Conceived and designed the experiments: XW JZ ST. Performed the experiments: XW. Analyzed the data: XW. Contributed reagents/materials/analysis tools: ZY. Wrote the paper: XW IH.
The authors have declared that no competing interests exist.
Avian H5N1 influenza viruses cause severe disease and high mortality in infected humans. However, tissue tropism and underlying pathogenesis of H5N1 virus infection in humans needs further investigation. The objective of this work was to study viremia, tissue tropism and disease pathogenesis of H5N1 virus infection in the susceptible ferret animal model. To evaluate the relationship of morbidity and mortality with virus loads, we performed studies in ferrets infected with the H5N1 strain A/VN/1203/04 to assess clinical signs after infection and virus load in lung, brain, ileum, nasal turbinate, nasal wash, and blood. We observed that H5N1 infection in ferrets is characterized by high virus load in the brain and and low levels in the ileum using real-time PCR. In addition, viral RNA was frequently detected in blood one or two days before death and associated with symptoms of diarrhea. Our observations further substantiate pathogenicity of H5N1 and further indicate that viremia may be a bio-marker for fatal outcomes in H5N1 infection.
In 1997, the avian influenza A virus subtype H5N1, which is a highly pathogenic H5N1 avian influenza initially confined to poultry, crossed the avian-human species barrier
The primary pathologic process that causes death is fulminant viral pneumonia
To evaluate virologic characteristics in susceptible ferrets, 24 ferrets (12/group) were challenged with either 1 FLD50 (50% ferret lethal dose) or 10 FLD50 of the H5N1 strain A/VN/1203/04 on Day 0, since these doses were known to cause symptomatic infection to varying degrees in the susceptible animal models. All ferrets were observed to be clinically normal on Day 0. Clinical signs of infection were initially observed on Day 2 postinfection with most animals exhibiting signs by Days 3 or 4 postinfection. Clinical signs observed in both groups included diarrhea, nasal discharge, hypoactivity and recumbency (
Day Postinfection | ||||||||||||
Group | Clinical Sign | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
10 FLD50 | Diarrhea | 2 |
2 | 1 | 2 | 1 | 1 | |||||
Discharge | 5 | 7 | 9 | 7 | 7 | 3 | 2 | 1 | ||||
Hypoactive | 1 | 1 | ||||||||||
Recumbent | 1 | 1 | ||||||||||
Found dead | 2 | 1 | 3 | 1 | 1 | 1 | ||||||
1 FLD50 | Diarrhea | 1 | 1 | 1 | 2 | 2 | 1 | 1 | ||||
Discharge | 3 | 4 | 9 | 7 | 6 | 5 | 4 | 2 | 2 | |||
Hypoactive | 3 | 4 | 1 | 1 | 1 | 1 | ||||||
Recumbent | 1 | 1 | 1 | |||||||||
Found dead | 1 | 1 | 1 | 1 |
Note:
indicates number of surviving ferrets exhibiting clinical sign on the day postinfection.
We examined all clinical symptoms during the course of infection to characterize the disease caused by H5N1 infection and to identify characteristics that explain the severity of infection. We observed high fevers (39.3∼41.5°C in the 10 FLD50 group and 38.3∼41.3°C in the 1 FLD50 group) on Day 2 or Day 7 postinfection (
There was a greater increase in weight loss and body temperatures for longer duration in the 10 FLD50 group compared to the 1 FLD50 group. In the latter group, ferrets regained body weight after Day 7 and their body temperature returned to normal after Day 8 post-infection, suggesting that some animals in this group recovered from the disease. Not surprisingly, the virus dose of 1 FLD50 was not sufficient to cause death in all ferrets, as the majority remained alive on Day 11 post-infection, the day of scheduled sacrifice. Using the FLD50 assay, we found that virus could be detected in the nasal washes more frequently in animals in the 10 FLD50 group compared with those in the 1 FLD50 group (
(A) Changes in body temperatures of infected ferrets. Ferrets were inoculated with either 1 FLD50 or 10 FLD50 of the viruses, and body temperatures were monitored daily by the use of subcutaneous implantable temperature transponders for 11 days postinfection. Each data point represents the mean value ± SD for the surviving ferret(s). Note: only one animal remained alive in the 10 FLD50 group (one value) on Day 9 postinfection. (B). Changes in weights of ferrets infected with the viruses. The weights of ferrets were measured daily. The loss or gain of weight was calculated for each ferret as the percent change in the initial mean starting weight on day 0. Values are the averages ± SD for the ferret(s) alive for each group. (C) Virus titer in nasal wash with FLD50 assay. Nasal washes were performed on Day 2, Day 4, and Day 6 postinfection in selection of ferrets in both groups using FLD50 assay.
The severity of clinical signs and symptoms, including diarrhea, induced by infection with H5N1 indicated a rapid spread of the virus4 confirmed by virus detection in multiple organs, including the brain (
Day Postinfection | ||||||
Group | Organ | 4 | 5 | 6 | 7 | 8 |
Brain | 8.51×107±1.16×108 |
6.23×107 |
3.88×107±5.27×107 | 3.80×107±4.30×107 | ||
10 FLD50 | Lung | 2.95×107±1.71×106 | 2.06×104 |
1.10×106±9.13×105 | 8.51×106±1.20×105 | |
N.T. | 1.88×104±2.20×104 | 3.40×104±2.28×104 | ||||
Ileum | 4.44×103±3.32×103 | 485±337 | ||||
Brain | 3.94×107 |
7.76×107 |
3.11×107±4.59×106 | |||
1 FLD50 | Lung | 2.70×103 |
1.09×105 |
3758±2822 | ||
N.T. | 9.33×104 |
5.50×105 |
8138±1094 | |||
Ileum | 4.36×103 |
3.47±1.08 |
Note:
Known concentrations of A/VN/1203/04 viral RNA (serially diluted: 108 to 10 fg) were used as templates and quantitative RT-PCR performed to generate a standard curve. 25∼30 mg of tissue from each organ was used for extraction of viral RNA and dissolved in 50 µl. 3 µl of the vRNA was used as template to perform real-time PCR. Each value represents the average concentration of six reactions based on the standard curve.
indicates value for 1 animal.
It has been previously reported that virus could be detected in blood in severely ill patients
Group | Animal code | Viremia |
Diarrhea day postinfection | Died |
15 | 2 | 3 | 4 | |
23 | 2 | 3 | 4 | |
28 | 2 | 6 | ||
10 FLD50 | 17 | 6 | 7 | |
32 | 6 | 4 | 7 | |
33 | 6 | 6 | 7 | |
34 | 6 | 8 | ||
1 FLD50 | 29 | 2 | 3 | 5 |
26 | 4 | 6 |
Note:
the day postinfection when virus was initially detected in the ferret blood sample.
the day postinfection when the animal was found dead.
H5N1 virus related influenza remains a relatively novel disease with poorly understood pathology and pathogenesis, and only a limited number of reports describing pathological findings in human H5N1 cases have been published
Thus far, several hundred human infections with avian H5N1 viruses have been confirmed. The A/VN/1203/04 strain that we used in our study is a highly pathogenic isolate
Avian H5N1 strain A/VN/1203/04 was obtained from the Centers for Disease Control and Prevention (Atlanta, GA) (Lot Number: E3/E2 1/18/07) and amplified in viable 10–11 day old embryonated hen's eggs (S&G Poultry, Clanton, AL). The virus was maintained at −80°C until use in the study.
50 µl of allantoic fluid were harvested from euthanized hen's eggs and added to a microtiter plate. 50 µl of 0.5% turkey (tRBC) were then added to all wells and plates were incubated for 30 minutes at room temperature. Plates were read for agglutination or non-agglutination. The 50% endpoint was determined by the method of Reed and Muench17 from virus dilutions testing positive for hemagglutinin activity in Turkey Red Blood Cells (tRBC). Data were expressed as 50% egg infectious dose (EID50) per milliliter.
24 adult male ferrets (Triple F Farms, Sayre, PA) (The Southern Research IACUC has approved the animal care and use proposal (ACUP) #08-10-074B by IACUC Chairman, Larry Bowen) that were 6∼7 months of age and were seronegative for representative currently circulating human influenza A strains prior to shipment were used for the study. Ferrets were lightly anesthetized with a solution of ketamine/xylazine/atropine formulated to provide doses of 25 mg/kg ketamine, 1.7 mg/kg xylazine, and 0.05 mg/kg of atropine to each animal. The animals were inoculated intranasally with one ml of virus, approximately 500 µl to each nare; 12 of ferrets with 1×50% of ferret lethal dose (FLD50), which equals to a concentration of 10×108.5 (EID50/ml) viruses and others with ten times of FLD50/ml viruses. Clinical signs of infection, weight, and temperatures were recorded daily. Nasal wash samples were collected from all ferrets on Days 0 (approximately 12 hours post-dose), 1, 2, 4, 6, and 8 for viral load determination. A total of 0.5 ml from each animal were collected and used to determine the viral load using the EID50 assay. Nasal washes were performed by slowly dripping 0.5 ml of sterile solution of 1% v/v BSA, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamicin in Dulbecco's Phosphate-Buffered Saline into each nare, while allowing the animal to sneeze and/or attempt to blow the solution out of the nostril into a sterile Petri dish. Nasal wash samples were made into aliquots in cryovials and placed on dry ice immediately after collection. Samples were stored at −80°C until used.
A representative section of the left caudal lobe of the lung, nasal turbinate, spleen, brain, and ileum tissue samples were collected from each euthanized or deceased animal for viral load determination. Samples were snapped frozen in liquid nitrogen and stored at −80°C until analyzed for viral load determination. On Days 0, 1, 2, 4, and 6 postinfection, blood samples were collected from surviving ferrets, and stored at −80°C until used.
Nasal washes were thawed and cleared by centrifugation. The resulting supernatants were serially diluted (log10) in DPBS (Dulbecco's Phosphate-Buffered Salines with antibiotics, 100 U/ml Penicillin, l00 µg/ml Streptomycin and 50 µg/ml Gentamicin) (Invitrogen, Carlsbad, CA). The 50% endpoint was determined by the method of Reed and Muench
Quantitative real-time RT-PCR was also used for detection of virus load in the brain, lung, nasal turbinates, ileum, and blood. Nucleic acids were isolated by using the QIAamp Viral RNA Mini Kit (Valencia, CA 91355) according to the manufacturer's protocol. We designed a set of primers and probes for the matrix gene, M, of the avian H5N1 influenza A virus, according to GenBank database. The forward primer was
The authors wish to acknowledge Dr. Krishna Devadas, Dr. Mathew Sandbulte and Dr. Hira Nakhasi for critical review of this manuscript. The authors also wish to acknowledge Southern Research Institute for performing the animal infections. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.