protein was abundantly cleaved by caspase 3 in these mammalian cells at a conserved DQVD motif exposed at the tip of its arm domain, and that cleaved HAZV-N was subsequently packaged into nascent virions. However, in stark contrast, we show for the first time that nairovirus infection of cells of the tick vector failed to induce apoptosis, as evidenced by undetectable levels of cleaved caspases and lack of cleaved HAZV-N. Our findings reveal that nairoviruses elicit diametrically opposed cellular responses in mammalian and tick cells, which may influence the infection outcome in the respective hosts.

A Minigenome Study of Hazara Nairovirus Genomic Promoters.

Hazara nairovirus (HAZV) is a trisegmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) in the order Bunyavirales The terminal roughly 20 nucleotides (nt) of its genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements: the extreme termini of both strands (promoter element 1 [PE1]) are conserved and virus specific and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment specific, with the potential to form double-stranded RNA (dsRNA), and the latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunyaviruses and arenaviruses in that they contain a short single-stranded region between the two regions of complementarity. Using a HAZV minigenome system, we found the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, is essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in light of our current understanding of the initiation of RNA synthesis.IMPORTANCE A minigenome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV, such as peribunyaviruses and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements: the extreme termini of both 3' and 5' strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion and the following sequences with the potential to anneal but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short single-stranded RNA (ssRNA) region between the two elements. The single-stranded nature of this region is an essential element of the promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.