Reassortant Cache Valley Virus Associated With Acute Febrile, Nonneurologic Illness, Missouri

Abstract

An adult male from Missouri sought care for fever, fatigue, and gastrointestinal symptoms. He had leukopenia and thrombocytopenia and was treated for a presumed tickborne illness. His condition deteriorated with respiratory and renal failure, lactic acidosis, and hypotension. Next-generation sequencing and phylogenetic analysis identified a reassortant Cache Valley virus.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. Since the virus was first isolated from Culiseta species mosquitoes in Cache Valley, Utah, it has been isolated from a broad range of mosquito species, including Aedes, Anopheles, Coquillettidia, Culex, and Culiseta genera, throughout parts of the United States (US), Canada, Central America, and the Caribbean [1, 2]. To date, 5 human cases of CVV disease have been reported in the literature [3–7]. All reported cases had either meningitis or meningoencephalitis, with 3 patients dying. All cases resided in or traveled to the upper Midwest or eastern half of the US prior to symptom onset. Here we report the first case of CVV disease with nonneurologic presentation occurring in an individual infected in Missouri with a reassortant strain of the virus.

CASE REPORT

The patient was a man in his 60s from northwest Missouri with a history of diabetes mellitus, coronary artery disease, and history of thymectomy for a malignant thymoma. He initially presented in mid-September 2015 with a 5-day history of generalized weakness, fevers (up to 40°C), chills, rigors, cough, abdominal pain, anorexia, nausea, diarrhea, and vomiting. In the 2 weeks before becoming ill, the patient reported being bitten by mosquitoes around his home and when fishing in a neighboring county. More than 2 weeks prior to his illness, he also reported pulling off an embedded tick.

On examination, he was noted to be alert and oriented with epigastric tenderness and trace ankle edema. His temperature was 37.9°C, blood pressure was 105/61 mm Hg, pulse rate was 100 beats/minute, respiratory rate was 20 breaths/minute, and oxygen saturation was 97% on room air. Initial laboratory findings showed leukopenia (3970 cells/μL) with 78% polymorphonuclear cells and 18% immature cells, thrombocytopenia (53 000 cells/μL), hyponatremia (sodium 131 mmol/L), elevated creatinine level (1.4 mg/dL [normal range, 0.6–1.2 mg/dL]), elevated lipase (314 U/L [normal range, 23–300 U/L), and normal liver function tests. He was initially started on doxycycline and piperacillin-tazobactam for possible acute pancreatitis and ehrlichiosis. Subsequent evaluation on the day of admission noted worsening thrombocytopenia (45 000 cells/μL) and increasing lactate dehydrogenase (maximum level, 1158 U/L). Antibiotic coverage was extended, adding vancomycin and levofloxacin, due to concern for worsening sepsis and the patient was transferred to a tertiary care center.

At the tertiary care center, the patient was febrile (temperature 38.3°C) with a respiratory rate of 15 breaths/minute and oxygen saturation of 92% on 5 L of oxygen. On physical examination, he was coherent and was noted to have left lower lung crackles and edema bilaterally in his lower extremities. Laboratory testing showed a normal leukocyte count (4900 cells/μL) and worsening thrombocytopenia (35 000 cells/μL). His creatinine level was 1.8 mg/dL, blood urea nitrogen was 50 μg/dL, and his aspartate aminotransferase (AST) level was 85 U/L (normal range, 17–59 U/L). Peripheral smear did not show significant schistocytes.

Within 12 hours, the patient’s condition declined with worsening dyspnea and increasing oxygen needs to maintain his saturations at 93%–97%. He also developed lactic acidosis and became acutely hypotensive (mean arterial pressures in the 40s) and delirious. He was transferred to the intensive care unit (ICU) for management of septic shock with hypoxemic respiratory failure of unknown etiology, where he was intubated and placed on vasopressors. A lumbar puncture performed on his second day in the ICU showed no evidence of inflammation, with a white blood cell count of 1 cell/μL. Head magnetic resonance imaging obtained 2 days later showed no abnormal enhancement. Chest radiographs and chest computed tomography with contrast noted intermittent bibasilar atelectasis with small bilateral pleural effusions. He continued to have thrombocytopenia (range, 49 000–61 000) and elevated AST (maximum level, 161 U/L) while in the ICU. The patient slowly improved and was extubated after 6 days. He was transferred to the medical ward and was eventually discharged to a rehabilitation facility 14 days after being admitted.

Results of comprehensive evaluations for infectious diseases—including serologic testing of acute and convalescent samples for Rocky Mountain spotted fever, tularemia, leptospirosis, herpes simplex viruses, and syphilis; molecular testing for Ehrlichia species and cytomegalovirus; and antigen testing for legionellosis—were negative except for evidence of previous spotted fever rickettsioses. Blood, sputum, and urine bacterial and fungal cultures were negative. Whole blood specimens collected 6 and 14 days after illness onset were sent to the Centers for Disease Control and Prevention (CDC) (Fort Collins, Colorado) for Heartland and Bourbon virus testing as part of an active institutional review board–approved protocol.

At CDC, blood and serum were tested for Heartland and Bourbon viral RNA and neutralizing antibodies by real-time reverse-transcription polymerase chain reaction (RT-PCR) and plaque reduction neutralization test (PRNT) with 6-well plates with confluent Vero E6 and Vero monolayers based on the growth characteristics of these viruses [8–10]. Additional confirmatory PRNTs were performed on Vero cells. Standard virus isolation methods were used with 200 μL of undiluted and 1:10 dilutions of blood or serum specimens inoculated onto confluent Vero cells in T25 flasks. Inoculated flasks were incubated at 37°C and reviewed for cytopathic effect (CPE) daily.

Supernatants collected from first passage of virus isolation cell cultures were subjected to next-generation sequencing (NGS) methods using the Ion Torrent PGM sequencer (Life Technologies, Grand Island, New York). Following identification of viral sequences, RT-PCR was designed to target the newly derived sequences and applied to the patient’s samples [11]. Alignments were generated on deduced nucleotide open-reading frames from multiple genomic segments of selected viruses of the same viral family by Clustal W codon within the MEGA 7 software package (http://www.megasoftware.net/). Phylogenetic reconstruction by Bayesian inference was completed using BEAST version 1.8.4 and segment reassortment was evaluated on concatenated genomes using the Recombination Detection Program (RDP) as previously described [12]. Generated sequences for small, medium, and large segments have been deposited into GenBank (S: MK861965; M: MK861966; L: MK861967).

Blood and serum showed negative results for Heartland and Bourbon viral RNA and antibodies. However, virus isolation methods showed substantial CPE at day 2 postinoculation in cells inoculated with specimens collected 6 days after illness onset. These findings were confirmed by repeated isolation attempts. NGS methods applied to cell culture supernatants from first passage isolations identified CVV. We observed 98% overall average nucleotide sequence identity with CVV in multiple genomic segments. Blood and serum samples were verified as the source of the CVV by RT-PCR detection of viral RNA in these samples. Finally, a 14-day serum sample tested by PRNT yielded a CVV-specific neutralizing antibody titer of 20 480.

Maximum clade credibility phylogenetic trees were generated for the nucleotide open-reading frame of each RNA segment (Figure 1). The small and medium RNA segment phylogenies placed the CVV isolated from this patient into lineage II with high support. The large RNA segment formed a well-supported monophyletic clade with lineage I CVV. Evaluation of concatenated genomes using RDP software confirmed that this CVV isolate represented an interlineage reassortant virus.

Figure 1.

Bayesian maximum clade credibility trees of Cache Valley viruses and selected orthobunyaviruses. Nucleotide coding sequences of the small (a), medium (b), and large (c) genome segments are presented. The virus sequenced in this study is labeled with an asterisk. Viruses are labeled with virus names, isolate designation where applicable, and GenBank accession numbers. Branches are labeled with the posterior probabilities, and scale bar depicts nucleotide substitutions per site. Abbreviation: CVV, Cache Valley virus.

Although CVV has been identified in mosquitoes throughout the US and human serosurveys have found seroprevalence rates ranging from 3% to 19% in selected areas [1, 2, 13, 14], there only have been 5 cases of CVV disease reported in the literature to date [3–7]. All 5 case-patients were adults who resided or traveled to the upper Midwest or eastern half of the US and were reported to have either meningitis or meningoencephalitis with documented cerebrospinal fluid (CSF) pleocytosis. Two of the 5 patients previously described were immunocompromised and had CVV identified in their brain tissues or CSF by RT-PCR and immunohistochemical staining several weeks to years following their acute neurologic illness [6, 7]. The other 3 patients were not immunocompromised and had CVV cultured from their CSF or blood from 2 to 9 days after illness onset and CVV antibodies detected several weeks after infection [3–5]. Our patient from Missouri had similar signs and symptoms of the previously described cases but did not have any overt neurologic signs other than delirium. He also had no evidence of enhancement on neuroimaging or evidence of CSF pleocytosis. His serum obtained 6 days after illness onset was culture positive while his day 14 serum sample contained high levels of CVV-specific neutralizing antibodies. Our case-patient was originally believed to have a tick-borne illness given his leukopenia and thrombocytopenia, but he failed to improve on broad-spectrum antibiotics and developed septic shock, similar to the initial cases of CVV disease in the US [3].

Recent phylogenetic analysis of CVV separated the virus isolates into 2 distinct lineages [2]. Lineage II viruses were first recognized in 2008 in Mexico and later identified in 2010 in the northeastern US. Since then, lineage II viruses were believed to have replaced lineage I viruses in the US [2]. The phylogenetic analysis of the virus sequence from our patient confirmed 2 distinct lineages of CVV with lineage II small and medium RNA segments, but lineage I large segment, indicative of a segment reassortment event. Segment reassortment is a well-documented phenomenon reported in arboviruses with segmented genomes [15]. This is the first detection of CVV representing the lineage I genome prior to 2014, and the first isolate to be identified as an interlineage reassortant. Further work is needed to determine if other reassortant viruses might be present in the same or other regions and what role the vectors and reservoirs might play in influencing how the viruses reassort.

Healthcare providers should consider CVV infection in patients with meningitis, encephalitis, or severe sepsis during late spring through late fall, particularly when tests are negative for more common infectious disease causes, and advise patients on mosquito bite prevention (eg, using repellent). Further case identification is needed to determine the disease incidence, seasonality, geography, and full clinical spectrum associated with CVV infections.