Introduction
Methods
Migratory water birds are key reservoirs of avian influenza viruses
(AIV). The fact that AIV are maintained in aquatic bird populations by
water-borne transmission is well established but information on
survival of AIV in aquatic habitats is not sufficient to completely
understand the epidemiology of these viruses. The present study was
designed to assess and compare the survival of AIV in different water
types: distilled water (DW), normal saline (NS) and surface water
(SW) at a wide range of temperatures, including very low
temperatures, for an extended period of time (months).
Three LPAI viruses were used in the trials (H4N6, H5N1, and H6N8). Tenacity was
tested in DW, NS and SW obtained from Lake Constance. The viruses were mixed
at a ratio of 1:10 for DW and NS and 1:100 for SW and incubated at –10, 0, 10, 20,
and 30 °C. Virus quantitation was carried out by titr ation on MDCK. Results were
confirmed by HA. The samples were tested for a maximum of 36 weeks. For all of
the groups, average infectivity titers of duplicate samples were calculated and serial
data thus obtained was analyzed by a linear regression model. The estimated
persistence of viral infectivity with a starting viral concentration of 106 as well as T90 values (time required for one log reduction in the virus titer) were calculated
using this model.
Results
Table 1. Comparison of T-90* values of three AIV in various water types at 5 different
temperatures
Germany/03 (H4N6)
Surface water analysis:
The total bacterial count in the SW was 1.15x102 colony forming
units/ml. which increased in the virus inoculated samples stored at
30, 20 and 10°C but did not change at 0 and -10 °C.
Virus persistence
Linear regression models to demonstrate the expected persistence
of the tested AIV in various water types at specified temperatures
are presented in Fig. 1,2,3. In order to have a better comparison of
all treatment groups, T-90 values are shown in Table 1.
Germany/05 (H5N1)
Germany/07 (H6N8)
Temperature
DW
NS
SW
DW
NS
SW
DW
NS
SW
30 °C
8
6
2
5
6
2
13
2
2
20 °C
15
12
4
14
13
3
37
7
3
10 °C
121
59
14
85
68
10
197
70
14
0 °C
443
190
31
383
185
35
558
169
34
-10 °C
642
321
55
576
236
58
594
233
66
*T-90: Time duration in days required for 90 % reduction in viral infectivity
DW = Distilled water, pH 7.80, NS = Normal saline, pH of 7.20, SW = Surface water collected from Lake Constance
Fig. 1: Linear regression models showing tenacity of avian influenza virus type H4N6 at various temperatures; () Surface water, () Normal saline, () Distilled water
Fig. 2: Linear regression models showing tenacity of avian influenza virus type H5N1 at various temperatures; () Surface water, () Normal saline, () Distilled water
Fig. 3: Linear regression models showing tenacity of avian influenza virus type H6N8 at various temperatures; () Surface water, () Normal saline, () Distilled water
Conclusions
The results of the present study suggest that AIV vary in their sensitivity to inactivation in aquatic habitats and their persistence is inversely proportional to the
water temperature. The water type plays a large role in tenacity and viral persistence is shorter in surface water as compared to sterile distilled water and
normal saline. Influenza viruses may remain infective in surface water for periods ranging from a few days to several months depending upon the environmental
temperature. These findings underline the importance of water as a factor in the maintenance and spread of AIV infections.
ACKNOWLEDGEMENTS:
Viruses used in this study were kindly provided by PD Dr. Timm Harder, Friedrich-Loeffler-Institut, Insel Riems, Germany. This study was financed by a grant
from the Ministerium für Ernährung und Ländlichen Raum Baden-Württemberg, Germany in the research program “Wildvögel und Vogelgrippe”.