Molecular Biology and Clinical Aspects of Lassa Virus

Structure of Lassa Virus

• The virus is a single-stranded RNA virus (RNA virus – virus whose genetic material is RNA, not DNA) belonging to the virus family Arenaviridae (– a family of enveloped RNA viruses).
• The virion is spherical particles (virion – complete infectious virus particle) with an average diameter of 90–110 nm (nanometer – very small unit of length).
• Lassa virus is a single-stranded RNA virus that is enveloped in lipid (– surrounded by a lipid membrane derived from host cell) with glycoprotein spikes (– protein molecules with sugar groups attached) protruding from the outside surface.
• Glycoproteins on the surface of the virion form T-shaped spikes (– spike-like projections) extending 7–10 nm from the envelope.

Figure: Structure of Lassa Virus, Source: Swiss Institute of Bioinformatics

Genome of Lassa Virus

• It contains two species of RNA (– two separate RNA segments) called the small and large units, and each unit has two genes at opposite ends that do not overlap (– ambisense genome organization).
• The small unit has some double stranded areas (– regions where RNA pairs with itself) that form stem-loop structures (– folded RNA structures important for regulation).
• The large species of RNA encodes for the Z protein (– zinc-binding regulatory protein) and L protein (– RNA-dependent RNA polymerase) at the 5’ and 3’ ends (– ends of RNA strand) respectively, and the small species of RNA encodes for glycoprotein and nucleoprotein (– protein that binds viral RNA) at the 5’ and 3’ ends respectively.
• Lassa virus consists of four lineages (– genetically distinct groups), which have a strain variation of 27% in nucleotides and 15% in amino acids (– genetic and protein-level variation).
• The large segment encodes a small zinc-binding protein (Z) (– protein involved in viral regulation) that regulates transcription (– synthesis of RNA) and replication (– copying of viral genome) and the RNA polymerase (L) (– enzyme that synthesizes RNA).
• The small segment encodes the nucleoprotein (NP) (– RNA-binding protein) and the surface glycoprotein precursor (GP) (– inactive form of spike protein), also known as the viral spike, which is proteolytically cleaved (– cut by enzymes) into the envelope glycoproteins GP1 and GP2 that bind to the alpha-dystroglycan receptor (– host cell surface receptor) and mediate host cell entry (– virus entering the cell).
• The gene that encodes for the nucleoprotein is 1,710 nucleotides long, and the protein has 569 amino acids (– building blocks of proteins).
• The gene that encodes for the glycoprotein is 1,473 nucleotides long.

Figure: Genome of Lassa Virus, Source: Swiss Institute of Bioinformatics

Epidemiology of Lassa Virus

• The Lassa virus is so named because, in 1969, it was first isolated and correlated as the causative agent (– disease-causing agent) of Lassa fever in a small town called Lassa in North-eastern Nigeria.
• Lassa fever is endemic (– constantly present in a region) in parts of West Africa including Sierra Leone, Liberia, Guinea, and Nigeria; however, other neighboring countries are also at risk, as the animal vector (– organism that transmits disease) for Lassa virus, the multimammate rat (Mastomys natalensis) (– natural reservoir host), is distributed throughout the region.
• Lassa virus consists of four lineages, three of these lineages are located in Nigeria, while the other can be found in Guinea, Liberia, and Sierra Leone.
• The number of Lassa virus infections per year in West Africa is estimated at 100,000 to 300,000, with approximately 5,000 deaths.
• In some areas of Sierra Leone and Liberia, it is known that 10%–16% of people admitted to hospitals every year have Lassa fever, which indicates the serious impact (– high disease burden) of the disease on the population of this region.

Figure: Epidemiology of Lassa Virus, Source: CDC

Transmission of Lassa Virus

• Humans contract the virus primarily through contact with the contaminated excreta (– urine or feces containing virus) of Mastomys natalensis (– multimammate rat, natural host) rodents, which is the natural reservoir (– organism that harbors the virus) for the virus.
• The virus is transmitted to humans through cuts and scratches (– breaks in skin) or inhaled via dust particles (– airborne transmission) in the air.
• Secondary transmission (– human-to-human spread) of the virus between humans occurs through direct contact with infected blood or bodily secretions (– fluids released from the body).

Replication of Lassa Virus

• The Lassa virus gains entry into the host cell by means of the cell-surface receptor alpha-dystroglycan (alpha-DG) (– host receptor protein), a versatile receptor for proteins of the extracellular matrix (– structural network outside cells).
• After virus enters the cell by alpha-dystroglycan mediated endocytosis (– receptor-driven uptake into cell), a low-pH environment (– acidic condition) triggers pH-dependent membrane fusion (– fusion of viral and host membranes) and releases the RNP complex (viral ribonucleoprotein – viral RNA bound to proteins) into the cytoplasm (– fluid part of the cell).
• Viral RNA is unpacked, and replication and transcription (– genome copying and RNA synthesis) initiate in the cytoplasm.
• As replication starts, both S and L RNA genomes (– small and large viral RNA segments) synthesize the antigenomic S and L RNAs (– complementary RNA copies), and from the antigenomic RNAs, genomic S and L RNAs are synthesized.
• Both genomic and antigenomic RNAs (– original and complementary strands) are needed for transcription and translation (– RNA and protein synthesis).
• S RNA encodes GP (– glycoprotein) and NP (viral nucleocapsid protein – RNA-binding protein), and L RNA encodes Z (– regulatory protein) and L proteins (– RNA polymerase).
• The primary transcription (– first round of mRNA synthesis) first transcribes mRNAs (– messenger RNAs) from the genomic S and L RNAs, which code NP and L proteins, respectively.
• Transcription terminates (– stops) at the stem-loop (SL) structure (– folded RNA structure) within the intergenomic region (– region between genes).
• Arenaviruses (– virus family of Lassa virus) use a cap-snatching strategy (– stealing caps from host mRNA) to gain cap structures (– protective RNA modifications) from the cellular mRNAs, mediated by the endonuclease activity of the L polymerase (– RNA-cutting function) and the cap-binding activity of NP (– cap recognition by nucleoprotein).
• Antigenomic RNA transcribes viral genes GPC and Z, encoded in genomic orientation (– gene direction), from S and L segments, respectively.
• After translation of GPC (– glycoprotein precursor), it is post-translationally modified (– chemically altered after synthesis) in the endoplasmic reticulum (– protein processing organelle).
• GPC is cleaved (– cut enzymatically) into GP1 and GP2 at the later stage of the secretory pathway (– protein transport system).
• Cleaved glycoproteins (– processed spike proteins) are incorporated into the virion envelope (– viral outer membrane) when the virus buds and releases (– exits the cell) from the cell membrane.

Pathogenesis of Lassa Virus

• When initiating an infection, the Lassa virus attaches to a receptor on the cell surface with the glycoprotein GP-1 (– attachment protein).
• Its initial sites of replication include dendritic cells (DC) (– antigen-presenting immune cells) and macrophage–monocyte cells (– immune cells involved in phagocytosis), and it is then delivered throughout the entire body.
• Infected DC fail to secrete proinflammatory cytokines (– immune signaling molecules), do not upregulate costimulatory molecules (– signals needed for T-cell activation) such as CD40, CD80, and CD86, and poorly induce proliferation of T cells (– multiplication of immune cells).
• Lassa virus prevents a host’s innate immune system (– first line of immune defense) by NP activity (– immune-suppressive action of nucleoprotein).
• Patients infected with LASV (– Lassa virus) produce IgM and IgG antibody isotypes (– early and long-term antibodies).
• Neutralizing antibodies (– antibodies that block infection) appear months after acute infection (– initial severe phase) is resolved, and the titers (– antibody concentration) are often low.
• The neutralizing antibody titers continue to rise even several months after convalescence (– recovery period) has been established, which may indicate constant stimulation of B cells (– antibody-producing cells) due to low levels of virus persistence (– remaining virus in body).
• Antibodies in seroconverted individuals (– people who developed antibodies) are specific to GPC, NP, and, likely, Z protein (– viral regulatory protein).

Clinical Manifestations of Lassa Virus

• The incubation period (– time between infection and symptoms) of Lassa fever ranges from 6–21 days.
• The spectrum of clinical effects (– range of disease severity) manifested in Lassa fever ranges from asymptomatic (– without symptoms) infection to multi-organ system failure (– failure of multiple organs) and death.
• The onset of the disease, when it is symptomatic, is usually gradual, starting with fever, general weakness, and malaise (– general feeling of discomfort).
• After a few days, headache, sore throat, muscle pain, chest pain, nausea, vomiting, diarrhea, cough, and abdominal pain may follow.

• In severe cases, facial swelling, fluid in the lung cavity (– pleural effusion), bleeding from the mouth, nose, vagina or gastrointestinal tract (– hemorrhagic manifestations), and low blood pressure (– hypotension) may develop.
• Shock (– circulatory failure), seizures (– abnormal electrical brain activity), tremor (– involuntary shaking), disorientation (– confusion), and coma (– loss of consciousness) may be seen in the later stages.
• Death from Lassa fever most commonly occurs 10 to 14 days after symptom onset.

Diagnosis of Lassa Virus

• Detection of IgM and IgG antibodies (– early and long-term immune antibodies) as well as Lassa antigen (– viral protein) by enzyme-linked immunosorbent serologic assays (ELISA) (– antibody-based diagnostic test).
• The virus can be uncovered using reverse transcription PCR (RT-PCR) (– molecular method to detect viral RNA).
• Virus isolation by cell culture, however, this procedure should only be done in a high-containment laboratory (– biosafety level laboratory) with good laboratory practices (– safety protocols). Mice and guinea pigs have been evaluated as models of LASV infection (– experimental animals for study).
• Immunohistochemistry (– antibody-based tissue staining method), performed on formalin-fixed tissue specimens (– preserved tissues), can be used to make a post-mortem diagnosis (– diagnosis after death).

Treatment of Lassa Virus

• Ribavirin (– antiviral drug) is only effective if administered early in infection, within the first 6 days after disease onset.

Prevention and Control of Lassa Virus

• No vaccine (– preventive immunization) for Lassa fever is currently available for use in humans.
• Prevention by promoting good community hygiene (– cleanliness practices at community level) to discourage rodents (– disease-carrying animals) from entering homes.
• Effective measures include storing grain and other foodstuffs in rodent-proof containers, disposing of garbage far from the home, maintaining clean households, and keeping cats (– rodent control).
• Avoiding contact with blood and body fluids (– infection source) while caring for sick persons.
• In health-care settings, staff should always apply standard infection prevention and control precautions (– universal safety measures) when caring for patients, regardless of their presumed diagnosis. These include basic hand hygiene, respiratory hygiene, use of personal protective equipment (– PPE such as gloves and masks), safe injection practices, and safe burial practices.