African Strains Research Articles

Identification of tyrphostin AG879 and A9 inhibiting replication of chikungunya virus by screening of a kinase inhibitor library

Chikungunya virus (CHIKV) is a globally public health threat. There are currently no medications available to treat CHIKV infection. High-throughput screening of 419 kinase inhibitors was performed based on the cytopathic effect method, and six kinase inhibitors with reduced cytopathic effects, including tyrphostin AG879 (AG879), tyrphostin 9 (A9), sorafenib, sorafenib tosylate, regorafenib, and TAK-632, were identified. The anti-CHIKV activities of two receptor tyrosine kinase inhibitors, AG879 and A9, that have not been previously reported, were selected for further evaluation. The results indicated that 50% cytotoxic concentration (CC50) of AG879 and A9 in Vero cells were greater than 30 μM and 6.50 μM, respectively and 50% effective concentration (EC50) were 0.84 μM and 0.36 μM, respectively. The time-of-addition and time-of-removal assays illustrated that both AG879 and A9 function in the middle stage of CHIKV life cycle. Further, AG879 and A9 do not affect viral attachment; however, they inhibit viral RNA replication, and exhibit antiviral activity against CHIKV Eastern/Central/South African and Asian strains, Ross River virus and Sindbis virus in vitro.

Predominance of low pathogenic avian influenza virus H9N2 in the respiratory co-infections in broilers in Tunisia: a longitudinal field study, 2018–2020

Respiratory diseases are a health and economic concern for poultry production worldwide. Given global economic exchanges and migratory bird flyways, respiratory viruses are likely to emerge continuously in new territories. The primary aim of this study was to investigate the major pathogens involved in respiratory disease in Tunisian broiler poultry and their epidemiology. Between 2018 and 2020, broilers farms in northeastern Tunisia were monitored, and 39 clinically diseased flocks were sampled. Samples were screened for five viral and three bacterial respiratory pathogens using a panel of real-time PCR assays. The reemergence of H9N2 low pathogenic avian influenza virus (LPAIV) in commercial poultry was reported, and the Northern and Western African GI lineage strain was typed. The infectious bronchitis virus (IBV) GI-23 lineage and the avian metapneumovirus (aMPV) subtype B also were detected for the first time in broilers in Tunisia. H9N2 LPAIV was the most detected pathogen in the flocks tested, but rarely alone, as 15 of the 16 H9N2 positive flocks were co-infected. Except for infectious laryngotracheitis virus (ILTV), all of the targeted pathogens were detected, and in 61% of the respiratory disease cases, a combination of pathogens was identified. The major combinations were H9N2 + aMPV (8/39) and H9N2 + IBV (6/39), showing the high contribution of H9N2 LPAIV to the multifactorial respiratory diseases. This field survey provided evidence of the emergence of new respiratory viruses and the complexity of respiratory disease in Tunisia. A comprehensive and continuous surveillance strategy therefore is needed to better control respiratory pathogens in Tunisia.

Detection and molecular characterization of virulent Newcastle disease virus (subgenotype VII.2) in broiler chickens in Northern Vietnam.

Newcastle disease (ND) is a major viral disease of poultry worldwide. However, data on the molecular characterization of Newcastle disease virus (NDV) in Vietnam are limited. This study aimed to identify the molecular characteristics of NDV strains from the vaccinated chickens farmed in Northern Vietnam. We used reverse-transcription polymerase chain reaction (PCR), sequencing and phylogenetic analysis to characterize NDV strains from vaccinated chicken farms in Northern Vietnam. Seven out of 72 (9.7%) chicken tissue samples collected from seven chicken farms in the four cities/provinces in northern Vietnam were positive for the NDV genome by PCR method. The complete sequences of the fusion (F) and hemagglutinin-neuraminidase (HN) genes of NDVs isolated in the North of Vietnam from 2021 to 2022 were further evaluated. The results indicated that all seven Vietnamese isolates obtained were reported as virulent NDV strains with the amino acid (AA) sequence of the F0 protein proteolytic cleavage site motif (112RRRKRF117). Phylogenetic analysis revealed that they were grouped with other NDV class II from subgenotype VII.2, including the two previous Vietnamese NDV (2015), the Chinese (2017), and Southern African (2013) NDV strains. In addition, some AA substitutions were observed in the neutralizing epitopes of the F and HN proteins of the current Vietnamese NDV strains. The present findings provide useful information for future studies of the evolution of NDVs and improve strategies for ND-controlling programs in Vietnam.

Detection of SPV2 and SPCSV and their possible influence on the yield in multiple virus infections

AbstractThe prevalence of Sweet potato virus 2 (SPV2) and Sweet potato chlorotic stunt virus (SPCSV) in the Limpopo, Mpumalanga, and Gauteng provinces of South Africa was determined using serological and nucleic acid based methods. Cloning and sequencing part of the coat protein (CP) gene and the complete genome using next generation sequencing (NGS) were also conducted to characterize and determine the sequence variability in SPV2 isolates. Primers designed in the current study successfully detected and confirmed the prevalence of SPV2 in the three provinces surveyed. The pairwise comparison of the amino acid (aa) sequences encoding the CP gene region confirmed that isolates in this study are related to SPV2, sharing a 94% to 99% CP aa identity with those originally reported in South Africa. The East African (EA) strain of SPCSV was also detected in the three provinces surveyed. Phylogenetic analysis using part of the CP encoding nucleotide (nt) sequences showed that the South African isolates correspond to but are distinct from the other EA strains of SPCSV used as reference sequences. Assessment of varieties and breeding lines’ response to multiple infection of potyviruses identified variety NC1560, breeding lines 2002‐21‐1 and 2006‐5‐4 as promising for inclusion in future breeding programs as sources of parent material for virus tolerance.

Pathogenicity and Pathological Characteristics of African Swine Fever Virus Strains from Pig Farms in South Korea from 2022 to January 2023.

Since the first African swine fever (ASF) outbreak occurred at a pig farm in South Korea in September 2019, as of 31 January 2023, 31 ASF cases have occurred at pig farms, while 2799 ASF virus (ASFV)-infected wild boars have been identified. The circulation of ASFV in wild boar populations poses a high risk of spillover to pig farms in the country. However, information on the changes in the pathogenicity of Korean ASFV strains from wild boars is not available. Investigating the pathogenicity of ASFV strains from pig farms is the only way to predict their alterations. In a previous study, no changes in the pathogenicity of ASFV strains circulating during 2019-2021 were identified through animal experiments. In this study, we chose two ASFV strains with potentially reduced pathogenicity among ten viruses obtained from pig premises from 2022 to January 2023 and estimated their pathogenicities and pathological characteristics. All the inoculated pigs died 8-10 days post-inoculation after showing pyrexia, depression, anorexia, and recumbency together with the common pathological lesions of enlarged hemorrhagic lymph nodes and splenomegaly with infarction. These results support that the pathogenicity among ASFV isolates in South Korea still remained unchanged during the study period.

Enduring pathogenicity of African strains of Salmonella on plastics and glass in simulated peri-urban environmental waste piles

In low- and middle-income countries, plastic has become a major constituent of landfills and urban dump sites. Environmental plastic pollution can also provide a novel surface for the formation of microbial biofilm, which often includes pathogenic bacteria and viruses. Here, under conditions simulating a peri-urban waste pile typical of an African informal settlement, we aimed to determine if pathogenic Salmonella spp. can retain their virulence following a prolonged period of desiccation on the surfaces of environmental plastic and glass. We show that clinically (and environmentally) relevant strains of Salmonella including S. Enteritidis, S. Typhimurium and S. Typhi can persist on plastic and glass for at least 28-days and that temperature (which increases with the depth of an urban waste pile) is a key determinant of this survival. All three strains of Salmonella retained their pathogenicity (determined by using a Galleria mellonella model of infection) following their recovery from the plastisphere indicating that plastics in the environment can act as reservoirs for human pathogens and could facilitate their persistence for extended periods of time. Pathogens colonising environmental plastic waste therefore pose a heightened public health risk, particularly in areas where people are frequently exposed to plastic pollution.

Rescue and characterization of the first West African Marburg virus 2021 from Guinea

Marburg virus (MARV) is a causative agent of a severe hemorrhagic fever with high fatality rates endemic in central Africa. Current outbreaks of MARV in Equatorial Guinea and Tanzania underline the relevance of MARV as a public health emergency pathogen. In 2021, the first known human MARV case was confirmed in Guinea, West Africa. Since no infectious virus could be isolated from that fatal case in 2021, we generated recombinant (rec) MARV Guinea by reverse genetics in order to study and characterize this new MARV, which occurred in West Africa for the first time, in terms of its growth properties, detection by antibodies, and therapeutic potential compared to known MARV strains. Our results showed a solid viral replication of recMARV Guinea in human, bat, and monkey cell lines in comparison to other known MARV strains. We further demonstrated that replication of recMARV Guinea in cells can be inhibited by the nucleoside analogue remdesivir. Taken together, we could successfully reconstitute de novo the first West African MARV from Guinea showing similar replication kinetics in cells compared to other central African MARV strains. Our reverse genetics approach has proven successful in characterizing emerging viruses, especially when virus isolates are missing and viral genome sequences are incomplete.

Comparative genomics identifies conserved and variable TAL effectors in African strains of the cotton pathogen Xanthomonas citri pv. malvacearum.

Strains of Xanthomonas citri pv. malvacearum cause bacterial blight of cotton, a potentially serious threat to cotton production worldwide, including in sub-Saharan countries. Development of disease symptoms, such as water soaking, has been linked to the activity of a class of type 3 effectors, called TAL (transcription activator-like) effectors, which induce susceptibility genes in the host's cells. To gain further insight into the global diversity of the pathogen, to elucidate their repertoires of TAL effector genes and to better understand the evolution of these genes in the cotton-pathogenic xanthomonads, we sequenced the genomes of three African strains of X. citri pv. malvacearum using nanopore technology. We show that the cotton-pathogenic pathovar of X. citri is a monophyletic lineage containing at least three distinct genetic subclades, which appear to be mirrored by their repertoires of TAL effectors. We observed an atypical level of TAL effector gene pseudogenization, which might be related to resistance genes that are deployed to control the disease. Our work thus contributes to a better understanding of the conservation and importance of TAL effectors in the interaction with the host plant, which can inform strategies for improving resistance against bacterial blight in cotton.

Genetic Characteristics of the Virus Detected in the First Mpox Imported Case in Tokyo, Japan.

Mpox, caused by the mpox virus (MPXV), produces symptoms similar to those of smallpox when transmitted to humans. Since 1970, this disease has been endemic, particularly in Africa. However, since May 2022, the number of patients without a history of travel to endemic areas has increased rapidly globally. Under these circumstances, in July 2022, two different real-time PCR methods were used on specimens brought to the Tokyo Metropolitan Institute of Public Health. MPXV was detected in the skin samples, and it was inferred that the virus was a West African strain. Furthermore, a more detailed analysis of the genetic characteristics of the detected MPXV using next-generation sequencing revealed that the MPXV detected in Tokyo was strain B.1, which corresponds to the same strain that is prevalent in Europe and the USA. This suggests that mpox reported for the first time in Japan was imported and related to outbreaks in Europe and the USA. Therefore, it is necessary to continue monitoring outbreaks in Japan in conjunction with global epidemics.

Gene-by-environment interactions influence the fitness cost of gene copy-number variation in yeast.

Variation in gene copy number can alter gene expression and influence downstream phenotypes; thus copy-number variation provides a route for rapid evolution if the benefits outweigh the cost. We recently showed that genetic background significantly influences how yeast cells respond to gene overexpression, revealing that the fitness costs of copy-number variation can vary substantially with genetic background in a common-garden environment. But the interplay between copy-number variation tolerance and environment remains unexplored on a genomic scale. Here, we measured the tolerance to gene overexpression in four genetically distinct Saccharomyces cerevisiae strains grown under sodium chloride stress. Overexpressed genes that are commonly deleterious during sodium chloride stress recapitulated those commonly deleterious under standard conditions. However, sodium chloride stress uncovered novel differences in strain responses to gene overexpression. West African strain NCYC3290 and North American oak isolate YPS128 are more sensitive to sodium chloride stress than vineyard BC187 and laboratory strain BY4743. Consistently, NCYC3290 and YPS128 showed the greatest sensitivities to overexpression of specific genes. Although most genes were deleterious, hundreds were beneficial when overexpressed-remarkably, most of these effects were strain specific. Few beneficial genes were shared between the sodium chloride-sensitive isolates, implicating mechanistic differences behind their sodium chloride sensitivity. Transcriptomic analysis suggested underlying vulnerabilities and tolerances across strains, and pointed to natural copy-number variation of a sodium export pump that likely contributes to strain-specific responses to overexpression of other genes. Our results reveal extensive strain-by-environment interactions in the response to gene copy-number variation, raising important implications for the accessibility of copy-number variation-dependent evolutionary routes under times of stress.

Genome editing of an African elite rice variety confers resistance against endemic and emerging Xanthomonas oryzae pv. oryzae strains.

Bacterial leaf blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), threatens global food security and the livelihood of small-scale rice producers. Analyses of Xoo collections from Asia, Africa and the Americas demonstrated complete continental segregation, despite robust global rice trade. Here, we report unprecedented BB outbreaks in Tanzania. The causative strains, unlike endemic African Xoo, carry Asian-type TAL effectors targeting the sucrose transporter SWEET11a and iTALes suppressing Xa1. Phylogenomics clustered these strains with Xoo from Southern-China. African rice varieties do not carry effective resistance. To protect African rice production against this emerging threat, we developed a hybrid CRISPR-Cas9/Cpf1 system to edit all known TALe-binding elements in three SWEET promoters of the East African elite variety Komboka. The edited lines show broad-spectrum resistance against Asian and African strains of Xoo, including strains recently discovered in Tanzania. The strategy could help to protect global rice crops from BB pandemics.

Comparative Infections of Zika, Dengue, and Yellow Fever Viruses in Human Cytotrophoblast-Derived Cells Suggest a Gating Role for the Cytotrophoblast in Zika Virus Placental Invasion.

The Zika virus (ZIKV) is teratogenic and considered a TORCH pathogen (toxoplasmosis [Toxoplasma gondii], rubella, cytomegalovirus, herpes simplex virus [HSV], and other microorganisms capable of crossing the blood-placenta barrier). In contrast, the related flavivirus dengue virus (DENV) and the attenuated yellow fever virus vaccine strain (YFV-17D) are not. Understanding the mechanisms used by ZIKV to cross the placenta is necessary. In this work, parallel infections with ZIKV of African and Asian lineages, DENV, and YFV-17D were compared for kinetics and growth efficiency, activation of mTOR pathways, and cytokine secretion profile using cytotrophoblast-derived HTR8 cells and monocytic U937 cells differentiated to M2 macrophages. In HTR8 cells, ZIKV replication, especially the African strain, was significantly more efficient and faster than DENV or YFV-17D. In macrophages, ZIKV replication was also more efficient, although differences between strains were reduced. Greater activation of the mTORC1 and mTORC2 pathways in HTR8 cells infected with ZIKV than with DENV or YFV-17D was observed. HTR8 cells treated with mTOR inhibitors showed a 20-fold reduction in ZIKV yield, versus 5- and 3.5-fold reductions for DENV and YFV-17D, respectively. Finally, infection with ZIKV, but not DENV or YFV-17D, efficiently inhibited the interferon (IFN) and chemoattractant responses in both cell lines. These results suggest a gating role for the cytotrophoblast cells in favoring entry of ZIKV, but not DENV and YFV-17D, into the placental stroma. IMPORTANCE Zika virus acquisition during pregnancy is associated with severe fetal damage. The Zika virus is related to dengue virus and yellow fever virus, yet fetal damage has not been related to dengue or inadvertent vaccination for yellow fever during pregnancy. Mechanisms used by the Zika virus to cross the placenta need to be deciphered. By comparing parallel infections of Zika virus strains belonging to the African and Asian lineages, dengue virus, and the yellow fever vaccine virus strain YFV-17D in placenta-derived cytotrophoblast cells and differentiated macrophages, evidence was found that Zika virus infections, especially by the African strains, were more efficient in cytotrophoblast cells than dengue virus or yellow fever vaccine virus strain infections. Meanwhile, no significant differences were observed in macrophages. Robust activation of the mTOR signaling pathways and inhibition of the IFN and chemoattractant response appear to be related to the better growth capacity of the Zika viruses in the cytotrophoblast-derived cells.

Myrtus communis in Europe threatened by the pandemic and South African strains of the myrtle rust pathogen Austropuccinia psidii (Sphaerophragmiaceae, Pucciniales)

Austropuccinia psidii, the causal agent of myrtle rust, has emerged as a significant threat to Myrtaceae in planted and natural woody ecosystems. The first detection of A. puccinia in South Africa was from severely infected ornamental Myrtus communis. This raised concern that M. communis, the sole Myrtaceae species native to Europe and an important component of vegetation in Mediterranean regions, could be threatened by the rust. In light of the potential threat to this unique species, seed was collected from 12 Italian provenances of M. communis, including mainland and island (Sardinia and Sicily) populations. We assessed the susceptibility of these provenances to both the pandemic and South African strains of A. psidii. In Colombia, where the pandemic strain of A. psidii is native, seedlings rapidly became infected by natural inoculum. In South Africa, a preliminary screening of seedlings by artificial inoculation with a single-uredinium isolate produced high levels of disease. Finally, plants of each of the 12 provenances were planted and monitored in Florence, Italy. To date, these showed no signs of disease, but will continue to be monitored. This study highlights the significant threat that both the pandemic and South African strains of A. puccinia pose to M. communis in Europe.

Differentiation of African Swine Fever Virus Strains Isolated in Estonia by Multiple Genetic Markers.

The African swine fever virus (ASFV) was first detected in Estonia, in September 2014. In the subsequent three years, the virus spread explosively all over the country. Only one county, the island of Hiiumaa, remained free of the disease. Due to the drastic decrease in the wild boar population in the period of 2015-2018, the number of ASFV-positive cases among wild boar decreased substantially. From the beginning of 2019 to the autumn of 2020, no ASFV-positive wild boar or domestic pigs were detected in Estonia. A new occurrence of ASFV was detected in August 2020, and by the end of 2022, ASFV had been confirmed in seven counties in Estonia. Investigations into proven molecular markers, such as IGR I73R/I329L, MGF505-5R, K145R, O174L, and B602L, were performed with the aim of clarifying whether these cases of ASFV were new entries or remnants of previous epidemics. The sequences from the period of 2014-2022 were compared to the Georgia 2007/1 reference sequence and the variant strains present in Europe. The results indicated that not all the molecular markers of the virus successfully used in other geographical regions were suitable for tracing the spread of ASFV in Estonia. Only the B602L-gene analysis enabled us to place the ASFV isolates spreading in 2020-2022 into two epidemiologically different clusters.

Endophytic Effect of the South African Beauveria bassiana Strain PPRI 7598 on the Population Growth and Development of the Russian Wheat Aphid, Diuraphis noxia

The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is one of the main pests of small-grain cereal crops, including bread wheat, Triticum aestivum (Poaceae). In a series of glasshouse experiments, we evaluated the systemic effect of endophytic Beauveria bassiana strain PPRI 7598 on D. noxia biotype RWASA1 using three South African wheat cultivars, Gariep, Molopo, and Kariega. The objectives of the study were: (a) to determine the effect of endophytism on RWA reproduction and population growth, and (b) to assess the effect of the endophyte on aphid biomass and T. aestivum response to D. noxia herbivory using a damage rating index. Plant endophytic colonisation was confirmed before each trial using a B. bassiana-selective medium. Three independent trials were performed 10 days apart under glasshouse conditions. The effect of the endophyte-treated versus non-treated plants varied significantly in terms of net reproductive rate (R0) and the intrinsic rate of increase (rm) of the D. noxia population. Overall, the endophyte significantly reduced D. noxia R0 by approximately 14 nymphs/female and decreased the aphid mass by 13% in treated plants, whereas the mean aphid mass increased by 17% in control plants in all pooled cultivars. These findings demonstrated the endophytic potential of B. bassiana strain PPRI 7598 for suppression of D. noxia populations in RWASA1-susceptible cultivars. The integration of B. bassiana endophytism with host plant resistance may counteract biotype development and support a more sustainable approach towards RWA control in integrated pest management programmes.

A minisatellite-based MLVA for deciphering the global epidemiology of the bacterial cassava pathogen Xanthomonas phaseoli pv. manihotis.

Cassava Bacterial Blight (CBB) is a destructive disease widely distributed in the different areas where this crop is grown. Populations studies have been performed at local and national scales revealing a geographical genetic structure with temporal variations. A global epidemiology analysis of its causal agent Xanthomonas phaseoli pv. manihotis (Xpm) is needed to better understand the expansion of the disease for improving the monitoring of CBB. We targeted new tandem repeat (TR) loci with large repeat units, i.e. minisatellites, that we multiplexed in a scheme of Multi-Locus Variable number of TR Analysis (MLVA-8). This genotyping scheme separated 31 multilocus haplotypes in three clusters of single-locus variants and a singleton within a worldwide collection of 93 Xpm strains isolated over a period of fifty years. The major MLVA-8 cluster 1 grouped strains originating from all countries, except the unique Chinese strain. On the contrary, all the Xpm strains genotyped using the previously developed MLVA-14 microsatellite scheme were separated as unique haplotypes. We further propose an MLVA-12 scheme which takes advantage of combining TR loci with different mutation rates: the eight minisatellites and four faster evolving microsatellite markers, for global epidemiological surveillance. This MLVA-12 scheme identified 78 haplotypes and separated most of the strains in groups of double-locus variants (DLV) supporting some phylogenetic relationships. DLV groups were subdivided into closely related clusters of strains most often sharing the same geographical origin and isolated over a short period, supporting epidemiological relationships. The main MLVA-12 DLV group#1 was composed by strains from South America and all the African strains. The MLVA-12 scheme combining both minisatellite and microsatellite loci with different discriminatory power is expected to increase the accuracy of the phylogenetic signal and to minimize the homoplasy effects. Further investigation of the global epidemiology of Xpm will be helpful for a better control of CBB worldwide.

Infection of the maternal-fetal interface and vertical transmission following low-dose inoculation of pregnant rhesus macaques (Macaca mulatta) with an African-lineage Zika virus.

Congenital Zika virus (ZIKV) infection can result in birth defects, including malformations in the fetal brain and visual system. There are two distinct genetic lineages of ZIKV: African and Asian. Asian-lineage ZIKVs have been associated with adverse pregnancy outcomes in humans; however, recent evidence from experimental models suggests that African-lineage viruses can also be vertically transmitted and cause fetal harm. To evaluate the pathway of vertical transmission of African-lineage ZIKV, we inoculated nine pregnant rhesus macaques (Macaca mulatta) subcutaneously with 44 plaque-forming units of a ZIKV strain from Senegal, (ZIKV-DAK). Dams were inoculated either at gestational day 30 or 45. Following maternal inoculation, pregnancies were surgically terminated seven or 14 days later and fetal and maternal-fetal interface tissues were collected and evaluated. Infection in the dams was evaluated via plasma viremia and neutralizing antibody titers pre- and post- ZIKV inoculation. All dams became productively infected and developed strong neutralizing antibody responses. ZIKV RNA was detected in maternal-fetal interface tissues (placenta, decidua, and fetal membranes) by RT-qPCR and in situ hybridization. In situ hybridization detected ZIKV predominantly in the decidua and revealed that the fetal membranes may play a role in ZIKV vertical transmission. Infectious ZIKV was detected in the amniotic fluid of three pregnancies and one fetus had ZIKV RNA detected in multiple tissues. No significant pathology was observed in any fetus; and ZIKV did not have a substantial effect on the placenta. This study demonstrates that a very low dose of African-lineage ZIKV can be vertically transmitted to the macaque fetus during pregnancy. The low inoculating dose used in this study suggests a low minimal infectious dose for rhesus macaques. Vertical transmission with a low dose in macaques further supports the high epidemic potential of African ZIKV strains.

The efficacy of an inactivated avian influenza H5N1 vaccine against an African strain of HPAI H5N8 (clade 2.3.4.4 B)

ABSTRACT Highly pathogenic avian influenza (HPAI) viruses from the Goose/Guangdong/96-lineage emerged in Southeast Asia and subsequently spread to the Middle East, Africa and Europe, infecting a range of birds and mammals (including humans). This lineage of H5 viruses can efficiently establish itself in wild birds after circulating among gallinaceous poultry, facilitating reassortment with low pathogenic avian influenza (LPAI) virus strains, enhancing dispersal over long distances and contributing to endemicity. The detection of HPAI H5N8 virus (clade 2.3.4.4B) in 2017 in the Mpumalanga Province of South Africa marked the beginning of an epidemic that devastated the South African poultry industry. Vaccines were tested to assess protection against the circulating field strain. This article describes the performance of a reverse genetics inactivated H5N1 vaccine from Zoetis (RG-H5N1), with 96.1% identity to the circulating HPAI H5N8 virus. Two locally formulated benchmarks, one containing an H5N8 antigen homologous to the field strain (Benchmark-H5N8), the other containing a heterologous (87.6% identity to field virus) LPAI H5N1 antigen (Benchmark-H5N1), were included for comparison. Efficacy was assessed in specific pathogen-free (SPF) chickens using a prime-boost approach (injections at days 21 and 45), followed by a challenge with a South African HPAI H5N8 isolate (70 days of age). The Zoetis RG-H5N1 vaccine and Benchmark-H5N8 outperformed the Benchmark-H5N1 in terms of humoral response against the H5N8 antigen and reduction of shedding. The Zoetis RG-H5N1 vaccine protected 100% of the chickens against clinical disease and death. This study confirmed that antigenically matched inactivated vaccines could induce robust protection and markedly reduce viral shedding. RESEARCH HIGHLIGHTS Conditionally licensed vaccine protected against HPAI H5N8 (clade 2.3.4.4B). Complete protection against clinical disease and mortality. Drastic reduction of viral shedding after challenge.

Development of a CRISPR/Cas12a-recombinase polymerase amplification assay for visual and highly specific identification of the Congo Basin and West African strains of mpox virus.

Human mpox is a zoonotic disease, similar to smallpox, caused by the mpox virus, which is further subdivided into Congo Basin and West African clades with different pathogenicity. In this study, a novel diagnostic protocol utilizing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a nuclease (CRISPR/Cas12a)-mediated recombinase polymerase amplification (RPA) was developed to identify mpox in the Congo Basin and West Africa (CRISPR-RPA). Specific RPA primers targeting D14L and ATI were designed. CRISPR-RPA assay was performed using various target templates. In the designed CRISPR-RPA reaction system, the exponentially amplified RPA amplification products with a protospacer adjacent motif (PAM) site can locate the Cas12a/crRNA complex to its target regions, which successfully activates the CRISPR/Cas12a effector and achieves ultrafast trans-cleavage of a single-stranded DNA probe. The limit of detection for the CRISPR-RPA assay was 10 copies per reaction for D14L- and ATI-plasmids. No cross-reactivity was observed with non-mpox strains, confirming the high specificity of the CRISPR-RPA assay for distinguishing between the Congo Basin and West African mpox. The CRISPR-RPA assay can be completed within 45 min using real-time fluorescence readout. Moreover, the cleavage results were visualized under UV light or an imaging system, eliminating the need for a specialized apparatus. In summary, the developed CRISPR/RPA assay is a visual, rapid, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Congo Basin and West African mpox in resource-limited laboratories.

Effect of Viral Strain and Host Age on Clinical Disease and Viral Replication in Immunocompetent Mouse Models of Chikungunya Encephalomyelitis.

The alphavirus chikungunya virus (CHIKV) represents a reemerging public health threat as mosquito vectors spread and viruses acquire advantageous mutations. Although primarily arthritogenic in nature, CHIKV can produce neurological disease with long-lasting sequelae that are difficult to study in humans. We therefore evaluated immunocompetent mouse strains/stocks for their susceptibility to intracranial infection with three different CHIKV strains, the East/Central/South African (ECSA) lineage strain SL15649 and Asian lineage strains AF15561 and SM2013. In CD-1 mice, neurovirulence was age- and CHIKV strain-specific, with SM2013 inducing less severe disease than SL15649 and AF15561. In 4-6-week-old C57BL/6J mice, SL15649 induced more severe disease and increased viral brain and spinal cord titers compared to Asian lineage strains, further indicating that neurological disease severity is CHIKV-strain-dependent. Proinflammatory cytokine gene expression and CD4+ T cell infiltration in the brain were also increased with SL15649 infection, suggesting that like other encephalitic alphaviruses and with CHIKV-induced arthritis, the immune response contributes to CHIKV-induced neurological disease. Finally, this study helps overcome a current barrier in the alphavirus field by identifying both 4-6-week-old CD-1 and C57BL/6J mice as immunocompetent, neurodevelopmentally appropriate mouse models that can be used to examine CHIKV neuropathogenesis and immunopathogenesis following direct brain infection.