Case Report: Metagenomic Next-Generation Sequencing for Diagnosis of Human Encephalitis and Endophthalmitis Caused by Pseudorabies Virus

PurposeThe objective of our study was to report a case of encephalitis and endophthalmitis caused by pseudorabies virus (PRV), identified using metagenomic next-generation sequencing (mNGS).Case PresentationA 54-year-old worker, from a swine slaughterhouse, developed signs of severe encephalitis, including fever, disturbance of consciousness, hypopnea, and status epilepticus, after finger injury at work. The PRV sequences were successfully identified from the blood, cerebrospinal fluid (CSF), and aqueous humor of the patient through mNGS, which was further verified using a Sanger sequencing.ConclusionOur case emphasizes the importance of mNGS in early diagnoses of infectious diseases, and gives a clue that PRV can spread across species and infect human. It is necessary to carry out a skin protection and education about disease prevention for people who have close contact with swine.

A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. Importance Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There’s a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

The recombinant pseudorabies virus expressing porcine deltacoronavirus spike protein is safe and effective for mice

Background Porcine deltacoronavirus (PDCoV) is a new pathogenic porcine intestinal coronavirus, which has appeared in many countries since 2012. PDCoV disease caused acute diarrhea, vomiting, dehydration and death in piglets, resulted in significant economic loss to the pig industry. However, there is no commercially available vaccine for PDCoV. In this study, we constructed recombinant pseudorabies virus (rPRVXJ-delgE/gI/TK-S) expressing PDCoV spike (S) protein and evaluated its safety and immunogenicity in mice. Results The recombinant strain rPRVXJ-delgE/gI/TK-S obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster syrian kidney-21 (BHK-21) cells and is safe to mice. After immunizing mice with rPRVXJ-delgE/gI/TK-S, the expression levels of IFN-γ and IL-4 in peripheral blood of mice were up-regulated, the proliferation of spleen-specific T lymphocytes and the percentage of CD4+ and CD8+ lymphocytes in mice spleen was increased. rPRVXJ-delgE/gI/TK-S showed good immunogenicity for mice. On the seventh day after booster immunity, PRV gB and PDCoV S specific antibodies were detected in mice, and the antibody level continued to increase, and the neutralizing antibody level reached the maximum at 28 days post- immunization (dpi). The recombinant strain can protect mice with 100% from the challenge of virulent strain (PRV XJ) and accelerate the detoxification of PDCoV in mice. Conclusion The recombinant rPRVXJ-delgE/gI/TK-S strain is safe and effective with strong immunogenicity and is expected to be a candidate vaccine against PDCoV and PRV.

First Detection of NADC34-like PRRSV as a Main Epidemic Strain on a Large Farm in China

The newly emerged sublineage 1.5 (NADC34-like) porcine reproductive and respiratory syndrome virus (PRRSV) has posed a direct threat to the Chinese pig industry since 2018. However, the prevalence and impact of NADC34-like PRRSV on Chinese pig farms is unclear. In the present study, we continuously monitored pathogens—including PRRSV, African swine fever virus (ASFV), classical swine fever virus (CSFV), pseudorabies virus (PRV), and porcine circovirus 2 (PCV2)—on a fattening pig farm with strict biosecurity practices located in Heilongjiang Province, China, from 2020 to 2021. The results showed that multiple types of PRRSV coexisted on a single pig farm. NADC30-like and NADC34-like PRRSVs were the predominant strains on this pig farm. Importantly, NADC34-like PRRSV—detected during the period of peak mortality—was one of the predominant strains on this pig farm. Sequence alignment suggested that these strains shared the same 100 aa deletion in the NSP2 protein as IA/2014/NADC34 isolated from the United States (U.S.) in 2014. Phylogenetic analysis based on open reading frame 5 (ORF5) showed that the genetic diversity of NADC34-like PRRSV on this farm was relatively singular, but it had a relatively high rate of evolution. Restriction fragment length polymorphism (RFLP) pattern analysis showed that almost all ORF5 RFLPs were 1-7-4, with one 1-4-4. In addition, two complete genomes of NADC34-like PRRSVs were sequenced. Recombination analysis and sequence alignment demonstrated that both viruses, with 98.9% nucleotide similarity, were non-recombinant viruses. This study reports the prevalence and characteristics of NADC34-like PRRSVs on a large-scale breeding farm in northern China for the first time. These results will help to reveal the impact of NADC34-like PRRSVs on Chinese pig farms, and provide a reference for the detection and further prevention and control of NADC34-like PRRSVs.

Meclizine Inhibits Pseudorabies Virus Replication by Interfering With Virus Entry and Release

Pseudorabies virus (PRV) is a pathogen that causes substantial economic losses to the swine industry. With the emergence and widespread of PRV variants since 2011 in China, current commercial vaccines cannot provide complete protection against PRV infection. Therefore, antiviral drugs may work as an alternative way to control and prevent PRV. In this study, the inhibitory effects and underlying molecular mechanisms of meclizine against PRV were studied. Meclizine displayed a significant inhibitory effect against PRV when it was added before, simultaneously with, or after virus infection. The inhibitory effect of meclizine occurred during viral entry and cell-to-cell spreading but not at viral attachment into PK-15 cells. Meclizine also inhibited viral particle release at the late stage of infection. The antiviral effect of meclizine was tested in mice, and the results showed that meclizine reduced the severity of clinical symptoms and the viral loads in tissues, and delayed the death, after PRV challenge. The above results indicated that meclizine had an inhibitory effect on PRV. Our findings will contribute to the development of potential therapeutic drugs against PRV infection.

Superinfection Exclusion of Alphaherpesviruses Interferes with Virion Trafficking

Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection interferes with secondary viral infections within that same cell. Although SIE has been observed across many viruses, it has remained relatively understudied. A recently characterized glycoprotein D (gD) -independent SIE of alphaherpesviruses presents a novel mechanism of co-infection restriction for Herpes Simplex Virus Type 1 (HSV-1) and Pseudorabies virus (PRV). In this study, we evaluated the role of multiplicity of infection (MOI), receptor expression, and trafficking of virions to gain greater insight into potential mechanisms of alphaherpesvirus SIE. We observed that high MOI secondary viral infections were able to overcome SIE in a manner that was independent of receptor availability. Utilizing recombinant viruses expressing fluorescent protein fusions, we assessed virion localization during SIE through live fluorescent microscopy of dual-labeled virions and localization of capsid assemblies. Analysis of these assemblies confirmed changes in the distribution of capsids during SIE. These results indicate that SIE during PRV infection inhibits viral entry or fusion while HSV-1 SIE inhibits infection through a post-entry mechanism. Although the timing and phenotype of SIE is similar between alphaherpesviruses, the related viruses implement different mechanisms to restrict coinfection.

Serological Investigation and Genetic Characteristics of Pseudorabies Virus in Hunan Province of China From 2016 to 2020

Pseudorabies (PR), caused by variant pseudorabies virus (PRV), is an economically important viral disease in China. Recently, PRV infection in humans has also received attention worldwide. To investigate the PRV infection in Hunan province, China, we collected a total of 18,138 serum specimens from 808 PRV-vaccinated pig farms cross this region during 2016–2020, and we detected the presence of PRV glycoprotein B (gB) and gE-specific antibodies. The enzyme-linked immunosorbent assay (ELISA) results revealed that 80.47% (14,596/18,138, 95 CI 79.9–81.0) and 23.55% (4,271/18,138, 95 CI 22.9–24.2) of serum samples were positive for PRV gB and gE-specific antibodies, respectively. Further analysis indicated that the seroprevalence of wild PRV infection was associated with the season and breeding scale (p < 0.01). In addition, five PRV strains were isolated from PRV-positive samples in Vero cells and the virus titers varied from 106.5 to 107.51 TCID50/0.1 ml. The phylogenetic analysis revealed that one isolate was a classical strain of PRV genotype II, and four other isolates belonged to the variants of genotype II. Collectively, the data indicate that the prevalence of PRV remains high in pigs in Hunan province, and the variant PRV strains are the major genotypes affecting the development of the pig industry.

The Valproic Acid Derivative Valpromide Inhibits Pseudorabies Virus Infection in Swine Epithelial and Mouse Neuroblastoma Cell Lines

Pseudorabies virus (PRV) infection of swine can produce Aujeszky’s disease, which causes neurological, respiratory, and reproductive symptoms, leading to significant economic losses in the swine industry. Although humans are not the natural hosts of PRV, cases of human encephalitis and endophthalmitis caused by PRV infection have been reported between animals and workers. Currently, a lack of specific treatments and the emergence of new PRV strains against which existing vaccines do not protect makes the search for effective antiviral drugs essential. As an alternative to traditional nucleoside analogues such as acyclovir (ACV), we studied the antiviral effect of valpromide (VPD), a compound derived from valproic acid, against PRV infection in the PK15 swine cell line and the neuroblastoma cell line Neuro-2a. First, the cytotoxicity of ACV and VPD in cells was compared, demonstrating that neither compound was cytotoxic at a specific concentration range after 24 h exposure. Furthermore, the lack of direct virucidal effect of VPD outside of an infected cell environment was demonstrated. Finally, VPD was shown to have an antiviral effect on the viral production of two strains of pseudorabies virus (wild type NIA-3 and recombinant PRV-XGF) at the concentrations ranging from 0.5 to 1.5 mM, suggesting that VPD could be a suitable alternative to nucleoside analogues as an antiherpetic drug against Aujeszky’s disease.

Evasion of I Interferon-Mediated Innate Immunity by Pseudorabies Virus

Type I interferon (IFN-I) mediated innate immunity serves as the first line of host defense against viral infection, ranging from IFN-I production upon viral detection, IFN-I triggered signaling pathway that induces antiviral gene transcription the antiviral effects of IFN-I induced gene products. During coevolution, herpesviruses have developed multiple countermeasures to inhibit the various steps involved to evade the IFN response. This mini-review focuses on the strategies used by the alphaherpesvirus Pseudorabies virus (PRV) to antagonize IFN-I mediated innate immunity, with a particular emphasis on the mechanisms inhibiting IFN-I induced gene transcription through the JAK-STAT pathway. The knowledge obtained from PRV enriches the current understanding of the alphaherpesviral immune evasion mechanisms and provides insight into the vaccine development for PRV control.