Reverse Genetics of Murine Rotavirus: A Comparative Analysis of the Wild-Type and Cell-Culture-Adapted Murine Rotavirus VP4 in Replication and Virulence in Neonatal Mice.

Small-animal models and reverse genetics systems are powerful tools for investigating the molecular mechanisms underlying viral replication, virulence, and interaction with the host immune response in vivo. Rotavirus (RV) causes acute gastroenteritis in many young animals and infants worldwide. Murine RV replicates efficiently in the intestines of inoculated suckling pups, causing diarrhea, and spreads efficiently to uninoculated littermates. Because RVs derived from human and other non-mouse animal species do not replicate efficiently in mice, murine RVs are uniquely useful in probing the viral and host determinants of efficient replication and pathogenesis in a species-matched mouse model. Previously, we established an optimized reverse genetics protocol for RV and successfully generated a murine-like RV rD6/2-2g strain that replicates well in both cultured cell lines and in the intestines of inoculated pups. However, rD6/2-2g possesses three out of eleven gene segments derived from simian RV strains, and these three heterologous segments may attenuate viral pathogenicity in vivo. Here, we rescued the first recombinant RV with all 11 gene segments of murine RV origin. Using this virus as a genetic background, we generated a panel of recombinant murine RVs with either N-terminal VP8* or C-terminal VP5* regions chimerized between a cell-culture-adapted murine ETD strain and a non-tissue-culture-adapted murine EW strain and compared the diarrhea rate and fecal RV shedding in pups. The recombinant viruses with VP5* domains derived from the murine EW strain showed slightly more fecal shedding than those with VP5* domains from the ETD strain. The newly characterized full-genome murine RV will be a useful tool for dissecting virus-host interactions and for studying the mechanism of pathogenesis in neonatal mice.

In vivo rescue of arboviruses directly from subgenomic DNA fragments.

Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals. We previously developed the "Infectious Subgenomic Amplicons" (ISA) method, which enables the rescue of single-stranded positive sense RNA viruses in vitro by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct in vivo generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method in vivo. Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2- fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20 ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses. This method provides an efficient strategy for in vivo rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains in vivo. It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.

Generation of Defective Interfering Particles of Morbilliviruses Using Reverse Genetics.

RNA viruses generate defective genomes naturally during virus replication. Defective genomes that interfere with the infection dynamics either through resource competition or by interferon stimulation are known as defective interfering (DI) genomes. DI genomes can be successfully packaged into virus-like-particles referred to as defective interfering particles (DIPs). Such DIPs can sustainably coexist with the full-length virus particles and have been shown to negatively impact virus replication in vitro and in vivo. Here, we describe a method to generate a clonal DI genome population by reverse genetics. This method is applicable to other RNA viruses and will enable assessment of DIPs for their antiviral properties.

Applying Reverse Genetics to Study Measles Virus Interactions with the Host.

The study of virus-host interactions is essential to achieve a comprehensive understanding of the viral replication process. The commonly used methods are yeast two-hybrid approach and transient expression of a single tagged viral protein in host cells followed by affinity purification of interacting cellular proteins and mass spectrometry analysis (AP-MS). However, by these approaches, virus-host protein-protein interactions are detected in the absence of a real infection, not always correctly compartmentalized, and for the yeast two-hybrid approach performed in a heterologous system. Thus, some of the detected protein-protein interactions may be artificial. Here we describe a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect protein partners during the infection (AP-MS in viral context). This way, virus-host protein-protein interacting co-complexes can be purified directly from infected cells for further characterization.

An improved system to generate recombinant canine distemper virus.

BACKGROUND: Canine distemper virus (CDV) is a pathogen with the capability of cross-species transmission. It has crossed the species barrier to infect many other species, and its host range is expanding. The reverse genetic platform, a useful tool for scientific research, allows the generation of recombinant viruses from genomic cDNA clones in vitro. METHODS: To improve the reverse genetic system of CDV, a plasmid containing three independent expression cassettes was constructed for co-expression of the N, P, and L genes and then transfected with a full-length cDNA clone of CDV into Vero cells. RESULTS: The results indicated that the established rescue system has the advantages of being more convenient, easy to control the transfection ratio, and high rescue efficiency compared with the conventional reverse genetics system. CONCLUSION: This method not only reduces the number of transfection plasmids, but also improves the rescue efficiency of CDV, which could provide a reference for the recovery of other morbilliviruses.

VP4 Mutation Boosts Replication of Recombinant Human/Simian Rotavirus in Cell Culture.

Rotavirus A (RVA) is the leading cause of diarrhea requiring hospitalization in children and causes over 100,000 annual deaths in Sub-Saharan Africa. In order to generate next-generation vaccines against African RVA genotypes, a reverse genetics system based on a simian rotavirus strain was utilized here to exchange the antigenic capsid proteins VP4, VP7 and VP6 with those of African human rotavirus field strains. One VP4/VP7/VP6 (genotypes G9-P[6]-I2) triple-reassortant was successfully rescued, but it replicated poorly in the first cell culture passages. However, the viral titer was enhanced upon further passaging. Whole genome sequencing of the passaged virus revealed a single point mutation (A797G), resulting in an amino acid exchange (E263G) in VP4. After introducing this mutation into the VP4-encoding plasmid, a VP4 mono-reassortant as well as the VP4/VP7/VP6 triple-reassortant replicated to high titers already in the first cell culture passage. However, the introduction of the same mutation into the VP4 of other human RVA strains did not improve the rescue of those reassortants, indicating strain specificity. The results show that specific point mutations in VP4 can substantially improve the rescue and replication of recombinant RVA reassortants in cell culture, which may be useful for the development of novel vaccine strains.

Research Note: Rapid generation of a novel fowl adenovirus serotype 11 reverse genetic platform.

Fowl adenovirus serotype 11 (FAdV-11) is one of the main causative agents of inclusion body hepatitis (IBH) in broilers. Outbreaks of FAdV-11-related IBH have been increasingly reported in China and many other geographical areas worldwide. However, the critical virulence factors of FAdV-11 remain uncertain due to the lack of technical platforms for efficient manipulation of FAdV-11 genome. Here, we reported the establishment of a FAdV-11 reverse genetic system based on a novel FAdV-11 Chinese isolate FJSW/2021 using the exonuclease combined with RecET (ExoCET), Redαß recombineering and ccdB counter-selection techniques for the first time. A recombinant FAdV-11 was rescued efficiently by using the established reverse genetic platform through swapping the ORF11 gene of the FAdV-11 FJSW/2021 with the ZsGreen fluorescent protein expression cassette. This study provides an effective technical platform for identifying virulence factors of FAdV-11 and developing recombinant FAdV-11-vectored vaccine candidates.

Genomic profile of eGFP-tagged senecavirus A subjected to serial plaque-to-plaque transfers.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. This virus possesses a positive-sense, single-stranded RNA genome, approximately 7200 nt in length, composed of a single 5' untranslated region, encoding region and 3' untranslated region. In this study, a recombinant SVA tagged with enhanced green fluorescent protein (eGFP) sequence, rSVA-eGFP, was rescued from its cDNA clone using reverse genetics. The passage-5 (P5) rSVA-eGFP was totally subjected to 55 rounds of consecutive fluorescent plaque-to-fluorescent plaque (FP-FP) transfers, and one extra common passaging in vitro. The P61 viral stock was analyzed by next-generation sequencing. The result showed ten single-nucleotide mutations (SNMs) in the rSVA-eGFP genome, including nine transitions and only one transversion. The P61 progeny still showed a complete eGFP sequence, indicating no occurrence of copy-choice recombination within the eGFP region during serial FP-FP transfers. In other words, this progeny was genetically deficient in the recombination of eGFP sequence (RES), namely, an RES-deficient strain. Out of ten SNMs, three were missense mutations, leading to single-amino acid mutations (SAAMs): F15V in L protein, A74T in VP2, and E53R in 3D protein. The E53R was predicted to be spatially adjacent to the RNA channel of 3D protein, perhaps involved in the emergence of RES-deficient strain. In conclusion, this study uncovered a global landscape of rSVA-eGFP genome after serial FP-FP transfers, and moreover shed light on a putative SAAM possibly related to the RES-deficient mechanism.

Impact of the H274Y Substitution on N1, N4, N5, and N8 Neuraminidase Enzymatic Properties and Expression in Reverse Genetic Influenza A Viruses.

The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.

Production of OSU G5P[7] Porcine Rotavirus Expressing a Fluorescent Reporter via Reverse Genetics.

Rotaviruses are a significant cause of severe, potentially life-threatening gastroenteritis in infants and the young of many economically important animals. Although vaccines against porcine rotavirus exist, both live oral and inactivated, their effectiveness in preventing gastroenteritis is less than ideal. Thus, there is a need for the development of new generations of porcine rotavirus vaccines. The Ohio State University (OSU) rotavirus strain represents a Rotavirus A species with a G5P[7] genotype, the genotype most frequently associated with rotavirus disease in piglets. Using complete genome sequences that were determined via Nanopore sequencing, we developed a robust reverse genetics system enabling the recovery of recombinant (r)OSU rotavirus. Although rOSU grew to high titers (~107 plaque-forming units/mL), its growth kinetics were modestly decreased in comparison to the laboratory-adapted OSU virus. The reverse genetics system was used to generate the rOSU rotavirus, which served as an expression vector for a foreign protein. Specifically, by engineering a fused NSP3-2A-UnaG open reading frame into the segment 7 RNA, we produced a genetically stable rOSU virus that expressed the fluorescent UnaG protein as a functional separate product. Together, these findings raise the possibility of producing improved live oral porcine rotavirus vaccines through reverse-genetics-based modification or combination porcine rotavirus vaccines that can express neutralizing antigens for other porcine enteric diseases.

A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation.

Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology. IMPORTANCE: The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

Development of reverse genetics system for Guanarito virus: substitution of E1497K in the L protein of Guanarito virus S-26764 strain changes plaque phenotype and growth kinetics.

Guanarito virus (GTOV) is the causative agent of Venezuelan hemorrhagic fever. GTOV belongs to the genus Mammarenavirus, family Arenaviridae and has been classified as a Category A bioterrorism agent by the United States Centers for Disease Control and Prevention. Despite being a high-priority agent, vaccines and drugs against Venezuelan hemorrhagic fever are not available. GTOV S-26764, isolated from a non-fatal human case, produces an unclear cytopathic effect (CPE) in Vero cells, posing a significant obstacle to research and countermeasure development efforts. Vero cell-adapted GTOV S-26764 generated in this study produced clear CPE and demonstrated rapid growth and high yield in Vero cells compared to the original GTOV S-26764. We developed a reverse genetics system for GTOV to study amino acid changes acquired through Vero cell adaptation and leading to virus phenotype changes. The results demonstrated that E1497K in the L protein was responsible for the production of clear plaques as well as enhanced viral RNA replication and transcription efficiency. Vero cell-adapted GTOV S-26764, capable of generating CPE, will allow researchers to easily perform neutralization assays and anti-drug screening against GTOV. Moreover, the developed reverse genetics system will accelerate vaccine and antiviral drug development.IMPORTANCEGuanarito virus (GTOV) is a rodent-borne virus. GTOV causes fever, prostration, headache, arthralgia, cough, sore throat, nausea, vomiting, diarrhea, epistaxis, bleeding gums, menorrhagia, and melena in humans. The lethality rate is 23.1% or higher. Vero cell-adapted GTOV S-26764 shows a clear cytopathic effect (CPE), whereas the parental virus shows unclear CPE in Vero cells. We generated a reverse genetics system to rescue recombinant GTOVs and found that E1497K in the L protein was responsible for the formation of clear plaques as well as enhanced viral RNA replication and transcription efficiency. This reverse genetic system will accelerate vaccine and antiviral drug developments, and the findings of this study contribute to the understanding of the function of GTOV L as an RNA polymerase.

Construction of a Mycoviral Infectious Clone for Reverse Genetics in Botrytis cinerea.

The most important advances in our understanding of the viral life cycle, such as genome replication, packaging, transmission, and host interactions, have been made via the development of viral infectious full-length clones. Here, we describe the detailed protocols for the construction of an infectious clone derived from Botrytis virus F (BVF), a mycoflexivirus infecting the plant pathogenic fungus Botrytis cinerea, the determination of the complete sequence of the cloned mycovirus, the preparation of fungal protoplasts, and the transfection of protoplasts using transcripts derived from the BVF infectious clone.

A Breakthrough in Kitavirids: Genetic Variability, Reverse Genetics, Koch's Postulates, and Transmission of Hibiscus Green Spot Virus 2.

Hibiscus green spot virus 2 (HGSV-2), a member of the genus Higrevirus (family Kitaviridae), is a positive-stranded RNA virus associated with leprosis-like symptoms in citrus and green spots on leaves in hibiscus. HGSV-2 has only been reported in Hawaii, and while it is speculated that mites in the genus Brevipalpus might be responsible for its transmission, proper transmission assays have yet to be conducted. This study characterizes additional citrus and hibiscus isolates of HGSV-2 collected from two Hawaiian Islands. We constructed an infectious cDNA clone from a hibiscus isolate of HGSV-2 collected on Oahu and demonstrated its ability to infect several experimental hosts, including Phaseolus vulgaris, Nicotiana tabacum, and N. benthamiana, as well as natural hosts, Citrus reticulata and Hibiscus arnottianus. Bacilliform virions with varied sizes of 33 to 120 nm (length) and 14 to 70 nm (diameter) were observed in partially purified preparations obtained from agroinoculated leaves. Virus progeny from the infectious cDNA clone was found to be infectious after mechanical transmission to N. benthamiana and to cause local lesions. Finally, an isoline colony of the mite Brevipalpus azores had vector competence to transmit a citrus isolate of HGSV-2 collected from Maui to citrus and hibiscus plants, demonstrating the mite-borne nature of HGSV-2. The infectious cDNA clone developed in this study is the first reverse-genetics system for a kitavirid and will be fundamental to better characterize basic biology of HGSV-2 and its interactions with host plants and mite vectors.

A large sequenced mutant library - valuable reverse genetic resource that covers 98% of sorghum genes.

Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat-tolerant C4 species, requires a vast, large-scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large-scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)-induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis-regulatory elements. A comparison of the distribution of mutations in the large-scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS-induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5' UTR, gene body, and 3'-UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the Ka /Ks value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB (https://www.depts.ttu.edu/igcast/sorbmutdb.php) and SorghumBase (https://sorghumbase.org/). This current large-scale sequence-indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

Reverse Genetics of Bat Influenza A Viruses.

New World fruit bats were recently found to harbor two distinct and previously unknown influenza A viruses (IAVs) of the subtypes H17N10 and H18N11. Although viral genome sequences were detected in the liver, intestine, lung, and kidney of infected bats and the complete genome sequences have been isolated from their rectal swab samples, all attempts to isolate an infectious virus from bats in nature have failed. The lack of an infectious bat IAV isolate was overcome by reverse genetic approaches that led to the generation of an infectious virus in vitro. Using such synthetic bat IAVs enabled the identification of their unconventional cell entry via major histocompatibility complex II (MCH-II) molecules and their ability to replicate in mice, ferrets, and bats. Importantly, we also showed that these synthetic recombinant bat IAVs are not able to reassort with conventional IAVs, preventing the acquisition of enhanced transmission properties in non-bat species by reassortment with conventional IAVs. As authentic viruses are key for understanding the molecular biology of bat IAVs, in this chapter, we describe our recently established reverse genetics protocol for generating H17N10 and H18N11 in vitro. This step-by-step protocol starts with cloning of cDNA copies of the viral RNA segments into reverse genetics plasmids, followed by the generation of a highly concentrated stock and finally a method to determine viral titers.

Reverse Genetics Systems for Filoviruses.

Filoviruses are causative agents of severe hemorrhagic fevers with high case fatality rates in humans. For studies of virus biology and the subsequent development of countermeasures, reverse genetic systems, and especially those facilitating the generation of recombinant filoviruses, are indispensable. Here, we describe the generation of recombinant filoviruses from cDNA.

Reverse Genetics System for Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is an important mosquito-borne virus that can cause severe disease manifestations in humans including ocular damage, vision loss, late-onset encephalitis, and hemorrhagic fever. In ruminants, RVFV can cause high mortality rates in young animals and high rates of abortion in pregnant animals resulting in an enormous negative impact on the economy of affected regions. To date, no licensed vaccines in humans or anti-RVFV therapeutics for animal or human use are available. The development of reverse genetics has facilitated the generation of recombinant infectious viruses that serve as powerful tools for investigating the molecular biology and pathogenesis of RVFV. Infectious recombinant RVFV can be rescued entirely from cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis and generate live-attenuated vaccines. In this chapter, we will describe the experimental procedures for the implementation of RVFV reverse genetics.

Reverse Genetics Systems for the De Novo Rescue of Diverse Members of Paramyxoviridae.

Paramyxoviruses place significant burdens on both human and wildlife health; while some paramyxoviruses are established within human populations, others circulate within diverse animal reservoirs. Concerningly, bat-borne paramyxoviruses have spilled over into humans with increasing frequency in recent years, resulting in severe disease. The risk of future zoonotic outbreaks, as well as the persistence of paramyxoviruses that currently circulate within humans, highlights the need for efficient tools through which to interrogate paramyxovirus biology. Reverse genetics systems provide scientists with the ability to rescue paramyxoviruses de novo, offering versatile tools for implementation in both research and public health settings. Reverse genetics systems have greatly improved over the past 30 years, with several key innovations optimizing the success of paramyxovirus rescue. Here, we describe the significance of such advances and provide a generally applicable guide for the development and use of reverse genetics systems for the rescue of diverse members of Paramyxoviridae.

Plasmid-Based Lassa Virus Reverse Genetics.

Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public health problem in their endemic regions. The Old World (OW) mammarenavirus Lassa virus (LASV) is estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of Lassa fever (LF) cases, a disease associated with high morbidity and mortality. No licensed vaccines are available to combat LASV infection, and anti-LASV drug therapy is limited to the off-label use of ribavirin whose efficacy remains controversial. The development of reverse genetics approaches has provided investigators with a powerful approach for the investigation of the molecular, cell biology and pathogenesis of mammarenaviruses. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in viral genome replication and gene transcription, assembly, and budding, which has facilitated the identification of several anti-mammarenavirus candidate drugs. Likewise, it is possible now to rescue infectious recombinant mammarenaviruses from cloned cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of viral pathogenesis. Reverse genetics have also allowed the generation of mammarenaviruses expressing foreign genes to facilitate virus detection, to identify antiviral drugs, and to generate live-attenuated vaccine (LAV) candidates. Likewise, reverse genetics techniques have allowed the generation of single-cycle infectious, reporter-expressing mammarenaviruses to study some aspects of the biology of HF-causing human mammarenavirus without the need of high security biocontainment laboratories. In this chapter, we describe the experimental procedures to generate recombinant (r)LASV using state-of-the-art plasmid-based reverse genetics.