A 54-year-old male, who worked as a butcher of the slaughterhouse in Hunan Province, China, was admitted to the Neurology Intensive Care Unit (NICU) of Second Xiangya Hospital, Central South University, due to recurrent fever for a duration of 8 days and convulsions with impaired consciousness for 3 days. He obtained some minor cuts on his left hand during the autopsy process of a dead swine 14 days before his initial symptom occurred. The initial symptoms (day 1) presented with cough and fever (the highest temperature of 39 C). No other neurological symptoms were presented at that time. He was then admitted to the local hospital and was diagnosed with upper respiratory tract infection, receiving an antibiotic treatment. However, his condition was not improving. On day 5, after initial symptoms, he developed fuzzy consciousness, headache, fecal, and urinary incontinence. Plain brain computer tomography (CT) was normal. Analysis of cerebrospinal fluid (CSF) showed white blood cells (WBC) of 4 x 106/L (0-5 x 106/L) and protein content of 0.63 g/L (0.15-0.45 g/l) (Table 1). The results of CSF further indicated that it was an early phase of viral meningitis. He was given an antiviral treatment (Acyclovir and Ribavirin) and antibiotic therapy (Piperacillin/tazobactam). On day 6, he developed facial and limb convulsions and was treated with anti-epileptic therapy (sodium valproate and midazolam). He was given an emergency orotracheal intubation and ventilator-assisted ventilation, due to shortness of breath. On day 8, after the initial symptoms, he was referred to our hospital due to status epilepticus and disturbance of consciousness. On admission, he presented with a body temperature of 39 C, heart rate of 107 bpm (beats per minute), blood pressure of 155/94 mmHg, and oxygen saturation (SpO2) of 99% at an inspired oxygen concentration of 30% with mechanical ventilation, and no spontaneous breaths were detected. Glasgow Coma Scale (GCS) was E1VTM1. Pupils were both 3 mm in diameter. The left pupil was sluggishly reactive to light and the reflex of the right one disappeared. Although he had neck stiffness and Kernig signs, long tract signs, such as hyperreflexia, Hoffman's signs, as well as Babinski signs were not detected. Some minor cuts were found on his left hand (Supplementary Figure 1). Laboratory findings were WBC 12.01 x 109/L (3.5-9.5 x 109/L), hemoglobin (Hb) 127 g/dl (115-150 g/dl), sodium 148.3 mmol/L (135-145 mmol/l), potassium 3.4 mmol/L (3.5-5.5 mmol/l), creatinine 89 mumol/L (62-115 mumol/L), albumin 39.7 g/L (40-55 g/l), alanine transaminase (ALT) 90.6 U/L (9-50 U/l), and aspartate transaminase (AST) 49.3 U/L (15-40 U/l). Serological tests for HIV, TP, hepatitis B/C, and TB (T-SPOT) were negative. The cytomegalovirus antibody IgG (CMV-IgG) of 19.300 IU/mL, herpes simplex virus type I antibody (HSV) IgG of 26.76 COI (positive), rubella virus antibody IgG (RU-IgG) of 20.800 IU/mL, and Toxoplasma gondii antibody IgG (TOX-IgG) of <0.130 IU/ml were found. The IgM against all pathogens was negative. Lumbar puncture indicated an opening pressure of 290 mmH2O (80-180 mmH2O), CSF showed WBC of 10 x 106/L (0-5 x 106 /L), protein of 0.37 g/L (0.15-0.45 g/L) and glucose of 3.05 mmol/L (synchronous blood glucose of 9.7 mmol/L) (Table 1). Besides, CSF latex agglutination test (LAT), microbiological tests including the Gram/acid-fast bacillus (AFB) were negative, as well as the autoimmune encephalitis antibodies including NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABABR, IgLON5, DPPX, GAD65, GlyR1, DRD2, mGluR5, mGluR1, and Neurexin-3a, which were tested based on a cell-based assay (Kingmed Diagnostics Co., Ltd.). On day 8, Plain CT brain imaging was still normal after initial symptoms (admission day) (Figure 1A). With the suspicion of viral meningitis on admission, he was treated with Acyclovir (500 mg, Q8h), 20% Mannitol (125 ml, Q8h), Glycerol fructose (250 ml, Q12h), Piperacillin/tazobactam (4.5 g, Q8h), fluid therapy and nutritional support. The epileptic seizures of the patient were difficult to bring under control, propofol was used intravenously to stop status epilepticus. Meanwhile, oral antiepileptic therapy including Levetiracetam and Oxcarbazepine were applied. On day 11, after initial symptoms, pathogen reads PRV was detected in CSF, blood, and aqueous humor, using PACEseq mNGS, so we accordingly added Phosphonoformate (3 g, Q8h) combined with Acyclovir (500 mg, Q8h) to enhance the antiviral treatment. However, the condition of the patient did not see any significant improvement. On day 15, after initial symptoms, Plain CT brain imaging showed patchy hypo-density in the bilateral frontotemporal lobe (Figure 1B). Methylprednisolone Sodium Succinate (120 mg, Qd) was intravenously given for 7 days, followed by oral prednisone (60 mg, Qd) treatment. There was no improvement of the consciousness of the patient. Even when all the sedatives used to control epileptic were stopped, he was still in deep coma. On day 19, CT brain imaging showed multiple patchy hypo-density in right basal ganglia, left thalamus, bilateral frontotemporal lobe, and bilateral insular lobe (Figure 1C). On day 24, the lesion site of CT brain is basically the same as before (Figure 1D), which indicated that the present treatment might not be effective. During hospitalization, the patient was unable to wean off mechanical ventilation, although the pulmonary infection had been basically healed. Given that the prognosis of the patient was extremely poor, the patient was voluntarily discharged to local hospital for palliative treatment.
A PACEseq mNGS detection (Hugobiotech, Beijing, China) was performed. The QIAamp DNA Micro Kit (QIAGEN) was used to extract DNA. The library was then built using QIAseq  Ultralow Input Library Kit (Illumina, San Diedo, CA, USA). The library quality was assessed using Qubit (Thermo Fisher) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and was finally sequenced on Nextseq 550 platform (Illumina, San Diedo, CA, USA). Short, low-quality, and low-complexity reads were removed from the raw data. The human DNA was also filtered out by alignment to human reference database (hg38) using bowtie 2. The remaining reads were finally aligned to Microbial Genome Databases (ftp://ftp.ncbi.nlm.nih.gov/genomes/) by BWA. A total of 652 unique reads mapped to the Suid alphaherpesvirus 1 genome out of 15,970 microbial reads (Figure 2A) with a genome coverage rate of 25.3% (Figure 2B) in CSF sample, whereas only one unique read of Suid alphaherpesvirus 1 in blood sample was detected, which might be due to the low concentration of viral DNA in blood. Notably, mNGS of aqueous humor sample detected 1,566 unique reads (0.42%, 1,566/368,878) (Figure 2C) aligned to the Suid alphaherpesvirus 1 genome with a genome coverage rate of 35% (Figure 2D). Furthermore, PCR targeting glycoprotein B (gB) gene of Suid alphaherpesvirus 1 showed that fragments of about 194 bp in length were successfully amplified from CSF and aqueous humor samples (Figure 2E). The identity of the fragment sequence obtained from a Sanger sequencing, against the reference sequence of Suid alphaherpesvirus 1 (GenBank accession no. MK806387.1) was 99% (Figure 2F). However, a negative PCR result was found in blood sample due to the low viral load. The above results confirmed the presence of Suid alphaherpesvirus 1 in CSF and aqueous humor samples.